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https://openalex.org/W4311673964	https://www.nature.com/articles/s41598-022-25985-7.pdf	English	null	Family caregivers’ perspectives of cultural beliefs and practices towards mental illness in Zambia: an interview-based qualitative study	Scientific reports	2,022	cc-by	8,905	Family caregivers’ perspectives of cultural beliefs and practices towards mental illness in Zambia: an interview‑based qualitative study F Sichimba1 A C Janlöv2 & A Khalaf 2* OPEN F. Sichimba 1, A.‐C. Janlöv 2 & A. Khalaf 2* Many elements of mental illness, including accessibility and utilization of mental health care, are influenced by societal cultural ideas. In Zambia, for example, traditional healers are not recognized, yet a large segment of the population continues to use their services due to their conviction. Despite this, studies on cultural beliefs and practices regarding mental illness in Zambia are scarce. Thus, this study is contextualized in Lusaka, Zambia, with the goal of filling a gap in the knowledge by exploring the cultural beliefs and practices surrounding mental illness as experienced by family caregivers caring for a next-of-kin with mental illness. Using a qualitative exploratory design, a purposeful sample of 15 family caregivers of next-of-kins diagnosed with mental illness were recruited. Data were collected via individual interviews, and analyzed using qualitative thematic analysis. The analysis revealed four main themes: (1) prevailing beliefs on cause of mental illness; (2) encountering social support and neglect; (3) recognizing the need of professional help; and (4) seeking culturally influenced help. Findings show that traditional attributions (for example, being bewitched, demon possession and sexual relations with uncleansed widows) are deeply embedded in beliefs and descriptions of what causes mental illness. These beliefs were found to influence not only help-seeking practices but also how people perceived and related to families. Given that beliefs influence caregiver help-seeking, these findings have implications for culturally sensitive practice. The study recommends that public health practitioners consider cultural beliefs and practices when developing health promotion programs, and public health messages. Abbreviations CSO Central Statistics Office COVID-19 Coronavirus disease 2019 MDAC Mental Disability Advocacy Centre WHO World Health Organization Abbreviations CSO Central Statistics Office COVID-19 Coronavirus disease 2019 MDAC Mental Disability Advocacy Centre WHO World Health Organization Culture and its impact on mental health has recently become a topic in Public Health debates. In the last two decades, there has been renewed interest in understanding cultural beliefs on mental illness1. However, cultural beliefs and practices around mental illness in Zambia remain largely unknown yet socio-cultural beliefs can contribute to the risk of stigmatization among people living with mental illness and affects mental health service utilization2. In Zambia, there are limited mental facilities with only seven general hospitals offering psychiatric services. www.nature.com/scientificreports www.nature.com/scientificreports 1Department of Psychology, University of Zambia, P.O. Box 32379, Lusaka, Zambia. 2Faculty of Health Sciences, Kristianstad University, Elmetorpsvägen 15, 291 88 Kristianstad, Sweden. email: atika.khalaf@hkr.se www.nature.com/scientificreports/ Literature has shown that, though mental health problems in the traditional African belief systems are attributed to spiritual sources13,14, there is a lack of attention to traditional understanding of health and belief systems in most of the studies conducted on mental illness. The Social Ecological Model by Bronfenbrenner15, underscores the importance of social environment in understanding illness. According to model, the social environment, namely the microsystem, mesosystem, exosystem, macrosystem and chronosystem, has an influence on human behavior and development. Thus, the Social Ecological Model provokes public health practitioners to look beyond the individual and focus on beliefs informed by culture if mental illness and help- seeking behavior are to be understood. This study uses the ecological model to understand caregiver’s beliefs and practice as influenced by the environment.i pl y Culture is defined as a "set of explicit and implicit guidelines that individuals inherit as members of a particular society, and that tells them how to view the world, experience it emotionally, and behave in it in relation to other people, supernatural forces or gods, and the natural environment”. Culture also provides the "current generation with a way of transmitting these guidelines to the next generation—by the use of symbols, language, art, and rituals"16. The coming of European missionaries and colonists in the early nineteenth century brought with it several changes in values, not only in the way of life but also in medical provision. This change meant everything that was Zambian was evil, primitive, and at worst uncivilized17. Thus, traditional African understanding of health care was pushed outside conventional medical service provision and discourse. Mental health provision was also not spared from these changes as conventional mental health practice was given prominence much to the exclusion of traditional cultural viewpoint of mental illness18. Despite the prevalence of modern mental health services, culture continues to influence how people conceptualize the cause of mental illness in general, which may explain the variation in preferred treatment methods. However, most studies on mental illness tend to focus on professional facts and knowledge while ignoring the belief systems that people inherit from their cultures. Opare-Henaku and Utsey19 in their study among the Akan people of Ghana underscored that mental illness is a culturally laden phenomenon and beliefs about aetiology vary across cultures. www.nature.com/scientificreports/ to provide care for sick family members6. Family caregivers are primary carers for people living with mental disorders and serve as source of emotional, physical (helping to eat, bath, take medication and bedside caring of individual is a norm) and provide much needed financial support associated with mental health treatment7. Despite widespread involvement of family in caring for next-of-kin living with mental illness, no studies to our knowledge have explored cultural beliefs and practices (the way they handle and deal with persons with mental illness) among family caregivers in Zambia are limited.h to provide care for sick family members6. Family caregivers are primary carers for people living with mental disorders and serve as source of emotional, physical (helping to eat, bath, take medication and bedside caring of individual is a norm) and provide much needed financial support associated with mental health treatment7. Despite widespread involvement of family in caring for next-of-kin living with mental illness, no studies to our knowledge have explored cultural beliefs and practices (the way they handle and deal with persons with mental illness) among family caregivers in Zambia are limited.h g y g Globally, about one billion people live with mental illness8. The changing global landscape has exacerbated the mental health challenge still further. For example, armed conflicts, HIV, poverty, globalization, local changes, and now COVID 19 exacerbate the risk of mental illness. By 2015, the World Health Organization reported that one in every four people is likely to be afflicted with mental illness and this figure was projected to rise by 15% in the year 2020. According to the World Health Organization, one out of every eight persons is impacted by mental disorders9. In Africa, the situation remains unclear owing to the lack of statistics on the mental illness burden in most countries. In Zambia, for example, there are no statistics on mental illness; nonetheless, hospital evidence suggests that 3.61 and 1.8 per 10,000 people are serviced by the hospitals’ catchment area for acute psychotic episodes and schizophrenia, respectively10.h p y p p p y The impact of mental illness in most developing countries is worsened by less comprehensive coverage of the mental health system which often covers a small population11. This situation is further compounded by cultural beliefs that limit people from accessing mental health services as the majority prefer to seek help from traditional healers12. Family caregivers’ perspectives of cultural beliefs and practices towards mental illness in Zambia: an interview‑based qualitative study F Sichimba1 A C Janlöv2 & A Khalaf 2 OPEN Coupled with this, the country also has a deficit of trained personnel with literature showing that as of 2014, the country had only three psychiatrists for a population of 13 million3. However, despite the limited psychiatric hospitals, mental services such as assessment, rehabilitation, inpatient and outpatient treatment services are available but not utilized the way they should due to reliance on traditional medicine4. y y Family involvement in patient care is well-documented in literature5. In most developing societies, caring for the sick by family members is widespread. For example, in Zambia, family caregivers are traditionally expected \| https://doi.org/10.1038/s41598-022-25985-7 Scientific Reports \| (2022) 12:21388 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Similarly, Kirmayer and Swartz20 points to the inherent weakness in the medical model as it failed to consider culture, thus advocating the need to understand the social underpinnings and local variability of mental illness. p g y Studies on cultural beliefs about mental illness in Zambia are scarce. According to Ventevogel et al.21, scholars cannot afford to ignore cultural understanding if holistic mental health care is to be provided. Aim. The aim of this study was to explore cultural beliefs and practices of family caregivers caring for a next- of-kin with mental illness. Methods Study design. A qualitative exploratory design was used for this study because of its ability to broaden our understanding of people’s beliefs, experiences, and behavior22. Methods In addition, four of the participants were single; ten were married and one divorcee. In terms of education level; three had primary education, five had secondary education, and seven had tertiary education. The median caregiver age was 48 years, with the minimum age of 25 years and maximum age of 56 years. Seven out of the 15 caregivers were taking care of their children (of which five were sons and two were daughters) while four caregivers were taking care of their brothers, three caregivers were taking care of their spouses, and one provided care for a nephew. Data collection method. The information sheets were distributed by the principal investigator to the family caregivers accompanying their next-of-kin to the targeted hospital’s outpatient department. Family caregivers who met the inclusion criteria and were considered to be eligible participants were then recruited. Before indicating their interest in taking part in the study, the family caregivers were also provided verbal information and urged to ask the researcher any questions they might have. Participants were given the option to select an appropriate day and time for the interviews when they registered to participate.h pp p y y g p p Individual interviews were used to collect data for the study. The use of in-depth interviews is a common data collection approach in health services research25; it enabled the researchers to explore caregiver’s beliefs and practices. An interview guide (Table 1) was developed in line with the research aim of this study and later piloted to ascertain its usefulness in answering the central research question. Following the pilot research, no changes to the interview guide were made. The guide provided topics within which the interviewer (the Principal Investigator) freely asked questions, probed, and made follow-up questions where clarification, further explanation, or illustrative examples were needed. Open-ended questions allowed participants to freely express their opinions while also allowing the researcher to collect detailed descriptions of beliefs and practices. Examples of questions see interview guide below:h g The interviews were conducted at Chainama Hills Mental Hospital, in a quiet private room, free from distraction. Each interview lasted between 45 and 60 min and was digitally recorded and transcribed by the first author. When no new information was given by the last four participants, the data was assumed to have reached saturation and no new interviews were conducted. Data analysis. Methods Give me any suggestions based on what you have been through that you think will help families caring for next of kin living with mental illness? Table 1. Interview guide. 1. Can you please tell me about what the term mental illness mean to you? 2. Share with me what happened when your next of kin first showed signs of the illness Probe: Kindly share your experiences before and after you found out? What you thought about the condition and how it came about What people said about condition? Treatments that were sought and why? What made you decide to take your next of kin to the hospital? 3. In your community/neighborhood, tell me how people view mental illness? Probe: Kindly share your experiences before and after you found out? What you thought about the condition and how it came about What people said about condition? Treatments that were sought and why? What made you decide to take your next of kin to the hospital? 4. Please share with me the reactions of neighbors, relatives and other members of the community when they found out that your next of kin had mental illness 5. Give me any suggestions based on what you have been through that you think will help families caring for next of kin living with mental illness? Table 1. Interview guide. caring for their loved one living with mental illness, and familiarity with the subject under study. Additionally, the purposive sampling aimed for a recruitment of a more diverse caregiver sample such as gender, age, relationship to the person with mental illness, and type of mental illness in their care. No formal data was collected about the mental illness diagnoses, but according to the family caregivers their next-of-kin suffered from a range of mental illnesses including schizophrenia, bipolar disorder, and substance-related psychosis. caring for their loved one living with mental illness, and familiarity with the subject under study. Additionally, the purposive sampling aimed for a recruitment of a more diverse caregiver sample such as gender, age, relationship to the person with mental illness, and type of mental illness in their care. No formal data was collected about the mental illness diagnoses, but according to the family caregivers their next-of-kin suffered from a range of mental illnesses including schizophrenia, bipolar disorder, and substance-related psychosis. Of the 15 caregivers who participated, nine were male and six were female. Methods Study design. A qualitative exploratory design was used for this study because of its ability to broaden our nderstanding of people’s beliefs, experiences, and behavior22. Setting. The research was carried out in Lusaka, Zambia. The estimated total population of the city stands at approximately 1.2 million and is one of the fastest growing populations in Africa23. Lusaka hosts the biggest mental hospital in Zambia. The hospital is the country’s largest referral mental health facility with a bed capacity of 210. Mental health services provided by hospital include psychiatric treatment, rehabilitation, and general care services to both inpatient and outpatient. Schizophrenia, affective disorders, alcohol-related issues, psychotic episodes, and organic brain abnormalities are among the mental illnesses treated in the hospital4. As of 2017, Zambia has only 6 psychiatrists, 3 psychologists and 1.43 mental health nurses per 100,000 people24. Just like the rest of Zambia, some barriers to mental health services utilization in Lusaka include dependence on traditional healers, poverty, lack of awareness and culture4. Participants. To explore beliefs and practices, a total of fifteen (15) participants who were living with and caring for a next-of-kin diagnosed with mental illness were recruited in September, 2021 from Chainama Hills Mental Hospital in Lusaka, the out-patient department’s waiting room. Purposive sampling methodology allowed the researchers to select only caregivers who met the inclusion criteria of being primary caregivers, https://doi.org/10.1038/s41598-022-25985-7 Scientific Reports \| (2022) 12:21388 \| www.nature.com/scientificreports/ T bl I d 1. Can you please tell me about what the term mental illness mean to you? 2. Share with me what happened when your next of kin first showed signs of the illness Probe: Kindly share your experiences before and after you found out? What you thought about the condition and how it came about What people said about condition? Treatments that were sought and why? What made you decide to take your next of kin to the hospital? 3. In your community/neighborhood, tell me how people view mental illness? Probe: Kindly share your experiences before and after you found out? What you thought about the condition and how it came about What people said about condition? Treatments that were sought and why? What made you decide to take your next of kin to the hospital? 4. Please share with me the reactions of neighbors, relatives and other members of the community when they found out that your next of kin had mental illness 5. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Table 2. An overview of the sub-themes and themes regarding experiences of family caregivers’ beliefs and practices when caring for next-of-kin with mental illness diagnosis. Sub-themes Themes Viewing supernatural forces as cause of mental illness Prevailing beliefs about cause of mental illness Attributing cause of mental illness to individual’s behavior Viewing life circumstances as cause of mental illness Receiving support from others Encountering social support and neglect Struggling with culturally influenced attitudes Trying to manage next of kin’s behavior Recognizing the need of professional help Trying to manage own tiredness Receiving advice from well-wishers Seeking spiritual intervention Seeking culturally influenced help Seeking traditional help as culturally advised Seeking medical help is a fast and convenient solution Table 2. An overview of the sub-themes and themes regarding experiences of family caregivers’ beliefs and practices when caring for next-of-kin with mental illness diagnosis. Sub-themes Themes Viewing supernatural forces as cause of mental illness Prevailing beliefs about cause of mental illness Attributing cause of mental illness to individual’s behavior Viewing life circumstances as cause of mental illness Receiving support from others Encountering social support and neglect Struggling with culturally influenced attitudes Trying to manage next of kin’s behavior Recognizing the need of professional help Trying to manage own tiredness Receiving advice from well-wishers Seeking spiritual intervention Seeking culturally influenced help Seeking traditional help as culturally advised Seeking medical help is a fast and convenient solution Table 2. An overview of the sub-themes and themes regarding experiences of family caregivers’ beliefs and practices when caring for next-of-kin with mental illness diagnosis. themes were formed in line with a manifest/descriptive level of analysis (see the Supplemental file for examples of the analysis). hemes were formed in line with a manifest/descriptive level of analysis (see the Supplemental file for examples f the analysis). Ethical approval. The research was carried out in accordance with the Helsinki Declaration’s recommendations27. Ethical approval and permission for this study were obtained from the University of Zambia, School of Humanities and Social Sciences Ethical committee (number HSSREC:2021-MAY-024). Prior to data collection, all participants were availed information about the study and its purpose both orally and in written, after which consent was obtained. In addition, study participants were informed of the voluntary nature of their participation, right to withdraw from the research without any explanation and were assured that no harm would result on account of their participation28. www.nature.com/scientificreports/ Participants were also assured that the data obtained was for research purposes only, their confidentiality would be secured, and that no personal details were going to be reported27. Because of the sensitive nature of the mental illness, it was envisaged that interviews might evoke psychological stress; thus, the author had a referral system in place for family caregivers that needed counseling a while after. Follow-up with the referral services after the study revealed that no participant used the service. Literature has shown that reflexivity involves critical self-reflections of how the researcher’s position, social background, and assumptions impact the research process29. In qualitative research, where the researcher has control over the process adopting a reflexive stance was important. Having been born and bred in Zambia, and familiar with prevailing attitudes, it was important to take a reflexive stance. The researcher strived to not influence the study with self-bound values and pre-understanding biases. Thus, to record the researchers’ pre-understanding and reflections, a diary was employed before the data collection process. This diary was not used as a data collection method and thus not analyzed further. Methods According to Braun and Clarke26, thematic analysis involves identification, analysis, and reporting of patterns (themes) within the data. Because thematic analysis is subjective, a triangulation process was initiated in which all three authors participated separately, in different phases of the analysis process. Identifying meaning units, condensing and coding them, and developing subthemes and, finally, themes were all part of this process26. The first step was to thoroughly read the transcripts in order to become acquainted with the data. Thereafter, statements with specific meaning to the research goal (meaning units) were identified and condensed. The condensed units aided in the development of the codes. Following that, the analyzed codes were grouped based on similarities and differences to form subthemes, which were then grouped into themes. All three authors then reviewed the analyzed data to ensure agreement on the final sub-themes and themes. The Scientific Reports \| (2022) 12:21388 \| https://doi.org/10.1038/s41598-022-25985-7 www.nature.com/scientificreports/ illness spell is a route used by jealous people to thwart ones’ progress or success. Caregivers explained when someone is successful and doing well, or making progress in their work or business, people who are jealous are able to cast a mental illness spell through spiritual possession. illness spell is a route used by jealous people to thwart ones’ progress or success. Caregivers explained when someone is successful and doing well, or making progress in their work or business, people who are jealous are able to cast a mental illness spell through spiritual possession. Attributing cause of mental illness to individual’s behavior. Caregivers’ narratives mirrored the belief that the afflicted individual is responsible and wholly to be blamed for their mental illness on account of their behavior. Thirteen of the fifteen caregivers explained that bad deeds such as disregard for authority of elders, adultery, substance use, failure to follow procedures when using charms as well as stealing someone’s property caused mental illness. For example, unfaithfulness in marriage was said to attract the wrath of God which manifested in form of mental illness. Also, lack of special regard for elders was reported to attract a bad omen of mental illnesses by eleven caregivers. Lastly, overuse of substances such as alcohol, drugs, and other substances were irresponsible behaviors mentioned by five caregivers that they believed led to mental illness. One of the caregivers pointed out that: “drinking beer too much and smoking too much … causes mental illness” (Caregiver 4). Viewing life circumstances as cause of mental illness. Seven caregivers shared the beliefs that life circumstances can lead to mental illness. This culturally influenced belief reflected mental illness as stemming from life situations that were beyond individual control such as heredity, poverty, and life stressors. Caregivers reported that challenges of life such as poverty, losing a loved one, and general stress of life made people to over-think and worry resulting into mental illness. A participant reflected this belief saying: “Problems of life can make one think too much and this can lead to mental illness” (Caregiver 11). Additionally, three caregivers described how mental illness runs in some families, thus people who have the misfortune of being born in such families find themselves suffering from mental illness. For example, as one caregiver noted: “Mental illnesses are generational. Encountering social support and neglect. Encountering social support and neglect in caregiving yielded from receiving support from others and struggling with culturally influenced negative attitudes. Encountering social support and neglect. Encountering social support and neglect in caregiving yielded from receiving support from others and struggling with culturally influenced negative attitudes. Receiving support from others. More than half of caregivers acknowledged getting help from close friends, family members, and neighbors. Support from others included emotional, material, and financial support to the family. Practices such as being emotionally supported through encouragement, gestures of goodwill, provision of material, and financial support in form of helping caregivers with transport or helping them to buy prescribed drugs and taking care of the next-of-kin were all expressed by the interviewed participants. For example, one caregiver said: “My neighbors have been helpful. They encourage me, provide advise on how to take care of her and usually help to bring her to the hospital, sometimes with money” (Caregiver 8). Struggling with culturally influenced negative attitudes. While some caregivers shared supportive tendencies, neglectful tendencies were also reported. Eight caregivers described practices mirroring struggle with culturally influenced negative attitudes targeted towards the family as well as the person with mental illness. For instance, a caregiver described: “People blame my husband for his illness, claiming that he went to a Satanist to seek riches, but the charms backfired. Others even avoid our home because they believe we are Satanists.” (Caregiver 6).h fi y g Further, caregivers reported blaming practices as common and experienced on a daily basis. They described how their sick next-of-kin suffered mistreatment and were frequently viewed as useless, ineffective, and unproductive. Consequently, society did not perceive mentally ill persons as human beings and treated them as outsiders. Caregivers also indicated that their next-of-kin were subjected to public ridicule, thus caregivers often locked them in the house to shield them. Stigma was described as an everyday experience for their next-of-kin as they were perceived as aggressive and could erupt anytime; thus, the practice was to avoid them. Given these practices, caregivers reported struggling every day to negotiate negative attitudes, practices, and stereotypical beliefs even within their own families and community in a quest to take care of their ill next-of-kin. Having a person with mental illness in their homes changed not only how people related to them but also how they were perceived as a family. Recognizing the need of professional help. This theme is contextualized on the practice around decision-making in seeking professional help from the hospital. www.nature.com/scientificreports/ It could be that the illness has been in the family and someone is unfortunate to have inherited it” (Caregiver 1). Results Th h The thematic analysis yielded themes and sub-themes related to beliefs about and practices around mental illness by family caregivers (note that the term caregivers henceforth means family caregivers). One theme emerged on beliefs about mental illness: namely, Prevailing beliefs about cause of mental illness (with three subthemes). On the practices, three themes were identified: Encountering support and neglect (with two subthemes), Recognizing the need of professional help (with three subthemes), and Seeking culturally influenced help (with three subthemes). An overview of the themes and subthemes is presented in Table 2. Prevailing beliefs about cause of mental illness. Within the theme Prevailing beliefs about cause of mental illness, three sub-themes were identified related to caregiver beliefs around mental illness: Viewing supernatural forces as cause of mental illness, Attributing cause of mental illness to individual’s behavior, and Viewing life circumstances as cause of mental illness. Viewing supernatural forces as cause of mental illness. The family caregivers reflected beliefs in supernatural forces beyond the individual as being responsible for mental illness. Supernatural reasons included the disregard for the tradition of sexual cleaning and spiritual possession. Majority of the caregivers alluded to the belief that having sex with uncleansed widow or widower, attracted spell and in some instances even the ghost of a dead spouse in form of mental illness. **“When one loses a spouse, they must be cleansed by the dead spouse’s family. If one is not cleansed or refuses to be cleansed and someone has intercourse with such a widow or widower, they will develop a mental illness.” (Caregiver 14) Closely linked to supernatural beliefs was spiritual possession. Ten of the fifteen family caregivers expressed that jealousy and enmity from friends, family, or neighbors cause mental illness. Caregivers reported that mental Scientific Reports \| (2022) 12:21388 \| https://doi.org/10.1038/s41598-022-25985-7 www.nature.com/scientificreports/ Encountering social support and neglect. Encountering social support and neglect in caregiving yielded from receiving support from others and struggling with culturally influenced negative attitudes. Examples regarding advice given included managing the condition, cause of illness and where to seek help. “People from church are encouraging and always keep us going by praying for her and the family. They remind us that God never fails and that he heals all illnesses. Their support and advice made us bring her to the hospital.” (Caregiver 15). Seeking culturally influenced help. The theme revealed various practices around culturally influenced help-seeking. Help-seeking practices revolved around taking the next-of-kin for different sources of help informed by caregiver beliefs about cause of the illness. When mental illness was spiritual in nature, spiritual intervention was sought, while mental illness linked to bewitchment and failure to perform cultural rites, help was sought from traditional healers. Mental illness caused by misuse of substances, heredity and life stressors, meant that medical help was sought. The subthemes that emerged here were; Seeking spiritual intervention, Seeking traditional help is culturally advised, and Seeking medical help—a fast and convenient solution. Interestingly, caregiver’s narratives showed that even those who sought medical help or traditional help first also reported using spiritual healing alongside other treatments. g p y g p Interestingly, caregiver’s narratives showed that even those who sought medical help or traditional help first lso reported using spiritual healing alongside other treatments. Seeking spiritual intervention. Despite caregivers reporting various help-seeking practices, spiritual healing through their religious affiliation was reported by all the fifteen caregivers as the first line of treatment sought. Caregivers reported that their faith in God and assurance that God cures all illnesses motivated them to take their loved ones for prayers. “People said the condition was spiritual. My family advised me to seek spiritual help by taking the patient for prayers.” (Caregiver, 14). Seeking traditional help is culturally advised. Caregivers who attributed mental illness to supernatural forces and bewitchment sought traditional help. Caregivers narrated that traditional treatment helped to cure mental illness caused by supernatural forces and bewitchment. Thirteen caregivers who reported that mental illness was caused by a curse, or spell due to individuals’ bad deeds or misfortune sought traditional help. For example, one caregiver narrated that: “You know our society you cannot run away African medicine. People constantly tell you …look for a witchdoctor because this illness is from people.” (Caregiver 9). Seeking medical help—a fast and convenient solution. Encountering social support and neglect. Encountering social support and neglect in caregiving yielded from receiving support from others and struggling with culturally influenced negative attitudes. Three subthemes were identified; Trying to manage next-of-kin’s behavior, Trying to manage own tiredness as well as Receiving advice from well-wishers as being at the center of the caregivers’ decision to seek professional help from the hospital. Trying to manage next‑of‑kin’s behavior. The difficulties in managing next-of-kin’s behavior were experienced as a culturally sensitive issue and were thus a common reason for seeking help. All the caregivers interviewed reported failing to manage and cope with the demands of the illness at home. They reported that the next- of-kin’s disruptive behavior such as undressing in public and aggression was not only culturally unacceptable but draining. For aggressive behavior, six caregivers narrated that the decision to take their next-of-kin to the hospital was when they noted that the person was a danger to themselves and a danger to the caregiver. Thus, the consequent fear and the need for safety motivated family caregivers to take their next-of-kin to the hospital. For example, one caregiver narrated “He became too aggressive, always beating me; I feared he was going to kill me.” (Caregiver 1). https://doi.org/10.1038/s41598-022-25985-7 Scientific Reports \| (2022) 12:21388 \| www.nature.com/scientificreports/ Trying to manage own tiredness. The caregiver quest to manage their own tiredness was also reported to have contributed to the decision to take the next-of-kin to the hospital. Tiredness, being stressed, exhausted, and inability to cope was widely expressed by caregivers as having informed the decision to take the patient to the hospital among caregivers. As stated by one caregiver: “She became too much, we tied her up with a chain, she never wanted to stay at home, running away from home, pouring water on herself, I became tired … that’s how they brought her to the hospital” (Caregiver 3). More than half of the caregivers reported that, not knowing the source of mental illness was not only emotionally difficult, but also frustrating to them. They also reported being overwhelmed by the illness and being constantly tired influenced the decision to take the next-of-kin to the hospital. Receiving advice from well‑wishers. Receiving advice from well-wishers (church mates, neighbors and friends) was perceived as a culturally common practice and it was also instrumental in the caregiver’s decision to take the next-of-kin to the hospital. Nine caregivers reported that advice from well-wishers and medical personnel made them take their next-of-kin to the hospital. Encountering social support and neglect. Encountering social support and neglect in caregiving yielded from receiving support from others and struggling with culturally influenced negative attitudes. In addition, caregivers who attributed the cause of mental illness to life circumstances such as life stressors and heredity sought medical help first. Medical help was also sought by two caregivers who held beliefs that medical help was faster and more convenient. For example, one caregiver said that: “I decided to bring him to the hospital knowing that medical personnel can be able to examine him and know what to do faster.” (Caregiver 3). Scientific Reports \| (2022) 12:21388 \| www.nature.com/scientificreports/ with previous research that has shown a link between socio-cultural beliefs and help-seeking2. Thus, if holistic mental health care is to be offered, practitioners must be sensitive to and familiarize themselves with the cultural beliefs that may limit persons’ need for support in the culture in which they work. Furthermore, public health messages that aim at promoting mental health should take into account social-cultural views.i g p g A somewhat surprising finding is that even when the next-of-kin got professional medical treatment at the hospital, family caregivers continued to seek other forms of non-medical treatment such as traditional and spiritual help through prayer. This practice might point to how deeply cultural beliefs are engrained. It could well be that pervasive cultural attributions to mental illness engulfed in beliefs may advertently explain why caregiver prefer spiritual and traditional healing methods as they align with contextual philosophies. As noted by Kajawu et al.30 in Africa that have found that people seek African traditional treatment methods as it tends to meet cultural expectations as opposed to biomedical help. This finding highlights the need to incorporate socio-cultural knowledge in mental illness interventions that may help family caregivers in seeking appropriate help early and improve access to modern psychiatric services.i Another finding reveals stigmatizing beliefs and practices around mental illness. Traditional beliefs held informed how communities respond and act towards families taking care of a next-of-kin living with mental illness. Notwithstanding the positive support that the participating caregivers had received, the finding revealed a lack of support, and avoidance of the family as common. Taking care of a next-of-kin living with mental illness was experienced as taxing due to negative responses attributed to beliefs held on mental illness. Beliefs such as mental illness is a punishment of the individual by God and results from vices such as “immorality‟ or stealing made families susceptible to stigmatization. These beliefs and constructions that caregivers are forced to face most likely add to their burden and stress. In the African culture, vices such as adultery, and stealing, are strongly discouraged31 as they are considered to attract bad omen such as mental illness to those who engage in the vice. Given this culture, it is not surprising that beliefs about the cause of mental illness reflected personal responsibility as informed by culture and put the blame sorely on the individual for their mental illness. www.nature.com/scientificreports/ The blame and shame beliefs around mental illness seem to lead to negative stereotyping, and discrimination on those living with mental illness and their families. Thus, in keeping with this finding, stigma directed at caregivers made them live in a constant state of stress, worsening their already worsened mental state due to the stress of taking care of their relative. This finding supports Ayalew et al.32 study conducted in Ethiopia, which found that stigma contributed to the caregiver burden which harmed the care provided by the family to the person with mental illness. The findings from this study lend support to the socio-ecological theory given that perceived negative practices around mental illness are influenced by beliefs held, which are contextual. Applying Bronfenbrenner15 socio-ecological model on the current study’s findings that negative practices for example stigma operate as a process embedded in socio-cultural beliefs informed by context and influence caregiver practices. Our findings indicate that mental illness-related stigma might be a barrier to mental illness medical treatment and management in a hospital setting and can hinder caregivers from seeking appropriate help. This finding resonates with literature that has reported mental illness-related stigma as a barrier to mental illness treatment33. It is therefore imperative to develop culturally appropriate public health education programs on mental illness that target beliefs to curb social rejection and stigma. Further, to reduce social rejection and stigma, sensitization programs must form more effective partnerships with and target communities, churches, and traditional leadership such as chiefs and faith healers34,35. Furthermore, incorporating traditional and local healing into Zambian health systems may increase cultural relevance, which may promote early help seeking and help reduce social rejection. Methodological considerations. To ensure the trustworthiness of the findings, four dimensions as suggested by Lincoln and Guba36 namely credibility, transferability, conformability, and dependability; all of which have been adhered to throughout the research process. To help achieve credibility, i.e. the degree to which the research portrays the genuine meanings of the research participants36, the researchers recruited only primary caregivers who were knowledgeable about the condition of their next-of-kin. The credibility of the study can also be considered strengthened by following Braun and Clarke26 steps to thematic analysis and outlining the procedure used to develop codes, sub-themes, and themes. The researchers additionally attempted to reduce risks to trustworthiness by being reflective in order to avoid researcher bias and by cross-checking conclusions with other sources of findings. www.nature.com/scientificreports/ Transferability was considered by providing the study context and describing the recruitment process. To avoid the risk of bias due to pre-understanding, the principle investigator was reflective throughout the process of data collection and analysis. In efforts to ensure the conformability of the study the researchers presented direct quotes from caregivers, thus showing that the findings were supported by the data. Further, outlining the research procedure and clearly documenting findings could be seen as strengthening the study’s dependability. Discussionh This study explores cultural beliefs and practices related to mental illness among family caregivers caring for a next-of-kin living with mental illness. Overall, the study revealed varied beliefs and practices around mental illness rooted in socio-cultural explanations. The beliefs, help-seeking behaviors, stigma, and care load are the main findings discussed here.hi i The findings of this study provide important insight into caregivers’ socio-cultural perspectives on mental illness beliefs and practices. The social ecological model of human development proposed by15 emphasizes the interdependence of individual and contextual systems. The results of this study reveal that caregiver beliefs and help-seeking practices on mental illness among family caregivers in Lusaka are entrenched in socio-ecological understanding such as supernatural and spiritual beliefs, in line with the ecological model15. The findings support Bronfenbrenner’s viewpoint, since caregiver’s conceptualization of cause, and decisions about seeking help mirrored the discourse in the socio-cultural context. Caregivers who held religious beliefs, for example, sought divine intervention through spiritual help, whereas those who linked mental illness to life circumstances sought medical help, and those who believed in witches sought traditional help. Noteworthy, some of the beliefs on the cause of mental illness such as sexual relations with uncleansed spouses and contempt for the authority of elders are culturally specific to Zambia and some countries in Africa. The findings in this study are consistent Scientific Reports \| (2022) 12:21388 \| https://doi.org/10.1038/s41598-022-25985-7 www.nature.com/scientificreports/ References Cross-national comparison of Middle Eastern university students: Help-seeking behaviors, attitudes toward helping professionals, and cultural beliefs about mental health problems. Community Ment. Health J. 45(1), 26–36 (2009). p y 35. Gearing, R. E. et al. Adaptation and translation of mental health interventions in Middle Eastern Arab countries: A systematic review of barriers to and strategies for effective treatment implementation. Int. J. Soc. Psychiatry 59(7), 671–681 (2013). 36. Lincoln, Y. S. & Guba, E. G. Naturalistic Inquiry (Sage, 1985). 35. Gearing, R. E. et al. Adaptation and translation of mental health interventions in Middle Eastern Arab countries: A systematic review of barriers to and strategies for effective treatment implementation. Int. J. Soc. Psychiatry 59(7), 671–681 (2013). 36 Lincoln Y S & Guba E G Naturalistic Inquiry (Sage 1985) f 36. Lincoln, Y. S. & Guba, E. G. Naturalistic Inquiry (Sage, 1985). Conclusionsh The study revealed that cultural beliefs such as spiritual possession, witchcraft, and failure to perform traditional rite are at the center of family caregiver’s attributions of cause of mental illness. This study also found that cultural beliefs shaped practices around help seeking. Given the finding that stigma is dominate, this study recommends that public health practitioners should take into consideration the cultural beliefs and practices in designing appropriate methods for health promotion programs, public health messages, and medical education. Thus, future research should look at how traditional forms of care can be incorporated into conventional medical practice. https://doi.org/10.1038/s41598-022-25985-7 Scientific Reports \| (2022) 12:21388 \| www.nature.com/scientificreports/ References First do no harm: An exploration of researchers’ ethics of conduct in Big Data behavioral studies. PLoS One 15(11), e0241865 (2020).l 9. Kalu, M. E. How does “subjective I” influence a qualitative research question, theoretical approach and methodologies?. Glob. J Pure Appl. Sci. 25(1), 97–101 (2019). pp ( ), ( ) Kajawu, L., Chingarande, S. D., Jack, H., Ward, C. & Taylor, T. What do African traditional medical practitioners do in the treatment f mental disorders in Zimbabwe? Int J Cult Ment Health 9(1) 44–55 (2016) pp ( ), ( ) 30. Kajawu, L., Chingarande, S. D., Jack, H., Ward, C. & Taylor, T. What do African traditional medical practitioners do in the treat of mental disorders in Zimbabwe?. Int. J. Cult. Ment. Health 9(1), 44–55 (2016). pp 30. Kajawu, L., Chingarande, S. D., Jack, H., Ward, C. & Taylor, T. What do African trad of mental disorders in Zimbabwe?. Int. J. Cult. Ment. Health 9(1), 44–55 (2016). j , , g , , J , , , y , of mental disorders in Zimbabwe?. Int. J. Cult. Ment. Health 9(1), 44–55 (2016). g, G. E. African culture and values. Phronimon 16(2), 97–111 (201 32. Ayalew, M., Workicho, A., Tesfaye, E., Hailesilasie, H. & Abera, M. Burden among caregivers of people with mental illness at Jimma University Medical Center, Southwest Ethiopia: A cross-sectional study. Ann. Gen. Psychiatry 18(1), 1–11 (2019). y p y y y 33. Knaak, S., Mantler, E. & Szeto, A. Mental illness-related stigma in healthcare: Barriers to ac solutions. In Healthcare Management Forum Vol 30, 2, 111–116 (SAGE Publications, 2017). 3. Knaak, S., Mantler, E. & Szeto, A. Mental illness-related stigma in healthcare: Barriers to access and care and evidence-based solutions. In Healthcare Management Forum Vol 30, 2, 111–116 (SAGE Publications, 2017). 33. Knaak, S., Mantler, E. & Szeto, A. Mental illness-related stigma in healthcare: Barriers to access and care and evidence- solutions. In Healthcare Management Forum Vol 30, 2, 111–116 (SAGE Publications, 2017). g 34. Al-Krenawi, A., Graham, J. R., Al-Bedah, E. A., Kadri, H. M. & Sehwail, M. A. Cross-national comparison of Middle Eastern university students: Help-seeking behaviors, attitudes toward helping professionals, and cultural beliefs about mental health problems. Community Ment. Health J. 45(1), 26–36 (2009). 34. Al-Krenawi, A., Graham, J. R., Al-Bedah, E. A., Kadri, H. M. & Sehwail, M. A. Author contributions F.S. is the Principal Investigator and responsible for data collection, analysis, and drafting the manuscript. A.C. contributed to interview guide development, analysis, discussion and critically reviewing of the manuscript. 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Additional information Supplementary Information The online version contains supplementary material available at https://doi.org/ 10.1038/s41598-022-25985-7. Correspondence and requests for materials should be addressed to A.K. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. 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Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. Funding To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2022 https://doi.org/10.1038/s41598-022-25985-7 Scientific Reports \| (2022) 12:21388 \|
https://openalex.org/W4310089392	https://zenodo.org/records/7369993/files/ESTUDO%20DE%20MONITORAMENTO%20PARA%20MAQUINA%20DE%20IMAGEM%20RADIOL%C3%93GICA%20VOLTADO%20A%20ENGENHARIA%20CL%C3%8DNICA%20E%20SEUS%20EFEITOS.%20%E2%80%93%20ISSN%201678-0817.pdf	Portuguese	null	ESTUDO DE MONITORAMENTO PARA MAQUINA DE IMAGEM RADIOLÓGICA VOLTADO A ENGENHARIA CLÍNICA E SEUS EFEITOS.	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	9,760	REGISTRO DOI: 10.5281/zenodo.7369991 André Luiz Silva Cavalcante; Gabriel Pereira dos Santos; Rafael Santos Chemello; Orientador: Bruno Jesus dos Santos. ESTUDO DE MONITORAMENTO PARA MAQUINA DE IMAGEM RADIOLÓGICA VOLTADO A ENGENHARIA CLÍNICA E SEUS EFEITOS. REGISTRO DOI: 10.5281/zenodo.7369991 Resumo: Hoje sabemos que o principal desafio que se encontra em equipamentos hospitalares é sua manutenção para o correto funcionamento, diminuir falhas e paradas nos exames que prejudicam diretamente o público (paciente) e aumenta o custo e diminuindo o faturamento, assim buscando sempre aumentar a disponibilidade do equipamento. A grande questão é diminuir o tempo desde a ocorrência de uma falha até a sua resolução, estas de responsabilidade da engenharia clínica que a todo buscam novas tecnologias para aumentar a eficiência em sua gestão hospitalar. Este artigo vem de encontro a esta demanda, visando apresentar estudo e desenvolvimento de um protótipo para um sistema de monitoramento de maquinas de imagem radiológicas em hospitais ou laboratórios, com o foco principal no monitoramento de parâmetros da máquina de ressonância magnética, onde utilizamos para estudo a máquina da fabricante Siemens Healthineers, modelo Magnetom Aera, instalada no Hospital 9 de Julho, em São Paulo – SP. Onde através do monitoramento desde parâmetros e disponibilizamos para a engenharia clinica informando de alertas que ocasionem a parada da máquina, iremos descrever durante o artigo que é possível diminuir o tempo de parada e aumentar a disponibilidade do equipamento para exames, tendo ganho para o paciente, para o hospital seja monetário ou de credibilidade quanto para o fabricante do equipamento. Palavras-chave: Ressonância Magnética, Monitoramento, engenharia clínica, disponibilidade. Monitoring Study for Radiological Imaging Machines focused on Clinical Engineering and its effects. Abstract: Today we know that the main challenge that is found in hospital equipment is its maintenance for the correct functioning, to reduce failures and stoppages in the exams that directly harm the public (patient) and increase the cost and decreasing the billing, thus always seeking to increase the availability of the equipment.The big question is to reduce the time from the occurrence of a failure to its resolution, these being the responsibility of clinical engineering who are always looking for new technologies to increase efficiency in their hospital management. This article meets this demand,aiming to present the study and development of a prototype for a monitoring system for radiological imaging machines in hospitals or laboratories, with the main focus on monitoring the parameters of the magnetic resonance machine, where we use the machine from the manufacturer Siemens Healthineers, Magnetom model, to study The age,installed at Hospital 9 de Julho, in São Paulo – SP. Where through the monitoring of parameters and we make available to clinical engineering informing of alerts that cause the machine to stop, we will describe during the article that it is possible to reduce the stop time and increase the availability of the equipment for exams,having gained for the patient, for the hospital, either monetary or in terms of credibility, as well as for the equipment manufacturer. Keywords: MRI, Monitoring, clinical engineering, availability 1. Introdução Nos últimos anos houve um desenvolvimento acentuado de novas tecnologias voltadas à área da saúde, seja em melhorias nos equipamentos médicos- assistenciais (EMAs) e apoio ao diagnóstico, surgimento da telemedicina, entre outros avanços. Sendo que a difusão dessas tecnologias no setor de saúde proporciona notáveis melhorias para a saúde e qualidade de vida da população (SORENSON et al., 2008). Nesse contexto que se insere o Engenheiro Clínico. De acordo com a American College of Clinical Engineering (ACCE, 2017), o Engenheiro Clínico é o profissional que utiliza seus conhecimentos em engenharia e práticas de gerenciamento de tecnologias da saúde, para apoiar e melhorar os cuidados prestados aos pacientes. Para exercer tal função, esse profissional deve participar do planejamento, avaliação, seleção e processo de implementação de uma nova tecnologia (MYTTON, 2010), tendo em vista que seus conhecimentos diversos na área da saúde, são de extrema importância para compatibilizar as várias frentes atuantes num projeto hospitalar. Com o intuito de auxiliar o engenheiro clinico na busca pela melhoria na gestão hospitalar de equipamentos medico assistências, voltado a área de maquinas de imagem radiológica, com o foco na máquina de ressonância magnética, surge o proposito e desenvolvimento deste estudo. Desenvolver um sistema de baixo custo que auxilie a equipe em situação de falha do equipamento para que possa tomar a decisão mais eficiente, antes mesmo da parada por completo do equipamento, assim de diminuir o tempo de indisponibilidade que temos hoje. E através dos resultados apresentados neste artigo que é possível através de um sistema de baixo custo dedicado a tal função, aumentar a eficiência da equipe de engenharia clínica na atuação e correções de falhas em equipamentos de ressonância magnética, aumentando a disponibilidade para exames consequentemente aumentando o ganho e diminuindo custos para o hospital. ressonância magnética, aumentando a disponibilidade para exames consequentemente aumentando o ganho e diminuindo custos para o hospital. 1.2. Objetivos Geral e especifico De maneira geral o estudo apresentado neste artigo, tem como função auxiliar a engenharia clínica a aumentar sua eficácia no trabalho assistencial em equipamentos de imagem radiológica, auxiliando-os principalmente no monitoramento destes equipamentos para em caso de falha possam tomar a melhor decisão para a resolução do problema. 1.3. Objetivo Específico O estudo visa apresentar sistema de monitoramento de baixo custo e fácil aplicação para equipamentos de ressonância magnética em hospitais ou laboratórios, auxiliando principalmente as equipes da engenharia clinica a saberem de maneira antecipada em caso de problemas, até mesmo antes da parada do funcionamento por completa da máquina, a fim de tornar a atuação e resolução do problema em caso de falha mais rápida e diminuir o tempo de indisponibilidade que o equipamento possuiu hoje. Tendo ganho monetário ao hospital ou laboratório pela não indisponibilidade por um período longo do equipamento, ganho para o paciente que precisa fazer o exame e ganho ao fabricante que com as informações já passada de maneira antecipada pela engenharia clínica, tornara as manutenções corretivas mais eficientes. 1.1. Justificativa Hoje o principal desafio da Engenharia clínica em hospitais é a redução do tempo de indisponibilidades dos equipamentos médicos-assistenciais, a indisponibilidade causa vários problemas para o hospital, como a perda de faturamento de exames, perda de confiabilidade dos seus clientes e acúmulo de custos com o equipamento, uma máquina em pleno funcionamento ou com o tempo de parada minimizado é capaz de cobrir seus custos. Claro que medidas para diminuir a indisponibilidade não são simples, o estudo aprofundado de cada equipamento é necessário, entender as causas de paradas e seus efeitos é fundamental para propor novas soluções, auxiliados principalmente de tecnologia para agilizar e orientar para a diminuição da parada do equipamento. Onde atualmente os equipamentos médicos-assistenciais apesar de possuírem monitoramento do sistema e acusarem suas falhas, ainda pecam na necessidade de informar a engenharia clínica do erro ocorrido, não é nada direto onde a maioria dos casos só saberão da falha quando a máquina parar por completo. Isso acarreta na demora da resolução do problema, podendo parar a máquina por completo por horas, até a chegada de um técnico da fabricante que irá avaliar a situação, entender o problema e solucioná-lo. Por fim, percebeu-se a necessidade de propor um sistema de monitoramento mais direto voltado a engenharia clínica, monitorando os principais pontos de funcionamento e erros dos equipamentos médico-assistenciais, a fim de quando ocorrer a falha a equipe do hospital saiba o que ocorreu antes mesmo da parada do equipamento e atuando para a correção. Vale ressaltar que para o desenvolvimento deste estudo utilizando como base a máquina de ressonância magnética de supercondutores, por terem um sistema mais complexo de funcionamento e monitoramento, principalmente por conta de seu sistema de resfriamento por hélio líquido. mais complexo de funcionamento e monitoramento, principalmente por conta de seu sistema de resfriamento por hélio líquido. 2.1. Sistema de máquinas de imagem radiológica Neste tópico do artigo iremos discutir e nos aprofundar no funcionamento das maquinas de imagem radiológicas estudadas, não sobre o ponto de vista de fenômenos físicos que operam a máquina por não ser o foco do estudo em questão, mas sim de suas ações práticas vivenciadas no dia a dia de quem opera e faz manutenções no equipamento, a fim de entendermos o seu funcionamento, características de seu sistema de monitoramento, controles, alertas e situações que os sistemas apresentam, com foco principal nas seguintes máquinas: a) Ressonância Magnética; b) Tomografia; a) Ressonância Magnética; b) Tomografia; a) Ressonância Magnética; b) Tomografia; b) Tomografia; 2. Revisão Bibliográfica Este capítulo foi escrito com objetivo de subsidiar o tema proposto neste estudo e orientar a pesquisa, sobretudo na sustentação dos resultados e na discussão apresentada. Tais assuntos estão assim organizados: Sistema de máquinas de imagem radiológica evidenciando a Ressonância Magnética (RM) e Tomografia com foco principal em sistemas RM a ser implementado durante este estudo, atuação da engenharia clínica, Manutenção de RM e Fatores que podem afetar sua disponibilidade e custos com equipamento de RM. 2.1.1. Ressonância Magnética Por definição, a RM (Ressonância Magnética) é a propriedade física por núcleos de determinados elementos que, quando submetidos a um campo magnético forte e excitados por ondas de rádio (RF) em determinada frequência (Frequência de Larmor), emitem rádio sinal, o qual pode ser captado por uma antena e transformado em imagem. (FERRARINI, 2009, p.2) Para exames de MRI (Magnetic Resonance Imaging), é necessário a presença de um campo magnético para manipulação de prótons presente no corpo humano. Campo este que é gerado através de um magneto, centrado na máquina de MRI, que funciona de forma similar a um imã em larga escala. Em aplicações biomédicas, estes magnetos possuem três variações: magnetos Permanentes, magnetos Resistivos e magnetos Supercondutores. (SOUZA, 2017, p.37). Onde dos equipamentos disponível no mercado cerca 75% são supercondutores, 23% são permanentes e 2% são resistivos, sendo suas principais características: 2.1.1.1. Magnetos Permanentes Figura 2 – Como exemplo de magnetos permanentes Ressonância Supertar 0.35T Shimadzu Fonte: SHIMADZU (2014) Fonte: SHIMADZU (2014) Tabela 1 – Características dos principais tipos de magnetos Fonte: Meireles, Souto, 2011, p.76 Fonte: Meireles, Souto, 2011, p.76 Onde apesar da variedade de sistemas de obtenção de imagem por RM disponível, os instrumentos têm os mesmos subsistemas básicos, que podem ser divididos em: • Magneto principal; • Magneto principal; • Bobinas de gradiente de campo magnético; obinas de gradiente de campo magnético; ransmissor e receptor de radiofrequência; rocessador de Imagem; istemas de computadores. Figura 1 – Componentes básicos e a “arquitetura” de um sistema de ressonância magnética Fonte: Meireles, Souto, 2011, p.75 • Transmissor e receptor de radiofrequência; • Processador de Imagem; • Sistemas de computadores. • Sistemas de computadores. Figura 1 – Componentes básicos e a “arquitetura” de um sistema de ressonância magnética ressonância magnética Fonte: Meireles, Souto, 2011, p.75 Fonte: Meireles, Souto, 2011, p.75 2.1.1.1. Magnetos Permanentes 2.1.1.1. Magnetos Permanentes Os magnetos permanentes são construídos por grandes blocos de material ferromagnético, que conservam o magnetismo após serem expostos a outro campo magnético. O material mais comumente utilizado para a sua produção é uma liga de alumínio, níquel e cobalto, conhecida como ALNICO, com aspecto semelhante ao de uma ferradura simples. (Meireles, Souto, 2011, p.76) Onde os dois pólos magnéticos são próximos de forma paralela, onde este espaçamento dos pólos é formado um campo magnético uniforme suficiente para o uso em imagem médica, com a força do campo variante podendo chegar até 0,3 Tesla. Para Souza (2017, p.37) dentre suas vantagens, estão os baixos custos de compra iniciais, comparado aos sistemas com magnetos supercondutores, além de maior conforto para os pacientes que sofrem de claustrofobia, por conta de seu formato ser mais aberto e compacto. Figura 2 – Como exemplo de magnetos permanentes Ressonância Supertar 0.35T Shimadzu 2.1.1.2. Magnetos Resistivos Magnetos resistivos consiste basicamente em anéis de fios condutores de corrente, ele é muito mais leve que os grandes pole-shoes ferromagnéticos utilizados nos magnetos permanentes. Porém, embora seu custo inicial seja comparativamente baixo, os custos operacionais de um magneto resistivo são bem altos dada a enorme energia necessária para manter o campo magnético. Para que o campo magnético funcione, a fonte de energia do sistema tem de permanecer ligada. (WESTBROOK, ROTH, TALBOT, 2013, p.266) Para que o campo magnético funcione, a fonte de energia do sistema tem de permanecer ligada. (WESTBROOK, ROTH, TALBOT, 2013, p.266) A potência máxima do campo em um sistema como esse é inferior a 0,2 T ou 0,3 T, pelo fato de que qualquer aumento no campo magnético demanda elevação da corrente. Isso criaria maior resistência nos circuitos, o que, por sua vez, aumentaria a temperatura a um nível que no final destruiria o eletromagneto. (WESTBROOK, ROTH, TALBOT, 2013, p.265) Figura 3 – Como exemplo de magnetos resistivos Ressonância MROpen Evo 0,35T ASG superconductors Fonte: ASG superconductors (2022) Figura 3 – Como exemplo de magnetos resistivos Ressonância MROpen Evo 0,35T ASG superconductors p Fonte: ASG superconductors (2022) Fonte: ASG superconductors (2022) 2.1.1.3. Magnetos Supercondutores Magnetos supercondutores que serão o nosso foco de aplicação do sistema de monitoramento por exigirem um maior cuidado de operação e controle, para Diaz (1998, p.23) atualmente são os mais usados nos equipamentos de RM, são refrigerados a temperaturas de supercondutores (4º K, usando He e Nitrogênio líquido); nesta temperatura a resistência elétrica é quase nula. Os magnetos supercondutores geram um campo magnético constante, altamente homogêneo e intenso; as desvantagens são os altos custos de manutenção e refrigeração. Os magnetos supercondutores, que são disponibilizados no mercado, têm como princípio de funcionamento eletroímãs que operam perto da temperatura zero absoluto (4,2 °K ou 270 °C), com pouca resistência em seus fios. Correntes elétricas muito fortes geram um campo magnético alto sem estimular calor significativo, tornando fundamental o uso de criogênicos (hélio líquido e nitrogênio) para atingir as temperaturas ultra baixas necessárias. Os equipamentos de RM de campo aberto, em sua maioria, são do tipo magneto permanentes ou resistivos e não requerem criogênicos. Com isso, são de baixa manutenção e intensidades de campo também mais baixas, gerando menos sinal. (HASHEMI; BRADLEY JUNIOR; LISANTI, 2004). Figura 4 – Exemplo de Magnetos supercondutores, fabricante Siemens modelo Magnetom Area 1.5T Fonte: SIEMENS (2022) Fonte: SIEMENS (2022) Conforme mencionamos anteriormente o sistema de uma máquina de ressonância magnética é dividido em diversos componentes que fazem atuação em conjunto para seu controle e funcionamento, podendo ser alocado é dividido em três principais salas dentro de um laboratório ou hospital. Seguindo o Aparecida (2013) em seu manual para a família Magneton da Siemens um sistema é composto por Sala de exame, Sala de controle e Sala de equipamento, que seus componentes e características dimensionais podem ser visualizadas na tabela abaixo: Tabela 2 – Tabela de componentes e sua dimensão para as salas que compõem um RM Tabela 2 – Tabela de componentes e sua dimensão para as salas que compõem um RM Fonte: APARECIDA (2013) Conforme apresentado no estudo YAMANAKA (2019) a sala de exames é camada que podemos dizer exterior da ressonância magnética como componentes principais o Magneto e a mesa onde irá o paciente, vale ressaltar que o ambiente deve ser totalmente blindada por conta do seu forte campo magnético, onde vemos seus principais componentes na figura 5. Conforme apresentado no estudo YAMANAKA (2019) a sala de exames é camada que podemos dizer exterior da ressonância magnética como componentes principais o Magneto e a mesa onde irá o paciente, vale ressaltar que o ambiente deve ser totalmente blindada por conta do seu forte campo magnético, onde vemos seus principais componentes na figura 5. Como a sala de exames fica completamente isolada por causa das blindagens, o entre forro dessa sala fica inacessível. Portanto, é vetada a passagem de qualquer tipo de instalação nesse local, pois qualquer necessidade de manutenção ocasionará a parada do equipamento. Um simples reparo no entre forro acarretaria a perda de hélio da máquina, na abertura das blindagens e na parada na realização dos exames de ressonância magnética, um dos mais rentáveis para o hospital. (YAMANAKA, p.28, 2019) Tabela 3 – Dimensionamento Sala Ressonância Magnética (1) Fonte: Adaptado YAMANAKA (2019) Tabela 3 – Dimensionamento Sala Ressonância Magnética (1) Fonte: Adaptado YAMANAKA (2019) Já para a sala de controle conforme descrito por APARECIDA (2013) é o local para a operação do software para a realização dos exames pelos técnicos responsáveis, através de sistema computadorizado com monitor e teclado, o monitor com um painel plano mostra uma interface gráfica com o usuário que possibilita a alimentação dos parâmetros de exame e, ainda, o posicionamento gráfico dos cortes. Além da aquisição de dados e da visualização das imagens recém- adquiridas, o console do operador viabiliza o acesso a várias técnicas de manipulação de imagens. (WESTBROOK, ROTH, TALBOT, 2013, p.286) Continuaremos desde ponto dando ênfase a sala de equipamento, onde é lá que se encontra o sistema eletrônico e sistema de resfriamento da máquina de RM que será monitorado neste projeto. Segundo manual de APARECIDA (2013) ela expõe que a sala de equipamento ou sala de controle possui três principais gradientes distribuídos em três gabinetes conforme figura 5. Primeiro gabinete de gradientes, onde o gabinete de gradiente contém os componentes eletrônicos de potência para gerar os gradientes do campo magnético. Tabela 2 – Tabela de componentes e sua dimensão para as salas que compõem um RM (APARECIDA, p.64, 2013) Segundo o seu gabinete de controle, onde o gabinete de controle inclui componentes eletrônicos diferentes para operar o sistema de RM, (APARECIDA, p.64, 2013) e seu separados do sistema, onde o separador do sistema contém componentes eletrônicos e equipamento de resfriamento para fornecer uma potência de resfriamento adequada ao sistema. Se estiver instalado um sistema de arrefecimento especial (chiller), o separador de sistema não será necessário. Figura 5 – (1) Gabinete de gradiente (2) Gabinete de controle (3) Separador do sistema Fonte: APARECIDA (2013) Figura 5 – (1) Gabinete de gradiente (2) Gabinete de controle (3) Separador do sistema Figura 5 – (1) Gabinete de gradiente (2) Gabinete de controle (3) Separador do sistema Gabinete de gradiente (2) Gabinete de controle (3) Separado sistema Fonte: APARECIDA (2013) Fonte: APARECIDA (2013) Fonte: APARECIDA (2013) Fonte: APARECIDA (2013) Já no estudo realizado por YAMANAKA (2019) buscando os principais requisitos de instalação de equipamentos médico-assistenciais, realizou levantamento a fim de verificar a estrutura mínima necessária para os equipamentos de RM, afinal como existem várias marcas e modelos no mercado, componentes para a operação do sistema pode se diferenciar, mas com o levantamento vemos que os principais componentes dentro da sala de equipamento são: • Quadros de força Ressonância magnética; • Transformadores para RM; • Estabilizador para RM; • Nobreak para console RM; • Tubo Quench Externo; • Chiller para Ressonância Magnética. • Chiller para Ressonância Magnética. Tabela 4 – Dimensionamento Sala Ressonância Magnética Fonte: Adaptado YAMANAKA (2019) Fonte: Adaptado YAMANAKA (2019) Assim com base em todas as referências apresentadas podemos entender que o sistema de controle e operação de uma máquina de RM, não é simples e exige vários níveis de atuação, principalmente quando falamos de supercondutores que necessitam de um controle muito específico de temperatura, onde novas tecnologias podem auxiliar neste controle e monitoramento para garantir o seu melhor funcionamento. 2.1.2. Tomografia Essa técnica, que se baseia em raios-X, foi utilizada para aplicações clínicas ainda no início da década de 70, uma vez que torna possível examinar o encéfalo e, com maior clareza, os limites do sistema ventricular e as partes ósseas do crânio. O aparelho consiste em uma fonte de raios-X que é acionada ao mesmo tempo em que realiza um movimento circular ao redor da cabeça do paciente, emitindo um feixe de raios-X em forma de leque. No lado oposto a essa fonte, está localizada uma série de detectores que transformam a radiação em um sinal elétrico que é convertido em imagem digital. (Junior; Yamashita, 2001, p.1) Na unidade de escaneamento, encontram-se à mesa na qual será posicionado o paciente, a fonte de raios X e os detectores. Para obtenção das imagens, a fonte (tubo de raios X) e os detectores são acoplados de forma a desenvolver o movimento de rotação sobre o paciente, o qual permanece imóvel. O feixe de raios X passa por uma secção axial do paciente atingindo os detectores, os quais reconheceram a radiação atenuada que emergiu do corpo, (Cecília; Costa; Xavier, p. 15) conforme imagem abaixo: Figura 6 – – Representação esquemática das partes básicas de um tomógrafo. Fonte: CECÍLIA; COSTA; XAVIER () Fonte: CECÍLIA; COSTA; XAVIER () Fisicamente um dos maiores equipamentos de imagem e assim como a ressonância magnética, apresenta grande variação entre fornecedores. Indica-se a preparação da infraestrutura básica para instalação do equipamento, sendo a finalização da sala realizada após escolha do fornecedor. (YAMANAKA, p.31, 2019) Apesar de não ser tão complexo, também é um equipamento com suas peculiaridades, com vários modelos diferentes com necessidades de instalação distintas. Em alguns modelos, por exemplo, existe a necessidade de sala técnica para acomodar os equipamentos de apoio, que, caso não prevista, geraria um transtorno para adicioná-la ao projeto. Porém, o mais comum são interferências mais básicas na obra, como a necessidade de canaletas para acomodação dos cabos de alimentação elétrica do equipamento. Essas canaletas são embutidas no piso, e tem cerca de 10 cm de altura, o que pode gerar problemas estruturais na laje caso o equipamento não esteja no andar térreo. Se abaixo do equipamento houver outro andar, será necessário que essa canaleta seja passada pelo entreforro do andar inferior, realizando-se furos na laje para a sua passagem. 2.1.2. Tomografia (YAMANAKA, p.33, 2019) Onde podemos notar conforme apresentado que a tomografia apesar de possuir um equipamento menos complexo que a ressonância magnética ainda exige seus cuidados e monitoramento constante de seu fluxo de trabalho, podendo ser auxiliados através de novas tecnologias. 2.2. Atuação da Engenharia Clínica A Engenharia Clínica segundo Marquez (2015) têm como objetivo principal implementar atividades através de conhecimentos baseados em engenharia e gestão aplicadas às tecnologias referentes a saúde e, consequentemente, aos equipamentos médico assistenciais. Engenharia de Manutenção é um ramo da engenharia clínica e, é conceituado como a capacidade de manter a disponibilidade dos seus equipamentos médico assistenciais de tal forma que, possa ser sempre empregada a utilização de uma técnica eficaz e com o menor custo possível. Para que isso ocorra, o Engenheiro de Manutenção juntamente com o gestor, determina estratégias de manutenções para que possam ocorrer menos avarias aos mesmos e ainda, evitar que ocorram acidentes dentro dos EASs (Estabelecimentos Assistenciais de Saúde) (Silva, 2015, p.72) Para um EAS, é de fato interessante e oportuno que possa ser estudado o porquê no âmbito da existência de uma minimização da disponibilidade dos seus equipamentos, e com isto, averiguar quais são os reais motivos desta minimização para que, sucessivamente possase tentar neutralizar e corrigir as principais causas que levam a acarretar este fator. Entre as principais ocorrências que se leva à minimização dos equipamentos médicoassistenciais, estão: erros de operação, falhas internas de infraestruturas, manutenções imperfeitas, projetos internos com inexatidões (Silva, 2015, p.72) Tratando-se de equipamentos médico-assistenciais que são utilizados na área da saúde, a realização de manutenções é de fato imprescindível para o bom desempenho e qualidade de atendimento à população, além de, afetar a capacidade de realização dos trabalhos dos profissionais da área. Ademais, o papel da manutenção dos equipamentos médicoassistenciais é fundamental para a disponibilidade e custos dos mesmos (C.M.Godoi, 2016, p.162) 2.3. Manutenção de RM e Fatores que podem afetar sua disponibilidade Neste momento buscamos em outras fontes e estudos evidenciar a importância de um sistema para auxiliar as manutenções de RM, sendo elas tanto preditivas, preventivas e corretivas. Evidenciando também como a disponibilidade do sistema está diretamente ligado a manutenções que são prestadas e como uma gestão mais rápida e eficaz é fundamental Em Souza, Coelli (2001) foi realizado estudo buscando os impactos gerados na indisponibilidade de um equipamento de RM devido a fatores de infraestrutura e técnicos que muitas vezes não eram de conhecimento dos operadores. Onde os autores puderam concluir que através de uma política de planejamento de manutenções, treinamentos diretos aos operadores com uma gestão contínua é possível diminuir a indisponibilidades dos equipamentos. Já em Garcia (2002) em seu estudo sobre a percepção da gestão de materiais médicoassistenciais no processo de trabalho puderam notar que uma das principais causas de uma gestão insatisfatória e indisponibilidade do equipamento é a ausência de manutenções de equipamentos médico- assistenciais rápidos e eficazes. Portanto, acredita-se que fatores como boas gestões e disponibilidades de equipamentos médicos estão diretamente ligadas (Claudio, 2019, p.72). Onde para C. M. Godoi (2016) a manutenção preventiva de um equipamento visa principalmente a sua disponibilidade, onde só é possível através de programas de gerenciamento de manutenções. No estudo de Silva, Ferreira (2019) os autores desenvolveram software Ferramenta para Auxiliar a Gestão de Manutenção da Engenharia Clínica nos Hospitais. Com os resultados é possível que este tipo de software seja uma alternativa muito viável para solucionar problemas com paradas longas e gerenciamento da disponibilidade do equipamento, porém é válido ressaltar que devido ao avanço rápido de tecnologias ainda é fundamental o treinamento constante das equipes para atualização. CARVALHO (2007) em seu estudo pela análise HAZOP (análise de risco onde se visa identificar os reais e principais perigos e problemas de operabilidade na instalação), para que se pudesse identificar as prováveis falhas ou condições que ocorreram as falhas no equipamento. Após a realização desta análise, na seção conclusão do trabalho, notou-se que o equipamento estava com um desempenho melhor do que se esperava, pois se acreditava que o aparelho não estava realizando devidamente sua função, e ainda que, a manutenção preventiva era o principal responsável pelo bom desempenho da máquina. 2.3. Manutenção de RM e Fatores que podem afetar sua disponibilidade Onde no estudo realizado por CLAUDIO (2019) visando apresentar dados reais sobre as manutenções e disponibilidade de equipamentos de RM, com levantamento de dois anos de custos de manutenção, operação e causas de indisponibilidade do equipamento, podese concluir que as principais paradas das máquinas estudadas, são ocasionais por paradas de manutenções preventivas que são previamente organizadas e fazem parte da rotina, troca de componentes e gradientes já essas corretivas que demandam mais tempo da parada do equipamento. Figura 6 – Relação entre as categorias de interrupções no serviço e os EASs estudados Fonte: CLAUDIO (2019) Portanto, vemos que equipamentos de RM exigem manutenções constantes desde elas preventivas e corretivas, onde nota-se em diversos pontos dos estudos apresentados as manutenções preventivas são o que traz os maiores benefícios para a indisponibilidade de equipamento de RM, portanto somente ser contornado através de gestão de treinamento constantes para equipes, além de participação cada vez mais ativas de novas tecnologias para identificação de problemas. 2.4. Custos com equipamentos de Ressonância magnética Devido ao alto custo que é necessário para operação dos equipamentos de RM, a todo momento as EASs estão em busca de novas soluções para redução de custos, principalmente com manutenções. Não no estudo realizado por SANTIAGO (2007) buscando realizar uma análise das variáveis que impactam no custo do serviço de ressonância magnética entre dois hospitais, conseguiram observar que fatores de manutenções impactaram os hospitais com elevados custos, outro ponto desenvolvido durante o estudo que a disponibilidade e operabilidade do equipamento influencia diretamente os custo e concluíram que enquanto o equipamento está operando realizando exames e cumprindo suas rotinas os valores cobrados cobriria os gastos do equipamento. Novamente evidenciado no estudo de SILVA (2012) cujo o objetivo foi apresentar os critérios para a incorporação de uma máquina de RM na rede pública, observou-se que a depender da quantidade de exames realizados os custos podem ser equilibrados perante a operação, trazendo como um investimento de longo prazo, os custos relacionados com o equipamento seriam sanados. Onde no estudo realizado por CLAUDIO (2019) visando apresentar dados reais sobre as manutenções e disponibilidade de equipamentos de RM, com levantamento de dois anos de custos de manutenção, operação e causas de indisponibilidade do equipamento, podese concluir que do período abril de 2017 a dezembro de 2018 para um dos setores da pesquisa, o principal gasto foi com a substituição de peças. A partir de onde o equipamento saiu de sua garantia, e totalizou R$ 28.780,00 com gastos de mão de obra, R$ 376.264,92 com gastos de peças para o aparelho. O equipamento ainda apresenta uma previsão de gasto de R$ 11,320.00 referente a visita técnica para troca de um módulo de gradiente, ou seja, apenas com mão de obra, e R$ 164.378,42 da peça, portanto, o custo total com operação para a máquina desde o término do contrato de manutenção é de R$ 580.743,34. (CLAUDIO, 2019, p.128) Tabela 5 – Gastos com operação do equipamento de RM Fonte: CLAUDIO (2019) Tabela 5 – Gastos com operação do equipamento de RM Fonte: CLAUDIO (2019) Já outros custos relacionados à operação da máquina de RM, conforme mencionado a ressonância magnética funciona através do consumo de hélio líquido, fazendo reposições de acordo com o consumo efetivo. 2.4. Custos com equipamentos de Ressonância magnética A engenharia clínica também faz o controle do abastecimento dos equipamentos, de modo que informou que há mensalmente o gasto de em média R$ 5.000,00 (mensal) com o reabastecimento do hélio líquido da ressonância magnética. (SANTIAGO, 2007, p.85) Onde apesar de defasado o valor mensal que poderia ter o consumo de hélio líquido em equipamentos de RM, fora máquinas mais modernas que reduziram estes consumos, podemos ter uma base de custo a mais que a máquina gera mensalmente para o nosso estudo. 3. Materiais e Métodos Nesta seção, apresentaremos os materiais e métodos utilizados para o desenvolvimento do sistema de monitoramento na máquina de ressonância magnética, evidenciando as características técnicas do sistema proposto, seu processo de implementação e sua atuação junto a engenharia clínica foco do trabalho proposto. Os sistemas estão divididos em: Os sistemas estão divididos em: 3.1 Localização e análise do sistema No intuito de mitigarmos o tempo de parada da máquina de ressonância magnética já descritas anteriormente e conseguirmos um sistema mais estável possível, nosso projeto consiste na leitura dos parâmetros mais importantes para o funcionamento do sistema de refrigeração de uma ressonância magnética, podendo ser implantado em tomografias e outros equipamentos de imagem radiológica que, porém, não são tão dependentes de infraestrutura de refrigeração. Baseando-se nos modelos de tomografia Somatom Definition, fabricante Siemens Healthineers, podemos ter dois modelos de refrigeração, por ar- condicionado ou sistema de Chiller do próprio hospital. O monitoramento para modelos com Chiller seriam implementados para a temperatura e para a sala técnica do mesmo, com controle de temperatura e umidade. Por exemplo para hemodinâmicas, o controle de temperatura e umidade na sala de exames e na sala técnica, já bastam para garantir um bom funcionamento. Nas ressonâncias magnéticas o sistema de refrigeração é mais complexo, devido a forma com que o equipamento foi desenvolvido e pelos materiais utilizados em sua fabricação. Podemos dividir a refrigeração do equipamento em alguns sistemas. Neste caso estamos utilizando como estudo uma ressonância magnética da empresa Siemens Healthineers, modelo Magnetom Aera, instalada no Hospital 9 de Julho, em São Paulo – SP b) Sistema secundário Sistema de refrigeração do fabricante no caso estudo SIEMENS, onde se utiliza do sistema primário para resfriar o sistema secundário, com um trocador de calor instalado em um dos armários técnicos. Após a troca de calor, a bomba de água, instalada no mesmo armário do trocador, refrigera alguns sistemas do equipamento, como gradientes e compressor de Hélio. A temperatura do sistema secundário deve ficar em 20°C, no equipamento que estamos usando de estudo: a) Sistema primário Sistema de refrigeração instalado e gerenciado pelo hospital e/ou laboratório, porém alguns equipamentos podem ser gerenciados pelo próprio fabricante. No equipamento utilizado no estudo, a equipe de engenharia hospitalar gerencia a manutenção do sistema. manutenção do sistema. Consiste em um sistema de Chiller com os parâmetros solicitados pelo fabricante, 6° a 12°C de temperatura com um fluxo de 90 a 120 litros por minuto. A figura abaixo mostra o sistema de entrada e saída da água com o monitoramento instalado pelo fabricante do sistema Figura 7 – Sistema primário de refrigeração abastecido pelo hospital Fonte: Autoria própria Figura 7 – Sistema primário de refrigeração abastecido pelo hospital Figura 7 – Sistema primário de refrigeração abastecido pelo hospi Fonte: Autoria própria Fonte: Autoria própria Figura 8 – Sistema secundário de refrigeração localizado sala de equipamento Fonte: Autoria própria Fonte: Autoria própria Fonte: Autoria própria c) Compressor de Hélio Figura 9 – Compressor de Hélio c) Compressor de Hélio Como a ressonância magnética utiliza supercondutores para seu funcionamento, este material necessita de um elemento de refrigeração com temperatura mais baixa para este processo. Este elemento é o Hélio, em estado líquido e gasoso. Por isso utiliza-se de um compressor de hélio para o ciclo do líquido dentro do equipamento. Figura 9 – Compressor de Hélio Fonte: Autoria própria Fonte: Autoria própria Fonte: Autoria própria A refrigeração da ressonância magnética é realizada de forma correta, quando os três sistemas estão operando normalmente. A refrigeração da ressonância magnética é realizada de forma correta, quando os três sistemas estão operando normalmente. 3.1.1 Falhas na refrigeração A falha mais comum neste sistema, com exceção de quedas de energia elétrica da rede, seria em seu sistema primário de refrigeração. No momento em que a temperatura sobe ou o fluxo diminui, a troca térmica com o sistema secundário é menor, causando a subida de temperatura e de pressão. Com essa falha o compressor automaticamente desliga. Com o compressor desligado, o equipamento funciona por cerca de 30 minutos até a parada do mesmo. Desde a falha no sistema primário, desligamento do compressor e parada do equipamento, alguns alarmes são acionados para o operador do equipamento, mas a engenharia clínica é geralmente acionada após a falha no exame. Com isso, nosso projeto irá monitorar três parâmetros importantes destes sistemas: • Temperatura do primário • Temperatura do secundário • Funcionamento do compressor 3.2. Sistema de monitoramento O sistema que iremos desenvolver consiste em três sensores, sendo dois de temperatura e um de vibração. Os sensores de temperatura ficarão instalados nas tubulações dos sistemas primários e secundários. Quando o compressor está em funcionamento, conseguimos verificar uma vibração na carcaça, o sensor de vibração será instalado na mesma, para o monitoramento. No microcontrolador iremos programar os status e alarmes para a equipe de Engenharia Clínica e/ou áreas interessadas. Os alarmes serão divididos em duas partes. A primeira é uma página HTML em tempo real onde irá informar o status dos sistemas com os sensores instalados. A segunda é um sistema de aviso de alarme utilizando mensagem de celular e/ou e-mail, para em caso de elevação da temperatura ou falha no compressor. Portanto o sistema será montado da seguinte forma: 3.2.1. Componentes do sistema Para implementação do sistema teste do estudo desenvolvido, utilizamos os seguintes componentes: • Microcontrolador ESP32 • Microcontrolador ESP32 Figura 11 – Microcontrolador ESP32 Tabela 6 – Instalação dos sensores Tabela 6 Instalação dos sensores Fonte: Autoria própria Podemos verificar o funcionamento conforme fluxograma abaixo: Figura 10 – Fluxograma de atuação do sistema Fonte: Autoria própria Fonte: Autoria própria Fonte: Autoria própria Fonte: Autoria própria Podemos verificar o funcionamento conforme fluxograma abaixo: Podemos verificar o funcionamento conforme fluxograma abaixo: Figura 10 – Fluxograma de atuação do sistema Podemos verificar o funcionamento conforme fluxograma abaixo: Figura 10 – Fluxograma de atuação do sistema Figura 10 – Fluxograma de atuação do sistema Fonte: Autoria própria Fonte: Autoria própria Fonte: Autoria própria 3.2.1. Componentes do sistema Figura 11 – Microcontrolador ESP32 Fonte: ESPRESSIF (2022) Fonte: ESPRESSIF (2022) Fonte: ESPRESSIF (2022) • Sensor de temperatura DHT11 Figura 12 – Sensor de temperatura DHT11 Fonte: AOSONG (2022) nsor de vibração SW-420 Figura 13 – Sensor de vibração SW-420 Fonte: SEED STUDIO (2022) Fonte: AOSONG (2022) Fonte: AOSONG (2022) Fonte: AOSONG (2022) Fonte: AOSONG (2022) • Sensor de vibração SW-420 Figura 13 – Sensor de vibração SW-420 Figura 13 – Sensor de vibração SW-420 Fonte: SEED STUDIO (2022) Fonte: SEED STUDIO (2022) Nos testes conseguimos instalar os sensores de temperatura no encanamento primário e secundário e o sensor de vibração no compressor, utilizando o monitor serial da plataforma Arduino, conseguimos o seguinte resultado: Figura 14 – Monitor serial do Arduino Fonte: Autoria própria Conseguimos a leitura do encanamento com uma pequena diferença de temperatura pois o modelo de sensor não é apropriado para esta medida. Para o monitoramento do compressor, verificamos que a resposta condiz com o funcionamento. 3.3. Implantação Como nosso sistema é não invasivo, a montagem física do sistema não depende de nenhuma empresa terceira. Na sala técnica precisaremos somente de uma tomada para alimentar o sistema. A montagem física consiste em instalar um sensor de temperatura na tubulação de entrada de água do sistema primário e na tubulação de saída do sistema secundário. Figura 15– Instalação do sensor de temperatura no encanamento Figura 15– Instalação do sensor de temperatura no encanamento Fonte: Autoria própria Fonte: Autoria própria Fonte: Autoria própria Para o compressor iremos instalar o sensor de vibração em cima da carcaça, no ponto de maior vibração. Figura 16 – Instalação do sensor no compressor Fonte: Autoria própria Fonte: Autoria própria A placa de interface será instalada ao lado de qualquer ponto com tomada na sala técnica. A placa de interface será instalada ao lado de qualquer ponto com tomada na sala técnica. Essa placa necessita de uma conexão wi-fi próxima, pois utilizaremos para as interfaces de alarmes e status em tempo real. Utilizando como estudo a Engenharia Clínica do Hospital 9 de Julho, um painel está instalado na sala dos técnicos, onde seria configurado o acesso da página HTML com interface dashboard para o status em tempo real do funcionamento dos sistemas. O sistema de alarme por celular e/ou e-mail serão utilizados os servidores mqtt, conectados ao microcontrolador e os alarmes irão para os celulares e e-mails cadastrados. Podemos dividir a implantação nas seguintes etapas: 1° Visita a sala técnica do equipamento e análise da estrutura para instalação 2° Configuração da rede wireless junto ao cliente do microcontrolador 3° Montagem mecânica dos sensores e testes 1° Visita a sala técnica do equipamento e análise da estrutura para instalação 2° Configuração da rede wireless junto ao cliente do microcontrolador 3° Montagem mecânica dos sensores e testes 4° Configuração do painel na sala da engenharia clínica ou área de interesse informada 5° Configuração dos dispositivos remotos que receberão os alarmes 4. Resultados e Discussão Inicialmente, após apresentados os aspectos teóricos e os procedimentos utilizados para o desenvolvimento do trabalho, são relevantes a apresentação dos resultados obtidos e a partir destes resultados, traçar discussões. Com isto este tópico visa apresentar os resultados obtidos com o protótipo implementado, quais foram as vantagens obtidas com esse sistema, quais foram as impressões da engenharia clínica com o novo sistema e comparar o protótipo desenvolvido com outras tecnologias que estão surgindo no mercado. 3.3.1 Dificuldades na instalação Durante nossa pesquisa, conseguimos verificar junto à equipe de engenharia clínica que a maior dificuldade na instalação seria da conexão de internet com o microcontrolador, pois as redes de hospitais são protegidas contra acessos externos. Portanto essa configuração deve ser realizada junto a equipe de T.I local. Outro ponto que teríamos de dificuldade é com a operação do novo sistema para a engenharia clínica, afinal um sistema novo é necessário treinamento e adaptações, seria necessário a elaboração de um treinamento e apresentação para a engenharia clínica, afim de tornar o sistema de conhecimento geral e o atuar em caso de disparada dos alarmes, como não é um sistema de várias camadas e de difícil compreensão, treinamento teórico e prático seria suficiente para absorção. Fonte: Autoria própria Nesta análise de um caso real, podemos notar que ocorreu uma falha 00h52, acionando o primeiro alarme de temperatura, onde podemos verificar que com o erro de temperatura afetou o compressor que automaticamente desligou e ciclo de hélio no equipamento parou. Figura 18 – Alarmes de falha no sistema conforme log do equipamento 4.1 Análise de situação real de falha Em um caso prático, da falha da refrigeração da máquina de ressonância magnética até o momento em que a engenharia clínica é acionada, vários problemas ocorrem e que podem ser mitigados com este sistema. Em um caso prático, da falha da refrigeração da máquina de ressonância magnética até o momento em que a engenharia clínica é acionada, vários problemas ocorrem e que podem ser mitigados com este sistema. Iremos analisar esta falha com um caso real que vivenciamos ao longo de nosso estudo. O equipamento que estamos usando como base de estudo Siemens Iremos analisar esta falha com um caso real que vivenciamos ao longo de nosso estudo. O equipamento que estamos usando como base de estudo Siemens Healthineers, modelo Magnetom Aera, salva todo o histórico de funcionamento dos equipamentos com isso também os erros e falhas em um log, por um período de tempo para análise dos engenheiros da fabricante que prestam manutenção, conforme figura 17. Healthineers, modelo Magnetom Aera, salva todo o histórico de funcionamento dos equipamentos com isso também os erros e falhas em um log, por um período de tempo para análise dos engenheiros da fabricante que prestam manutenção, conforme figura 17. Figura 17 – Log de erros do equipamento Figura 17 – Log de erros do equipamento Fonte: Autoria própria Conforme descrito pela equipe de engenharia clínica após algumas ações, o equipamento voltou a operar às 08hrs da manhã, vale ressaltar que em casos de falhas que tivesse como consequências de mais erros no equipamento que posteriormente necessitasse de peças para reposição, a empresa fabricante teria que ser acionada para a manutenção corretiva e o equipamento continua sem operação até a ação da mesma. Fonte: autoria própria Vale se analisar o tempo do momento que o equipamento teve o alarme de falha até a resolução do problema por conta da engenharia clínica, foram mais de 7hs do equipamento sem funcionamento que poderiam ocasionar outros problemas, no caso em questão por ter acontecido de noite a mesma não teve prejuízos para pacientes e nem para o hospital, mas em um caso que ocorresse durante o dia em horário de exames ocasionaria em transtornos para o hospital. Vale se analisar o tempo do momento que o equipamento teve o alarme de falha até a resolução do problema por conta da engenharia clínica, foram mais de 7hs do equipamento sem funcionamento que poderiam ocasionar outros problemas, no caso em questão por ter acontecido de noite a mesma não teve prejuízos para pacientes e nem para o hospital, mas em um caso que ocorresse durante o dia em horário de exames ocasionaria em transtornos para o hospital. Figura 18 – Alarmes de falha no sistema conforme log do equipamento Fonte: Autoria própria Como o equipamento em questão não é utilizado durante o período noturno para exames a equipe de engenharia clinica foi acionada somente até as 07hrs da manhã, provavelmente quando algum operador da máquina tentou opera-la e notou que a mesma estava em estado de falha, o equipamento permaneceu com o estado de erro durante todo este período de tempo. Como o equipamento em questão não é utilizado durante o período noturno para exames a equipe de engenharia clinica foi acionada somente até as 07hrs da manhã, provavelmente quando algum operador da máquina tentou opera-la e notou que a mesma estava em estado de falha, o equipamento permaneceu com o estado de erro durante todo este período de tempo. Figura 19 – Log de erros do equipamento as 03hrs da manhã 4.2. Atuação do sistema proposto Conforme descrevemos com a análise real de uma falha de temperatura em uma máquina de ressonância magnética, o grande problema é que a falha só é reportada a engenharia clínica do hospital quando ela realmente acontece e o equipamento já está sem operação, que como conforme vimos o problema pode se persistir por horas. Em um caso que ocorram durante o dia em período de exames, conforme foi reportado pela equipe de engenharia clínica, a primeira ação que é análise do que está ocorrendo a falha no equipamento demora cerca de 30 minutos e pelo menos mais 1 hora até a resolução do problema, caso seja possível para a engenharia clínica, caso seja algo mais específico e necessite da atuação de uma equipe de manutenção do fabricante o período até a resolução do problema pode não ter prazo. Com o sistema de monitoramento proposto, atuando 24hs e 7 dias por semana, em caso de falha de temperatura sendo ela no primário ou no secundário que ocasione a parada do equipamento, a equipe de engenharia clínica já será acionada sem a necessidade que o operador comunique a falha. Se utilizamos como comparação o erro de temperatura que apresentado ocorreu no período noturno do hospital, a falha seria comunicada a engenharia clínica que a solucionaria durante o próprio período noturno, não comprometendo a disponibilidade do equipamento para exames que ocorreriam durante o período da manhã. Em caso de parada durante exames, a equipe já atuaria em menos de 30 minutos dos quais são hoje para a resolução do problema e como o sistema já indica a falha a manutenção seria ainda mais eficaz pois antes mesmo de ter contato com o equipamento a engenharia clínica já saberia o que ocorreu com a mesma, reduzindo também o período de 1 hora que utilizam hoje. Onde até mesmo em caso que necessitem do acionamento da equipe da fabricante para a troca de algum componente, seria mais eficaz pela troca de informações com a engenharia clínica. De maneira geral o sistema atua em aumentar a disponibilidade do equipamento, com um sistema direto de monitoramento avisando de falhas para a equipe responsável, aumentando a gestão hospitalar do equipamento, onde com as falhas e seu histórico além de auxiliá-los nas manutenções corretivas, auxiliam também nas manutenções preventivas, podendo atuar onde o equipamento mais acusou falhas. 4.3. Analise da engenharia clínica Em nosso estudo com o objetivo central sempre esteve voltado a auxiliar a equipe de engenharia clínica em sua gestão hospitalar, neste caso para as máquinas de ressonância magnética, pedimos para que a mesma nos descrevesse o quanto este sistema seria importante para a melhoria de gestão. “A implantação do monitoramento da temperatura e funcionamento do compressor no sistema de ressonância magnética, traz para a engenharia clínica benefícios na redução de tempo para o atendimento imediato, facilitando a comunicação entre os setores de infraestrutura, reduzindo ou evitando os impactos na parada do equipamento, até mesmo danos adicionais gerados por temperatura de água. Além disso, torna-se um fator no requisito de segurança do paciente, zelando pela experiência e qualidade durante a realização do seu exame. ” – Amanda Cipriano, Técnica Engenheira clínica, hospital nove de julho, 18 de novembro 2022. 4.4. Tecnologia encontrada no mercado Hoje o principal sistema que encontramos no mercado que se assemelhe com o que estamos propondo neste estudo é o sistema de monitoramento online 24hs de Ressonância da fabricante TRIGAS Serviços de Criogenia, onde o foco do sistema é o monitoramento dos principais parâmetros da máquina de RM, conforme figura 21. Figura 21 – Parâmetros monitorados de um RM desenvolvido pela TRIGAS Figura 21 – Parâmetros monitorados de um RM desenvolvido pela TRIGAS Fonte: Adaptado de TRIGAS (2022) Fonte: Adaptado de TRIGAS (2022) Onde os principais pontos que a empresa apresenta como efeitos de um equipamento parado de ressonância magnética, são: • Custo com Manutenção corretiva; • Cancelamento da agenda de exames; • Menos exames = menor faturamento; • Insatisfação dos clientes. • Insatisfação dos clientes. Figura 22 – Efeitos de um equipamento de ressonância parado Figura 22 – Efeitos de um equipamento de ressonância parado Fonte: Adaptado de TRIGAS (2022) Fonte: Adaptado de TRIGAS (2022) Assim a TRIGAS (2022) desenvolveu uma interfase que monitora os arquivos de log de erros da máquina e carrega novos arquivos no servidor, enviando alerta para os responsáveis, estes alertar sendo via SMS ou e-mail com o foco em emergência como a parada do compressor e baixo nível de hélio na ressonância. Figura 23 – interface Trigas de monitoramento Fonte: Adaptado de TRIGAS (2022) Fonte: Adaptado de TRIGAS (2022) Com a implementação do sistema a TRIGAS (2022), lista como as principais vantagens de ter esse sistema são: • Redução com manutenção; • Redução com manutenção; • Aumento de produtividade e quantidade de exames; Sem cancelamento de agendas e mais satisfação para seus clientes; • Acompanhamento dos principais parâmetros da RMN, via celular, tablet e notebook; • Acompanhamento dos principais parâmetros da RMN, via celular, tablet e notebook; • Alerta aos responsáveis da clínica, informando alterações e condições e/ ou valor parametrizado. Figura 24 – Vantagens apresentado pelo sistema Trigas Figura 24 – Vantagens apresentado pelo sistema Trigas 4.5. Discussões e considerações do sistema O sistema proposto neste estudo é desenvolvido em protótipo, em comparação aos já desenvolvidos em mercado, como o monitoramento da fabricante Trigas, possuem as mesmas preocupações, diminuir o tempo de indisponibilidade da máquina, diminuindo custos e aumentando os ganhos para o hospital ou laboratório e os dois entendem que uma atuação direta e disparos de alertas voltados aos responsáveis pelas manutenções, no caso a engenharia clínica, é o mais eficaz para minimizar as preocupações. A grande diferença nas tecnologias propostas é o nível de implementação e custo ao hospital com o sistema, enquanto a da fabricante Trigas busca apresentar o monitoramento junto aos logs das máquinas, que exigiria uma parceria com as fabricantes para ter acesso a esse sistema que consequentemente um custo agregado muito maior. O sistema proposto visa ser de baixo custo e de fácil implementação, sem ser invasivo a máquina tanto em sua parte de software, hardware e equipamentos, com isso tendo ganhos de maior facilidade e rápida implementação, podendo atuar em quase todas as máquinas de ressonância magnética independente do fabricante. Claro que ao mesmo tempo que possui esta vantagem, o sistema possui o monitoramento de menos parâmetros do sistema, como o caso do nível de hélio que é necessário ser mais invasivo a máquina. Que vai de encontro com o foco e atuação da engenharia clínica na gestão hospitalar, sendo ela conceituada como a capacidade de manter a disponibilidade dos seus equipamentos médico assistenciais de tal forma que, possa ser sempre empregada a utilização de uma técnica eficaz e com o menor custo possível. (Silva, 2015, p.72) O sistema proposto visa ser de baixo custo e de fácil implementação, sem ser invasivo a máquina tanto em sua parte de software, hardware e equipamentos, com isso tendo ganhos de maior facilidade e rápida implementação, podendo atuar em quase todas as máquinas de ressonância magnética independente do fabricante. Claro que ao mesmo tempo que possui esta vantagem, o sistema possui o monitoramento de menos parâmetros do sistema, como o caso do nível de hélio que é necessário ser mais invasivo a máquina. Que vai de encontro com o foco e atuação da engenharia clínica na gestão hospitalar, sendo ela conceituada como a capacidade de manter a disponibilidade dos seus equipamentos médico assistenciais de tal forma que, possa ser sempre empregada a utilização de uma técnica eficaz e com o menor custo possível. (Silva, 2015, p.72) 5. Conclusões Em vista de todos dos pontos levantados durante o desenvolvimento deste artigo, podemos chegar às seguintes conclusões: O presente estudo visa apresentar uma demanda e propor uma solução a engenharia clínica que em todo o seu processo de gestão hospitalar sempre está em busca de novas soluções para aprimorar o seu processo, sempre com critérios para aumentar a disponibilidade de equipamentos com a melhor solução técnica e dispondo-se do menor recurso possível. Em primeiro momento o trabalho busca através de material teórico evidenciar os principais pontos de operação dos equipamentos de imagem radiológica, tendo foco principal nas ressonâncias magnéticas de supercondutores, que é possível através do mesmo compreender suas características, seu funcionamento e principais e seus componentes que compõem o sistema e quais os parâmetros necessários para a operação do equipamento. Posteriormente evidenciando quais os principais focos que envolvem a engenharia clínica e seu objetivo dentro da gestão médico hospitalar. Para assim chegar a necessidade de manutenções dentro dos hospitais, sejam elas preventivas quanto corretivas a fim evidenciando também que a disponibilidade de um equipamento está diretamente ligada às manutenções prestadas e como uma solução rápida e eficaz é fundamental. Com isso propomos um sistema de monitoramento não invasivo dos parâmetros de temperatura dos dutos primário e secundário com sensores de temperatura acoplados aos mesmo, além do status de operação do compressor, através de um sensor vibração que conseguiremos assim verificar se o mesmo está operando ou não. Todos os dados enviados até um microcontrolar que através de versão web estará a todo momento atualizando a engenharia clínica O sistema tem integração com a engenharia clínica em formato de dashboard para a fácil compreensão, apresentando todos os parâmetros monitorados, e em caso de falha enviará um alerta para o setor, assim ganhando tempo e agilidade na resolução da falha encontrada. Conforme apresentado em resultados, evidenciando um caso prático, com o sistema proposto a engenharia clínica ganhará agilidade para a resolução do problema, podendo atuar antes mesmo que o equipamento pare por completo, aumentando assim a disponibilidade e ganhos ao hospital. Para futuros estudos é interessante analisar a interessam da engenharia clínica teria com o novo sistema, se seria bem adaptado ao dia a dia da equipe, avaliar possíveis treinamentos para agilizar ainda mais o atendimento ao equipamento. 5. Conclusões Além de possível integração de relatórios de falhas, para que a própria engenharia clínica possa avaliar possíveis causas de falhas recorrentes e propor manutenções preventivas ainda mais eficazes. 6. Referências Bibliográficas Aparecida, Naci. INSTRUÇÕES DE USO Equipamento de Ressonância Magnética MAGNETOM Avanto, marca SIEMENS. MAGNETOM Family Manual do Usuário – Sistema de RM syngo MR E11. MS/ANVISA Nº. de Registro: 10345162003, 2013 C. M. Godoi. ANÁLISE DA DISPONIBILIDADE DE EQUIPAMENTOS MÉDICO ASSISTENCIAIS APÓS REESTRUTURAÇÃO DA PROGRAMAÇÃO DE MANUTENÇÃO PREVENTIVA EM UM HOSPITAL PÚBLICO DE GRANDE PORTE E ALTA COMPLEXIDADE. Dissertação (Mestrado em Engenharia Biomédica). Universidade Federal de Uberlândia. Uberlândia. 2016. p.162. CARVALHO, J. J. A. D. Estratégias de Manutenção Hospitalar: Aplicação à Ressonância Magnética Nuclear. Dissertação (Mestrado em Manutenção Industrial). Departamento de Engenharia Mecânica e Gestão Industrial da Faculdade de Engenharia da Universidade do Porto. Porto, 2007. p.83-95. Claudio, João ANÁLISE DE DADOS DE MANUTENÇÃO PARA AVALIAÇÃO DA DISPONIBILIDADE DE EQUIPAMENTOS DE RESSONÂNCIA MAGNÉTICA EM ESTABELECIMENTOS ASSISTENCIAIS DE SAÚDE. Uberlândia MG, p.159, 2019. Monografia (Pós-Graduação) – Universidade Federal de Uberlândia Coelho, R. R; et al. Influência das Estratégias de Manutenção na Disponibilidade Física de Equipamentos de Moagem de uma Mineradora. Anais. In: Simpósio de Engenharia de Produção. 14. Bauru. 2017. Diaz, Eduardo TÉCNICAS DE DIAGNÓSTICOS POR IMAGENS: RESSONÂNCIA MAGNÉTICA NUCLEAR. Porto Alegre RS, 25p., 1998. Departamento de Biofísica – Universidade Federal do Rio Grande do Sul. Ferrarini, Maria C. Imagem por ressonância magnética: princípios básicos. Santa Maria RS, 9p., 2009. Dissertação (Doutorado) – Universidade Federal de Santa Maria. Garcia, Domingues; et al. Gestão de Material Médico-Hospitalar e o Processo de Trabalho em um Hospital Público. Revista Brasileira de Enfermagem, v.65, n.2, 2012. https://doi.org/10.1590/S0034-71672012000200021 HASHEMI, Ray Hashman; BRADLEY JUNIOR, William G; LISANTI, Christopher J. MRI: The Basic. 2. ed. Philadelphia: Lippincott Williams & Wilkins, 2004. Júnior, E. A Yamashita, H ASPECTOS BÁSICOS DE TOMOGRAFIA ASPECTOS BÁSICOS DE TOMOGRAFIA COMPUTADORIZADA E RESSONÂNCIA C. M. Godoi. ANÁLISE DA DISPONIBILIDADE DE EQUIPAMENTOS MÉDICO ASSISTENCIAIS APÓS REESTRUTURAÇÃO DA PROGRAMAÇÃO DE MANUTENÇÃO PREVENTIVA EM UM HOSPITAL PÚBLICO DE GRANDE PORTE E ALTA COMPLEXIDADE. Dissertação (Mestrado em Engenharia Biomédica). Universidade Federal de Uberlândia. Uberlândia. 2016. p.162. COMPUTADORIZADA E RESSONÂNCIA MAGNÉTICA. São Paulo Rev Bras Marquez, P. B. et al. Proposta de Medidas de Avaliação de Desempenho para o Centro de Engenharia Clínica e Bioequipamentos do HCFMRP-USP. In: Simpósio de Gestão em Organizações de Saúde. Revista: Medicina (Ribeirão Preto. Online). v.48, n.1, 2015. https://doi.org/10.11606/issn.2176-7262.v48i1p65-76 MYTTON, O. T.; VELAZQUEZ, A.; BANKEN, R.; et al. Introducing new technology safely. Quality and Safety in Health Care, v. 19, n. Suppl 2, p. i9–i14, 2010. Disponível em: http://qualitysafety.bmj.com/lookup/doi/10.1136/qshc.2009.038554. Acesso em: 10 nov. 2019. Meireles, Fernanda. F Souto, Marcelo. N. MANUAL DE TÉCNICAS EM RESSONANCIA MAGNETICA. Rio de Janeiro: Ed. Rubio, 2011 Meireles, Fernanda. F Souto, Marcelo. N. MANUAL DE TÉCNICAS EM RESSONANCIA MAGNETICA. Rio de Janeiro: Ed. Rubio, 2011 Meireles, Fernanda. F Souto, Marcelo. N. MANUAL DE TÉCNICAS EM RESSONANCIA MAGNETICA. Rio de Janeiro: Ed. Rubio, 2011 SANTIAGO, J. S. Análise das Variáveis que Impactam no Custo do Serviço de Ressonância Magnética nas Entidades Hospitalares: Um Estudo Comparativo nos Hospitais da UnB e da UFRN. Dissertação (Mestre em Ciências Contábeis). Universidade Federal do Rio Grande do Norte. 2007. p.154. SORENSON C, Drummond M, Kristensen FB, Busse R. How can the impact of health technology assessments be enhanced? World Health Organization on behalf of the European Observatory on Health Systems and Policies; 2008. Available from: http://www.euro.who.int/__data/assets/pdf_file/0019/73225 /E93420.pdf. Acessado 10 nov. 2019. SILVA, M. L. Critérios Norteadores para a Tomada de Decisão Quanto à Incorporação de um Equipamento de Ressonância Magnética em um Hospital Pediátrico, da Rede Pública Estadual, em Santa Catarina. Dissertação (Mestrado em Epidemiologia). Universidade Federal do Rio Grande do Sul. Porto Alegre, 2012 SILVA, G. L. Análise da Relação entre Disponibilidade e Manutenção dos Mamógrafos e Tomógrafos do HCU-UFU. Trabalho de Conclusão de Curso (Departamento de Engenharia Elétrica – Biomédica). Universidade Federal de Uberlândia, Uberlândia, 2015. p.72 (Departamento de Engenharia Elétrica – Biomédica). Universidade Federal de Uberlândia, Uberlândia, 2015. p.72 Silva, L. M; Ferreira, A. C. M. Sistemas de Cadastro e Manutenção de Equipamento Médico-Hospitalar: Proposta para o Desenvolvimento como uma Ferramenta para Auxiliar a Gestão de Manutenção da Engenharia Clínica nos Hospitais. Fatec Bauru. http://fatecbauru.edu.br/mtg/source/Sistema%20de%20cadastro%20de%20equip am ento%20m%C3%A9dico-hospitalar%20.pdf>. Acesso em: 09 de julho de 2019. Souza, Lucas ESTUDO E ANÁLISE DA USABILIDADE E FATORES HUMANOS NA INSTALAÇÃO DO EQUIPAMENTO DE RESSONÂNCIA MAGNÉTICA SIEMENS MAGNETOM AERA. Brasília DF, 97p., 2017. Monografia (Bacharelado) – Universidade de Brasília – UnB Faculdade UnB Gama – FGA Souza, A. F; Coelli, F. C. Redução da Indisponibilidade de Equipamentos de RM Através de Política de Treinamento. Revista Brasileira de Inovação Tecnológica em Saúde. v.1, n.3, 2011. TRIGAS, S.C. MONITORAMENTO ONLINE 24HS DE SUA RESSONÂNCIA Disponível em: < http://www.trigasbrasil.com/monitoramento/index.html> Acesso em: 21 nov. 2022 Westbrook, Catherine. Roth, Carolyn. Talbot, John RESSONÂNCIA MAGNÉTICA APLICAÇÕES PRÁTICAS. Rio de Janeiro: Ed. Guanabara, Koogan, 2013. YAMANAKA, O. M. Orientações sobre os Principais Requisitos de Instalação de Equipamentos Médico-Assistenciais, Uberlândia, p.48, 2019, Dissertação (Bacharelado) – Universidade Federal de Uberlândia Faculdade de Engenharia Elétrica. ← Post anterior ← Post anterior RevistaFT A RevistaFT é uma Revista Científica Eletrônica Multidisciplinar Indexada de Alto Impacto e Qualis “B”. Periodicidade mensal e de acesso livre. Leia gratuitamente todos os artigos e publique o seu também clicando aqui. Contato Queremos te ouvir. 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https://openalex.org/W4390511578	https://environmentalmicrobiome.biomedcentral.com/counter/pdf/10.1186/s40793-023-00545-2	English	null	A unified compendium of prokaryotic and viral genomes from over 300 anaerobic digestion microbiomes	Environmental microbiome	2,024	cc-by	14,021	Abstract Background The anaerobic digestion process degrades organic matter into simpler compounds and occurs in strictly anaerobic and microaerophilic environments. The process is carried out by a diverse community of micro- organisms where each species has a unique role and it has relevant biotechnological applications since it is used for biogas production. Some aspects of the microbiome, including its interaction with phages, remains still unclear: a better comprehension of the community composition and role of each species is crucial for a cured understanding of the carbon cycle in anaerobic systems and improving biogas production. Results The primary objective of this study was to expand our understanding on the anaerobic digestion microbi- ome by jointly analyzing its prokaryotic and viral components. By integrating 192 additional datasets into a previ- ous metagenomic database, the binning process generated 11,831 metagenome-assembled genomes from 314 metagenome samples published between 2014 and 2022, belonging to 4,568 non-redundant species based on ANI calculation and quality verification. CRISPR analysis on these genomes identified 76 archaeal genomes with active phage interactions. Moreover, single-nucleotide variants further pointed to archaea as the most critical members of the community. Among the MAGs, two methanogenic archaea, Methanothrix sp. 43zhSC_152 and Methanoculleus sp. 52maCN_3230, had the highest number of SNVs, with the latter having almost double the density of most other MAGs. Conclusions This study offers a more comprehensive understanding of microbial community structures that thrive at different temperatures. The findings revealed that the fraction of archaeal species characterized at the genome level and reported in public databases is higher than that of bacteria, although still quite limited. The identification of shared spacers between phages and microbes implies a history of phage-bacterial interactions, and specifically lysogenic infections. A significant number of SNVs were identified, primarily comprising synonymous and nonsynony- mous variants. Together, the findings indicate that methanogenic archaea are subject to intense selective pressure and suggest that genomic variants play a critical role in the anaerobic digestion process. Overall, this study provides †Guido Zampieri and Laura Treu contributed equally to this work. Centurion et al. Environmental Microbiome (2024) 19:1 https://doi.org/10.1186/s40793-023-00545-2 Centurion et al. Environmental Microbiome (2024) 19:1 https://doi.org/10.1186/s40793-023-00545-2 Environmental Microbiome Open Access © The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecom- mons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. A unified compendium of prokaryotic and viral genomes from over 300 anaerobic digestion microbiomes Victor Borin Centurion1, Alessandro Rossi1, Esteban Orellana1, Gabriele Ghiotto1, Balázs Kakuk2, Maria Silvia Morlino1, Arianna Basile3, Guido Zampieri1†, Laura Treu1† and Stefano Campanaro1 †Guido Zampieri and Laura Treu contributed equally to this work. Correspondence: Guido Zampieri guido.zampieri@unipd.it Laura Treu laura.treu@unipd.it Full list of author information is available at the end of the article Correspondence: Guido Zampieri guido.zampieri@unipd.it Laura Treu laura.treu@unipd.it Full list of author information is available at the end of the article Introductionh The increasing demand for energy and the depletion of fossil fuels have shifted attention towards alterna- tive energy production processes. One such process is anaerobic digestion (AD), which has a lower environ- mental impact, while supporting the concept of circular economy by utilizing a variety of end-products including food, agricultural, industrial and municipal wastes. AD is a natural process that breaks down complex organic matter into simpler compounds, occurring in ecological niches with low oxygen or strictly anaerobic conditions, such as bogs, sediments and the guts of herbivores [1]. This process is also utilised at industrial-scale activities for biogas production. The breakdown is carried out by a microbial community that can range in complexity from a few species to an extremely complex microbiome con- sisting of thousands of species [2–4]. The biogas obtained from industrial reactors typically contains a mixture of methane (CH4) and carbon dioxide (CO2), with trace lev- els of hydrogen sulfide (H2S), ammonia (NH4 +), hydrogen (H2), and various volatile organic compounds, depend- ing on the feedstock and on the functional activity of the microbiota [5]. fi y The rise of metagenomics in the last two decades has revealed the important role viruses play in shap- ing microbial communities [11]. In many environments viruses are responsible for selective pressure, lateral gene transfer, and nutrients recycling, all of which impact the microbiota. Understanding their role at community level is crucial and offers an opportunity to fine-tune the AD process. Despite the important role viruses play, the viral community of AD has received little attention [12, 13]. This gap is due to difficulties associated with viral metagenomics compared to the prokaryote investigation. To separate the viral fraction of the community from the microbial is challenging, as viruses represent a small fraction of the total genetic material. Therefore, viruses are more understudied than bacteria and archaea. How- ever, research has shown that phages, in particular, are involved in microbiome dynamics and process stability, regulating microbial abundance and diversity in full-scale biogas units [14]. From a biotechnological perspective, CH4 is the most significant constituent of the biogas generated during the methanogenesis step of the AD process and is produced by methanogenic Archaea [6, 7]. AD is carried out by a diverse community of microorganisms, where each spe- cies has a unique role in highly specialized and complex microbiomes [8]. © The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecom- mons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Centurion et al. Environmental Microbiome (2024) 19:1 Page 2 of 19 Centurion et al. Environmental Microbiome a more balanced and diverse representation of the anaerobic digestion microbiota in terms of geographic location, temperature range and feedstock utilization. Keywords Anaerobic digestion, Metagenomics, Bacteriophage, Biogas, Functional annotation, Microbe, Metagenome-assembled genome composition, temperature, organic loading rate, hydrau- lic retention time, and other physicochemical parameters [9, 10]. Controlling these parameters can offer unique opportunities for microbial selection or manipulation to improve the process efficiency.h Introductionh First, complex organic matter is trans- formed by hydrolytic bacteria into soluble organic com- pounds. Second, acidogenic bacteria transform the latter into intermediates (e.g., volatile fatty acids), with syn- trophic acetogenesis playing an essential role in break- ing compounds down into the simplest molecules. Third, methanogenesis is performed by archaea using ace- tate, methylamine and/or CO2 and H2 to produce CH4. Understanding the composition and role of each species in the community is crucial for a better understanding of the carbon cycle in anaerobic systems and improving biogas production. However, the isolation of many spe- cies using classical microbiological techniques can be challenging, making metagenomics an ideal alternative for characterizing the community complexity. In addi- tion to the microbiome, the efficiency of AD is dependent on several interconnected factors, including feedstock Metagenome sequencing has become a valuable tool to gain insights into the genetic repertoire of non-cultivable biogas community members [15]. Advances in sequenc- ing throughput and computational techniques nowadays allow the recovery of Metagenome-Assembled Genomes (MAGs) from highly diverse environments [16]. These MAGs are obtained through binning together assem- bled contigs with similar sequence composition, depth of coverage, and taxonomic affiliations [17–19]. Despite limitations regarding completeness and contamination [20], MAGs are useful proxies for studying microbial and viral genomes present in the system, providing insights into taxonomy, functional properties, and dynamics of the microbiome. Several studies have attempted to gain insights into the AD microbiome for biogas production [15, 21], but the limited geographic distribution of the samples and the lack of a global analysis on the viral frac- tion have prevented a complete characterization of the microbiome. To address this gap, a global meta-analysis study was performed to tentatively assess the impact of physicochemical parameters, microbiota, and reactor characteristics on the AD process. The aim of this study Centurion et al. Environmental Microbiome (2024) 19:1 Centurion et al. Environmental Microbiome Page 3 of 19 completeness lower than 50% and contamination higher than 10% were filtered out. The two sets of medium–high quality MAGs were combined and used as input for the dereplication step. MAGs were dereplicated with dRep v3.2.2 [33] using parameters coherent with the previ- ously proposed definition of species [34]: 95% average nucleotide identity on at least 50% of the MAG genome. To ensure sequencing depth homogeneity, libraries were subsampled to 8 million reads and mapped onto the MAGs with Bowtie2 v2.5.0 [28]. Taxonomy and functional prediction Taxonomy and functional prediction GTDB-Tk v1.4.1 [40] was used to taxonomically assign prokaryotic MAGs, with the release 95 of Genome Tax- onomy Database (GTDB) serving as reference database. Prodigal v2.6.3 [41] was used to predict protein-encod- ing genes, and functional annotation was carried out with eggnog-mapper v2.1.7 [42] on dereplicated MAGs and viral genomes. The completeness of KEGG modules was determined for each MAG using KEMET (release version July 2022) [43] with the parameter ’-a eggnog’ utilized to incorporate the eggNOG annotations. MAG functions were assessed by manually inspecting specific KEGG modules related to functional classes of AD: methanogenesis (M00357, M00567); beta-oxidation (M00087); anaerobic carbon metabolism (M00173, M00377, M00618); nitrogen metabolism (M00528, M00529, M00530, M00531, M00615, M00804); sulfate reduction (M00176, M00596). For phylogenetic analy- sis of MAGs, PhyloPhlAn 3.0.51 [44] was used with the parameters—diversity high and—fast, and PhyloPhlAn database. The resulting Newick file was visualized using iTOL v6.7 [45]. Taxonomic assignment of phages was carried out using PhaGCN v2.0 [46] considering con- tigs above 5kbp (–len 5000). Introductionh For each prokaryotic MAG, the relative abundance and reads counts were calculated with coverM (https://github.com/wwood/ CoverM) using the “genome” subcommand. Alpha- diversity was calculated with the “estimate_richness” function of Phyloseq v1.40.0 R package [35] using the mapped reads counts on the MAGs. Beta-diversity was calculated with ExpressBetaDiversity v1.0.10 [36] using Bray–Curtis dissimilarity and the relative abundances of prokaryotic MAGs. Virus predicting software PPR-Meta v1.1 [37], CheckV v0.7.0 (end_to_end program) [38], and VIBRANT v1.2.0 [39] were launched on the assem- bled contigs. A contig was assigned as viral if the predic- tion was independently confirmed by at least two of the applied programs. All the scaffolds predicted as viral but shorter than 5 kbp were removed to reduce the number of mispredicted and partially assembled viral genomes. was to complement and consolidate previous results and establish a more comprehensive reference database of microbial and viral genomes. In addition, strain-resolved metagenomics was applied to reveal fine-scale evolution- ary mechanisms, functional dynamics, and strain-level metabolic variation, potentially contributing to the selec- tion within a microbial community. Collection of samples and metadata In order to expand the Biogas Microbiome database [15], 192 additional metagenomic datasets were retrieved from SRA using the SRA toolkit fastq-dump software v2.10.8 [22]. The present version of the database now includes details regarding the 314 metagenomes derived from AD biogas reactors, which were identified through searches on SRA and the literature (Additional file 1). Only data- sets published between 2014 and 2022 were considered, with metadata taken from the respective experiments when available. Temperature information was present in most experiments and classified as psychrophilic (0–19 °C), mesophilic (20–40 °C) and thermophilic (41– 56 °C). The metadata for the additional datasets can be checked in Additional file 1. Reads were filtered and qual- ity checked as previously described [23] using a two-step procedure: at first Trimmomatic v0.39 [24] was used in its paired-end mode, using the parameters leading = 20, trailing = 20, slidingwindow = 4:20 and minlen = 70, at second, a further check was performed to remove adapters and contaminants associated to phiX174 with BBDuk v38.86 [25]; filtering was performed using the files “adapters.fa” and “phix174_ill.ref.fa” as references, and the parameters k = 21 ktrim = r mink = 11 hdist = 2 threads = 4. Assembly, binning, and coverage Metagenome assembly was performed independently for all the samples of one experiment using MEGAHIT v1.2.9 [26]. Assembly statistics were obtained using QUAST v5.0.2 [27]. Bowtie2 [28] was used to map reads back to the contigs for the creation of coverage files for the binning analysis. Every assembly was binned both with MetaBAT2 v2.12.1 [29] and MaxBin v2.2.7 [30] using standard parameters. Previous experiments, which underwent analysis using MetaBAT2 only, were binned with MaxBin. 11,781 MAGs previously generated by Ma and colleagues [21] were downloaded from “http://dx. doi.org/https://doi.org/10.5524/100842” and included in the database. Completeness and contamination of MAGs generated in this study and in MAGs generated by Ma and colleagues were assessed with CheckM v1.1.2 [31] and CheckM2 v0.1.2 [32] for comparative assessments across different versions of the software. MAGs with Page 4 of 19 Centurion et al. Environmental Microbiome (2024) 19:1 Centurion et al. Environmental Microbiome each phylum was compared against the SNVs/Mbp of all MAGs containing SNVs. CRISPR detection [ MinCED v0.4.2 [48] was used to identify clustered regu- larly interspaced short palindromic repeats (CRISPR) spacers in the dereplicated MAGs. A BLAST v2.6.0 [49] search for viral sequences recovered in this study against the spacers was performed using parameters including -task blastn-short, -gapopen 10, -gapextend 2, -penalty -1, -word_size 7, -perc_identity 100, as previ- ously reported [50]. The results were filtered to include only those that matched the whole length of the spacer. To visualize interactions between Phages and Archaea, HoloViews v1.15.4 (https://github.com/holoviz/holov iews), along with Pandas (v2.3.1) and NumPy (v1.21) packages in python v3.6.13 were used. The phage cover- age was estimated using CoverM v0.6.1 with the “contig” subcommand. To compare the presence of phages in dif- ferent experiments for each virMAG, the abundance was divided by the total number of reads in each sample. To determine the relationship between PTR and pro- cess temperature for individual MAGs, those detected in at least five different temperature values spanning a mini- mum range of 10 °C were selected, in order to consider a reasonable temperature spectrum. For the selected MAGs, second-order ordinary polynomial models were fitted with temperature and PTR as independent and dependent variables, respectively, using the lm func- tion in R v3.6. To correct for multiple testing, the p-val- ues were adjusted for linear and quadratic coefficients using the Benjamini–Hochberg method. Fits that had at least one significant coefficient at a 0.1 FDR level were selected. Finally, preferential temperature ranges were calculated by considering PTR values greater or equal to the median PTR of each MAG independently, and then taking maximum and minimum temperatures for the conditions where these were detected. Only MAGs hav- ing at least three PTR values available were considered in this case. each phylum was compared against the SNVs/Mbp of all MAGs containing SNVs. Species replication rates across the 8 million reads subsamples were estimated using CoPTR v1.1.4 with default parameters [47]. This approach estimates the slope of read coverage on genomes or MAGs assum- ing that fast-replicating microbes are associated with increasing DNA abundance the closer a sequence is to the origin of replication. Such a slope is named peak-to- trough ratio (PTR) and quantifies DNA synthesis and generation rate in terms of an adimensional coverage decay rate, which in turn is a proxy of microbial repli- cation rate. To guarantee robust PTR estimates, MAGs with minimum 5,000 aligned reads were selected for this analysis. Statistical analysish The Pearson correlation between Chao1, Shannon alpha-diversity indices, temperature, and checkM and checkM2 quality estimates was calculated in R v4.2.1 utilizing ggpubr v0.6.0. The cumulative abundance of archaeal MAGs was defined as the sum of all samples relative abundance with complete and one block miss- ing of M00357 (Methanogenesis, acetate = > methane) and M00567 (Methanogenesis, CO2 = > methane) KEGG modules. The read counts of MAGs with complete and one block missing M00357 and M00567 KEGG modules were filtered and values normalized as relative abun- dance. The data were then plotted using the Complex- Heatmap v2.12.1 R package [53]. Strain analysish The analysis of variants was performed using the tool InStrain v1.6.3 [51], specifically the module “profile”. To do this, a scaffold-to-bin-file establishing correspond- ences between contigs and 160 MAGs was generated using the parse_stb.py script from the dRep v3.2.2, and the ORF prediction file obtained using Prodigal v2.6.3. InStrain profile was launched with additional parameters: “–min_genome_coverage 1”, “–skip_plot_generation”, “–min_mapq 2”, “–min_read_ani 0.98” and “–skip_mm_ profiling”. A MAG was deemed present in the sample only if it exhibited a breadth of coverage greater than 0.5. Variants were then filtered based on the procedure outlined in Ghiotto et al. (2023) [52]. The Mann–Whit- ney U-test was employed to evaluate the accumulation of variants within a specific phylum in comparison to the overall population. The distribution of SNVs/Mbp for Expansion of the global anaerobic digestion microbiome database To create a more balanced representation of MAGs from various countries, the previous iteration of the Biogas Microbiome database featured sequences from 18 studies, containing 123 samples [15]. However, it was biased towards samples collected from Denmark, which accounted for 68% of the database, mostly deriving from laboratory-scale biogas reactors and batch tests. In the present version, we added 24 studies to the database, resulting in a total of 314 samples. Notably, a significant number of samples and MAGs, derived from Chinese biogas plants, were included in a recent study and imple- mented in the new database [21]. This expanded Biogas Centurion et al. Environmental Microbiome (2024) 19:1 (2024) 19:1 Centurion et al. Environmental Microbiome Page 5 of 19 Fig. 1 Geographic and microbial diversity of the expanded Biogas Microbiome database. The expanded anaerobic digestion microbiome database includes 314 samples distributed across 11 countries, with Denmark and China being the origin of most samples. Information about all the samples can be visualized in Additional file 1 Fig. 1 Geographic and microbial diversity of the expanded Biogas Microbiome database. The expanded anaerobic digestion microbiome database includes 314 samples distributed across 11 countries, with Denmark and China being the origin of most samples. Information about all the samples can be visualized in Additional file 1 Microbiome database now encompasses samples col- lected in eleven countries (Fig. 1, Additional file 1), with China and Denmark being the primary contributors, accounting for 75% of the total number of samples. Sam- ples were categorized according to reactor type, with 111 described as “lab scale” or “batches” operated in six coun- tries, 104 described as “full-scale biogas plants”, and 99 as “(semi)continuous lab reactors” or “CSTR” (Additional file 1). majority of the samples in this study. However, psychro- philic temperatures generally slow down the metabolic activity of anaerobic microorganisms. This can result in a prolonged retention time for organic matter in the digester and lead to a decrease in methane yield per unit of organic matter compared to mesophilic or thermo- philic conditions [56, 57]. Prokaryotic community compositionh Other 45 less widespread MAGs were here assigned to Limno- chordia DTU010 and several species of the same order have also recently been identified across a numerous independent set of full-scale plants in sensible abundance [61], further raising our interest in this uncharacterized taxon. Besides, MAGs belonging to the phylum Candi- datus Atribacteria were widespread but with a low rela- tive abundance (less than 0.1%). This finding suggests that methanogenic Archaea and Synergistetes, for which only a small number of MAGs have been identified in the database, play crucial roles and are very flexible, being able to adapt to a variety of environmental conditions. The two most widespread Archaea, Methanothrix sp. 43zhSC_152 and Candidatus Methanoculleus thermohy- drogenotrophicum 31mySI_10, were detected in 49 and 42 samples, respectively, with relative abundance ≥ 1%. Similarly, the two most common Synergistetes MAGs, Synergistaceae sp. 24abBP_148 and Acetomicrobium fla- vidum 43zhSC_162, were present in 35 and 15 samples at relative abundance ≥ 1%. In fact, members of the Syn- ergistaceae taxon were previously found to show ace- tate-oxidizing ability, which may work in syntrophy with hydrogenotrophic methanogens for methane production [62, 63]. This crucial cooperation activity may explain the similar common widespread distribution of these two phyla. On the other hand, some Candidate phyla and Planctomycetes have a scattered distribution. Genome size is correlated with the number of genes and, on aver- age, Planctomycetes have the second-highest genome size. This suggests that the ability to colonize many differ- ent samples is not necessarily related to species gene con- tent. The highest archaea/bacteria ratio was found in a thermophilic batch reactor fed with a synthetic medium containing methanol (Yan_2020_Batch_2, Additional file 1). The Archaea in Yan and colleagues [62] is repre- sented by one MAG from the Euryarchaeota phylum with 100% completeness which could not be taxonomi- cally assigned with higher specificity, indicating the need for a more detailed taxonomic investigation on some Archaea branches. Ph k t i t ti i di t h d d t ti had more than 50% mapped reads, indicating that this version of the Biogas Microbiome database predomi- nantly characterizes the microbiome. The batch experi- ment enriched culture with cellulose (Jia_2018_Batch_12 and 13) [58] had the highest proportion of mapped reads, with about 94% of bacterial MAGs mapped. Prokaryotic community compositionh i To gather data, only basic statistics were collected from the metadata of different publications. The data were evenly distributed across different temperatures, with 4% being psychrophilic, 51% mesophilic, and 38% ther- mophilic; 22 samples had no clearly reported operating temperature or reactor type. This study provides a more comprehensive representation of microbiota structure and species growing over a wider range of temperatures, compared to previous investigations [15]. Temperature is an essential factor in determining the diversity of the community, as it was found to be inversely correlated with Chao1 and Shannon alpha-diversity indices (Pear- son’s r = − 0.4, p = 1·10–12) (Additional file 2: Fig. 1). The three samples with the highest diversity were collected from reactors operated at the lowest temperature, such as Ma_2021_BGP_27, 29, and 34, with temperatures rang- ing from 14 to 17 °C and having Chao1 ∼4,566 and Shan- non ∼6.37 (Fig. 1). Previous studies have reported that psychrophilic temperatures have more diversity of meth- anogenic Archaea [54] and fluctuation in rare biosphere taxa [55] than mesophilic reactors, which represented the The binning process generated 11,831 MAGs, which, when combined with the database by Ma and colleagues [21], resulted in a total of 26,612 MAGs. To reduce redun- dancy, 4,568 MAGs were selected for clustering based on ANI calculation and quality analysis with CheckM. Of these, 2,217 (48.5%) were classified as high quality (completeness > = 90%; contamination < = 10%) while 2,351 (51.5%) were of medium quality (90% > complete- ness > = 50%; contamination < = 10%) (see Availability of Data and Materials). These results were validated with CheckM2 and presented a high correlation with those of CheckM (R2 = 0.7). This concordance is especially rel- evant as it demonstrates the accuracy of CheckM2 on a large dataset, while still validating quality assessments obtained with CheckM. Specifically, the estimated com- pleteness was higher for 59% of the MAGs, lower for 40.8%, and 0.24% showed the same value as CheckM. Notably, out of the 387 Candidatus MAGs, 293 (75.7%) had higher completeness scores with CheckM2, and 57 (14.7%) had completeness scores that were two times higher than CheckM. Additionally, 76% of the samples Centurion et al. Environmental Microbiome (2024) 19:1 Page 6 of 19 Centurion et al. Environmental Microbiome syntrophic interactions with methanogens [60]. Prokaryotic community compositionh pp Taxonomic investigation on the prokaryotic commu- nity revealed that members of Firmicutes dominate, com- prising 38% of the MAGs in the database, followed by Proteobacteria (12%) and Bacteroidetes (10%). Archaea are represented by 198 species (4.3%), mainly from the Euryarchaeota phylum (76.3%), while Candidatus Bath- yarchaeota and Candidatus Diapherotrites are repre- sented by 13 and 12 members, respectively. Previous metataxonomy-driven investigations [15] identified 53 MAGs belonging to the Euryarchaeota phylum while the other Candidatus phyla were not represented. Moreover, among the archeal population, Euryarchaeota were simi- larly identified as the predominant phylum in another large-scale study examining the microbiome of 80 anaer- obic digesters [59]. Among all the MAGs identified, only a small number (3.7%) were assigned at species level (152 Bacteria and 19 Archaea), while 19.3% were assigned at genus level (794 Bacteria; 87 Archaea). These results confirm that the fraction of archaeal species already characterized at genome level and reported in public repositories of microbial genomes is higher (9.6%) than that of bacteria (3.5%), but still very low. Analysis performed on MAG relative abundance in all the samples provided insight into the distribution of microbial species in the database. By calculating the number of samples in which each MAG abundance was greater than 0.01, 0.1, and 1%, Firmicutes, Euryarchaeota, Bacteroidetes, Proteobacteria, and Synergistetes were identified to be more widespread, while others, such as Fibrobacteres, Ignavibacteriae, Crenarchaeota, and many Candidatus phyla had a more scattered distribution (Additional file 2: Fig. 2). Similar results were obtained using 0.1% or 0.01% as the relative abundance threshold to define the presence of a MAG (see Availability of Data and Materials). Strikingly, all the four bacterial strains enriched in previously characterized biofilm commu- nities developed on the gas injection systems of biogas upgrading reactors were among the top widespread MAGs across samples at all relative abundance thresh- olds [60]. These MAGs include Firmicutes 50dbBF_058, Firmicutes 50dbBF_049, Firmicutes 50dbBF_057, and Synergistaceae sp. 24abBP_148. In particular, the most widely detected MAG Firmicutes 50dbBF_058 is alterna- tively classified as Limnochordia DTU010 and was pre- viously found to possess the glycine cleavage system and the glycine synthase-reductase pathway for CO2 reduc- tion, which are considered important for establishing Phage‑prokaryote interactions indicate shared adaptation strategiesi A total of 79,922 viral genomes were identified after dereplication, and 32% of them were successfully classi- fied. The composition of the viral community was largely dominated by tailed bacteriophages of the class Cau- doviricetes, as established by previous works [12, 13, 64, 65], comprising 97.6% of all viruses. Families Strabo- viridae, Peduoviridae, and Herelleviridae were the most represented in the dataset, with 2,927, 2,887, and 2,825 members, respectively. However, the family with the Centurion et al. Environmental Microbiome (2024) 19:1 Page 7 of 19 Centurion et al. Environmental Microbiome greatest coverage in the 268 samples was Peduoviridae, followed by Straboviridae and Drexlerviridae (Additional file 2: Fig. 3B, see Availability of Data and Materials for full statistics). The virome of the 314 samples analyzed represented between 8 × 10–4 and 0.86% of the total com- munity. As expected, 11 samples from an enrichment of viromes experiment [12] had a higher proportion of phages (0.35–0.86%). However, the virus with the high- est proportion, virMAG MPIJXP02F1_92297, was found at low abundance in these samples, while thermophilic reactors fed with exhausted gasses and carbohydrates had a higher abundance of this virus. Although PhaGCN was not able to taxonomically identify this phage, the presence of enzymes related to DNA binding domains (pfam13443), transposase related to bacteriophages (COG5421), and DNA-binding transcriptional regulator (COG3655) confirmed its classification as a viral genome. greatest coverage in the 268 samples was Peduoviridae, followed by Straboviridae and Drexlerviridae (Additional file 2: Fig. 3B, see Availability of Data and Materials for full statistics). The virome of the 314 samples analyzed represented between 8 × 10–4 and 0.86% of the total com- munity. As expected, 11 samples from an enrichment of viromes experiment [12] had a higher proportion of phages (0.35–0.86%). However, the virus with the high- est proportion, virMAG MPIJXP02F1_92297, was found at low abundance in these samples, while thermophilic reactors fed with exhausted gasses and carbohydrates had a higher abundance of this virus. Although PhaGCN was not able to taxonomically identify this phage, the presence of enzymes related to DNA binding domains (pfam13443), transposase related to bacteriophages (COG5421), and DNA-binding transcriptional regulator (COG3655) confirmed its classification as a viral genome. Permanova analysis revealed a significant correlation (p < 0.01) between the temperature of the reactor and the presence of different phages (Additional file 2: Fig. 3A). Fig. 3 Phylogenetic tree and virome interaction. A The tree is represented in an inverted orientation, with branches color-coded according to phylum taxonomy. Legend is clockwise oriented starting from Euryarchaeota phylum The outermost ring exhibits the phylogenetic classification of the various taxa (Phylum), followed by subsequent rings that display the log-normalized coverage for each MAG (Coverage), the percentage of guanine-citosine (GC%), and the number of interactions with phages (Phages). Red asterisks indicate MAGs with more than 32 phage interaction signals. Phages that infect more than two MAGs are indicated by inner lines. B Relationships between different archaeal genera and the phages that infect them, the numbers indicate the total number of genomic sequences (See figure on next page.) Functional analysis of the microbial community Functional analysis of the microbial community To understand the biological drivers of AD, functional annotation was integrated with taxonomic abundance, stratifying KEGG modules at different taxonomic levels. In order for a microbial consortium to perform a meta- bolic process, the simultaneous presence of all its func- tional units is required, though some gaps may exist in MAGs. As a result, only MAGs with complete and one-block-missing modules were considered (Addi- tional file 3 for Archaea, Additional file 4 for Bacteria). Some functional modules are of particular interest in AD communities (Materials and methods, Taxonomic and functional prediction. Besides the obvious role of methanogenesis,beta-oxidation is relevant for fatty acid degradation when feedstocks are particularly rich in lipids. Additionally, modules involved in anaerobic car- bon metabolismnitrogen metabolism, and sulfate reduc- tion were also considered due to their influence on the AD process efficiency. and on the interactions between Bacteria and Archaea. For example, ammonia nitrifi- cation influences methane production since NH4 can strongly affect both the methanogenesis process, and the growth of methanogens, including for example Methano- bacterium, Methanosarcina, and Methanospirillum spp. [69, 70]. In the majority of the analyzed experiments, the archaeal abundance was lower than bacteria, which lim- ited the detection of CRISPR. Despite this limitation, 76 archaeal genomes were identified to possess CRISPR arrays with more than 4 spacers. Further analysis of these genomes revealed that the genera Methanother- mobacter and Methanosarcina were the most infected, accounting for 70% of all recorded virus-archaea inter- actions (Fig. 2B). Phages infecting Methanothermobac- ter and Methanosarcina archaea do not belong to the same family. In fact, those with the highest abundance infecting Methanothermobacter are found in a set of batch experiments (Kougias_2016_Batch) [68], while phages infecting Methanosarcina are less dominant but distributed across a greater number of samples, Phage‑prokaryote interactions indicate shared adaptation strategiesi One of the most abundant phages (virMAG MPIBJC05F1_637440, family Vertoviridae) is present in 174 different samples from 9 countries and only two hosts were identified: Firmicutes sp. 28xzH2_85 and Clostridiaceae sp. 31mySI_51, with similar GC% and taxonomically close. Despite the specificity of this virus, the host’s widespread distribution allowed its spread in the AD system across different environmental conditions. including batch experiments (Ma_2021_BGP_52) and biogas plants fed with sweet potato [21] and manure [12]. phages and bacteria suggests a history of phage-bac- terial interactions, and specifically lysogenic infec- tions [66]. Most phages had only one host, but virMAG 384269_AS06 (Herelleviridae family) shared 40 dif- ferent spacers with one bacterial MAG, Candidatus Anammoximicrobium sp. 06rmzA_251. Both have sim- ilar GC percentages (62.61–64.4) and are found exclu- sively in one experiment [67], suggesting they shared a long period of co-evolution, with the incorporation of new spacers in the bacteria and the ability to evade the recognition of CRISPR system by phages. In contrast, virMAG 27752_AS02 (family Straboviridae) was found in spacers of 8 different MAGs belonging to Firmi- cutes Phyla, order Clostridiales, with the exception of one MAG of Tissierellia class. This phage has a broader range of infection and can be present in different condi- tions, from samples with only blood agar (BA) medium and casein (Zhu_2019_CSTR_2) to complex feedstock such as raw municipal biowaste (Tsapekos_2021_ CSTR_5). One of the most abundant phages (virMAG MPIBJC05F1_637440, family Vertoviridae) is present in 174 different samples from 9 countries and only two hosts were identified: Firmicutes sp. 28xzH2_85 and Clostridiaceae sp. 31mySI_51, with similar GC% and taxonomically close. Despite the specificity of this virus, the host’s widespread distribution allowed its spread in the AD system across different environmental conditions. Phage‑prokaryote interactions indicate shared adaptation strategiesi Considering taxonomically classified phages with rela- tive abundance over 0.3%, 27 out of 47 families were influenced by temperature. Rudiviridae predominated in reactors below 25 degrees, while Suoliviridae (n = 527) and Winoviridae (n = 256) were most represented among the 10 dominant families in mesophilic environments. In thermophilic environments, Peduoviridae, Herelleviri- dae, Casjensviridae, and Drexlerviridae had the highest representation among the 16 dominant families. Detection of some evidence regarding previous infec- tions can be used to study virus-host interactions. CRISPR spacers in particular were used to identify 2901 interactions between 546 MAGs and 1,822 viruses (Fig. 2A). The presence of shared spacers between Fig. 2 Microbial diversity of the expanded Biogas Microbiome database. Microbial community similarity across samples expressed by beta-diversity. Outer circles indicate the operational temperature and the community alpha-diversity as expressed by Chao1 and Shannon index Fig. 2 Microbial diversity of the expanded Biogas Microbiome database. Microbial community similarity across samples expressed by beta-diversity. Outer circles indicate the operational temperature and the community alpha-diversity as expressed by Chao1 and Shannon index 2 Microbial diversity of the expanded Biogas Microbiome database. Microbial community similarity across samples expressed circles indicate the operational temperature and the community alpha-diversity as expressed by Chao1 and Shannon index Centurion et al. Environmental Microbiome (2024) 19:1 Page 8 of 19 Centurion et al. Environmental Microbiome including batch experiments (Ma_2021_BGP_52) and biogas plants fed with sweet potato [21] and manure [12]. phages and bacteria suggests a history of phage-bac- terial interactions, and specifically lysogenic infec- tions [66]. Most phages had only one host, but virMAG 384269_AS06 (Herelleviridae family) shared 40 dif- ferent spacers with one bacterial MAG, Candidatus Anammoximicrobium sp. 06rmzA_251. Both have sim- ilar GC percentages (62.61–64.4) and are found exclu- sively in one experiment [67], suggesting they shared a long period of co-evolution, with the incorporation of new spacers in the bacteria and the ability to evade the recognition of CRISPR system by phages. In contrast, virMAG 27752_AS02 (family Straboviridae) was found in spacers of 8 different MAGs belonging to Firmi- cutes Phyla, order Clostridiales, with the exception of one MAG of Tissierellia class. This phage has a broader range of infection and can be present in different condi- tions, from samples with only blood agar (BA) medium and casein (Zhu_2019_CSTR_2) to complex feedstock such as raw municipal biowaste (Tsapekos_2021_ CSTR_5). Archaeal community Out of 198 archaeal MAGs, 70 (35%) have either com- plete or one missing block for methane production from acetate (M00357; acetoclastic methanogens), with most belonging to the class Methanomicrobia (72%), followed by Methanobacteria (24%). Considering methane pro- duction from CO2 (M00567; hydrogenotrophic metha- nogenesis), the proportion of Methanomicrobia is even higher, with 55 MAGs out of 72 (76%). Out of the total 89 MAGs with either one of the methane production modules, 72% have both methane production modules, of which 69% belong to the Methanomicrobia class and 19% to Methanobacteria. Both classes are reported in Page 9 of 19 Page 9 of 19 Centurion et al. Environmental Microbiome (2024) 19:1 Centurion et al. Environmental Microbiome (See legend on previous page.) Centurion et al. Environmental Microbiome (2024) 19:1 Page 10 of 19 Centurion et al. Environmental Microbiome literature as hydrogenotrophic methanogens, which is the most widespread pathway in Archaea [71]. Except for Candidatus Methanoculleus thermohydrogenotrophi- cum 31mySI10, which has a well-defined taxonomic affiliation, all the Candidatus archaeal MAGs harbor incomplete (or two block missing) methanogenic mod- ules (Fig. 3A), indicating the need for a more detailed functional investigation. Actually, a significant portion of genes found in archaea, ranging from approximately 30% to as high as 80%, code for proteins labeled as ’hypo- thetical proteins’. This is primarily due to the challenges in isolating and culturing most archaea in the labora- tory, which makes experimental characterization of their gene repertoire difficult [72]. In contrast, the three most abundant archaeal MAGs in all the samples (Candidatus Methanoculleus thermohydrogenotrophicum 31mySI10, Methanotrix sp. 43zhSC152, and Methanothermobacter wolfeii 31mySI 58) have both modules complete. How- ever, only Methanothrix exhibits both functional meth- ane pathways in vivo experiments [73]. Methanoculleus and Methanothermobacter are known hydrogenotrophs [74], and there is no evidence that they use acetoclas- tic pathways. This emphasizes the necessity for specific archaea annotations to ensure more precise genomes characterization (Fig. 4).h [80], and municipal biowaste [81]. However, the most abundant samples with both modules [4, 74, 79] were fed with acetate and propionate.h The observed variations in methanogenesis and aceto- clastic functions across different biogas reactors indicate a significant influence on the microbial community com- position on biogas production. The enrichment of metha- nogenic species in certain samples, facilitated by specific experimental conditions such as the use of hydrochar, suggests a potential for increased methane production. (See figure on next page.) Fig. 4 Methanogenesis modules in archaeal MAGs. A Phylophlan tree with all archaeal MAGs encoding the methanogenesis modules. MAGs quality (completeness and contamination) and their cumulative abundance (%) are displayed in the three innermost circles. The MAGs more frequently identified in the samples of the AD database are highlighted in light purple. B Heatmap reporting the samples with more than 10% relative abundance of complete and one block missing methanogenesis modules. The barplot represents the relative abundance of each genus in the samples and was calculated by taking into account only MAGs with complete modules, and those with one block missing. M00357: methanogenesis, acetate = > methane; M00567: methanogenesis, CO2 = > methane Archaeal community The confirmation that reactors fed with organic acids, particularly acetate, enrich specific microbial species, irrespective of temperature conditions, suggests a stra- tegic approach for optimizing biogas production. These findings emphasize the importance of tailoring substrate selection and reactor conditions to enhance the perfor- mance of microbial communities, providing valuable insights for improving overall biogas yields in anaerobic digestion processes. Bacterial communityh y The results obtained from the functional analysis indi- cate that three KEGG modules are widespread in the AD bacterial community: beta-oxidation (M00087), dis- similatory nitrate reduction to ammonium (M00530), and assimilatory sulfate reduction to sulfide (M00176) (Additional file 2: Fig. 4). The presence of the “dissimi- latory nitrate reduction to ammonium” function in 71% of phyla suggests that denitrification is widespread in the AD community, and according to this, the two-step process of nitrate conversion to ammonia and finally to nitrogen through denitrification of nitrate to nitrogen module (M00529) is very common. Despite other studies reported that the bacterial dissimilatory nitrate reduction to ammonium pathway only dominates under low nitrate availability and in sulfide-free environments [82, 83], the present data suggest that species using nitrate (rather than oxygen) as electron acceptor are dominant in the AD microbiome. A previous study used a CSTR to remove linear alkylbenzene sulfonate (LAS) present in commer- cial laundry wastewater (Delforno_2020_CSTR_1); in this investigation a higher abundance of dissimilatory nitrate reduction to ammonium, denitrification, and beta-oxidation modules were identified (Additional file 2: g The estimation of the relative abundance of each KEGG module in each sample was obtained by considering the complete modules and those with one block missing. According to this calculation, samples “Kouzuma_2017_ CSTR_4” and “Shi_2021_Batch_1” had the highest values for methanogenesis KEGG modules (M00357, M00567) (Fig. 3B). In the “Shi_2021_Batch” experiment, hydro- char was used to enrich methanogenic species including Methanobacterium, Methanolinea, and Methanothrix genera, and increase methane production. Conversely, “Kouzuma_2017_CSTR_2 and 4” and most samples of “Zhu_2020_CSTR” had the highest abundance of aceto- clastic functions, represented by Methanosarcina, and Methanothermobacter taxa. The presence of a diverse range of feedstock compositions can be observed in sam- ples where methane production modules exhibit a rela- tive abundance of over 10%, including acetate [4, 74–76], casein [75], glucose [75], oleate [67, 77], manure [67, 77–79], lactate [4], butyrate [4], propionate [4, 79], sludge Centurion et al. Environmental Microbiome (2024) 19:1 Page 11 of 19 Centurion et al. Environmental Microbiome g. 4 (See legend on previous page.) Fig. 4 (See legend on previous page.) Fig. 4 (See legend on previous page.) Fig. 4 (See legend on previous page.) Centurion et al. Environmental Microbiome (2024) 19:1 Page 12 of 19 Page 12 of 19 Centurion et al. Environmental Microbiome (2024) 19:1 Centurion et al. Environmental Microbiome Fig. 4). Microbial replication rates are linked to their functional capabilities f Nitrogen metabolism was frequently identified in some taxa including the candidate Zixibacteria divi- sion and Acidobacteria. For example, half of the MAGs assigned to the candidate Zixibacteria division have the complete function of nitrification (M00528) and denitrification (M00529), reflecting their metabolic ver- satility [88]. Regarding the Acidobacteria phylum, nitri- fication (M00528) and complete nitrification (M00804) modules were identified in 50% and 25% of the MAGs, respectively; despite this finding the nitrification ability was not proven in isolates of the Acidobacteria phylum [89, 90]. The Acidobacteria nitrification function was more abundant in thermophilic manure-supplemented biogas plants with high biogas production and low pH (Campanaro_2018_BGP_3).l To characterize microbial dynamics across AD systems, we calculated the peak-to-trough ratio (PTR) of the MAGs coverage, which estimates DNA synthesis and generation rate [47]. Sample-specific PTRs were obtained for 782 MAGs in the dataset, corresponding to those genomes meeting the minimal coverage requirement in at least one sample. As a result, PTR values were deter- mined in fewer than 20 samples for most MAGs, while for others, e.g. Methanothrix sp. 43zhSC_152, Synergista- ceae sp. 24abBP_148, Bacteroidales sp. 28xzH2_30, and various members of Firmicutes, PTRs were estimated for over a hundred samples, reflecting their widespread abundance. Resulting PTR values generally have a long- tail distribution, with a median of 0.37 and exceeding 2 in some cases, with varying spread across different tax- onomic groups (Additional file 2: Fig. 5). Within these distributions, both coarse- and fine-grain trends match- ing previous knowledge were observed. Firstly, acetoclas- tic and hydrogenotrophic methanogens exhibited a low mean PTR, consistently with direct measures of the repli- cation rate in isolates (Additional file 2: Fig. 6). Secondly, slightly lower mean PTR was observed in batch reactors, consistently with the limited duration and efficiency of the processes therein compared to CSTRs and full-scale plants. When considering individual microorganisms, significant PTR trends for MAGs classified at genus or species level are in some cases in agreement with docu- mented growth temperature preferences, where robust PTR distributions could be recovered (Fig. 5) [93, 94]. In particular, microorganisms of the Methanoculleus genus are cultivated at a temperature between 28° and 37 °C, and Methanoculleus sp. 52maCN_3230 has an estimated optimal growth temperature in approximately the same interval. Porphyromonadaceae sp. 02xzSI_42 reaches the highest PTR values around 40 °C, which is its recommended cultivation temperature [93]. Bacterial communityh Beta-oxidation is the second step of LAS degrada- tion and is mainly performed by Synergistes and Syntro- phus, which are widespread genera in LAS degradation reactors [84–86]. Wastewater frequently contains sev- eral nitrogen-rich compounds, including nitrate, nitrite, and ammonia [87], which may have contributed to the enrichment of bacteria capable of converting nitrogen to different oxidation states. Arnon-Buchanan cycle module, involving a reverse cit- ric acid cycle for CO2 fixation, was the most abundant in samples inoculated with sludge (Macedo_2020_CSTR and Zhang_2020_Batch), while the WL and acetogen modules were more abundant in a CSTR sample for the AD of saccharides with a feedstock of volatile fatty acid mixture (Zhu_2019_CSTR_5). Arnon-Buchanan cycle module, involving a reverse cit- ric acid cycle for CO2 fixation, was the most abundant in samples inoculated with sludge (Macedo_2020_CSTR and Zhang_2020_Batch), while the WL and acetogen modules were more abundant in a CSTR sample for the AD of saccharides with a feedstock of volatile fatty acid mixture (Zhu_2019_CSTR_5). Microbial replication rates are linked to their functional capabilities Similarly, Methanosarcina thermophila 28xzH2_79 has detect- able non-null PTR values predominantly close to 55 °C, even though some outliers were found in the mesophilic range. PTR distribution for other species, including Methanobacterium sp. 29adLB_146, Aminobacterium sp. 23ysBP_18, and Syntrophorhabdus sp. 42zhAM_214, p AD biogas production is heavily influenced by the organic substrates used. Some of these substrates may contain inhibitors such as sulfides, which can negatively affect the microbiome and decrease the AD process effi- ciency [91]. For example, sulfate-reducing bacteria (SRB) encoding proteins involved in the assimilatory sulfate reduction to sulfide function can compete with hydrog- enotrophic archaea for hydrogen, and generate H2S as final product [65, 66]. In particular, a batch reactor fed with cellulosic and xylan biomass (Jia_2018_Batch) showed the highest representation of assimilatory sul- fate reduction to sulfide across MAGs. Although the H2S concentration was not reported, all the batch experi- ments produced low concentration of CH4 (0.2–1.5 mM), suggesting that the process predominantly shifted to sulfate reduction [55]. Indeed, 68% of “Jia_2018_Batch” library read counts mapped to the Clostridium butyricum 37jiCB_291 MAG, a known SRB [83]. Overall, a global Biogas Microbiome database can be useful to infer puta- tive inhibitory in the AD process by analyzing the path- ways of the MAGs identified. i In contrast, key carbon metabolism modules includ- ing the Arnon-Buchanan cycle, the WL pathway, and the acetogenesis are complete or have only one block missing in less than 25% of the total MAGs suggesting these are shell modules in the AD system [92]. Nevertheless, these metabolic routes for carbon were identified in a range of phyla including Actinobacteria, Chloroflexi, Firmicutes, Ignavibacteriae, Proteobacteria, and Spirochaetes. The Centurion et al. Environmental Microbiome (2024) 19:1 Centurion et al. Environmental Microbiome Page 13 of 19 Fig. 5 Relationship between PTR and temperature for individual MAGs. Shown values refer to significant relationships as assessed by quadratic polynomial fits, based on a FDR-adjusted p-value threshold of 0.1 Fig. 5 Relationship between PTR and temperature for individual MAGs. Shown values refer to significant relationships as assessed by quadratic polynomial fits, based on a FDR-adjusted p-value threshold of 0.1 long tail around large values (Additional file 2: Fig. 5). More specifically, MAGs classified as Syntrophobacte- rales seem to have fast replication rates in several con- ditions. Microbial replication rates are linked to their functional capabilities 21ysBP_11 and Methanosarcina flavescens 22ysBP_46 are among the fast-growing archaea with PTR consistently above 2, making Methanomicrobiales sp. 21ysBP_11 a potential promising candidate for cultivation and isolation. In gen- eral, most MAGs exhibit their highest replication rates within a 5 °C range (preferential temperature range), indicating that temperature tightly controls replication efficiency (Additional file 2: Fig. 7). In fact, mesophilic and thermophilic communities tend to present distinct composition and diversity, as also seen above. Yet, a rel- evant number of bacterial and archaeal MAGs show their highest PTRs over a 20 °C window, suggesting that some species are able to better adapt across temperature regimes. harboring genetic variants. On the other hand, the pres- ence of Methanoculleus sp. 52maCN_3230 was limited to 23 mesophilic samples, and thus, the high number of genetic variants observed in this MAG could be due to different factors in comparison to Methanotrix. f In order to obtain a more reliable representation of the genomic variability, the number of SNV per MAG was normalized, both according to the genome length and to the number of samples where the MAG was identi- fied with coverage higher than 1 (Fig. 6B). This analy- sis highlighted Methanoculleus sp. 52maCN_3230 and Methanomicrobiales sp. 19jrsB_18 as outliers with more than 10,000 SNVs/Mbp per sample (Fig. 6B). This find- ing is of particular interest because, while Methanoc- ulleus sp. 52maCN_3230 is quite common in the AD samples, Methanomicrobiales sp. 19jrsB_18 was identi- fied in two samples only. The genomic location of non- synonymous variants identified in both methanogens allowed linking them to the gene and to the functional pathways. Interestingly, 2.5% of the Methanomicrobiales sp. 19jrsB_18 variants and 9.1% of the Methanoculleus sp. 52maCN_3230 variants were associated with core genes of the hydrogenotrophic methanogenesis, includ- ing heterodisulfide reductase (hdr), methyl-coenzyme M reductase (mcr) and formylmethanofuran dehydrogenase (fwd). Overall, these results suggest that variants with a crucial role in the adaptation of methanogenic archaea to reactors operated in different conditions. Numerous genomic variants delineate microbial population heterogeneity To date, there is a lack of information in literature regard- ing the genetic heterogeneity of AD-relevant species [74]. The presence of the same microbial species in reac- tors characterized by different process parameters can allow to identify variants impacting the adaptation pro- cess, as well as a more detailed characterization of spe- cies at strain level. Variant identification was performed by aligning shotgun reads of each experiment back to the MAGs obtained from the binning process, and this approach led to the identification of 10.5 millions single nucleotide variants (SNVs). The high number of variants revealed a high genetic heterogeneity in the microbial population, and variants characterization allowed their classification as synonymous (60.5%), nonsynonymous (28.9%), intergenic (10.4%), and multigenic (0.02%). Of the 3,050 MAGs containing SNVs, only eight exhib- ited more than 500,000 SNVs, two of which were the methanogenic archaea Methanothrix sp. 43zhSC_152 and Methanoculleus sp. 52maCN_3230, which harbor 938,000 and 661,000 SNVs, respectively (see Availabil- ity of Data and Materials). The high number of variants observed in Methanothrix sp. 43zhSC_152 could be due to the fact that this MAG is widespread, being present in 96 of the examined samples; possibly, the presence of this species in many reactors with highly different conditions have led to the differentiation of a large number of strains f In order to determine if some phyla were statistically more impacted by variants, the taxonomic assignment of each MAG at phylum level was considered along with the number of SNVs/Mbp. Results of the Mann– Whitney U-test on SNVs/Mbp distribution showed that eight phyla had a significant enrichment in the number of variants (p-value < 0.05) (Fig. 6C), with Candidatus Cloacimonetes (p = 0.0003), Euryarchaeota (p = 0.0025), Atribacterota (p = 0.0307) and Synergistetes (p = 0,0023), having a number higher than expected, and Firmicutes (p = 0.0380), Planctomycetes (p = 0.0145), Verrucomicro- bia (p = 0.0043) and Lentisphaerae (p = 0.0015), having a lower number. In general, the results obtained for Eur- yarchaeota suggest that methanogenic archaea are under a strong selective pressure and harbor a large amount of genetic variability. Microbial replication rates are linked to their functional capabilities This taxon harbors sulfate reducers, which can efficiently metabolize substrates such as pyruvate, metha- nol, and glucose and outcompete methanogens when sufficient sulfate is available [95, 96]. These results are confirmed by the long tail of PTRs associated with assim- ilatory sulfate reduction (Additional file 2: Fig. 6), and show their maximum values in the 25–40 °C range, in agreement with the largely mesophilic representation of these genera [94]. Although replication rate is generally influenced by a variety of factors, together, these patterns support the overall soundness of obtained distributions. Besides, other less obvious trends were found. While Verrucomicrobia, Fibrobacteres, and Planctomycetes are among the phyla with the largest mean PTR, subsets of Proteobacteria and Bacteroidetes show a more noticeable Centurion et al. Environmental Microbiome (2024) 19:1 Page 14 of 19 Page 14 of 19 Centurion et al. Environmental Microbiome thus support the rapid proliferation capability of these microbes. Other of such fast-growing microbial groups are Bacteroidales (e.g. Proteiniphilum sp. 29adLB_192), with relevant proteolytic role [97], and microbes involved in fatty acid oxidation (Additional file 2: Fig. 7). These groups could represent species with high energy genera- tion capability from the breakdown of such macromol- ecules. Moreover, archaeal phyla also have a wide range of replication rates, with some MAGs exhibiting high PTRs. In particular, Methanomicrobiales sp. 21ysBP_11 and Methanosarcina flavescens 22ysBP_46 are among the fast-growing archaea with PTR consistently above 2, making Methanomicrobiales sp. 21ysBP_11 a potential promising candidate for cultivation and isolation. In gen- eral, most MAGs exhibit their highest replication rates within a 5 °C range (preferential temperature range), indicating that temperature tightly controls replication efficiency (Additional file 2: Fig. 7). In fact, mesophilic and thermophilic communities tend to present distinct composition and diversity, as also seen above. Yet, a rel- evant number of bacterial and archaeal MAGs show their highest PTRs over a 20 °C window, suggesting that some species are able to better adapt across temperature regimes. thus support the rapid proliferation capability of these microbes. Other of such fast-growing microbial groups are Bacteroidales (e.g. Proteiniphilum sp. 29adLB_192), with relevant proteolytic role [97], and microbes involved in fatty acid oxidation (Additional file 2: Fig. 7). These groups could represent species with high energy genera- tion capability from the breakdown of such macromol- ecules. Moreover, archaeal phyla also have a wide range of replication rates, with some MAGs exhibiting high PTRs. In particular, Methanomicrobiales sp. Numerous genomic variants delineate microbial population heterogeneity The metabolic roles of species belong- ing to Candidatus Cloacimonetes and Synergistetes are still not completely clear, however, it was previously reported that some of them are characterized by ace- togenesis [98], or can compete for acetate utilization with Methanosaeta [99].i To get a first glimpse on the strains composition, a phy- logenetic analysis was conducted on Methanothrix sp. 43zhSC_152, Methanothermobacter wolfeii 31mySI 58 and Candidatus Methanocullus thermohydrogenotrophicum Centurion et al. Environmental Microbiome (2024) 19:1 Page 15 of 19 31mySI_10, the three archeal species with the high- est number of MAGs. These three species have differ- ent properties in the database: Methanotrix has a high number of MAGs and a worldwide distribution, the oth two have less MAGs and are more abundant in Eur pean reactors (see Availability of Data and Material Fig. 6 Overview of variants distribution among different taxonomic groups. A Number of MAGs associated with each phylum. B Number of SNVs/Mbp in each phylum for MAGs with more than 100 SNVs; each dot represents a MAG. Results are reported for each phylum with more than five MAGs, while all the others are reported as “Other”. C Statistical analysis comparing the median SNV density calculated for each phylum and the average value reported for the global Biogas Microbiome database. Statistically significant results are marked with asterisks: “”p < = 0.05, “”p < = 0.01, “”p < = 0.001 and “*”p < = 0.0001 Fig. 6 Overview of variants distribution among different taxonomic groups. A Number of MAGs associated with each phylum. B Number of SNVs/Mbp in each phylum for MAGs with more than 100 SNVs; each dot represents a MAG. Results are reported for each phylum with more than five MAGs, while all the others are reported as “Other”. C Statistical analysis comparing the median SNV density calculated for each phylum and the average value reported for the global Biogas Microbiome database. Statistically significant results are marked with asterisks: “”p < = 0.05, “”p < = 0.01, “”p < = 0.001 and “**”p < = 0.0001 Fig. 6 Overview of variants distribution among different taxonomic groups. A Number of MAGs associated with each phylum. B Number of SNVs/Mbp in each phylum for MAGs with more than 100 SNVs; each dot represents a MAG. Results are reported for each phylum with more than five MAGs, while all the others are reported as “Other”. Numerous genomic variants delineate microbial population heterogeneity C Statistical analysis comparing the median SNV density calculated for each phylum and the average value reported for the global Biogas Microbiome database. Statistically significant results are marked with asterisks: “”p < = 0.05, “”p < = 0.01, “”p < = 0.001 and “***”p < = 0.0001 number of MAGs and a worldwide distribution, the other two have less MAGs and are more abundant in Euro- pean reactors (see Availability of Data and Materials). 31mySI_10, the three archeal species with the high- est number of MAGs. These three species have differ- ent properties in the database: Methanotrix has a high Centurion et al. Environmental Microbiome (2024) 19:1 Page 16 of 19 Centurion et al. Environmental Microbiome with prokaryotes, also via means of cross-linking tech- niques and co-occurrence. Phylogenetic analysis revealed a regional distribution of MAGs for Methanotrix with a distinct cluster of MAGs deriving from Chinese biogas plants. Interestingly, all the MAGs recovered for Methanothrix sp. 43zhSC_152 are mesophilic, and all the MAGs of M. wolfeii 31mySI 58 are thermophilic, while Candidatus M. thermohydrog- enotrophicum 31mySI_10 is more flexible and is present in some mesophilic and thermophilic samples (Addi- tional file 2: Fig. 8). This finding contradicts previous data reporting this species only in thermophilic conditions [100], and suggests a different habit for this methanogen, which is able to adapt to mesophilic conditions as well. The temperature is not the main driver of strains differen- tiation for Candidatus M. thermohydrogenotrophicum, while the high phylogenetic distance among some M. wolfeii and Ca. M. thermohydrogenotrophicum MAGs suggest a possible impact of environmental conditions on their strains differentiation. Two M. wolfeii MAGs iden- tified in samples subjected to high H2 concentrations, 15tlH2_55 and 50dbBF_040, appeared quite distant from the others suggesting a selective pressure determined by environmental conditions. Abundance of microbial species competing with meth- anogens for H2 utilization, such as SRBs, has highlighted how a well-characterized MAG database allows to better understand the impact of the microbiome in reducing the performance in terms of biogas production. Finally, the investigation of SNVs impacting the genes involved in key functional processes has laid the foundations to study the evolution of AD microbiome and its role in reactor performance, suggesting that methanogenic archaea are under strong selective pressure. Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s40793-023-00545-2. g Additional file 1. Datasets and metadata information, assembly statistics and diversity values Additional file 2. Figure S1. Relationship between alpha-diversity and temperature. Figure S2. Phylum distribution across samples. Figure S3. Phage abundance overview. Figure S4. Bacterial functional modules. Figure S5. PTR distribution across phyla. Figure S6. PTR distribution in main functional modules. Figure S7. Strain distribution in the main methanogens. Figure S8. Phylogenetic analysis of the MAGs identified for three of the most common methanogens. Supplementary Figure S9. GC content distribution across phage families. Additional file 3. Archaeal MAGs with complete and one-block-missing M00357 and M00567 and their relative abundance across samples. Additional file 4. Bacterial MAGs with complete and one-block-missing modules for selected functions and their relative abundance. For every considered function, the cumulative abundance of all the MAGs satisfying these criteria within any identified phylum is reported for each sample. Additional file 4. Bacterial MAGs with complete and one-block-missing modules for selected functions and their relative abundance. For every considered function, the cumulative abundance of all the MAGs satisfying these criteria within any identified phylum is reported for each sample. Inspection of the phage repertoire provided a first over- view and, through the analysis of the CRISPR elements, a first characterization of phage-bacterial interactions and co-evolution. This analysis allowed to build the first ver- sion of the viral Biogas Microbiome database, currently represented only by DNA phages. The RNA phage frac- tion still remains to be identified and will be one of the next targets. While the viral component characterisation proved to be extremely complex, the presence of a com- bined database of prokaryotes and phages will certainly allow in the future a better tracking of their interactions Numerous genomic variants delineate microbial population heterogeneity Abbreviations AD Anaerobic digestion MAGs Metagenome-assembled genomes KEGG Kyoto Encyclopedia of Genes and Genomes PTR Peak-to-trough ratio CSTR Continuous stirred-tank reactor CRISPR Clustered regularly interspaced short palindromic repeats PFAM Protein families database COG Clusters of Orthologous Groups of proteins database BA Blood Agar WL Wood-Ljungdahl pathway LAS Remove linear alkylbenzene sulfonate SRB Sulfate-reducing bacteria SNV Single nucleotide variants Abbreviations AD Anaerobic digestion MAGs Metagenome-assembled genomes KEGG Kyoto Encyclopedia of Genes and Genomes PTR Peak-to-trough ratio CSTR Continuous stirred-tank reactor CRISPR Clustered regularly interspaced short palindromic repeats PFAM Protein families database COG Clusters of Orthologous Groups of proteins database BA Blood Agar WL Wood-Ljungdahl pathway LAS Remove linear alkylbenzene sulfonate SRB Sulfate-reducing bacteria SNV Single nucleotide variants Conclusions In this study, the metagenomic characterization of the microbial species involved in the AD process has been expanded through the analysis of a large number of dif- ferent reactor types operated under a range of conditions. In addition to expanding the number of species reported in the previous version of the Biogas Microbiome data- base by almost three times, the analysis was focused on archaea, one of the crucial components of the microbi- ome. The investigation of gene composition has led to a better characterization of archaea and their methano- genic metabolism; however there still remains a degree of uncertainty in the automatic association between gene composition and phenotype, which will require the development of new investigation methods based on gene expression or machine learning. Despite this huge increase in the number of catalogued species, the great diversity of this biotechnological niche has yet to be fully explored, especially with regard to bacterial species.i Supplementary Information Declarations 15. Campanaro S, Treu L, Rodriguez-R LM, Kovalovszki A, Ziels RM, Maus I, et al. New insights from the biogas microbiome by comprehensive genome-resolved metagenomics of nearly 1600 species originating from multiple anaerobic digesters. Biotechnol Biofuels. 2020;13:25. Funding Open access funding provided by Università degli Studi di Padova. This work was financially supported by the "Sviluppo Catalisi dell’Innovazione nelle Biotecnologie (MIUR ex D.M.738 dd 08/08/2019) of the Consorzio Interuni- versitario per le Biotecnologie (CIB)” and an EMBO Postdoctoral fellowship (to E.O. - ALT 739-2022). 8. Luo G, Fotidis IA, Angelidaki I. Comparative analysis of taxonomic, func- tional, and metabolic patterns of microbiomes from 14 full-scale biogas reactors by metagenomic sequencing and radioisotopic analysis. Biotechnol Biofuels. 2016;9:51. 9. Angelidaki I, Sanders W. Assessment of the anaerobic biodegradability of macropollutants. Rev Environ Sci Biotechnol. 2004;3:117–29. 9. Angelidaki I, Sanders W. Assessment of the anaerobic biodegradability of macropollutants. Rev Environ Sci Biotechnol. 2004;3:117–29. Received: 11 October 2023 Accepted: 21 December 2023 21. Ma S, Jiang F, Huang Y, Zhang Y, Wang S, Fan H, et al. A microbial gene catalog of anaerobic digestion from full-scale biogas plants. GigaS- cience. 2021;10:giaa164. Availability of data and materials 10. Mata-Alvarez J, Macé S, Llabrés P. Anaerobic digestion of organic solid wastes. An overview of research achievements and perspectives. Biore- sour Technol. 2000;74:3–16. 10. Mata-Alvarez J, Macé S, Llabrés P. Anaerobic digestion of organic solid wastes. An overview of research achievements and perspectives. Biore- sour Technol. 2000;74:3–16. The generated data represent the third stage of a continued effort to fully characterize the anaerobic digestion microbiome and make it fully avail- able online. To generate version V1 and V2 of the MAGs database, shotgun sequences were previously described in prior studies [15, 77]. 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https://openalex.org/W4394861585	https://www.scielo.br/j/ep/a/PXd7RgHdjTS6h8LLfV9Q3HC/?lang=pt&format=pdf	Portuguese	null	Metafísica do mercado, sujeito atomizado e corrosão da formação humana no neoliberalismo de Hayek	Educação e Pesquisa	2,024	cc-by	8,837	ARTIGOS ARTIGOS Metafísica do mercado, sujeito atomizado e corrosão da formação humana no neoliberalismo de Hayek Angelo Vitório Cenci1 ORCID: 0000-0003-0541-2197 Jelson Becker Salomão1 ORCID: 0000-0001-6461-3285 Regiano Bregalda2 ORCID: 0000-0003-0104-4163 Resumo O neoliberalismo pode ser mais bem compreendido como uma visão de mundo, que tem exercido notória hegemonia nas sociedades ocidentais, sobretudo, a partir do início dos anos 1980. Neste sentido, o presente texto trata de um dos principais expoentes do neoliberalismo – Friedrich A. Hayek – e das noções de ordem espontânea e de individualismo metodológico, conforme formuladas na sua própria versão da teoria neoliberal. O objetivo, com o esforço empreendido, é demonstrar que a relação existente na obra desse autor entre a defesa da ordem espontânea “metafísica” neoliberal do mercado e uma compreensão atomizada de sujeito tem, como resultado, a centralidade de uma sociabilidade de mercado, que tem implicações desligitimadoras e corrosivas para o sentido moderno iluminista da formação (cultural) humana. Dentre as possíveis consequências resultantes dessa relação, estão o enfraquecimento de temas chaves, tais como ação, liberdade, igualdade, justiça social e autonomia, sob o prisma de uma moralidade pós-convencional demandada pelas atuais sociedades complexas, plurais e desiguais. Dessa perspectiva, a conclusão aponta para a necessidade de recuperação do conceito filosófico de ação, o qual é formulado pela tradição filosófica clássica, de uma visão pós-metafísica e pós-tradicional de ordem social, de uma concepção pós-convencional de moralidade, bem como de uma concepção social de liberdade. 1 - Universidade de Passo Fundo (UPF), Passo Fundo, Rio Grande do Sul, Brasil. Contatos: angelo@upf.br; jelson@upf.br 2- Instituto de Teologia e Pastoral (Itepa), Passo Fundo, Rio Grande do Sul, Brasil. Contato: regiano_bregalda@hotmail.com 1 - Universidade de Passo Fundo (UPF), Passo Fundo, Rio Grande do Sul, Brasil. Contatos: angelo@upf.br; jelson@upf.br 2- Instituto de Teologia e Pastoral (Itepa), Passo Fundo, Rio Grande do Sul, Brasil. Contato: regiano_bregalda@hotmail.com https://doi.org/10.1590/S1678-4634202450264095 Thi t t i li d d C ti C tt ib ti t BY 4 0 Palavras-chave Metafísica do mercado – Ordem espontânea – Individualismo metodológico – Formaçã Metafísica do mercado – Ordem espontânea – Individualismo metodológico – Formação humana. https://doi.org/10.1590/S1678-4634202450264095 This content is licensed under a Creative Commons attribution-type BY 4.0 https://doi.org/10.1590/S1678-4634202450264095 This content is licensed under a Creative Commons attribution-type BY 4.0. 1 Educ. Pesqui., São Paulo, v. 50, e264095, 2024. Angelo Vitório CENCI; Jelson Becker SALOMÃO; Regiano BREGALDA Market metaphysics. Spontaneous order. Methodological individualism. Human training. Market metaphysics. Spontaneous order. Methodological individualism. Human training. Market metaphysics. Spontaneous order. Methodological individualism. Human training. 3 - Entendemos o neoliberalismo como uma visão de mundo que abrange uma determinada concepção de mercado, política, democracia, cultura, sujeito, educação e, sobretudo, um imaginário social. Abstract Neoliberalism may be best understood as a worldview that has exercised notorious hegemony in Western societies, especially since the early 1980s. In this sense, this text addresses one of the leading exponents of neoliberalism – Friedrich A. Hayek – and the notions of spontaneous order and methodological individualism, as formulated in his version of neoliberal theory. The objective of the undertaken effort was to demonstrate that the relationship existing in this author’s work between the defense of the neoliberal “metaphysical” spontaneous order of the market and an atomized understanding of the subject has, as a result, the centrality of a market sociability with de-legitimizing and corrosive implications for the modern Enlightenment sense of human (cultural) training. Among the possible consequences of this relationship is the weakening of key topics, such as action, freedom, equality, social justice, and autonomy, under the prism of a post- conventional morality demanded by today’s complex, plural, and unequal societies. From this perspective, the conclusion points to the need to recover the philosophical concept of action, formulated by the classical philosophical tradition, a post-metaphysical and post- traditional view of social order, a post-conventional conception of morality, and a social conception of freedom. Introdução O neoliberalismo de matriz hayekiana foi gestado desde a Segunda Guerra Mundial para proteger o capitalismo de qualquer forma de Estado de justiça social, especialmente das influências do keynesianismo (desenvolvimentista) e do socialismo. Ao colocar o mercado e a moral tradicional para governar e disciplinar os indivíduos, ao mesmo tempo em que procura maximizar sua liberdade e estabelecer tais esferas como meios de provisão das necessidades humanas, exclui completamente o Estado e a sociedade dessa tarefa. Por conseguinte, o neoliberalismo de Hayek, para muito além de um modelo específico de economia, constitui-se, avant la lettre, em uma visão de mundo e esse autor possivelmente seja, dentre seus fundadores, quem melhor a desenvolve.3 Isso se deve, sobretudo, à sua própria formação intelectual, de caráter amplo. Sua intenção original foi, como bem a interpreta Brown (2019), a de fazer com que o mercado e a moral tradicional – cristã, 2 Educ. Pesqui., São Paulo, v. 50, e264095, 2024. 2 Metafísica do mercado, sujeito atomizado e corrosão da formação humana no neoliberalismo de Hayek branca, conservadora e socialmente hierárquica – gerassem uma ordem e evolução espontâneas, governando e disciplinando os indivíduos de forma que a liberdade pessoal deles em sentido econômico fosse maximizada. Nessa moldura do projeto neoliberal, o social, a versão democrática da vida política, assim como o planejamento e a justiça social orientados pelo Estado teriam de ser radicalmente deslegitimados, demonizados e expurgados em nome da garantia da liberdade de mercado do sujeito. Hayek busca fortalecer a “esfera pessoal protegida” (o indivíduo atomizado), desmantelar a ideia de sociedade, tornar a justiça social uma “miragem”, destronar a política e domar a democracia. A implicação direta desse projeto para o campo educacional resulta que princípios consagrados pelo esclarecimento pedagógico moderno, tais como autonomia, igualdade social e solidariedade, assim como o caráter universal, gratuito e laico, e a finalidade essencialmente pública da educação passam a ser seriamente afetados. No entanto, se, por um lado, o neoliberalismo existente radicalizou muitos aspectos teorizados por seus fundadores, por outro, transformou-os, de modo a converter-se em uma espécie de patologia social ou patologia do social, gerando profundas desigualdades e novas e agudas formas de sofrimento social.4 No presente texto, importa-nos lançar um olhar crítico especificamente sobre os fundamentos do neoliberalismo clássico, tomando por base a obra de Hayek em dois de seus aspectos. 4- Entendemos por patologia social os fenômenos que afetam de modo generalizado as condições para a realização do bem viver das pessoas (a vida ética, em sentido hegeliano). A respeito do neoliberalismo como gerador de sofrimento social e a forma como deste se alimenta, ver especialmente Renault (2008) e Safatle, Junior e Dunker (2020). Introdução Inicialmente, tematizaremos o modo como o autor recorre, em sua visão de mundo, à metafísica de uma ordem espontânea do mercado que a tudo rege (1). Articulado a esse primeiro, o passo seguinte consiste em mostrar como a essa ordem espontânea do mercado está vinculada uma concepção atomizada de liberdade e de sujeito (2). Para concluir o percurso, extrairemos algumas possíveis consequências dos limites de sua concepção teórica no que tange à educação quando pensada como formação humana. A metafísica da ordem social espontânea do mercado Do mesmo modo que ocorre com a “mão invisível” de Adam Smith, a ordem espontânea do mercado leva o homem a “servir a um fim que não faz parte de suas intenções”. Contra a economia neoclássica, Hayek defende que fenômenos econômicos e sociais não podem ser compreendidos por nenhum conhecimento perfeito – como o da matemática –, que os indivíduos não produzem a ordem social a partir de suas escolhas racionais e que nenhum planejamento pode corrigir o mercado, uma vez que qualquer intervenção sobre ele resultaria em injustiças e na ineficácia do sistema econômico e social. A teoria de Hayek toma, como base, a concepção epistemológica de que a coordenação do conhecimento na sociedade pela lógica do mercado e sua “ordem espontânea” são o meio genuíno e possível pelo qual os indivíduos apropriam-se dos fragmentos de informações, visando direcioná-los às atividades que resultam em maior proveito para eles e, indiretamente, para todos. Outro fator chave para compreender o porquê dessa ordem espontânea ser vantajosa é sua lógica intrínseca, embora penosa, não de planejamento, mas de adaptação e de tendência à prosperidade: “Esta ordem nasce de si mesma. O fato de as normas se adequarem cada vez melhor para gerar ordem se deu não porque os homens compreenderam melhor sua função mas, porque os grupos que as mudaram de uma forma que os tornou cada vez mais capazes de se adaptar, prosperaram” (Hayek, 1995, p. 38). – como o da matemática –, que os indivíduos não produzem a ordem social a partir de suas escolhas racionais e que nenhum planejamento pode corrigir o mercado, uma vez que qualquer intervenção sobre ele resultaria em injustiças e na ineficácia do sistema econômico e social. A teoria de Hayek toma, como base, a concepção epistemológica de que a coordenação do conhecimento na sociedade pela lógica do mercado e sua “ordem espontânea” são o meio genuíno e possível pelo qual os indivíduos apropriam-se dos fragmentos de informações, visando direcioná-los às atividades que resultam em maior proveito para eles e, indiretamente, para todos. Outro fator chave para compreender o porquê dessa ordem espontânea ser vantajosa é sua lógica intrínseca, embora penosa, não de planejamento, mas de adaptação e de tendência à prosperidade: “Esta ordem nasce de si mesma. A metafísica da ordem social espontânea do mercado Na teoria de Hayek, a ordem espontânea e autorregulável do mercado – sua versão renovada da mão invisível de Adam Smith – tem lugar central e a ela é vinculado o argumento de que a ordem daí resultante constitui-se como a melhor e única forma adequada de organização social. Conforme já referimos anteriormente, o nosso intuito é mostrar que desse argumento decorre uma cosmovisão, uma teoria da sociedade, de indivíduo e de liberdade que possuem profundas consequências para a educação quando entendida como formação (cultural) humana. Essa cosmovisão exercerá larga influência, sobretudo, no que concerne ao capitalismo neoliberal implementado gradativamente, a partir dos anos 70 em diante do século passado, e tornado hegemônico como visão de mundo nas últimas décadas. Embora seu primeiro laboratório tenha sido o Chile, já nos anos 1970, na América Latina avançou principalmente nos anos 1990, impulsionado pelo “consenso de Washington” elaborado em 1989. Educ. Pesqui., São Paulo, v. 50, e264095, 2024. 3 Angelo Vitório CENCI; Jelson Becker SALOMÃO; Regiano BREGALDA Vale destacar que há, pelo menos, três campos principais de interlocução na base da teoria de Hayek, criticados por ele, a saber: (a) o racionalismo construtivista de tipo cartesiano, (b) a teoria econômica neoclássica e (c) o socialismo, reduzido e identificado diretamente a uma filosofia escatológica da história marxista. O que há subjacente às críticas feitas pelo autor a esses campos é seu ponto de partida metodológico, a saber, a visão de que o mercado é regido por uma ordem espontânea e o indivíduo é concebido como parte de um mundo complexo sobre o qual pouco pode compreender e interferir. Sua concepção do mercado como ordem espontânea, resultante de ações não intencionais – portanto, não racional –, remonta aos moralistas britânicos do século XVIII, como Bernard Mandeville, David Hume e Adam Smith. Este último já havia substituído a ideia então predominante do mercado e da sociedade como ordem racional pela de ordem natural, e a noção moderna (artificial) de contrato pela de mercado como fator constituidor das sociedades, estendendo a economia sobre todas as outras esferas da sociedade. O aspecto metodológico fundamental da teoria de Hayek é que essa ordem é explicada a partir do indivíduo como alguém que age sem nenhum tipo de intencionalidade ou desígnio prévio a ele. Educ. Pesqui., São Paulo, v. 50, e264095, 2024. A metafísica da ordem social espontânea do mercado O fato de as normas se adequarem cada vez melhor para gerar ordem se deu não porque os homens compreenderam melhor sua função mas, porque os grupos que as mudaram de uma forma que os tornou cada vez mais capazes de se adaptar, prosperaram” (Hayek, 1995, p. 38). A “ordem espontânea” do mercado caracteriza-se como não coercitiva. Isso se deve porque ela constitui-se sem a orientação de qualquer decisão política, materializando- se no mercado e na tradição moral. O mercado e a moral resultam de um processo não planejado e independente de qualquer interesse político. Tanto um quanto outro regulariam as interações sociais sem afetar a liberdade individual. Como argumenta oportunamente Brown (2019), na visão de Hayek, o erro da socialdemocracia ou de qualquer modelo de Welfare State residiria precisamente no esforço de querer substituir a ordem espontânea do mercado decorrente de um processo de evolução histórica, ancorada na tradição e instalada no costume, por projetos racionais de sociedade. Educ. Pesqui., São Paulo, v. 50, e264095, 2024. 4 Metafísica do mercado, sujeito atomizado e corrosão da formação humana no neoliberalismo de Hayek Esse erro de substituir a ordem espontânea por uma ordem criada conscientemente explicitaria uma incompreensão, porque quem a defende, “quanto à natureza dos seres humanos, da história, da mudança e da cooperação social, para não mencionar a justiça e a liberdade” (Brown, 2019, p. 131-132). Como veremos adiante, essa incompreensão deve- se à leitura inversa que Hayek faz – em relação às perspectivas iluministas ou “racional- construtivistas” – do avanço do processo civilizatório, que, para ele, vai do “coletivismo” instintivo primitivo para o evoluído “individualismo” garantido e garantidor do mercado e das tradições. De acordo com sua leitura, cooperação, solidariedade e justiça social indicariam resquícios de instintos remanescentes das sociedades tribais fechadas, pré- civilizadas e pouco evoluídas. p É oportuno destacar que Hayek (1985a), recorrendo aos gregos, distingue dois tipos de ordem, a saber, a criada pela mente humana (taxis) e a que se efetiva espontaneamente (kosmos). A primeira, de cunho racional-construtivista, é identificada com a economia planificada ou dirigida. Já a segunda seria associada a uma perspectiva evolucionista, própria de uma ordenação liberal de mercado. Hayek opera aqui com um modelo conceitual rígido (dicotômico) em que qualquer alternativa ao seu modelo de ordem neoliberal de mercado é identificada exclusivamente com economia planificada. 5- Como é possível perceber, e isso será retomado adiante, não lhe faltam razões para ter apoiado um regime como de Pinochet no Chile. Esse mesmo modelo está também na base da reivindicação explicita que o bolsonarismo faz no Brasil atual para justificar o clamor pela volta a um regime político ditatorial. A metafísica da ordem social espontânea do mercado Não deixa de ser anacrônico o recurso aos gregos para justificar seu conceito de ordem, uma vez que eles entendiam kosmos como ordem, sim, mas estática, eterna e imutável, não passível de evolução. Hayek critica a ordem racional derivada do modelo filosófico de Descartes e da Teoria econômica neoclássica, que ele denomina de razão construtivista e onipotente, em função de tanto um quanto outro terem a pretensão, a seu ver, completamente descabida, de construir ou de corrigir ordens econômicas e sociais, de modo a mudar a distribuição “natural” da renda. Na terminologia buscada dos gregos, essa ordem artificial por ser criada corresponderia à taxis. A mesma lógica é identificada por ele em Marx e no marxismo, pois o socialismo, amparado na ideia de uma ordem distinta – ancorada no desígnio de uma classe operária consciente –, é tomado como sinônimo de totalitarismo, fruto de uma razão onipotente orientada escatologicamente para um devir de superação da sociabilidade de mercado capitalista. A seu juízo e em seu modelo de argumentação maniqueísta, essa pretensão racional-construtivista do socialismo levaria inevitavelmente à servidão e ao totalitarismo. Vale observar, como o faz Ganem (2012), que a identificação do totalitarismo com o socialismo – e não com o fascismo – traduz seu não interesse em confrontar totalitarismo com democracia, mas de contrapor socialismo à ordem social de mercado. Para Hayek, o contraponto ao totalitarismo é o liberalismo e não a democracia, enquanto espaço singular da ação humana e da ação política em sentido mais específico.5 De acordo com sua posição, negar o (neo)liberalismo implicaria defender necessariamente uma volta a modelos ultrapassados, baseados na solidariedade e superados pela lógica do processo civilizatório. Seu conceito de liberdade é reduzido ao de liberdade negativa e o de justiça social a uma “miragem”. O neoliberalismo de Hayek reduz toda liberdade – e, sobretudo, a liberdade Educ. Pesqui., São Paulo, v. 50, e264095, 2024. 5 Angelo Vitório CENCI; Jelson Becker SALOMÃO; Regiano BREGALDA política – à liberdade econômica. É por essa razão que Hannah Arendt argumentara que o liberalismo colaborou efetivamente para a eliminação da noção de liberdade do âmbito político. A política, nesse caso, reduz-se, conforme alertara a autora, a ocupar-se quase que unicamente com a manutenção da vida e dos interesses desta. A metafísica da ordem social espontânea do mercado A seu ver, “onde a vida está em jogo, toda ação se encontra, por definição, sob o jugo da necessidade, e o âmbito adequado para cuidar das necessidades vitais é a gigantesca e sempre crescente esfera da vida social e econômica, cuja administração tem obscurecido o âmbito político desde os primórdios da época moderna” (Arendt, 2005, p. 202). A leitura de Arendt ajuda a apontar para o problema da estreiteza da teoria da ação hayekiana por esta não levar em conta o conceito grego de práxis, que tinha, como cerne, o aspecto da deliberação racional, justamente o que possibilitava o espaço da vida em comum entre os homens e a distinguia da esfera da produção. p ç Para compreender a “metafísica do mercado” de Hayek é necessário também dar- se atenção ao seu conceito de regras espontâneas. Ocorre que o autor sustenta uma teoria da evolução cultural falibilista, traduzida pela ideia de que os homens na história conduzem-se por um processo de experimentação. No fundo, são as regras de mercado e sua lógica própria que servem como parâmetro para todos os outros tipos de regras. A ordem espontânea hayekiana decorre, pois, da ação de homens seguidores de regras não controláveis, nem produzidas racionalmente pelo espírito humano. Desde sua ótica, os homens desconhecem o resultado coletivo de sua ação e as regras são transmitidas pela cultura mediante a experiência e a tradição. Há, todavia, uma contradição nessa formulação hayekiana ao sustentar que a ordem impessoal do mercado, em sua evolução cultural, igualmente espontânea, sustenta-se em regras escolhidas por indivíduos de maneira natural e espontânea, mas que não são frutos de sua vontade e lhes são intransparentes. Assim sendo, o que seria então possível aos homens escolherem dentro de uma ordem espontânea, inexorável e necessária, mas com resultados necessariamente vantajosos para si mesmos, a qual, de algum modo, todos estariam submissos? O que garante que ela seria benéfica para os indivíduos e que os levaria em direção a um processo de evolução cultural? A teoria de Hayek daria margem para imaginar-se alguma outra ordem de mercado possível, que não essa única – a neoliberal – adotada por ele? Certamente, a resposta a essas perguntas é negativa. 6- Essa tendência do neoliberalismo em acabar resultando em autoritarismo, por vezes, com traços claramente de “política fascista”, é bem perceptível no contexto contemporâneo (Stanley, 2018). A metafísica da ordem social espontânea do mercado Se não há alternativas ao mercado em termos de organização social, como defende Hayek, não seria possível nenhum outro imaginário social e político alternativo e o mercado tenderia a identificar-se com uma sociedade fechada e, pois, a assumir ares totalitários.6 Ora, esse tipo de sociedade era justamente o que Popper e Hayek queriam combater. São grandes as implicações desses limites presentes na teoria de Hayek. Como lembra Ganem, essa espécie de fatalismo ideológico, além de fragilizar o indivíduo, “anula as potencialidades de seu agir conjunto na política e o faz descrente de sua capacidade de irromper algo novo que lhe permita descortinar outras possibilidades no plano da História” (Ganem, 2009, p. 282). De modo muito oportuno, a autora destaca ainda que, Educ. Pesqui., São Paulo, v. 50, e264095, 2024. 6 Metafísica do mercado, sujeito atomizado e corrosão da formação humana no neoliberalismo de Hayek para Hayek, os sujeitos não seriam meramente passivos, mas ativos na medida em que elegeriam, orientados pela ordem espontânea e “em meio a sua ignorância, as regras que garantem a ordem e a manutenção do mercado” (Ganem, 2012, p. 114). Trata-se, no entanto, de um conceito de ação restringido pela ordem espontânea, ou seja, por uma espécie de poderosa força externa a eles próprios. O riquíssimo conceito de ação concebido como a capacidade de “novos começos”, advinda como novidade e em meio aos condicionamentos históricos, sustentado por Arendt como contraponto ao totalitarismo, passa longe da formulação hayekiana. Em Hayek, há o risco de o “novo” ser uma expressão do “sempre o mesmo”, identificado com a ordem espontânea e metafísica do mercado, que transcende a tudo e a todos, sem que se possa vislumbrar novas possibilidades ou outro imaginário social e político para além dela própria.7 Para Hayek, não são organizações, produto do “racionalismo construtivista”, mas “forças ‘auto-organizadoras’ e suprapessoais que geram as ordens espontâneas” (1985a, p. 146). Desse modo, ele confronta a auto-organização natural da ordem espontânea do mercado com toda e qualquer possibilidade de orientação deliberada e consciente da vida política, da sociedade e da economia. Hayek fica preso à camisa de força que ele próprio criou, na medida em que sua metafísica da ordem espontânea não consegue mais reservar espaço para a ação humana enquanto historicidade e espontaneidade que advêm dos próprios sujeitos. 7- Sobre a força e os limites do imaginário neoliberal, ver Casara (2021), especialmente cap.3. A metafísica da ordem social espontânea do mercado Trata-se de um conceito limitado de ação, conformado a regras que cumprem o papel de dar equilíbrio à ordem espontânea do mercado que a tudo e a todos transcende. O preço pago pelo reducionismo do conceito de liberdade à liberdade econômica é a impossibilidade de propor um conceito de liberdade política. É É bem verdade que a história humana não é feita sob circunstâncias da inteira escolha deliberada dos homens. Todavia, o modo como Hayek entende a ação dos sujeitos e a própria história depara-se com limites inequívocos. Mesmo que faça uma crítica veemente e generalizadora – identificando Marx e o socialismo com uma escatologia vinculada à inexorabilidade de leis históricas –, sua análise passa longe de algo crucial já presente em Marx. Vale lembrar o princípio marxiano apresentado em seu O 18 Brumário de Luis Bonaparte de que os homens fazem sua própria história, mas não o fazem como querem, pois ao fazerem têm de defrontar-se necessariamente também com circunstâncias de escolha legadas e transmitidas pelo passado. Neste sentido, reforça Marx: “E justamente quando parecem empenhados em revolucionarem a si e às coisas, em criar algo que jamais existiu, precisamente nesses períodos de crise revolucionária os homens conjuram ansiosamente em seu auxílio os espíritos do passado (...)” (Marx; Engels, 2003, p.7-8). Esse princípio fundamental da historicidade da ação humana e que traduz, ao mesmo tempo, a inseparabilidade do passado e a incontrolabilidade própria do seu curso presente não se coadunam nem com a metafísica da filosofia da história mecanicista do socialismo real – guiada por leis eternas e imutáveis –, nem com uma metafísica neoliberal da ordem espontânea do mercado. Marx refere-se a uma das condições intrínsecas à ação humana que é sua incontrolabilidade em razão das contingências que a perpassam e do passado que serve de referência a elas. Embora o processo histórico não possa ser controlado Educ. Pesqui., São Paulo, v. 50, e264095, 2024. 7 Angelo Vitório CENCI; Jelson Becker SALOMÃO; Regiano BREGALDA pelos homens, isso não significa que não possa ser influenciado pela vontade humana. Sob esse prisma, a própria ordem espontânea neoliberal do mercado é ininteligível se não compreendermos seu caráter intrinsecamente histórico. Neste sentido, o desvelamento da historicidade do mercado neoliberal – assentado no princípio da concorrência – é a antípoda da metafísica hayekiana do mercado. A metafísica da ordem social espontânea do mercado O mercado não é um ente que segue uma lógica inexorável e, pois, a-histórica, diante do qual os homens nada podem fazer além de seguir resignadamente suas regras abstratas e impessoais. Hayek coloca o mercado – e não a democracia liberal, como o será depois em Francis Fukoyama – como o fator que leva ao fim da História. A sociedade aberta, regida pelo jogo do mercado, representaria o fim da história. Como Hayek a interpreta de modo a-histórico, é bastante lógico concluir não ser mais possível existir experiências históricas para além dela. Mas, nesse caso, temos a absolutização da imanência mediante um fundamento último, de caráter metafísico, a saber, a ordem espontânea do mercado. Não deixa de ser curioso o fato de Hayek defender que o empenho de todos na solução de problemas ocorreria mediante a manutenção da ordem, como algo natural, sem rumar para um fim definido, um devir “escatológico”. i g O autor troca, pois, um racionalismo construtivista ou um fim escatológico, transcendente, por uma teleologia imanentista, a da autorregulação do mercado por si mesmo. Em outras palavras, de algum modo, o futuro já estaria realizado no presente, materializado pela ordem espontânea do mercado capitalista neoliberal. O fim transcendente seria substituído por um fim imanente, o que, na verdade, estaria muito distante da defesa de sociedades abertas. Por conseguinte, elas tenderiam a estar ilusoriamente abertas, uma vez enredadas na própria ordem espontânea e na sociabilidade omniabrangente do mercado em sua versão neoliberal. O que seria isso senão a substituição de uma metafísica da história por uma metafísica imanentista do mercado, erigido em única forma possível e abarcante de organização social? A verdadeira “miragem” residiria então na justiça social – como o autor sugere – ou na sua concepção de ordem auto-organizável, espontânea e impessoal do mercado que, no seu limite – como é possível observar-se hoje –, resulta facilmente no risco de anomia social e na necropolítica?8 Não por acaso, Wendy Brown (2019) chama atenção para o fato de que, embora o presente catastrófico não fora o resultado desejado pelo neoliberalismo, a consequência foi uma “criação frankensteiniana”. 8- Sobre a gestão da violência sobre a vida social e psíquica dos indivíduos no neoliberalismo ver Safatle (2020, p. 23s.). Sobre a necropolítica no contexto do atual neoliberalismo brasileiro, ver Franco (2021). O indivíduo atomizado como ponto de partida metodológico e antropológico Constitui um certo lugar comum afirmar que o indivíduo é o ponto de partida metodológico da teoria de Hayek. É bem verdade que assim o é, como também o é o jogo do mercado baseado nas trocas (catalaxia) que, juntos, traduzem uma determinada visão de sociabilidade e de mundo. Todavia, a nosso ver, o aspecto do individualismo metodológico hayekiano é indissociável também de uma concepção antropológica. Com efeito, uma Educ. Pesqui., São Paulo, v. 50, e264095, 2024. 8 8 Metafísica do mercado, sujeito atomizado e corrosão da formação humana no neoliberalismo de Hayek das chaves de leitura para compreender criticamente a teoria de Hayek consiste em ter presente como ele toma o indivíduo enquanto referência fundamental para a compreensão dos fenômenos da vida social e da vida humana em sentido amplo. É o indivíduo, mediante sua ação e seu comportamento, quem os determina, mas o faz dentro da lógica da ordem espontânea do mercado, que o guia de um modo intransparente e transcendente. Usando um trocadilho, se Norbert Elias (1994) concebe a sociedade humana como uma “sociedade dos indivíduos”, Hayek sustentará a ideia de “indivíduos sem sociedade”, maximizadores de seus interesses pessoais, “sujeitos que não devem nada a ninguém”9 e que submetem seus interesses apenas à lógica impessoal do mercado. Para o autor, os fenômenos sociais do presente são entendidos como individuais e ancorados em tradições morais que evoluíram de acordo com a lógica do mercado e não em conformidade com algum grau de vontade dos indivíduos. As tradições morais e o mercado são, pois, organizados e transmitidos espontaneamente mediante a tradição (ou a moral tradicional) e não pelo poder político. O mercado e a moral constituir-se-iam em formas suficientes e singulares de “provisão de recursos para as necessidades humanas” (Brown, 2019, p. 21). Há uma premissa epistemológica fundamental na base da teoria de Hayek, a saber: como o mundo, em sua totalidade, é incognoscível, somente podemos conhecer dele fragmentos. Mediante tal premissa, ele tira ardilosamente de cena a possibilidade de o cientista social buscar compreender a realidade a partir de um prisma de totalidade, que é o procedimento que permite alcançar-se uma visão crítica sobre ela. Com essa estratégia, Hayek neutraliza o sentido de toda a ação política, pois seria errônea epistemologicamente (devido à “limitação do conhecimento”) e resultaria totalitário politicamente, todo esforço de construir deliberadamente qualquer ordem sócio-político-econômica. 9- A esse respeito, Dardot e Laval (2016, p.366). Essa concepção de que só existe indivíduos é o que levará Margareth Thatcher a afirmar: “Não existe essa coisa de sociedade, o que há e sempre haverá são indivíduos”. O indivíduo atomizado como ponto de partida metodológico e antropológico Para o autor, o que orienta a ação do indivíduo atomizado social e politicamente dentro da ordem do mercado é as regras, visto que são elas que fornecem coesão e certa inteligibilidade à ordem espontânea, resultando, não intencionalmente, de um processo recorrente de ensaios e erro. Neste sentido, Hayek apresenta o mundo como decorrente de uma evolução guiada por regras que orientam os homens no interior de um processo de aprendizagem social e coletivo e que resultaria numa progressiva capacidade da sociedade de resolver problemas. “A evolução cultural seria na verdade similar a um processo de crescimento do conhecimento, um processo de aprendizagem social e coletivo que teria nas regras suas guias abstratas”, como destaca Ganem (2012, p. 109). As regras seriam aplicáveis indeterminadamente e teriam o papel de fortalecer o jogo das trocas no mercado. Caberia, então, ao Estado apenas garantir os fundamentos da ordem para que essas regras abstratas, regentes da ordem espontânea auto-organizada, permitissem à concorrência gerar riqueza e bem-estar social. Não se pode esquecer que, para Hayek, mesmo que não se saiba de antemão quais são essas regras, as regras de concorrência sempre serão as exitosas. Como é possível, então, justificá-las senão como regras necessárias?i i O indivíduo, fundamento dessa ordem, pode ser definido como um homem livre, que ignora as regras, que segue e que conhece de forma muito limitada, incompleta e fragmentadamente o mundo, assim como os resultados coletivos de sua ação. A sua Educ. Pesqui., São Paulo, v. 50, e264095, 2024. 9 Angelo Vitório CENCI; Jelson Becker SALOMÃO; Regiano BREGALDA liberdade é a liberdade econômica, negativa, caracterizada pela ausência da coação de outrem, assegurada pelo direito liberal. É, pois, livre economicamente e limitado epistemologicamente. A limitação do seu conhecimento sobre o mundo faz com que a ordem espontânea constitua-se, para ele, como uma categoria totalmente opaca e inacessível à razão. Isso se deve ao fato de que “o elevado número de variáveis presentes nos fenômenos sociais impossibilita o conhecimento de todas as circunstâncias que envolvem a ação dos indivíduos, o que confere uma natureza complexa aos fenômenos sociais [...]” (Ganem, 2009, p. 271). 10- A visão de Hegel seria um claro contraponto a de Hayek na medida em que seu conceito de “liberdade social”, associada ao de eticidade e, pois, à mediação dos indivíduos em instituições sociais, é infinitamente mais potente para explicar a relação entre indivíduos e sociedade inaugurada nas sociedades modernas. A esse respeito Hegel (2005), Honneth (2014) e Neuhouser (2000). 11- Esse problema leva certos autores a identificarem, atualmente, a forma jurídica do neoliberalismo com Estado de exceção. A esse respeito ver, entre outros, Casara (2018, 2021) e Valim (2017). 12- O conceito de liberdades substanciais desenvolvido a partir do enfoque das capabilities de Amartya Sen (2000) seria um forte contraponto à concepção de Hayek. O indivíduo atomizado como ponto de partida metodológico e antropológico Aspecto chave, neste sentido, é que as leis imanentes ao mercado – sustentadas nas regras necessárias da concorrência – revelam um processo sem sujeito, por um lado, e indivíduos sem sociedade, de outro.10 A consequência bastante plausível da maneira como Hayek subsume os indivíduos nas instituições é que, como lembra Gómez, parece não importar o ser humano, mas apenas as instituições geradas lentamente na forma de tradições pelo acaso evolutivo, e “tampouco importam as consequências mortais humanas, dado que são consequências não procuradas e que ocorrem ‘espontaneamente’” (2011, p. 171). O neoliberalismo hayekiano sustenta-se sobre uma ideia de indivíduo atomizado. A defesa radical de uma concepção de indivíduo sem sociedade – e, por vezes, contra a sociedade – obstaculiza a própria ideia de uma sociedade democrática. Ora, pergunta oportunamente Roriz: “É possível que essa combinação entre as demandas individuais (liberalismo) e a coesão social (democracia) consiga se resolver de forma razoável, sem que a coesão social se reduza apenas a uma soma gigantesca de interesses individuais?” (2011, p. 232). Que tipo de coesão social seria possível – se é que possível – nesse caso?11 Nessa antropologia atomista, o ser humano é entendido fundamentalmente como individualista, criador de normas e tradições, produto da evolução histórica da sociedade tribal fechada à grande sociedade aberta. Se olhada a partir de um prisma iluminista, essa ética é claramente heterônoma e convencional por identificar-se às regras funcionais da sociedade de mercado, imanentes à sua reprodução. A razão humana é limitada pela intransparência do conhecimento disperso na sociedade e a liberdade econômica individual de dispor da propriedade privada – negativa e abstrata – com garantia jurídico-estatal é seu valor central. O lugar de exercício por excelência da liberdade é o mercado, onde o indivíduo situa-se como agente maximizador de interesses mesmo na intransparência de sua ordem espontânea.12 Na base desse individualismo atomista está uma concepção antropológica nominalista, sustentada no mecanicismo hobbesiano e influenciada pelo individualismo metodológico de Popper. De acordo com essa concepção, a sociedade ou a realidade social seria constituída pela soma de indivíduos e ações. Coerente com essa premissa filosófica, para Hayek, a sociedade resume-se a um “conjunto de relações entre indivíduos ou grupos Educ. Pesqui., São Paulo, v. 50, e264095, 2024. O indivíduo atomizado como ponto de partida metodológico e antropológico 10 Metafísica do mercado, sujeito atomizado e corrosão da formação humana no neoliberalismo de Hayek organizados”, nada tendo a ver com algo deliberado, o que significaria cair em um “errôneo antropomorfismo” (Hayek, 1980, p. 11). Os fenômenos sociais só podem ser compreendidos a partir das ações individuais, nunca a partir de um prisma de totalidade. Hayek trata de ação espontânea dos homens (formalmente) livres, nunca da possibilidade de uma ação planejada pela vontade de homens livremente associados. Isso se deve ao fato de entender que “a colaboração espontânea entre os homens livres cria, com frequência, coisas muito maiores do que suas mentes individuais podem chegar a abarcar por completo” (Hayek, 1980, p. 7). q p g p p y p Hayek compreende a evolução histórica e o processo civilizatório como uma trajetória de avanço dos primitivos vínculos instintivos de solidariedade e de altruísmo para o individualismo, contestando Hobbes e sua tese de que teria existido originariamente um homem natural, intrinsecamente egoísta, e uma condição de guerra de todos contra todos. As considerações de Hayek sobre as sociedades primitivas distanciam-na da antropologia tanto de Hobbes, como já destacamos, quanto de Locke. Para este último (1994), o homem natural era livre e cada indivíduo considerava-se, dentro da perspectiva do “individualismo possessivo”, dono de sua própria pessoa e de suas capacidades, sem nada dever à sociedade por elas. A peculiaridade deste residia na sua concepção de indivíduo tomado como separado do todo social.13 Para Hayek, nas sociedades primitivas o indivíduo sequer seria considerado livre, pois pouco poderia fazer sem a aprovação dos demais. A seu juízo, a liberdade é uma criação da própria civilização, algo mais tardio, resultante de um processo de evolução. Diferentemente, o sentido da vida comum é algo instintivo, não histórico, e as regras sociais baseadas na solidariedade, que dão coesão social às sociedades tribais, por serem instintivas, não possuem valor moral. Elas seriam necessárias à sobrevivência dos grupos tribais, mas disfuncionais a uma sociedade de mercado (civilizada) evoluída em direção à liberdade econômica dos indivíduos. Para Hayek, civilização é sinônimo da passagem das normas instintivas coletivas, necessárias à garantia da sobrevivência do grupo/tribo, para normas impessoais de coordenação social. Antes de existirem essas normas de mercado, teria havido apenas instintos sociais que orientavam a ação em sociedades pré-civilizadas. A civilização apareceria com o surgimento do mercado. 13- A respeito desse individualismo, característico da teoria e prática política do século XVII, ver MacPherson (1979). O indivíduo atomizado como ponto de partida metodológico e antropológico Embora Hayek não identifique evolução biológica e evolução social, em sua análise, ele explica esta última como um curso natural em direção às sociedades de mercado que representariam sua fase mais evoluída ou o fim da história. O princípio do falibilismo e do evolucionismo é adaptado à lógica econômica e é a competição, não a contradição, que se constitui como o motor da história. Hayek entende que “não só toda evolução se baseia na competição como a contínua competição é necessária mesmo para preservar as realizações existentes” (1995, p. 46). Outro aspecto importante é que a racionalidade humana é resultado e não causa da evolução sociocultural. Hayek opera com um conceito extremamente reduzido de racionalidade e, pois, de ação. Reduz normas a normas de mercado apenas e toma-as como produzidas pela tradição em um processo espontâneo de melhorismo. Ao sustentar as regras ou normas de mercado na tradição, estas têm de ser entendidas necessariamente como tendo primazia em relação aos indivíduos em sentido de autonomia moral. Por Educ. Pesqui., São Paulo, v. 50, e264095, 2024. 11 Angelo Vitório CENCI; Jelson Becker SALOMÃO; Regiano BREGALDA conseguinte, fica fora de cena a possibilidade de uma moral pós-convencional. Não bastasse, as normas morais são autofundamentáveis, pois não podem ser submetidas a exame pela razão, uma vez que são o pressuposto e não o resultado da própria razão. Elas funcionam mesmo que não conheçamos o seu significado ou que não tenhamos a consciência de sua existência.14 Normas e tradições são, pois, resultado da evolução espontânea da sociedade, não da razão humana. p Foram as normas de mercado que trouxeram a civilização e que civilizaram os seres humanos, permitindo-lhes evoluir da sociedade tribal à sociedade ampliada. No lugar dos instintos de solidariedade que atavam os seres humanos ao grupo, tais normas permitem a coordenação espontânea das ações na forma de padrões inconscientes, derivados de hábitos e tradições solidamente estabelecidos. Hayek postula uma concepção de economia de mercado moderna, alicerçada em um modelo de moralidade tradicional, pré-iluminista, uma visão claramente heterônoma da moralidade. Esta não consegue alcançar nem o nível da liberdade reflexiva e seu princípio da autonomia moral, tal qual postulado por Rousseau, Kant e Mill, nem o da liberdade social, desenvolvido por Hegel e, posteriormente, por Marx. 14- Ver, a esse respeito, sobretudo o cap. 4 de seu livro Os fundamentos da liberdade. Considerações finais Ao longo deste percurso argumentativo, detivemo-nos em abordar duas dimensões centrais da obra de Hayek, levando em conta, fundamentalmente, o fato de este concebê- la não apenas como uma teoria econômica, mas, sobretudo, como visão de mundo. Vimos que há uma relação estreita entre a metafísica da ordem social espontânea do mercado e a sua concepção do indivíduo atomizado como ponto de partida metodológico e antropológico. Ora, de tal relação resultam importantes consequências para o campo da educação, quando pensada como formação (cultural) humana. Em primeiro lugar, na medida em que o indivíduo está inserido em uma ordem espontânea e intransparente, que tudo e a todos conduz, resta-lhe pouca margem de ação, tanto em sentido político quanto pedagógico. Hayek empobrece ou neutraliza, pois, o conceito filosófico de ação, chave para pensar uma formação humana própria ao contexto de sociedades complexas, plurais e profundamente desiguais como as neoliberais contemporâneas. A dimensão clássica da deliberação, tal como formulada desde Aristóteles, é neutralizada, assim como a ideia arendtiana da ação como possibilidade radical de “novos começos”. Por conseguinte, acaba havendo, em sua obra, a primazia de um conceito muito restrito de liberdade – a negativa –, se comparado ao de liberdade social de Hegel, Dewey e Honneth, prejudicando qualquer forma de pensá-lo em sentido ampliado e articulado às instituições, tais como é o caso da escola e do Estado. Trata-se de instituições que possibilitam práticas normativas, em que a autonomia individual é alcançada de modo intersubjetivo mediante relações de reconhecimento recíproco, de acordo com a fórmula hegeliana do “estar consigo mesmo no outro”. Um segundo aspecto, ligado a esse primeiro, é que a redução do conceito de ação à esfera econômica e à moralidade tradicional convencional ou, quiçá, pré-convencional, implica o esvaziamento do sentido político daquele. Como é possível vida social sem vida política e, pois, sem haver espaço para a formação democrática da vontade? Ao mesmo tempo em que o neoliberalismo requer um Estado forte para a manutenção da ordem social e o funcionamento dos mercados, fragiliza o Estado social e imprime uma forma tecnocrática à política democrática, esboroando suas demandas. Quando a noção de interesse público limita-se à proteção da liberdade privada e da segurança individual, a própria questão da formação democrática da vontade é diretamente afetada pela “antipolítica” (Brown). e só. A igualdade reduz-se à igualdade formal, mesmo que o contexto em que as pessoas encontrem-se seja de profunda desigualdade (injustiça) substancial ou material. e só. A igualdade reduz-se à igualdade formal, mesmo que o contexto em que as pessoas encontrem-se seja de profunda desigualdade (injustiça) substancial ou material. O indivíduo atomizado como ponto de partida metodológico e antropológico Para Hayek, a liberdade econômica (negativa) – o único nível alcançado por ele – ancora-se na moralidade tradicional e em suas regras heterônomas, que antecedem a razão humana. Os indivíduos teriam sempre de orientar-se pelas tradições morais, sociais e econômicas, necessárias para a reprodução da civilização. O paradoxo reside em entender o mercado capitalista como instituição moderna e como fim da história e, ao mesmo tempo, em alicerçá-lo em uma estrutura moral tradicional, de tipo pré-moderno. Além disso, não desenvolve satisfatoriamente nem uma caracterização aprofundada das sociedades tradicionais, nem das modernas. Outro traço antropológico do pensamento de Hayek (1983) é o de que as desigualdades sociais e econômicas, em uma sociedade de mercado, são consequência do próprio exercício da liberdade e da competição entre indivíduos desiguais. A liberdade não tem a ver com a igualdade e, inclusive, produz desigualdade em muitos aspectos. O autor deixa muito claro que “o grande objetivo da luta pela liberdade sempre foi a igualdade perante a lei” e também que a esse tipo específico de igualdade é o único que “conduz à liberdade e a única que podemos obter sem destruir a liberdade” (1983, p. 92). Por conseguinte, “a liberdade não só não tem relação alguma com qualquer outro tipo de igualdade como também tende, em muitos casos, a produzir desigualdade” (Hayek, 1983, p. 92). Isso se deve ao fato de que, segundo conclui, “se os efeitos da liberdade individual não demonstrassem que certos modos de vida levam a resultados melhores do que outros, provavelmente seria impossível justificá-la” (Hayek, 1983, p. 92). Em outros termos, o que justifica reduzir a igualdade à igualdade perante à lei (liberdade formal) é que esta é a única que possibilita estruturar-se no princípio da competição. Para sustentar esse argumento, Hayek precisa abstrair todo o complexo campo das diferenças – por exemplo, de classe, racial, de gênero, capacidades, etc. – entre pessoas, grupos e classes sociais. Ele é claro na conclusão de seu argumento: “É essencial à reivindicação de igualdade perante a lei que as pessoas sejam tratadas do mesmo modo, embora sejam diferentes umas das outras” (1983, p. 93). Igualdade resume-se à igualdade de todos perante à lei, Educ. Pesqui., São Paulo, v. 50, e264095, 2024. 12 Metafísica do mercado, sujeito atomizado e corrosão da formação humana no neoliberalismo de Hayek Educ. Pesqui., São Paulo, v. 50, e264095, 2024. Considerações finais É por essa razão que, com o apoio do Estado, a educação não pode ser ‘‘abandonada totalmente à arbitrariedade dos particulares``, como alertara Durkheim (2009, p. 61), mas deve criar condições para assegurar aos cidadãos a capacidade de compartilhar ideais guiados por valores democráticos, sem os quais a coesão social seria praticamente inviável. O próprio Durkheim já defendera que a educação deve ter por base determinados princípios que sejam comuns a todos e que cabe ao Estado esclarecê-los e possibilitar que sejam aprendidos nas escolas. A educação seria uma esfera propícia Educ. Pesqui., São Paulo, v. 50, e264095, 2024. 13 Angelo Vitório CENCI; Jelson Becker SALOMÃO; Regiano BREGALDA para fomentar modos de agir necessários à formação da vontade democrática e, para tal, precisaria apostar no desenvolvimento de hábitos consonantes a uma cultura associativa. Um terceiro aspecto é que, ao prender-se a uma visão pré-iluminista da dinâmica da vida social e a um conceito reduzido de liberdade, Hayek, de algum modo, neutraliza outro conceito central à ação pedagógica e à formação humana, que é o de autonomia. Aos sujeitos da “grande sociedade” postulada por Hayek parece restar a possibilidade de conformarem-se heteronomamente às regras impessoais do mercado. Caberia, então, à sociabilidade espontânea de mercado o papel de conformar processos de socialização e de individualização consoantes à sua lógica, almejando ter, como único referencial normativo, o próprio mercado. Todavia, um tal modelo de sociabilidade é completamente insuficiente para dar conta de tais processos por seu caráter limitado e excludente. Por mais que o mercado em sentido neoliberal pretenda conformar uma subjetividade aos seus moldes, essa tarefa resulta muito problemática, inclusive, perigosa sob o ponto de vista formativo. Vale lembrar que, em termos de socialização/individualização, a lógica de tais processos desemboca, via de regra, em uma subjetividade “ilimitada” e em uma sociedade “sem lei”, traduzidas pela perda da importância do simbólico identificado com a linguagem e originado do limite imposto por terceiros, a ser internalizado pelo indivíduo (Casara, 2018). Por fim, um quarto ponto a destacar é a visão estritamente convencional da moral em Hayek e a incapacidade de inclusão do outro no ponto de vista dos agentes. Ora, autonomia moral e pedagógica em sentido pleno só existem quando a referência para a organização da vida social e da ação dos indivíduos for uma moralidade de tipo pós- convencional. Educ. Pesqui., São Paulo, v. 50, e264095, 2024. Considerações finais Normas e tradições não são somente o resultado da evolução espontânea da sociedade, mas também da razão humana, constituídas de modo contraditório e não linear, resultantes de um processo de aprendizagem humano mediante instituições e na relação com os outros. Ao neutralizar o caráter deliberativo da ação humana, já tão bem assentado pela filosofia política grega, Hayek acaba por enredar-se nas teias anacrônicas de uma moralidade convencional pré-iluminista, completamente distanciada das exigências formativas colocadas para sujeitos em sociedades complexas e que, dado sua interdependência, exige mais e mais uma moral pós-convencional como condição para a garantia da própria coesão social. Em tais sociedades, por mais plurais e desiguais que venham a ser, o parâmetro normativo passa pelo simbólico em seu nível mais desenvolvido já alcançado pela modernidade, a saber, a capacidade de colocar-se no lugar do outro e da consideração da igual dignidade de todos os seres humanos. Nessa perspectiva, tanto a criação da ordem social quanto da própria subjetividade passa pelo outro. Como lembra Honneth, o outro “não é uma limitação, mas uma condição da liberdade individual [...]”, pois ela só se efetiva na mediação com aquele (2014, p. 186, grifos nossos). Os sujeitos relacionam- se uns com os outros tendo de reconhecer a necessidade de complementaridade de suas respectivas metas, de modo a poder conceber o outro como outro de si mesmo. O desafio em termos de educação como formação humana consiste não apenas em ultrapassar a metafísica neoliberal da ordem espontânea do mercado, mas também o sujeito metafísico atomizado a ela vinculado e negado mediante a negação do outro de si mesmo. 14 Metafísica do mercado, sujeito atomizado e corrosão da formação humana no neoliberalismo de Hayek Metafísica do mercado, sujeito atomizado e corrosão da formação humana no neoliberalismo de Hayek Referências ARENDT, Hannah. A condição humana. Trad. de Roberto Raposo. 10. ed. Rio de Janeiro: Forense Universitária, 2007. ARENDT, Hannah. Entre o passado e o futuro. 5. ed. São Paulo: Perspectiva, 2005. ARENDT, Hannah. Origens do totalitarismo. São Paulo: Companhia das Letras, 2012. ARENDT, Hannah. Qu’est-ce que la politique? Paris: Éditions du Seuil, 1995. BROWN, Wendy. Nas ruínas do neoliberalismo: a ascensão da política antidemocrática no ocidente. São Paulo: Politeia, 2019. CASARA, Rubens. Contra a miséria neoliberal. São Paulo: Autonomia Literária, 2021. CASARA, Rubens. Sociedade sem lei: pós-democracia, personalidade autoritária, idiotização e barbárie. Rio de Janeiro: Civilização Brasileira, 2018. DARDOT, Pierre; LAVAL, Christian. A nova razão do mundo: ensaio sobre a sociedade neoliberal. São Paulo: Boitempo, 2016. DURKHEIM, Emile. Educação e sociologia. Lisboa: Edições 70, 2009. DURKHEIM, Emile. Educação e sociologia. Lisboa: Edições 70, 2009. ELIAS, Norbert. A sociedade dos indivíduos. Rio de Janeiro: Zahar, 1994. ESTÉVEZ, Jorge Vergara. La concepción del hombre de Friedrich Hayek. Revista de Filosofía, Santiago de Chile, v. 65, n. 1, p. 61-176, 2009. FRANCO, Fábio Luis. Governar os mortos: necropolíticas, desaparecimento e subjetividade. São Paulo: Ubu, 2021. GANEM, Angela. Crítica à leitura hayekiana da história: a perspectiva da ação política de Hannah Arendt. Nova Economia, Belo Horizonte, v. 19, n. 2, p. 267-284, 2009. Disponível em: http://www.scielo.br/scielo. php?script=sci_arttext&pid=S010363512009000200003&lng=en&nrm=iso. Acesso em 13 jan. 2021. http://dx.doi.org/10.1590/S0103-63512009000200003 GANEM, Angela. Hayek: da teoria do mercado como ordem espontânea ao mercado como fim da história. Política & Sociedade, Florianópolis, v. 11, n. 22, p. 93-117, nov. 2012. GÓMEZ, Moisés. Crítica al concepto de “orden espontáneo” de Hayek. Revista Realidad, San Salvador, n. 127, p. 163-176, 2011. HAYEK, Friedrich. A arrogância fatal: os erros do socialismo. Porto Alegre: Ortiz: Instituto de Estudos Empresariais, 1995. 15 Educ. Pesqui., São Paulo, v. 50, e264095, 2024. Angelo Vitório CENCI; Jelson Becker SALOMÃO; Regiano BREGALDA HAYEK, Friedrich. Direito, legislação e liberdade: normas e ordem. v. 1. São Paulo: Visão, 1985a. HAYEK, Friedrich. Direito, legislação e liberdade: a miragem da justiça social. v. 2. São Paulo: Visão, 1985b. HAYEK, Friedrich. Direito, legislação e liberdade: a ordem política de um povo livre. v. 3. São Paulo: Visão, 1985c. HAYEK, Friedrich. El individualismo: verdadero y falso. Estúdios Públicos, Santiago de Chile, n. 22, p. 1-28, out. 1986. Disponível em: https://www.cepchile.cl/cep/estudios-publicos/n-1-a-la-30/ estudios-publicos-n-22/individualismo-el-verdadero-y-el-falso. Acesso em: 30 fev. 2023. HAYEK, Friedrich. Os fundamentos da liberdade. São Paulo: Visão, 1983. HAYEK, Friedrich. Os fundamentos da liberdade. São Paulo: Visão, 1983. Educ. Pesqui., São Paulo, v. 50, e264095, 2024. Referências HEGEL, Wilhelm Friedrich. Principios de la filosofía del derecho. Madrid: Edhasa, 2005. HONNETH, Axel. El derecho de la libertad. Madrid: Katz, 2014. HONNETH, Axel. El derecho de la libertad. Madrid: Katz, 2014. LOCKE, John. Segundo tratado sobre o governo civil: ensaio sobre a origem, os limites e os fins verdadeiros do governo civil. Petrópolis: Vozes, 1994. MACPHERSON, Crawford Brough. A teoria política do individualismo possessivo: de Hobbes a Locke. Rio de Janeiro: Paz e Terra, 1979. MARX, Karl. O Dezoito de Brumário de Luiz Bonaparte. São Paulo: Centauro, 2003. NEUHOUSER, Frederick. Foundations of Hegel’s social theory: actualizing freedom. Cambridge: Harvard University Press, 2000. RENAULT, Emmanuel. Souffrances socialesParis: La Découverte, 2008. Educ. Pesqui., São Paulo, v. 50, e264095, 2024. RENAULT, Emmanuel. Souffrances socialesParis: La Découverte, 2008. RORIZ, João Henrique Ribeiro. A política como continuação da liberdade: Hannah Arendt e sua crítica à democracia liberal. Filosofia Unisinos, São Leopoldo, v. 12, n. 3, p. 228-240, set./dez. 2011. Disponível em: http://revistas.unisinos.br/index.php/filosofia/article/view/fsu.2011.123.03. Acesso em: 30 fev. 2023. SAFATLE, Vladimir. A economia é a continuação da psicologia por outros meios: sofrimento psíquico e neoliberalismo como economia moral. In: SAFATLE, Vladimir; SILVA JUNIOR, Nelson S.; DUNKER, Christian. Neoliberalismo como gestão do sofrimento psíquico. Belo Horizonte: Autêntica, 2020. (p. 17-46). SEN, Amartya. Desenvolvimento como liberdade. São Paulo: Companhia das Letras, 2000. STANLEY, Jason. Como funciona o fascismo: a política do “nós” e “eles”. Porto Alegre: L&PM, 2018. VALIM, Rafael. Estado de exceção: a forma jurídica do neoliberalismo. São. Paulo: Contracorrente, 2017. 16 16 Metafísica do mercado, sujeito atomizado e corrosão da formação humana no neoliberalismo de Hayek Metafísica do mercado, sujeito atomizado e corrosão da formação humana no neoliberalismo de Hayek Recebido em: 16.05.2022 Aprovado em: 19.12.2022 Editor: Prof. Dr. Marcos Sidnei Pagotto-Euzebio Editor: Prof. Dr. Marcos Sidnei Pagotto-Euzebio Editor: Prof. Dr. Marcos Sidnei Pagotto-Euzebio Angelo Vitório Cenci possui graduação em filosofia (LP) pela Universidade de Passo Fundo, mestrado em filosofia pela Pontifícia Universidade Católica do Rio Grande do Sul, doutorado e pós-doutorado em filosofia pela Universidade Estadual de Campinas. É professor no curso de filosofia e no Programa de Pós-Graduação em Educação da Universidade de Passo Fundo. Coordena o Núcleo de Pesquisas em Filosofia e Educação (NUPEFE-UPF) e o Grupo de Estudos em Ética, Democracia e Educação (GEEDE-UPF). Jelson Becker possui graduação em filosofia (LP) e mestrado em educação pela Universidade de Passo Fundo. É doutor em filosofia pela Universidade Federal de Santa Catarina. É professor adjunto no curso de filosofia da Universidade de Passo Fundo. Regiano Bregalda possui graduação em filosofia (L e B) pela Universidade de Passo Fundo, graduação em teologia pelo Instituto de Teologia e Pastoral, mestrado e doutorado em educação pela Universidade de Passo Fundo. Realizou o doutorado sanduíche na École des Hautes Études en Sciences Sociales-Paris e no Fonds Ricoeur. É professor no curso de teologia do Instituto de Teologia e Pastoral. 17 17
https://openalex.org/W3151375002	https://zenodo.org/records/5457814/files/Final_Manuscript1%20(1).pdf	English	null	A Novel Learning-Based Model Predictive Control Strategy for Plug-In Hybrid Electric Vehicle	IEEE transactions on transportation electrification	2,022	cc-by	13,081	A Novel Learning Based Model Predictive Control Strategy for Plug-in Hybrid Electric Vehicle Yuanjian Zhang, Member, IEEE, Yanjun Huang, Member, IEEE, Zheng Chen, Senior Member, IEEE Guang Li, Member, IEEE, and Yonggang Liu, Senior Member, IEEE electromechanical coupling characteristics in PHEVs, however, embarrass energy management into high nonlinear and complex, laying the knotty challenge waiting to be tackled. Abstract— The multi-source electromechanical coupling renders energy management of plug-in hybrid electric vehicles (PHEVs) highly nonlinear and complex. Furthermore, the complicated nonlinear management process highly depends on knowledge of driving conditions, and hinders the control strategies efficiently applied instantaneously, leading to massive challenges in energy saving improvement of PHEVs. To address these issues, a novel learning based model predictive control (LMPC) strategy is developed for a serial-parallel PHEV with the reinforced optimal control effect in real time application. Rather than employing the velocity-prediction based MPC methods favored in the literature, an original reference-tracking based MPC solution is proposed with strong instant application capacity. To guarantee the optimal control effect, an online learning process is implemented in MPC via the Gaussian process (GP) model to address the uncertainties during state estimation. The tracking reference in LMPC based control problem in PHEV is achieved by a microscopic traffic flow analysis (MTFA) method. The simulation results validate that the proposed method can optimally manage energy flow within vehicle power sources in real time, highlighting its anticipated preferable performance. Developing efficient energy management strategies for PHEVs to optimize energy saving is generally a rather difficult task, that needs to account for different constraints from powertrain, driving demand and road conditions. It is intricate to achieve the optimal trade-off among real-time performance, control effect and environmental adaptability. In past years, a variety of efforts have been made to achieve prominent progress in control strategy design of PHEVs. Existing energy management strategies for PHEVs can be mainly divided into the following four categories: rule based control strategies [4, 5], global optimization based strategies [6, 7], instantaneous optimization based strategies [8, 9] and machine learning based strategies [10, 11]. For these four types of strategies, despite their validated performance under some specific conditions, it is still intractable to achieve continuous high-quality performance in real-time control, and the potential of PHEVs in energy saving cannot be fully unlocked. Index Terms— Optimal control strategy, leaning based model predictive control (LMPC), Gaussian process (GP) model, microscopic traffic flow analysis (MTFA), plug-in hybrid electric vehicle (PHEV). Rule based control strategies, such as threshold methods [4] and fuzzy logic based strategies [5], can be easily constructed and implemented according to the expert knowledge in engineering practice. Yonggang Liu is with the State Key Laboratory of Mechanical Transmissions & School of Automotive Engineering, Chongqing University, Chongqing, 400044, China (e-mail: andylyg@umich.edu). Zheng Chen is with the Faculty of Transportation Engineering, Kunming University of Science and Technology, Kunming, 650500, China and also with the School of Engineering and Materials Science, Queen Mary University of London, London E1 4NS, U.K. (e-mail: chen@kust.edu.cn). This work was supported in part by the National Key R&D Program of China (No. 2018YFB0104000), in part by the National Natural Science Foundation of China (No. 61763021 and 51775063), and in part by the EU- funded Marie Skłodowska-Curie Individual Fellowships Project under Grant 845102-HOEMEV-H2020-MSCA-IF-2018. (Corresponding Authors: Zheng Chen, and Yonggang Liu) Guang Li is with the School of Engineering and Materials Science, Queen Mary University of London, London, E1 4NS, United Kingdom (e-mail: g.li@qmul.ac.uk). A Novel Learning Based Model Predictive Control Strategy for Plug-in Hybrid Electric Vehicle Yuanjian Zhang, Member, IEEE, Yanjun Huang, Member, IEEE, Zheng Chen, Senior Member, IEEE Guang Li, Member, IEEE, and Yonggang Liu, Senior Member, IEEE However, the expert-knowledge based control logics cannot govern powertrain operation in optimal zones all the time. Additionally, the threshold tuning is rather sensitive to driving conditions. Global optimization methods, e.g., dynamic programming (DP) [6] and Pontryagin's minimum principle (PMP) [7], can achieve optimal energy distribution with the known driving conditions. The characterized solving processes globally find the optimal solutions by ergodic search, incurring burdensome computation. On the other hand, the driving knowledge based searching manners make them quite difficult to be applied in real-time problems directly. The instantaneous optimization Yuanjian Zhang is with the School of Mechanical and Aerospace Engineering, Queen’s University of Belfast, BT9 5AG, Northern Ireland. (y.zhang@qub.ac.uk). Yanjun Huang is with the School of Automotive Studies, Tongji University, Shanghai, 201804, China. (huangyanjun404@gmail.com) I. INTRODUCTION N OWADAYS, plug-in hybrid electric vehicle (PHEVs) have boosted to be top-ranking solutions in auto industry to promote energy consumption economy and mitigate global warming concern [1, 2]. The tailored powertrain configurations endow PHEVs with extraordinary capacities to leisurely balance driving mileage, drivability, and energy reservation [3]. PHEV, as a complex system with multiple energy degrees of freedom, desperately demands properly designed control strategies to optimally manage energy flow within different sources for maximizing energy saving potentials. The specific N strategies, including equivalent consumption minimization strategy (ECMS) [8] and model predictive control (MPC) [9], are ideal solutions for PHEVs by providing the quasi-optimal effect with the reasonable abilities in real-time implementation. Despite the rational instantaneous control effect, the inner parameters of algorithms and solving mechanisms in these methods entail partial pre-knowledge of future driving conditions. The accuracy of driving conditions identification [8] and prediction [9] in these methods cannot be guaranteed with the limited amount of computation, thus discounting the instantly optimal implementation. Machine leaning based strategies, like Q-learning [10] and Deep Q-learning [11], declare to be optimally implemented without knowing the driving conditions beforehand. The remarkable validation results manifest the massive potential of these methods in PHEV control; however, the offline preparation of these methods costs much effort, and the real application in engineering practice still remains challenging. such as advanced vehicle dynamic control [20, 21], and internal combustion engine (ICE) advance control [22, 23]. The homologous application in PHEV energy management, however, is quite rare. By referring to the state-of-the-art discussion, an application- oriented control strategy is proposed for a serial and parallel PHEV by employing the learning based MPC (LMPC) algorithm and microcosmic traffic flow analysis (MTFA). Rather than preferring the prediction-velocity based MPC in our former work [24], the reference-tracking based MPC manner is selected as the basic scheme. In [24], a synthesized velocity prediction method is introduced to facilitate the achievement of optimal control results, however the computation efficiency and application capability in practical environment is discounted. By contrast, the LMPC, with super capacity in reference-tracking based adaptive control, is applied to accomplish the energy flow management in the vehicle, raising efficient optimal solution in real time via minimizing the tracking gap and energy consumption. For providing the tracking reference for the LMPC based control, MTFA is applied to estimate future driving state on the basis of shared multi-source information. I. INTRODUCTION The simulation results validate the feasibility of the proposed LMPC algorithm and the MTFA based future driving state estimation technique. Two main contributions are added in the literature: g g p g g The MPC based methods have exhibited strong capabilities in instantaneous applications, and can provide preferable control effect that is rather close to global optimization results. The built-in state estimation models can mitigate the tight dependence on future driving information to a certain extent. MPC has been widely accepted in energy management of PHEVs [12, 13], and the majority of the adopted MPC based strategies can be considered as the velocity-prediction based methods, in which the solving algorithms, like DP [12] and estimation of distribution algorithm (EDA) [13], can search the optimal decision sequences within the receding prediction horizon. Intuitively, the effect of these solutions highly depends on the accuracy of velocity prediction. Even though some methods, such as back propagation neural network (BP-NN) [14], Markov chain (MC) [15] and deep neural network (DNN) [16], have been validated effective in precisely forecasting future velocity under certain conditions, improper prediction lengths and insufficient model training may deteriorate the application effect of the MPC strategies. In fact, MPC has been applied in vehicle control via reference-tracking for a long while [17, 18]. The step length of reference-tracking based MPC strategies can be less than 10 ms [17]. In the reference- tracking based MPC, the complex control process in short horizons (e.g., the same or close to sampling time in engineering practice) can be achieved rapidly by efficient solvers with the target of simultaneously minimizing certain optimization targets and mitigating the difference between current state and reference [18]. Consequently, the reference- tracking based MPC attains more promising capacity in real- time application than the velocity-prediction based long-term MPC. With more accurate reference in shorter receding horizon, the reference-tracking based MPC can search optimal solutions with higher efficiency [19]. Therefore, it is more appropriate to apply the reference-tracking based MPC in energy management problems in PHEVs. In current stage, to the best of authors’ knowledge, the application of reference- tracking based MPC has been widely applied in different fields, 1) LMPC is applied in energy management within the powertrain of PHEV via reference tracking, and the robustness of LMPC in remedying uncertainties during state estimation is verified by the preferable energy management result. I. INTRODUCTION 2) MTFA is implemented to generate the reference for LMPC based strategy. Based on the diverse information shared through internet of vehicles (IoVs), MTFA offers accurate and detailed description on future driving state influenced by driving behaviors and surrounding environment. The remainder of this paper is organized as follows. The studied PHEV and model construction are described in Section II. Section III elaborates the LMPC based strategy for energy management of PHEV. Section IV discusses the simulation results and comparatively validates the superior performance of the proposed control strategy. The main conclusions are drawn in Section V. II. THE STUDIED 4WD PHEV AND MODEL CONSTRUCTION The studied PHEV, as illustrated in Fig. 1, is with a serial and parallel configuration. The hybrid powertrain, including ICE, generator and motor, is fabricated in the front axle. The PHEV can operate in several modes through the cooperative operation among ICE, generator and motor. ICE can either supply tractive power through driving the generator in serial mode, or drive the vehicle directly with the motor in parallel mode. The switch between serial and parallel mode is attained by controlling the engagement of the clutch between ICE and motor. In addition, the battery can be charged or discharged in both serial and parallel modes governed by the control strategy. The detailed parameters are listed in Table I. Fig. 1. The schematic of the serial and parallel PHEV configuration. TABLE I 1 _ _ 1 _ _ 2 _ _ _ 2 _ 2 2 0 / = / = fuel path eng t g fd t fd em fd t g t fd ele path em t g fd t f g g g d EV Serial T T i i Parallel T i i EV Serial T T i i Parallel                    (3) (3) where eng T and em T respectively denote the engine torque and electric motor torque; fd i is the final drive ratio; _ t fd  , _ 1 t g  and _ 2 t g  denote the mechanical transmission efficiency of final gear, gear 1 and gear 2, respectively. The mechanical transmission efficiencies of the mentioned three gears are respectively set to 0.91, 0.92, and 0.92 in the study. a. Vehicle Dynamic Model (5) Considering that the study is focused on only the longitudinal performance of PHEV, the lateral dynamics of PHEV is neglected. The vehicle longitude dynamics can be presented, as:  where wheel  denotes the rotating speed of wheel. where wheel  denotes the rotating speed of wheel. c. Motor/Generator Model 2 _ 1 1 ( ) cos sin 2 req d brk D wh a v ma T T C Av mgf mg R    = − − − −  (1) (1) The motor and generator in the serial and parallel PHEV are all permanent magnet synchronous motors (PMSMs). PMSMs can operate either in tractive mode (functioned as motor) or generator mode. The dynamic characteristics of PMSMs are neglected due to the fast-transient responses and optimal control target. Likewise, the static models are also employed to describe the power performance of PMSMs, as: where v and a denote the longitudinal velocity and acceleration, m and wh R represent the vehicle mass and wheel radius,  and f denote gradient and rolling resistance factor, D C and A are the aerodynamic drag factor and frontal area, g expresses the gravity acceleration, _ req d T and brk T represent the tractive torque from the hybrid powertrains and mechanical braking torque. The tractive torque from the powertrain can be calculated as: ( , ) , = ) = ( mot mot mot mot gen gen gen gen T T        (6) (6) where mot T and gen T represent the torque of motor and generator; mot  and gen  mean the rotation speed of motor and generator; mot  and gen  denote the efficiency in tractive mode and generator mode; mot  and gen  describe the static map to calculate efficiency of motor and generator, respectively. d. Battery Model _ _ _ req d fuel path ele path T T T = + (2) (2) _ _ _ req d fuel path ele path T T T = + where _ fuel path T and _ ele path T denote the tractive torque provided by the fuel path and electric path. In different operation modes, the tractive torque from fuel path and electric path can be expressed as: where _ fuel path T and _ ele path T denote the tractive torque provided by the fuel path and electric path. In different operation modes, the tractive torque from fuel path and electric path can be expressed as: b. Engine Model Fig. 1. The schematic of the serial and parallel PHEV configuration. TABLE I In this study, the ICE’s nonlinear performance is characterized by an efficiency map acquired from the benchmark test. The following equation shows the relationship between the engine net efficiency and torque, as: TABLE I COMPONENT PARAMETERS IN THE STUDIED PHEV ICE Displacement 2.0 [L] 16V DOHC Maximum Power 105[kW] @6200[rpm] Maximum Torque 165[Nm] @2500~6500 [rpm] Motor and Generator Maximum Power 124 [kW] Maximum Torque 307 [Nm] Lithium-ion Battery Nominal Capacity 15.7 [kWh] Nominal Voltage 300 [V] Gear Ratio Between ICE and final d rive (gear 1) 1 gi =0.803 Between motor and final drive (gear 2) 2 gi =2.45 Between ICE and genera tor (gear 3) 3 gi =1.934 a Vehicle Dynamic Model ( ) , eng eng eng eng eng lhv f T T Q m    = (4) (4) where eng  is the engine efficiency, eng  means the rotating speed of engine, lhv Q represents the fuel lower heating value, and f m expresses the fuel consumption rate. In serial mode, eng  is determined by interpolating the brake-special fuel consumption (BSFC) line according to specific torques. In parallel mode, eng  is calculated by: 1 1 = wheel fd g eng i i   (5) a. Vehicle Dynamic Model A. General Optimal Control Problem Formulation (12) subject to: subject to: 2 ( ) ( ), 1 , , ( 1) ( ) ( 1), , ] ( , 0 ) 0 [ T T T T c c c p T T T Q U k u k u k u k h u k i u k h i h h Q Q R R x x Qx    +  + −  + =  + − =    =    =    = ‖‖ (9) (9) where u , x and y are the control vector, state vector and control output; y means the reference for tracking; ch and p h denote the control and prediction horizon; A and B are the system matrices; Q and R are the weight matrixes. 1 M can be expressed, as: ( ) j j j t g z w = + (13) (13) where j w is the Gaussian noise with zero mean and diagonal variance, and 2 2 1 = ([ , , ]) d w n diag    . The measurement is normally distributed with: d. Battery Model A simple equivalent circuit model is employed to characterize the electrical performance of battery. The comprehensive performance of PHEV powertrain in random conditions. Accordingly, the LMPC based control strategy is designed in this paper, and the accuracy of state prediction is significantly prompted after validation. The LMPC, compared with ordinary MPC, holds more ideal performance in complex control problems with the enhanced adaptability to complicate nonlinear systems [25]. Instead of using the fixed state prediction model, Gaussian process (GP) is integrated into the prediction model [26], allowing continuous update of the state prediction model to improve the estimating accuracy. On this account, the reference tracking precision is promoted, and the controlling performance is boosted [27]. By referring to the state prediction function, the learning process can be formulated, as: temperature and ageing influence on battery performance is neglected for simplicity. The model consists of an open circuit voltage source, nonlinearly varying with state of charge (SOC), and an internal resistor connecting in series topology. The SOC can be calculated according to the simplified model, as: 4 2 oc oc int batt int batt V V R P SOC R Q − − = − (7) (7) where SOC is the battery SOC, Voc is the open circuit voltage of battery, Rint is the internal resistance of battery, Pbatt is the battery power, and Qbatt is the battery capacity. The battery cells in the studied PHEV are lithium-iron phosphate battery, with a nominal capacity of 52.3 Ampere-hour (Ah). The nominal voltage of battery pack is 300 V, with 90 cells connected in series topology. On the basis of the detailed powertrain modeling, the proposed LMPC based energy management will be addressed in the next section. ( 1) ( ( ), ( )) ( ( ( ) ( ))) d x k f x k u k B d z k w k + = + + (11) (11) where d is the leaning part of the prediction model that estimates the model error true d between the model and measurement, z is the parameter vector related to the learning part, w is the process noise, and d B is the weight matrix of the learning part. In the learning process in MPC, GP accounting for predicting values of leaning part is trained by the difference between the measured ( 1) x k + and predictions by (10). d. Battery Model The training data ( ) t k can be expressed as [28]: A. General Optimal Control Problem Formulation For attaining the optimal control by MPC through tracking reference, the nonlinear control problem can be generally defined as: 2 * * 2 0 ( ) argmin ( ) ( ) ( ) p h R Q i U k y k i y k u k i = = + − +  +  ‖ ‖ (8) ( ) ( ( 1) ( ( ), ( ))) ( ( )) ( ) d true t k B x k f x k u k d z k w k + = + − = + (12) where d B+ denotes the Morre-Penrose pseud-inverse matrix. The training data in (12) is the standard form of a regression task. Given the collected data ( ) D z , the GP model can stochastically estimate the regression results. With the updated state estimation by (11), MPC can be executed by some optimization methods, such as sequential quadratic programming (SQP) [29]. In GP, based on the parameter vector 1 [ , , ] z M n T T M z z z  =  and the measurement vector 1 [ , , ] d M n T T M t t t  =  , the statistical model from the system can be expressed, as: (8) ( ) ( ( 1) ( ( ), ( ))) ( ( )) ( ) d true t k B x k f x k u k d z k w k + = + − = + (12) where d B+ denotes the Morre-Penrose pseud-inverse matrix. ( ) ( ( 1) ( ( ), ( ))) ( ( )) ( ) d true t k B x k f x k u k d z k w k + = + − = + (12) where d B+ denotes the Morre-Penrose pseud-inverse matrix. C. Formulation of Optimal Energy Management in PHEV In LMPC based optimal energy management for PHEV, the control process to complete the optimal energy distribution is illustrated in Fig. 2, where the desired tractive torque _ req d T ordered from driver is regulated by the torque difference T generated from the receding optimization via LMPC. Then, the real required tractive torque _ req r T , together with the torque ordered from driver is regulated by the torque difference T generated from the receding optimization via LMPC. Then, the real required tractive torque _ req r T , together with the torque Fig. 2. Simplified architecture of the control strategy for PHEV. Fig. 2. Simplified architecture of the control strategy for PHEV. motor, respectively; and _ 3 t g  is the mechanical transmission efficiency of gear 3. Moreover, the control should be subject to: motor, respectively; and _ 3 t g  is the mechanical transmission efficiency of gear 3. Moreover, the control should be subject to: The reference-tracking based LMPC in PHEV achieves the optimal control via efficient torque regulation and distribution. The torque regulation efficiently guides driving intention expressed in hybrid powertrain by referring to the estimated reference velocity. To avoid confliction with the original driving behaviors and driving comfort, the estimated reference velocity should be in line with the real driving in future. Therefore, the reference velocity estimation needs to be attained by the methods that can precisely describe the dynamics of future driving. The optimal control in PHEV for optimal energy management by reference-tracking based LMPC can be formulated as: The reference-tracking based LMPC in PHEV achieves the optimal control via efficient torque regulation and distribution. opt a co t o v a e c e t to que egu at o a d d st but o . The torque regulation efficiently guides driving intention expressed in hybrid powertrain by referring to the estimated reference velocity. To avoid confliction with the original driving behaviors and driving comfort, the estimated reference velocity should be in line with the real driving in future. C. Formulation of Optimal Energy Management in PHEV min max _ min _ max _ min _ max , _ min , , _ max _ min _ max , _ min , , _ max ( ) ( ) ( ) ( ) ( ) ( ) batt batt batt eng eng eng mot gen mot gen mot gen eng eng eng mot gen mot gen mot gen SOC SOC t SOC P P t P T T t T T T t T w w t w w w t w                       (21) (21) Therefore, the reference velocity estimation needs to be attained by the methods that can precisely describe the dynamics of future driving. The optimal control in PHEV for optimal energy management by reference-tracking based LMPC can be formulated as: where the superscripts min and max denote the minimum and maximum value of variables. The internal state prediction models in LMPC need to be selected to describe key features of PHEV (e.g., vehicle dynamics and electricity consumption), as:  2 * 1 2 3 0 ( ) ( ) arg min ( ) ( ) ( ) p h batt f ref lhv i P t U k m t v t v t Q    = = + − +  (19) (19) where 1 , 2  and 3  are the weight ratio, and ref v is the reference velocity. B. Learning Based MPC The defined MPC based control problem in (8) can only achieve the optimal effect by precisely tracking the reference, which is supplied by the state prediction function, as: 2 ., [ ] (0, ) a a ZZ a t N K  + (14) (14) where a ZZ K is the Gram matrix of the data points with the kernel function a k . In this study, the selected specific kernel function is the squared exponential kernel function [30], as: y p ( 1) ( ( ), ( )) x k f x k u k + = (10) (10) From this point of view, the state prediction function is pivotal to capture system dynamics within prediction horizons and influence the control effect significantly. Furthermore, the online applications of MPC require that the state prediction function can describe future state accurately with less computation through simplifying the function, which, however, may lead to inferior control effects due to the raised prediction error. In addition, the powertrains in PHEVs are complex and nonlinear systems that are difficult to identify. As such, simple state prediction functions cannot accurately describe the 2 1 1 ( , ) exp( ( ) ( )) 2 T f k z z z z L z z  − = − − − (15) (15) where z z n n L   is the matrix with positive diagonal length. Given each output dimension (1, , ) d a n  , the posterior distribution in dimension a at a specific parameter group z is the Gaussian function, of which the means and variance can be calculated, as: 2 1 ., 2 1 ( ) ( ) [ ] ( ) ( ) a a a zz zz a a a a a a a zz zz zz a zz z K K I t z K K K I K    − −  = +   = − +  (16) distribution ratios among engine, motor and generator, is assigned to the powertrain to attain the energy distribution. B. Learning Based MPC The relationship between the desired tractive torque and real tractive torque as well as torque difference can be expressed as: (16) 2 1 ( ) ( ) a a a a a zz zz zz a zz z K K K I K  −   = − +  Now, the GP based regression analysis of the unknown function ( ) g z can be yielded, as: q p _ _ req r req d T T T = + (18) (18) ( ) ( ( ), ( )) d a d z N z z   (17) , ] d d n  , and 1 [ , , ] d d d d n  =   . According to the control process shown in Fig. 2, the control inputs of LMPC include the torque difference T, the power distribution raito 1  between engine and motor and the power distribution ratio 2  between auxiliary power unit (APU) (consisting of ICE and generator) and motor. Here, the power distribution ratio 1  and 2  is respectively implemented in parallel and serial mode. The instant vehicle velocity and battery SOC are the state variables. Note that the studied PHEV will switch into charge sustaining stage from depleting stage when battery SOC is lower than 0.27. (17) C. Formulation of Optimal Energy Management in PHEV batt P can be calculated as: 2 _ int _ int _ 1 1 ( ) ( ( ) ( ) cos ( ) sin ( ) )) 2 4 ( ( ( ( ) ( ) ( ( ) ( ( ( ) ( )) ( ) ) 2 req r dyn eny e D d dyn oc oc ba y tt d eny bat n t d z v t T t C Av t mgf t mg t B m V V R P SOC t B R Q t t t t d z t w t t      +  = − − − + − − = − +  2 _ _ _ 2 _ 1 _ _ _ 2 _ 9550 ( ) 9550 req d wheel t fd t g e bm batt req d wheel t fd t g e bm T n Serial Mode P t T n Parallel Mode            =    (20) (22) where _ d dyn B and _ d eny B denote the weight matrix, dyn w and where _ d dyn B and _ d eny B denote the weight matrix, dyn w and eny w are the process noise, dyn z and eny z are the parameter (20) vector, as: vector, as: where _ e bm  and _ e gm  denote the electric transmission efficiency between motor and battery as well as generator and where _ e bm  and _ e gm  denote the electric transmission efficiency between motor and battery as well as generator and int [ , ] [ , ] dyn en a y x x b tt z v a R z P   =  = (23) (23) where xv and x a denote the velocity and acceleration in longitude direction, int R and batt P represent the battery internal resistance and power, respectively. In the paper, the Gaussian model is trained based on the data acquired through the benchmark test. The data of velocity, acceleration in longitudinal direction, battery power, tractive torque, motor torque, ICE torque, and battery SOC are collected via dynamometer test based on the WLTC driving cycle. The battery internal resistance is measured on a battery cycler according to the test specifications. C. Formulation of Optimal Energy Management in PHEV Therefore, the functions to describe the specific impact can be formulated as: According to (28), jam  and h are the key parameters According to (28), jam  and h are the key parameters According to (28), jam  and h are the key parameters affecting vehicle velocity in vehicle flow. jam  and h , as a matter of fact, can be easily influenced by driving behaviors and driving environment, including traffic lights, crossroads, nearby buildings, etc. Therefore, the functions to describe the specific impact can be formulated as: 2 2 1 1 2 3 2 1 2 ( ) ( ) ( ) jam ts c pub f h t v A v AB v B v va A N N B N N     =  + + +  +  = +   = +  (30) (30) D. Reference Velocity Estimation by MTFA In IoVs, vehicles are equipped with enhanced communication techniques that can realize vehicle to vehicle (V2V) communication and vehicle to infrastructure (V2I) communication [36]. The velocity of each vehicle on the route segment can be easily shared among multi-vehicles. Thus, ts N , c N , pub N and f N can be statistically collected by infrastructures at road side and effectively disseminated among vehicles. Open data source like OpenStreetMap can also provide the environment information minutely [37], and we referred to the data therein for simulation. where ts N , c N , pub N and f N are the number of traffic lights, crossroads on the route segment and public buildings such as schools and hospitals and other buildings, respectively; 1v , 2v , 1 1 1 2 1 ( ) ( ) ( 1) ( ) ( ) ( ( ) ( )) n n n n n n x t x t mx t x t t x t x t  + + + + −   + = −   −   (24) (24) where  is the sensitivity factor, x denotes the vehicle position on the route, m is the vehicle mass, t is the time step, and  denotes the time lag. By solving (8), the velocity of target vehicle at time t can be attained, as: ( ) exp( ) ( ) jam f v t v t   = (25) (25) where fv is the drag-free velocity, jam  is the jam density, and  is the instant traffic flow density. The drag-free velocity can be physical characteristics of routes, which can be acquired through IoVs. The instant traffic flow density can be calculated as: D. Reference Velocity Estimation by MTFA The required accurate estimation of reference velocity can be fulfilled only by integrally considering the influence from driving behaviors and surrounding environment. MTFA excels at describing the mentioned connection [32]. Even though microscopic traffic analysis has been widely investigated, integrating it into vehicle control, especially MPC based control, has not been fully investigated. MTFA proposed in this paper has considered much impact from environment and particular driver behaviors, making it rather suitable for MPC based control in PHEV. In this paper, the general expression of MTFA can be written as: where ts N , c N , pub N and f N are the number of traffic lights, crossroads on the route segment and public buildings such as schools and hospitals and other buildings, respectively; 1v , 2v , 3v , 1  and 2  are system parameters; 1  and 2  are weight ratios. To obtain accurate values of the system parameters and weight ratios shown in (14), particle filters are employed to calculate the values based on the shared data from IoVs [34, 35]. According to (13) and (14), the reference velocity can be accurately estimated for the LMPC based energy management strategy. In IoVs, vehicles are equipped with enhanced communication techniques that can realize vehicle to vehicle (V2V) communication and vehicle to infrastructure (V2I) communication [36]. The velocity of each vehicle on the route segment can be easily shared among multi-vehicles. Thus, ts N , c N , pub N and f N can be statistically collected by infrastructures at road side and effectively disseminated among vehicles. Open data source like OpenStreetMap can also provide the environment information minutely [37], and we referred to the data therein for simulation. where ts N , c N , pub N and f N are the number of traffic lights, crossroads on the route segment and public buildings such as schools and hospitals and other buildings, respectively; 1v , 2v , 3v , 1  and 2  are system parameters; 1  and 2  are weight ratios. To obtain accurate values of the system parameters and weight ratios shown in (14), particle filters are employed to calculate the values based on the shared data from IoVs [34, 35]. According to (13) and (14), the reference velocity can be accurately estimated for the LMPC based energy management strategy. C. Formulation of Optimal Energy Management in PHEV In the reference-tracking based LMPC for energy management of PHEV, the prediction horizon and the control step are set to 1 s and 0.1 s, respectively; and the reference velocity is updated every 1 s. The time parameters are chosen based on the real control process in on- board vehicle control units. By contrast, the prediction length of the velocity-prediction based MPC is set to 20 s to 50 s [31]. To guarantee the real-time application performance, the lengths of control step are enlarged to 1 s or longer. However, too larger control steps may not be implementable in real-time control. Even though the proposed LMPC based control strategy is developed based on the described PHEV configuration shown in Fig. 1, it can also be implemented to other PHEVs with different configurations, and only the powertrain parameters shown in Table I, the engine and motor efficiency maps, and the transmitting efficiencies in both fuel and electric path need to be updated. In addition, the inequality constraints described in (21) should also be adjusted accordingly. 1 ( ) ( ) n i i n t v h t  = =  (26) (26) where iv is the instant speed of certain vehicle on route segment, n is the number of vehicles on route segment, and h is the average time headway. By substituting (10) into (9), the velocity of target vehicle can be written as: ( ) exp( ( ) ) f jam rs v t v h t v  = − (27) (27) where rs v denotes the average speed of vehicles on the route segment that can be calculated as: 1 n i i rs v v n =  = (28) (28) Then, equation (27) can be extended by Taylor function [33], as: 2 3 ( ( ) ) ( ( ) ) ( ) (1 ( )) 2! 3! jam rs jam rs f jam rs h t v h t v v t v h t v    = − + − (29) (29) According to (28), jam  and h are the key parameters affecting vehicle velocity in vehicle flow. jam  and h , as a matter of fact, can be easily influenced by driving behaviors and driving environment, including traffic lights, crossroads, nearby buildings, etc. IV. SIMULATION AND EVALUATION To validate the performance of the proposed strategy, a series of simulations are performed [38]. In this study, the performance of MTFA in reference velocity estimation, the capability of LMPC in reference-tracking based optimal energy management in PHEV, and the energy-saving mechanism of LMPC are comprehensively evaluated through the comparison study. Note that the simulation is conducted on a workstation with an Intel i7-8700 processor and 16 Gigabytes memory. evaluate the effect of the chosen velocity estimation methods by comparing the fuel economy of PHEV based on different controllers. During evaluation, the data for validating the MTFA and other baseline methods are extracted from the real traffic conditions according to the method described in [41]. g Fig. 3 illustrates the estimated profiles by different methods together with the raw speed data. The estimation length of different methods is all set to 1 s. To better present the behaviors of different methods, the zoomed-in figures sketching the estimated velocities around 800 s to 900 s are also provided. As can be found, most of the methods can achieve relatively high prediction accuracy, expect the simple PSD based method. The enlarged figures can partially facilitate the explanation of prediction accuracy discrepancy by different methods. The employed methods, include MTFA, LSTM and SVM, can capture more details than the simple PSD based method (as shown in the zoomed-in figures in Fig. 3). The promising abilities of LSTM and SVM in capturing future driving behaviors stem from the strong regression after fully understanding the inner connection between history data and future behaviors. While, the superior capability of MTFA to estimate future driving is incurred by the specific designed manners with the consideration of more environment influence. A. Performance Validation of MTFA in Reference Velocity Estimation The capability of MTFA is of the vital importance for LMPC based optimal energy management. The estimation accuracy of MTFA is comparatively studied among several methods, including participatory sensing data (PSD) based method [9], long-short term memory (LSTM) network [39] and support vector machine (SVM) [40]. Then, the stability and robustness of MTFA is verified among different road conditions. The city urban road (CR), highway (HW) and combined road (CW) conditions involving both CR and HW are selected for validation. The data for training models are derived from the collected data in real traffic test. IV. SIMULATION AND EVALUATION To guarantee fair performance comparison and evaluation, all models are trained and evaluated by the same data set. Finally, the MPC controllers incorporating different velocity estimation methods are leveraged for energy management of the studied PHEV shown in Fig. 1, trying to Fig. 3. Comparison in velocity estimation with different methods. The numerical comparing results among different velocity estimation methods on the specified driving cycle are listed in Table II, where RMSE and MAE denote the root mean square error and mean absolute error respectively, as: 2 1 1 ˆ ( ) m i i i RMSE y y m = = −  (31) 1 1 ˆ ( ) m i i i MAE y y m = = −  (32) where iy is the estimation value, ˆiy is the real value from the raw data. cal T is the online processing time of CPU. The RMSE and MAE in Table II highlight that that LSTM and SVM methods perform slightly better than MTFA. However, the minor weakness in prediction accuracy cannot prevent MTFA from being applied in real time due to much less online calculation time, compared with LSTM and SVM. Even though the simple PSD based method costs much less time (compared with other methods) because of its straightforward calculation manner, its prediction accuracy is obviously the worst among the chosen methods. The general comparison in Table II validates that MTFA is an ideal method to achieve the promising prediction accuracy in real time. To further investigate its stability and robustness in velocity estimation, the MTFA based method is tested to estimate velocities within 50 groups of data extracted from real traffic under different road conditions, as listed in Table III, where RMSEava, MAEava and Tcal_ava denote the average RMSE, MAE and CPU processing time; RMSEbest, MAEbest and Tcal_best denote the minimum RMSE, minimum MAE and shortest CPU processing time, respectively. Fig. 3. Comparison in velocity estimation with different methods. Fig. 3. Comparison in velocity estimation with different methods. from being applied in real time due to much less online calculation time, compared with LSTM and SVM. Even though the simple PSD based method costs much less time (compared with other methods) because of its straightforward calculation manner, its prediction accuracy is obviously the worst among the chosen methods. IV. SIMULATION AND EVALUATION The general comparison in Table II validates that MTFA is an ideal method to achieve the promising prediction accuracy in real time. To further investigate its stability and robustness in velocity estimation, the MTFA based method is tested to estimate velocities within 50 groups of data extracted from real traffic under different road conditions, as listed in Table III, where RMSEava, MAEava and Tcal_ava denote the average RMSE, MAE and CPU processing time; RMSEbest, MAEbest and Tcal_best denote the minimum RMSE, minimum MAE and shortest CPU processing time, respectively. The numerical comparing results among different velocity estimation methods on the specified driving cycle are listed in Table II, where RMSE and MAE denote the root mean square error and mean absolute error respectively, as: 2 1 1 ˆ ( ) m i i i RMSE y y m = = −  (31) 1 1 ˆ ( ) m i i i MAE y y m = = −  (32) (31) (32) where iy is the estimation value, ˆiy is the real value from the raw data. cal T is the online processing time of CPU. The RMSE and MAE in Table II highlight that that LSTM and SVM methods perform slightly better than MTFA. However, the minor weakness in prediction accuracy cannot prevent MTFA The results in Table III show that the MTFA can estimate the vehicle velocity stably, and the average RSME, MAE and CPU processing time under different road conditions are quite close. The stable performance verifies the rational robustness of the MTFA method. The reason can be attributed to that the MTFA method can comprehensively consider the impact on future driving from different perspectives, and abundant information inclusion and carefully designed information integration manners all contribute to the performance improvement. Moreover, the reason why the performance of the MTFA method in CR is better than that in HW is that the amount of shared data in CR is more than that in HW, and certainly more data lead to better prediction precision. and the solving manner is described minutely in [42]. Table V lists the fuel consumption by the ordinary MPC and LMPC with different velocity estimation methods. The MPC with SVM 10, SVM 20 and SVM 30 represents the velocity-prediction based ordinary MPC controller, in which the length of velocity prediction is 10 s, 20 s and 30 s, respectively. IV. SIMULATION AND EVALUATION Similarly, the LMPC with SVM 10, SVM 20 and SVM 30 expresses the velocity-prediction based LMPC controller, and the prediction length is the same as above. In addition, the MPC with MTFA and the LMPC with MTFA present the reference-tracking based ordinary MPC and LMPC controllers with MTFA as the velocity prediction algorithm, where the prediction length is set to 1 s. Note that the control step of all the mentioned methods is 0.1 s. data lead to better prediction precision. TABLE II Numerical Comparison in Prediction Accuracy Method RMSE MAE Tcal (s) Simple PSD 1.081 0.733 0.011 MTFA 0.711 0.235 0.018 LSTM 0.702 0.269 0.112 SVM 0.692 0.223 0.107 TABLE III Comparison of the MTFA Based Velocity Estimation Type ava RMSE ava MAE _ cal ava T best RMSE best MAE _ min cal T CR 0.731 0.249 0.021 s 0.705 0.228 0.016 s HW 0.793 0.262 0.026 s 0.787 0.259 0.021 s Com 0.766 0.254 0.023 s 0.707 0.227 0.014 s The comparison results in Table IV validate the performance of different velocity estimation methods in another perspective. The reference-tracking based MPC controllers employed in the studied PHEV all utilize the same solving algorithm (SQP), and the difference lies in only the integrated velocity estimation methods. By this manner, the capabilities of velocity estimation methods can be further assessed. The results in Table IV reveal that the fuel economy becomes better after the MPC controller integrates more accurate velocity estimation. The EFC in Table IV denotes the equivalent fuel consumption that includes the converted fuel consumption from electric energy. Considering the trade-off between the controlling effect and capability in real-time application, MTFA is most suitable for the MPC based strategy. Specifically, the difference in optimality between MPC with MTFA and MPC with SVM is only 0.1% when employing DP as the benchmark; however, the Tcal of MTFA is much less than that of SVM, highlighting its comprehensive feasibility in velocity prediction. IV. SIMULATION AND EVALUATION TABLE IV Impact on Fuel Economy by Different Estimation Methods Method EFC (L/100km) Optimality (%) p TABLE II p p TABLE II Numerical Comparison in Prediction Accuracy Method RMSE MAE Tcal (s) Simple PSD 1.081 0.733 0.011 MTFA 0.711 0.235 0.018 LSTM 0.702 0.269 0.112 SVM 0.692 0.223 0.107 TABLE III Comparison of the MTFA Based Velocity Estimation Type ava RMSE ava MAE _ cal ava T best RMSE best MAE _ min cal T CR 0.731 0.249 0.021 s 0.705 0.228 0.016 s HW 0.793 0.262 0.026 s 0.787 0.259 0.021 s Com 0.766 0.254 0.023 s 0.707 0.227 0.014 s is 0.1 s. The numerical results listed in Table V underpin the conclusion that the LMPC controller outperforms the ordinary MPC controller. The equivalent fuel consumption by the LMPC controller is less than that based on the ordinary MPC controller. The accurate inner state prediction with GP models in LMPC supplies more chances to find optimal solutions and contributes to better fuel economy. Table V also lists the difference between the velocity-prediction based controller and reference- tracking based controller. By referring to all ordinary MPC and LMPC controllers with different solving mechanisms, the reference-tracking based controller can lead to obvious superiority in real-time implementation. In the controllers with the reference-tracking based solving mechanism, the consumed calculation time in each step plus the time of reference estimation is shorter than the pre-given control step length, verifying its feasibility in instant application. For the velocity- prediction based controller, however, only the CPU processing time in each step is much longer than the pre-defined control step length, thereby avoiding from instant real-time application. To apply the velocity-prediction based controllers in real time, the control steps should be preset with longer interval (such as 1 s), which cannot satisfy the demand of engineering practice. Notably, the EFC by reference-tracking based controller is slightly more than that by the velocity-prediction based controllers. The reason of incurring minor difference is that the velocity-prediction based strategies consider impact of future driving on fuel economy within longer horizons, which is closer to the global optimal solutions. However, the maximum difference in EFCs is only 0.024L/100km, which can be neglected in instantaneous application. IV. SIMULATION AND EVALUATION Comparison of the MTFA Based Velocity Estimation Type ava RMSE ava MAE _ cal ava T best RMSE best MAE _ min cal T CR 0.731 0.249 0.021 s 0.705 0.228 0.016 s HW 0.793 0.262 0.026 s 0.787 0.259 0.021 s Com 0.766 0.254 0.023 s 0.707 0.227 0.014 s The comparison results in Table IV validate the performance of different velocity estimation methods in another perspective. of different velocity estimation methods in another perspective. The reference-tracking based MPC controllers employed in the studied PHEV all utilize the same solving algorithm (SQP), and the difference lies in only the integrated velocity estimation methods. By this manner, the capabilities of velocity estimation methods can be further assessed. The results in Table IV reveal that the fuel economy becomes better after the MPC controller integrates more accurate velocity estimation. The EFC in Table IV denotes the equivalent fuel consumption that includes the converted fuel consumption from electric energy. Considering the trade-off between the controlling effect and capability in real-time application, MTFA is most suitable for the MPC based strategy. Specifically, the difference in optimality between MPC with MTFA and MPC with SVM is only 0.1% when employing DP as the benchmark; however, the Tcal of MTFA is much less than that of SVM, highlighting its comprehensive feasibility in velocity prediction. p y y p TABLE IV Impact on Fuel Economy by Different Estimation Methods Method EFC (L/100km) Optimality (%) MPC-Simple PSD 2.664 95.07% MPC-MTFA 2.634 96.17% MPC-LSTM 2.633 96.21% MPC-SVM 2.631 96.27% DP 2.533 100 B. Reference-Tracking Based Optimal Control in PHEV To better demonstrate the difference in solving process between the ordinary MPC and LMPC, Table VI compares the solving performance of the ordinary MPC and LMPC. Both the ordinary MPC and LMPC execute the solution searching based on the reference-tracking principle, and exploit the proposed MTFA to estimate the reference. In Table VI, 2 0 S denotes the average variance relating with state-constraint violations, and e means the difference between the predicted state after one step prediction and real state. The results show that the ordinary MPC without correction in inner state prediction leads to more constraint violation than that by LMPC, which is mainly caused by the discretization error inherited from the inflexible state B. Reference-Tracking Based Optimal Control in PHEV The raised LMPC can bring better performance in theory because of its better capability in inner state prediction after integrating the GP model. To more clearly demonstrate the remarkable performance of LMPC, a number of comparisons are conducted. In this part, MPC denotes the ordinary MPC application without the learning based state prediction function, reach 96.21% of that by DP. The ECMS performs slightly worse than the LMPC based strategy, attaining 93.16% of that by DP. The CD-CS method, obviously, raises the worst performance among the applied methods, only reaching 80.23% that by DP. As shown in Fig. 4, the proposed LMPC based strategy behaves quite similarly with DP, and leads to close fuel consumption and battery power variation. Both DP and LMPC tend to coordinately distribute the power of ICE and battery, avoiding the operation of powertrain components in unfavorable area. The ECMS, partially incorporating environment information by tuning the equivalent factor [8], achieves better performance than the simple CD-CS method. The battery SOC by the simple rule based strategy drops fastest at the beginning of trip, and enables ICE to operate frequently after the vehicle untimely switches into the CS stage. By contrast, the LMPC based control strategy and DP can govern electric energy rationally consumed during the entire trip, and hinder frequent ICE operation engagement. The gap in energy consumption by different methods owns to the disperse integration level of environmental information. Given the full knowledge of driving cycle, DP can search the optimal solution to minimize the overall energy consumption. The LMPC, equipped with the prediction capabilities for future driving conditions in specified horizons, can attain quasi-optimal solutions and exhaustively reduce the energy consumption within the prediction trip. However, larger lengths of optimization horizons contribute to better control performance but with the price of computation intensity increase. The ECMS behaves worse than DP and LMPC, due to the limited ability in incorporating the time-varying influence incurred by driving environment in each step. The CD-CS scheme, even calibrated by expert knowledge and engineering practice, presents poor adaption to driving conditions and results in clumsy control effect model. Smaller index e indicates that the GP model boosts the state prediction in LMPC. Thus, one can conclude that the GP model improves the accuracy of state prediction, thereby prompting the overall performance obviously. prompting the overall performance obviously. C. Energy-Saving Mechanism of LMPC based Strategy The results comparison in Table V validates the promising performance of LMPC, compared with the ordinary MPC. To further reveal the mechanism of the proposed LMPC based strategy in energy saving, the comparison study between the LMPC based method and a number of baseline control methods is performed, and the adopted baseline control strategies include the CD-CS scheme, ECMS, and DP, which can be described as follows. 1) The CD-CS scheme [4]. The CD-CS controller is a simple rule based strategy, in which the operation mode switches from CD to CS stage when battery SOC is less than 0.25. In the CD stage, the battery supplies all the driving power except when the required tractive power is larger than an upper threshold; while in the CS stage, the ICE and battery work together to drive the vehicle. Fig. 4. Fuel consumption and battery SOC trajectories by different methods. Fig. 4. Fuel consumption and battery SOC trajectories by different methods. Fig. 5 present the powertrain operation modes by different methods on the given driving cycle. The proportions of operation time of each mode by DP and the LMPC based strategy are quite consistent. In contrast, the ECMS and CD-CS control strategy all switches into the CS stage before the trip ends, opposing to energy saving. The difference between DP and the novel LMPC in mode operation lies in that the LMPC algorithm prefers sporadic parallel mode, especially in sudden acceleration scenarios, due to the short prediction on future driving and the corresponding improper solving manner. To further compare the difference between the LMPC strategy and DP, the powertrain component performance by the two control 2) ECMS [8]. In the ECMS based controller, the ICE and battery power combination that can contribute to the minimum instant equivalent fuel consumption is chosen as the current control policy. The equivalent factor in ECMS is tuned for given driving conditions by the specially designed optimization algorithm [43]. 3) DP [6]. DP, as a well-known global optimization strategy, is chosen as the benchmark methods in most cases. In DP based controller, the control step and the numbers of state and control grid are respectively set to 0.1, 700 and 300 in this study. Fig. 4. Fuel consumption and battery SOC trajectories by different methods. Fig. 5 present the powertrain operation modes by different methods on the given driving cycle. B. Reference-Tracking Based Optimal Control in PHEV TABLE V Comparison of Fuel Economy by Ordinary MPC and LMPC with Different V elocity Estimation Methods Method EFC (L/100km) Optimality (%) Tcal (s) Tcal_step (s) MPC SVM 10 2.620 99.66 16622.10 0.330 SVM 20 2.617 99.77 19745.04 0.392 SVM 30 2.611 100 24983.52 0.496 MTFA 2.634 99.13 3072.57 0.061 LMP C SVM 10 2.589 99.67 16873.95 0.335 SVM 20 2.587 99.75 19996.89 0.397 SVM 30 2.581 100 25336.11 0.503 MTFA 2.603 99.16 3223.68 0.064 TABLE VI Solving Performance Comparison between MPC and LMPC Method 20 S e MPC-MTFA 5.15 1.14 LMPC-MTFA 1.06 0.08 p p g p y TABLE V Comparison of Fuel Economy by Ordinary MPC and LMPC with Different V elocity Estimation Methods C. Energy-Saving Mechanism of LMPC based Strategy The proportions of operation time of each mode by DP and the LMPC based strategy are quite consistent. In contrast, the ECMS and CD-CS control strategy all switches into the CS stage before the trip ends, opposing to energy saving. The difference between DP and the novel LMPC in mode operation lies in that the LMPC algorithm prefers sporadic parallel mode, especially in sudden acceleration scenarios, due to the short prediction on future driving and the corresponding improper solving manner. To further compare the difference between the LMPC strategy and DP, the powertrain component performance by the two control Fig. 5 present the powertrain operation modes by different methods on the given driving cycle. The proportions of operation time of each mode by DP and the LMPC based strategy are quite consistent. In contrast, the ECMS and CD-CS control strategy all switches into the CS stage before the trip ends, opposing to energy saving. The difference between DP and the novel LMPC in mode operation lies in that the LMPC algorithm prefers sporadic parallel mode, especially in sudden acceleration scenarios, due to the short prediction on future driving and the corresponding improper solving manner. To further compare the difference between the LMPC strategy and DP, the powertrain component performance by the two control Note that LMPC in following figures and tables denotes the raised LMPC based control strategy with the incorporation of MTFA, of which the main function is to provide reference velocity trajectories. The investigation in this part is addressed on a driving cycle extracted from real traffic data shown in [41]. Table VII lists the comparison on energy consumption by different methods. Apparently, the LMPC based strategy leads to less fuel consumption with the similar behaviors as DP. As can be found, the optimality of the LMPC based strategy can strategies are illustrated in Fig. 6. The powers of ICE, motor and generator by two methods are quite close, except some noticeable difference in acceleration stage, e.g. from 1401 s to 1681 s. DP, with the full knowledge of driving cycle, selects the serial assist mode to reach the minimum energy consumption in the whole trip. In contrast, LMPC is forced to run optimal solution searching within the narrow prediction horizon, enabling the direct ICE drive mode to avoid fast SOC decrease. C. Energy-Saving Mechanism of LMPC based Strategy foresees upcoming continuous acceleration, thus prejudging that the high load driving condition is more suitable for ICE operation and battery SOC sustaining. Instead, DP, endowed with longer view of future deceleration, integrally exploits the serial assist mode, controlling the ICE to operate in higher efficiency region, and makes full use of regenerative braking energy to replenish electricity consumed in assist mode, as observed in Figs. 5 and 8. Note that the regenerative braking mode during deceleration is marked as the EV mode in both CD and CS stage for simplicity. To sum up, the proposed LMPC strategy can lead to preferable energy management in PHEV based on the strong local optimization and the receding horizon prediction capability. g p , enabling the direct ICE drive mode to avoid fast SOC decrease. TABLE VII Energy Consumption by Different Methods Method Fuel Consumpti on (g) Electricity (k Wh) EFC (L/10 0km) Optim ality (%) CD-CS 717.41 5.88 3.09 80.23 ECMS 583.92 5.45 2.66 93.16 LMPC 552.93 4.66 2.57 96.21 DP 517.21 4.09 2.48 100 Fig. 5. Comparison of powertrain operation modes by different methods. EV- CD and EV-CS respectively denotes pure electric mode in CD and CS stage; SH-CD and SH-CS expresses serial mode in CD and CS stage; PH-CD means the parallel mode in CD stage. Fig. 6. Comparison of component performance by different methods. Figs. 7 to 9 illustrate the powertrain performance by DP and LMPC based strategy in a sudden acceleration and deceleration segment. As shown in Fig. 7, DP and the LMPC algorithm result in almost the same fuel consumption rate except in the initial and middle acceleration stages. Although ICE is started more frequently by DP than by novel LMPC, DP enables more efficient operation of ICE than the novel LMPC algorithm, due to the benefit from pre-knowledge of the whole driving cycle. The LMPC strategy, limited by the finite predicted horizon, can only perform local optimal control within a short range, and fails to discover the minimum energy consumption in the whole trip. The difference in operation modes, as shown in Figs. 8 and 9, supports the same conclusion. To be specific, DP prefers the serial assist mode, while the LMPC method urges to switch to the ICE direct drive mode. However, the LMPC strategy enabling the direct ICE drive mode to avoid fast SOC decrease. C. Energy-Saving Mechanism of LMPC based Strategy TABLE VII Energy Consumption by Different Methods Method Fuel Consumpti on (g) Electricity (k Wh) EFC (L/10 0km) Optim ality (%) CD-CS 717.41 5.88 3.09 80.23 ECMS 583.92 5.45 2.66 93.16 LMPC 552.93 4.66 2.57 96.21 DP 517.21 4.09 2.48 100 Fig. 5. Comparison of powertrain operation modes by different methods. EV- CD and EV-CS respectively denotes pure electric mode in CD and CS stage; SH-CD and SH-CS expresses serial mode in CD and CS stage; PH-CD means the parallel mode in CD stage TABLE VII Energy Consumption by Different Methods Method Fuel Consumpti on (g) Electricity (k Wh) EFC (L/10 0km) Optim ality (%) CD-CS 717.41 5.88 3.09 80.23 ECMS 583.92 5.45 2.66 93.16 LMPC 552.93 4.66 2.57 96.21 DP 517.21 4.09 2.48 100 p p y Fig. 7. The zoomed-in fuel consumption rate in sudden acceleration and deceleration conditions. Fig. 8. The zoomed-in operation modes by DP and LMPC strategy in sudden a cceleration and deceleration conditions. p p y Fig. 7. The zoomed-in fuel consumption rate in sudden acceleration and deceleration conditions. Fig. 7. The zoomed-in fuel consumption rate in sudden acceleration and deceleration conditions. Fig. 8. The zoomed-in operation modes by DP and LMPC strategy in sudden a cceleration and deceleration conditions. Fig. 9. The zoomed-in component performance by DP and LMPC based strate gy in sudden acceleration and deceleration conditions D. Validation in Other PHEVs with Different Configurations Fig. 7. The zoomed-in fuel consumption rate in sudden acceleration and deceleration conditions. Fig. 8. The zoomed-in operation modes by DP and LMPC strategy in sudden a cceleration and deceleration conditions. Fig. 8. The zoomed-in operation modes by DP and LMPC strategy in sudden a cceleration and deceleration conditions. Fig. 5. Comparison of powertrain operation modes by different methods. EV- CD and EV-CS respectively denotes pure electric mode in CD and CS stage; SH-CD and SH-CS expresses serial mode in CD and CS stage; PH-CD means the parallel mode in CD stage. the parallel mode in CD stage. Fig. 8. The zoomed-in operation modes by DP and LMPC strategy in sudden a cceleration and deceleration conditions. Fig. 8. The zoomed-in operation modes by DP and LMPC strategy in sudden a cceleration and deceleration conditions. Fig. 9. The zoomed-in component performance by DP and LMPC based strate gy in sudden acceleration and deceleration conditions D Validation in Other PHEVs with Different Configurations Fig. 6. REFERENCES [1] Y. He et al., "Multiobjective Co-Optimization of Cooperative Adaptive Cruise Control and Energy Management Strategy for PHEVs," IEEE Transactions on Transportation Electrification, vol. 6, no. 1, pp. 346-355, Mar 2020. [2] Y. Zhang et al., "Energy management strategy for plug-in hybrid electric vehicle integrated with vehicle-environment cooperation control," Energy, vol. 197, Apr 15 2020, Art. no. 117192. [3] X. Zeng and J. Wang, "Optimizing the Energy Management Strategy for Plug-In Hybrid Electric Vehicles With Multiple Frequent Routes," IEEE Transactions on Control Systems Technology, vol. 27, no. 1, pp. 394-400, Jan 2019. g TABLE VIII Energy Consumption by Different Strategies in PHEVs with Different Configurations TABLE VIII Energy Consumption by Different Strategies in PHEVs with Different Configurations Vehicle Type Control Strategy Average EFC (L/100km) Optimality (%) Serial PHEV CD-CS 18.02 81.63 LMPC-MTFA 15.38 95.52 DP 14.71 - Parallel PHEV CD-CS 3.54 82.29 LMPC-MTFA 3.04 95.73 DP 2.91 - Serial-Parallel P HEV CD-CS 3.07 82.13 LMPC-MTFA 2.63 95.79 DP 2.52 - 4WD PHEV CD-CS 4.51 87.42 LMPC-MTFA 4.13 95.65 DP 3.95 - [4] X. Li and A. Simos, "Torque-Leveling Threshold-Changing Rule-Based Control for Parallel Hybrid Electric Vehicles," IEEE Transactions on Vehicular Technology, Article vol. 68, no. 7, pp. 6509-6523, Jul 2019. [5] J. Li, Q. Zhou, H. Williams, and H. Xu, "Back-to-Back Competitive Learning Mechanism for Fuzzy Logic Based Supervisory Control System of Hybrid Electric Vehicles," IEEE Transactions on Industrial Electronics, vol. 67, no. 10, pp. 8900-8909, Oct. 2020. [6] J. Liu, Y. Chen, W. Li, F. Shang, and J. Zhan, "Hybrid-Trip-Model-Based Energy Management of a PHEV With Computation-Optimized Dynamic Programming," IEEE Transactions on Vehicular Technology, vol. 67, no. 1, pp. 338-353, Jan 2018. [7] S. Xie, S. Qi, and K. Lang, "A Data-Driven Power Management Strategy for Plug-In Hybrid Electric Vehicles Including Optimal Battery Depth of Discharging," IEEE Transactions on Industrial Informatics, vol. 16, no. 5, pp. 3387-3396, May 2020. Through the comparison from different perspectives, the raised reference-tracking based LMPC controller shows obvious advantages in energy management of PHEV, and leads to preferable control effect with fast real-time implementation capacity. The specific reference-tracking manner fastens the computation speed, and moreover the leaning based state predictor and multi-source fused reference estimator improve the control effect significantly. pp y [8] Q. Guo, Z. Zhao, P. Shen, X. Zhan, and J. Li, "Adaptive optimal control based on driving style recognition for plug-in hybrid electric vehicle," Energy, vol. 186, Nov 1 2019, Art. REFERENCES no. 115824. [9] Y. Zhang, L. Chu, Y. Ou, C. Guo, Y. Liu, and X. Tang, "A Cyber-Physical System-Based Velocity-Profile Prediction Method and Case Study of Application in Plug-In Hybrid Electric Vehicle," IEEE transactions on cybernetics, 2019-Aug-05 2019. [10] C. Liu and Y. L. Murphey, "Optimal Power Management Based on Q- Learning and Neuro-Dynamic Programming for Plug-in Hybrid Electric Vehicles," IEEE Transactions on Neural Networks and Learning Systems, vol. 31, no. 6, pp. 1942-1954, Jun 2020. C. Energy-Saving Mechanism of LMPC based Strategy Comparison of component performance by different methods. Figs. 7 to 9 illustrate the powertrain performance by DP and LMPC based strategy in a sudden acceleration and deceleration segment. As shown in Fig. 7, DP and the LMPC algorithm result in almost the same fuel consumption rate except in the initial and middle acceleration stages. Although ICE is started more frequently by DP than by novel LMPC, DP enables more efficient operation of ICE than the novel LMPC algorithm, due to the benefit from pre-knowledge of the whole driving cycle. The LMPC strategy, limited by the finite predicted horizon, can only perform local optimal control within a short range, and fails to discover the minimum energy consumption in the whole trip. The difference in operation modes, as shown in Figs. 8 and 9, supports the same conclusion. To be specific, DP prefers the serial assist mode, while the LMPC method urges to switch to the ICE direct drive mode. However, the LMPC strategy Figs. 7 to 9 illustrate the powertrain performance by DP and LMPC based strategy in a sudden acceleration and deceleration segment. As shown in Fig. 7, DP and the LMPC algorithm result in almost the same fuel consumption rate except in the initial and middle acceleration stages. Although ICE is started more frequently by DP than by novel LMPC, DP enables more efficient operation of ICE than the novel LMPC algorithm, due to the benefit from pre-knowledge of the whole driving cycle. The LMPC strategy, limited by the finite predicted horizon, can only perform local optimal control within a short range, and fails to discover the minimum energy consumption in the whole trip. The difference in operation modes, as shown in Figs. 8 and 9, supports the same conclusion. To be specific, DP prefers the serial assist mode, while the LMPC method urges to switch to the ICE direct drive mode. However, the LMPC strategy Fig. 9. The zoomed-in component performance by DP and LMPC based strate gy in sudden acceleration and deceleration conditions D. Validation in Other PHEVs with Different Configurations Even though the proposed LMPC strategy is developed for a serial-parallel PHEV, the feasibility of transplanting the raised method to other PHEVs with different configurations is also investigated in this study. The energy consumption by the LMPC with the MTFA based reference velocity estimation in different PHEVs is listed in Table VIII. C. Energy-Saving Mechanism of LMPC based Strategy In the comparison study, the selected serial PHEV, parallel PHEV, and 4WD PHEV are simulated and controlled by the designed algorithm. The detailed description on the configurations and component parameters can be found in our previous research [44-46]. In Table VIII, average EFC denotes the average equivalent fuel consumption of simulation on NEDC, UDDS, US06, HWFER, and JC 08 driving cycles. The average EFCs by the proposed LMPC based strategy is compared with those by the CD-CS scheme and DP. As can be found, the LMPC strategy presents stable performance in different PHEVs, showcasing its superior application capability and robustness. The maximum optimality resulted by the strategy reaches 95.79% of that by DP, and leads to significant promotion in all PHEVs, compared with the simple CD-CS algorithm. different PHEVs is listed in Table VIII. In the comparison study, the selected serial PHEV, parallel PHEV, and 4WD PHEV are simulated and controlled by the designed algorithm. The detailed description on the configurations and component parameters can be found in our previous research [44-46]. In Table VIII, average EFC denotes the average equivalent fuel consumption of simulation on NEDC, UDDS, US06, HWFER, and JC 08 driving cycles. The average EFCs by the proposed LMPC based strategy is compared with those by the CD-CS scheme and DP. As can be found, the LMPC strategy presents stable performance in different PHEVs, showcasing its superior application capability and robustness. The maximum optimality resulted by the strategy reaches 95.79% of that by DP, and leads to significant promotion in all PHEVs, compared with the simple CD-CS algorithm. As the future driving status tends to be easily affected by multiple factors from driving behaviors and environment, more effort will be devoted to velocity prediction in our future work. In addition, intelligent methods will be explored to incorporate more information from multi-sources for prediction precision promotion of future driving velocity. V. CONCLUSION [11] C. Song, H. Lee, K. Kim, and S. W. Cha, "A Power Management Strategy for Parallel PHEV Using Deep Q-Networks," in 2018 IEEE Vehicle Power and Propulsion Conference, 2018. In this paper, a novel learning based control strategy is presented for PHEVs. The reference-tracking based MPC with leaning ability is preferred in the control strategy, possessing qualified capacity in real-time application. To promote the control effect, the Gaussian process model is integrated into the inner state predictor of MPC to mitigate the adverse effect on state prediction. The reference velocity in the leaning based MPC strategy is estimated by MTFA. The particular velocity estimation method can reasonably analyze the behaviors of future driving without much cost in calculation. The simulation evaluation demonstrates that the proposed leaning based control strategy is well-suited for the application in engineering practice with near-optimal effect. Compared with the rule based control strategy, the raised method can improve the fuel economy by as high as 15.98%, and can achieve 96.21% fuel economy of that by DP. The algorithm is also validated effective in different types of PHEVs, manifesting its application capability and robustness. [12] S. Xie, X. Hu, Z. Xin, and L. Li, "Time-Efficient Stochastic Model Predictive Energy Management for a Plug-In Hybrid Electric Bus With an Adaptive Reference State-of-Charge Advisory," IEEE Transactions on Vehicular Technology, vol. 67, no. 7, pp. 5671-5682, Jul 2018. [13] H. Lim and W. Su, "Hierarchical Energy Management for Power-Split Plug-In HEVs Using Distance-Based Optimized Speed and SOC Profiles," IEEE Transactions on Vehicular Technology, vol. 67, no. 10, pp. 9312-9323, Oct 2018. [14] C. Sun, X. S. Hu, S. J. Moura, and F. C. Sun, "Velocity Predictors for Predictive Energy Management in Hybrid Electric Vehicles," IEEE Transactions on Control Systems Technology, Article vol. 23, no. 3, pp. 1197-1204, May 2015. [15] C. Yang, S. You, W. Wang, L. Li, and C. Xiang, "A Stochastic Predictive Energy Management Strategy for Plug-in Hybrid Electric Vehicles Based on Fast Rolling Optimization," IEEE Transactions on Industrial Electronics, vol. 67, no. 11, pp. 9659-9670, Nov 2020. [16] J. Q. Guo, H. W. He, J. K. Peng, and N. N. Zhou, "A novel MPC-based adaptive energy management strategy in plug-in hybrid electric vehicles," Energy, Article vol. 175, pp. 378-392, May 2019. [17] X. Wang, L. Li, and C. V. 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Springer, Cham, 2017. 45-69. [19] Zhao, Dezong, Richard Stobart, and Byron Mason. "Real-time energy management of the electric turbocharger based on explicit model predictive control." IEEE Transactions on Industrial Electronics 67.4 (2019): 3126-3137. [43] Mirjalili, Seyedali, and Andrew Lewis. "The whale optimization algorithm." Advances in engineering software 95 (2016): 51-67. [44] Zhang, Yuanjian, et al. Optimal Control Strategy for the Next Generation Range Extended Electric Bus. No. 2020-01-0844. SAE Technical Paper, 2020. [20] Zarkadis, Kinstantinos. Model predictive torque vectoring control with active trail-braking for electric vehicles. Diss. 2018. [45] Zhang, Yuanjian, et al. "Energy management strategy for plug-in hybrid electric vehicle integrated with vehicle-environment cooperation control." Energy 197 (2020): 117192. [21] Siampis, Efstathios, Efstathios Velenis, and Stefano Longo. "Rear wheel torque vectoring model predictive control with velocity regulation for electric vehicles." Vehicle System Dynamics 53.11 (2015): 1555-1579. [46] Zhang, Yuanjian, et al. "A novel optimal power management strategy for plug-in hybrid electric vehicle with improved adaptability to traffic conditions." Journal of Power Sources 489 (2021): 229512. [22] Moriyasu, Ryuta, et al. "Diesel engine air path control based on neural approximation of nonlinear mpc." Control Engineering Practice 91 (2019): 104114. [23] Di Cairano, Stefano, et al. "Model predictive idle speed control: Design, analysis, and experimental evaluation." IEEE Transactions on Control Systems Technology 20.1 (2011): 84-97. [24] Zhang, Yuanjian, et al. "A hierarchical energy management strategy based on model predictive control for plug-in hybrid electric vehicles." IEEE Access 7 (2019): 81612-81629. [25] Manzano, José María, et al. "Robust learning-based MPC for nonlinear constrained systems." Automatica 117 (2020): 108948. [26] C. E. V. CONCLUSION Rasmussen, "Gaussian processes in machine learning," in Advanced Lectures on Machine Learning, vol. 3176, O. Bousquet, U. VonLuxburg, and G. Ratsch, Eds. Berlin: Springer-Verlag Berlin, 2004, pp. 63-71. [27] Zhang, Kunwu, Qi Sun, and Yang Shi. "Trajectory Tracking Control of Autonomous Ground Vehicles Using Adaptive Learning MPC." IEEE Transactions on Neural Networks and Learning Systems (2021). [28] C. K. I. Williams and C. E. Rasmussen, "Gaussian processes for regression," in Advances in Neural Information Processing Systems 8: Proceedings of the 1995 Conference, vol. 8, D. S. Touretzky, M. C. Mozer, and M. E. Hasselmo, Eds. Cambridge: M I T Press, 1996, pp. 514-520. [29] M. Vajedi and N. L. Azad, "Ecological Adaptive Cruise Controller for Plug-In Hybrid Electric Vehicles Using Nonlinear Model Predictive Control," IEEE Transactions on Intelligent Transportation Systems, Article vol. 17, no. 1, pp. 113-122, Jan 2016. pp [30] D. Duvenaud, H. Nickisch, and C. E. Rasmussen, "Additive Gaussian Processes," in neural information processing systems, 2011, pp. 226-234. [31] S. B. Xie, X. S. Hu, Z. K. Xin, and J. Brighton, "Pontryagin's Minimum Principle based model predictive control of energy management for a plug-in hybrid electric bus," Applied Energy, Article vol. 236, pp. 893- 905, Feb 2019. [32] Kessels, Femke, Kessels, and Rauscher. Traffic flow modelling. Springer International Publishing, 2019. [33] Zhang, Zhen, et al. "Continuous monitoring of residual torque of loose bolt in a bolted joint." Procedia Engineering 188 (2017): 278-285. [34] D. Yuan, X. Lu, D. Li, Y. Liang, and X. Zhang, "Particle filter re-detection for visual tracking via correlation filters," Multimedia Tools and Applications, vol. 78, no. 11, pp. 14277-14301, Jun 2019. [35] W. Li, Z. Wang, Q. Liu, and L. Guo, "An information aware event- triggered scheme for particle filter based remote state estimation," Automatica, vol. 103, pp. 151-158, May 2019. [36] F. Lyu et al., "Intelligent Context-Aware Communication Paradigm Design for IoVs Based on Data Analytics," IEEE Network, vol. 32, no. 6, pp. 74-82, Nov-Dec 2018. [37] M. Haklay and P. Weber, "OpenStreetMap: User-Generated Street Maps," IEEE Pervasive Computing, vol. 7, no. 4, pp. 12-18, Oct-Dec 2008. [38] X. Ding et al., "Analytical and Experimental Evaluation of SiC-Inverter Nonlinearities for Traction Drives Used in Electric Vehicles," IEEE Transactions on Vehicular Technology, vol. 67, no. 1, pp. 146-159, Jan 2018. [39] Memarzadeh, Gholamreza, and Farshid Keynia. V. CONCLUSION "A new short-term wind speed forecasting method based on fine-tuned LSTM neural network and optimal input sets." Energy Conversion and Management 213 (2020): 112824. View publication stats View publication stats
W4388288028.txt	https://akjournals.com/downloadpdf/journals/032/73/S1/article-p119.pdf	en		The connection between growth imperative of monetary production economies, the realization problem and Kornai's surplus economy	Acta oeconomica	2,023	cc-by	4,129	Acta Oeconomica 73 (2023) S1, 119–127 DOI: 10.1556/032.2023.00037 The connection between growth imperative of monetary production economies, the realization problem and Kornai’s surplus economy ZSOLT GILÁNYIp Department of Economics, Budapest University of Technology and Economics, Magyar tudósok körútja 2, Budapest, H-1117 Hungary Received: May 16, 2023 • Revised manuscript received: August 13, 2023 • Accepted: August 26, 2023 © 2023 The Author(s) ABSTRACT Kornai challenged not only the dominant economic views of the socialist system, but also those of market economies. The former brought him fame, and the latter remained, so to speak, his scientific testament. He studied the systemic properties of different economic systems through the analytical grid of the sign of aggregate excess supply, which defined three categories: shortage economies, equilibrium economies and surplus economies. In this paper, we show that business plans postulated with the aim of realizing strictly positive net retained profits in nominal terms exclude equilibrium economies; these business plans imply a surplus economy. This definition of the business plan makes it possible to combine seemingly disparate results from different parts of the economic literature. An economy in which business plans are the rule and no economic agent can run a permanent negative budget is a surplus economy, which manifests itself in the phenomena of both growth imperative and realization problem. In short, all these phenomena are the manifestations of the same essence: the working of the business plans. KEYWORDS realization problem, nominal growth imperative, surplus economy, monetary production economy, business plan, Kornai JEL CLASSIFICATION INDICES E12, O42 p Corresponding author. E-mail: gilanyi.zsolt@gtk.bme.hu Unauthenticated \| Downloaded 05/21/24 01:56 AM UTC 120 Acta Oeconomica 73 (2023) S1, 119–127 1. INTRODUCTION The issue of sustainability has recently reignited the debate on growth, with the pivotal question being whether growth is an intrinsic feature of market economies. If growth is not an inherent systemic attribute of market economies, then both growth and a zero-growth equilibrium are possible states. Nevertheless, if growth is an inherent characteristic of market economies, then a zero-growth equilibrium is not a possible state of this economic system. The alternative of growth is a situation where at least one economic agent necessarily cannot realize their plans. Rosier (1991) calls this situation a crisis. Economic literature uses the term ‘growth imperative’ to refer to this inherent need for growth where the alternative to growth is crisis (as understood by Rosier). Another way of expressing the same idea is that in a zero-growth steady state there is at least one economic agent who necessarily cannot realize his plans: there is a realization problem of business plans1 (Smithin 2016). From the point of view of pure theory, the existence of the growth imperative implies that the models that incorporate the possibility of a zero-growth equilibrium are inadequate to represent market economies, which means goodbye to the mainstream macroeconomics. Kornai, the eternal rebel, always went against the prevailing views, both in socialism and in capitalism: “Just as the shortage economy is a characteristic attribute of the socialist system, so is the surplus economy of the capitalist system.” (2013: 51) “…What I am proposing is backed by a million facts of everyday life. It is almost as an extra that I try to justify them logically as well.” (op. cit.: 119) Few economists share Kornai’s views. Neither empirical facts nor logical arguments convince the profession. As to the empirical facts, “though the phenomena before their eyes and mine are identical, we read different things into them because our outlooks differ.” (op. cit.: 52). Kornai’s extra logical justiﬁcation is centred around the concept of competition: “It can easily be discerned logically that where there is no surplus there is no competition among sellers— the drive deriving from the presence of a saleable surplus is absent. Why should the vehicle industry or the telephone service under socialism bother itself with innovation if there are waiting lists for their obsolete models or for telephone lines?” (op. cit.: 126) Kornai had never given up his ambition to convince the profession that “a surplus economy is an immanent attribute of capitalism” (op. cit.: 118) as his last English-language book Dynamism, Rivalry, and the Surplus Economy showed. In this paper, we prove that the phenomena of the growth imperative, the realization problem and the surplus economy are the manifestations of the same essence. This essence is the working of business plans defined with the goal of realizing strictly positive net retained profits in nominal terms. In short, an economy in which business plans are the rule and no economic agent can run a permanent negative budget balance is a surplus economy, which manifests itself in the phenomena of the growth imperative and the realization problem. This limited aim clearly short-circuits any debate on the connection between real capitalist systems and theoretical models of surplus economies, that Kornai couldn’t pass by without saying: “The war economy proves that the “capitalist system → surplus economy” causal connection cannot be applied mechanically or ubiquitously. War temporarily brings conditions that 1 Smithin (2016) also refers to the realization problem as the widget problem. Unauthenticated \| Downloaded 05/21/24 01:56 AM UTC 121 Acta Oeconomica 73 (2023) S1, 119–127 restrict or stop the operation of the mechanism that generates the surplus economy and starts the mechanism that generates the shortage economy” (op. cit.: 150) It also short-circuits the problem of identifying the conditions that ensure the operation of the mechanism that the business plans are the rule – a major concern of Kornai. The method used in this paper is the opposite of that used by Kornai in Dynamism, Rivalry and the Surplus Economy. In this book he “… examines only … the real sphere of the economy. I do not deal in detail with the monetary sphere …” (op. cit.: 55) Instead, we focus exclusively on money ﬂows. This allows us to avoid many of the difﬁculties raised by Kornai; in particular, we avoid the problems of identifying aggregate excess supply in real terms. As a result, despite Kornai’s warnings, the notion of an excess supply economy seems to be a valid notion to represent the concept of a surplus economy. 2. LOCATING THE GROWTH IMPERATIVE AND THE REALIZATION PROBLEM IN AN ACCOUNTING FRAMEWORK To situate the concept of growth imperative in its most general form, let us consider a closed economy and start from the banal identity drawn from national accounting, which states that in any period t, gross domestic product Yt can be split up into consumption Ct plus gross investment It, where the bold typing refers to aggregates: Y t ¼ Ct þ I t (1) If net aggregate investments in period t are strictly positive, gross investments are: I t >At (2) where At is the depreciation in period t in the economy. As depreciation is the result of past investments, equation (2) holds for all periods if and only if gross investment is increasing. As the lower bound on consumption is zero, strictly positive net investment implies a tendency for gross domestic product to grow. In fact, gross domestic product Y may temporally decrease because a drop in consumption C may counterbalance the increase of gross investments I, but this drop cannot perpetuate because consumption cannot fall below the lower bound zero. In the aggregate, investments are always equal to savings; hence investments net of depreciation INt in period t always equal net savings: I Nt ¼ SNt (3) N All put together, if aggregate net saving in period t, S t, is strictly positive, aggregate net investment, INt, is also strictly positive. Hence, aggregate gross investment is increasing and by force of identity (1), gross domestic product abides the same property. No growth is equivalent with zero aggregate net saving, SNt 5 0; growth means strictly positive aggregate net saving SNt >0. Thus, a necessary and sufficient condition for growth in nominal or real terms is the existence of strictly positive aggregate net savings in nominal or in real terms regardless of the specific model with or without phony mathematics. The specific models are required to make the link between nominal and real growth. To avoid digressions, let us comply with the custom Unauthenticated \| Downloaded 05/21/24 01:56 AM UTC 122 Acta Oeconomica 73 (2023) S1, 119–127 by considering the exclusively proportional steady states (i.e.: cyclical paths excluded) and by supposing that none of the economic agents can run constant negative budget. These hypotheses make the link between strictly positive net retained earnings of one single economic agent and strictly positive aggregate net savings. This said, all models of the growth imperative must come to the same conclusion in one way or another: if aggregate net saving is strictly positive, it is possible to make plans that are consistent for all the economic agents i.e., all agents are in principle able to realize their plans. In the opposite case when aggregate net saving is not strictly positive, there are no plans that are compatible for all the economic agents i.e., there is necessarily at least one economic agent who is unable to realize his plan. To put it another way, in a non-growing state, there is a realization problem of business plans. To sum up, if none of the economic agents can run a permanently negative budget, then business plans defined with the goal of realizing strictly positive net retained earnings in nominal terms imply a nominal growth imperative for the economy. This means that in a nongrowing state, there is a realization problem of the business plans. 3. LOCATING KORNAI’S SURPLUS ECONOMY IN TERMS OF THE COMMON NOTION OF AGGREGATE EXCESS SUPPLY Kornai intentionally avoids using the term ‘excess supply system’ as he believes that, upon closer analysis, the concept of aggregate excess supply is void of real meaning: “I could list more statistical and observation difﬁculties and anomalies, but these objections may sufﬁce to explain why I prefer the “shortage economy – surplus economy” pair of concepts for describing the general market state of a particular country, and shrink from using the “excess-demand economy– excess-supply economy” pair of concepts that many ﬁnd easier to grasp.” (op. cit.: 85) Nevertheless, we will deliberately use the pair “excess demand economy – excess supply economy”, not only because “many ﬁnd it easier to grasp”, but also because we believe we can avoid Kornai’s legitimate concerns. To do this, we give the following deﬁnition of the excess supply economy: an excess supply economy is one in which the total aggregate excess supply2 in nominal terms is always positive. Aggregate supply in nominal terms is the value of planned nominal sales; aggregate demand in nominal terms is the value of planned nominal purchases. In other words, positive aggregate excess supply is simply the intention to realize more income than expenditures in the aggregate. Leaving aside the problems of observing ex ante plans, the aggregation of income plans (supply) and expenditure plans (demand) in monetary terms escapes the criticism of being as meaningless as the aggregation of supply and demand is in real terms. Clearly, in an excess supply economy, the total nominal aggregate excess supply can be neither zero, nor negative. By comparison, in an equilibrium economy, total nominal aggregate excess supply may always be zero, even if, for example, a positive sign is observed in a particular state. Also, zero total nominal aggregate excess supply does not mean that there is an It will soon become clear that “total” is not a redundancy in the deﬁnition. 2 Unauthenticated \| Downloaded 05/21/24 01:56 AM UTC 123 Acta Oeconomica 73 (2023) S1, 119–127 equilibrium on each market. For economists accustomed to the mainstream model settings, the definition of an excess supply economy does not say that there can be no equilibrium or even excess demand in any given period in a multi-period model; it says that there can never be full equilibrium. Having said that, Kornai’s message, as we understand it, is precisely that not just any economic system can be in a situation of equilibrium, total surplus or total shortage depending on the business cycle and other factors. A socialist system is always in a situation of total excess demand, just as a capitalist system is always in a situation of total excess supply in nominal terms,3 even if in a particular period one observes the opposite. According to the custom, the term “aggregate excess supply” refers to an aggregation in a single period. The term “total” refers to an aggregation of all plans in execution; thus, the aggregation implies the current and future periods related to the business plans.4 Fortunately, the problems of such aggregation can be avoided in a monetary economy if we wish only to determine the possible sign of the total aggregate excess supply in nominal terms. In this case, the aggregation is simply unnecessary. This is because the possible upper and lower bounds on money flows determine the possible sign of this nominal excess supply. So, all we must do, is find those limits before the money flows. This is what we will do in the next section. 4. THE CONNECTION BETWEEN THE GROWTH IMPERATIVE, THE AGGREGATE EXCESS SUPPLY SYSTEM AND THE REALIZATION PROBLEM In the first section, we have seen that business plans defined with the goal of realizing profits do not imply a growth imperative, while business plans defined with the goal of realizing strictly positive net retained earnings in nominal terms do imply a nominal growth imperative and a realization problem of the business plans. This latter understanding of business plans can be represented in Marx’s scheme as: M-(C-C0 )-M0 , where M < M0 5 and all variables refer to the same period. In the monetary circuit, these business plans are puzzling in the aggregate since it is impossible to withdraw M0 money from circulation (supply) during the model period t if only M money is put into circulation (demand) during the same period. Therefore, the additional (M0 -M) money must also be put into circulation. Speciﬁcally, this extra money cannot be put into circulation by entrepreneurs for the purpose of consumption, because fully distributed proﬁt is a proﬁt “literally drunk” (Smithin 2016: 4). In other words, M0 -(C-C00 )-M0 is not a business plan.6 3 For us, the war economy is not a capitalist economy, because the premise that business plans are the rule does not hold. 4 For example, a dealer buys two cars for $2000 to sell them at a proﬁt. If he sells them minimum at least at a proﬁt as planned, his business plan is realized, regardless of the number of cars sold. If this dealer only intends to buy the two cars, but has not yet done so, then his plan is not in progress. In short, there is no business plan in progress until there is an initial expenditure to make a proﬁt, and business plans are in progress until there is an intention to make a proﬁt on the initial expenditure. 5 0 C is another commodity than C, as well as M0 is different amount of money than M. 6 00 C includes the consumption of the entrepreneur. In the Kaleckian understanding of the entrepreneur, this consumption is directly the consumption of the entrepreneur. In the modern term, when the Kaleckian entrepreneur is divided into two analytical economic agents, the one who seeks to realize business plans (called entrepreneur) and the one who seeks consumption (called consumer or household), this consumption appears as distribution of dividends to households and household consumption. Unauthenticated \| Downloaded 05/21/24 01:56 AM UTC 124 Acta Oeconomica 73 (2023) S1, 119–127 Thus, the additional (M0 -M) money is put into circulation to realize business plans. If this is the case, the plans that started with an outﬂow of M money in period t can now be completed with a backﬂow of M0 money in the same period t. But the business plans that started with an outﬂow of (M0 -M) money in period t, are not and cannot be completed in period t. For the business plans that started with (M0 -M) outﬂows to possibly be realized, new business plans must be started in subsequent periods and so on. The possibility of perpetual realization of business plans is a Ponzi game, i.e., always new business plans must be started in subsequent periods. That is, there is never enough money in circulation in any period to complete all the ongoing business plans deﬁned with the goal of realizing strictly positive net retained earnings in nominal terms if none of the economic agents can run a permanently negative budget. This means that total supply (planned current and future income aggregated in nominal terms) is always greater than total demand (possible current and future expenditures aggregated in nominal terms). In short, the realization problem of business plans is another expression to say excess supply economy, which manifests itself in the nominal growth imperative. Economists immersed in stock-flow consistent modelling who have quickly skimmed the above argument may miss the point and object that there is not necessarily “missing money” in the system, since the net retained earnings do not necessarily imply the accumulation of money; rather, they generally imply the accumulation of other assets - ﬁnancial or real - paid for with money. Therefore, the “missing money” ﬂows back into the economy. These economists are right that money is generally not accumulated but spent. However, at least one dollar of every retained proﬁt is necessarily spent with the aim of making net proﬁts, otherwise the business plan ceases to be a business plan. If this is the case, any net proﬁt will generate expenses that will be part of new business plans. Net retained earnings are money earned from completed business plans; part of the accumulated assets - real7 or ﬁnancial - ﬁnanced from net retained earnings are necessarily part of new business plans to be realized in subsequent periods. The “missing money” is available precisely because not all business plans have been completed, but new plans are being implemented. This dynamism makes the static setting inaccurate to represent excess supply economies. Kornai warns that this dynamism is a primordial feature as the first word of his last book shows. 5. CONCLUDING REMARKS Kornai challenged not only the mainstream economic views in the socialist system, but also those prevailing in the market economies. The first brought him fame, and the second remained, so to speak, his scientific testament. He studied the systemic properties of the different economic systems. His analytical grid was to classify the economic systems according to the systemic property of the sign of the aggregate excess supply, which defined three categories: excess demand economies, equilibrium economies and excess supply economies: 7 The purchase of real assets as part of a business plan is called investment. Unauthenticated \| Downloaded 05/21/24 01:56 AM UTC 125 Acta Oeconomica 73 (2023) S1, 119–127 Classiﬁcation of economies in Kornai’s own modelling framework Sign of the excess supply Negative Zero Positive Systemic property Shortage economy/Excess demand economy Core Discussion Soft budget constraints Kornai (1980): Economics of Shortage Kornai (1992): The socialist system Equilibrium economy Criticism of proﬁt maximization Kornai (1971): Anti-equilibrium Surplus economy/Excess supply economy Competition Kornai (2013): Dynamism, rivalry, and the surplus economy In his monumental books Kornai (1980, 1992) showed that socialist production plans imply shortage economies. Mainstream economics showed that in the market economies, business plans postulated with the aim of realizing proﬁts imply the property of an equilibrium system. In the Anti-Equilibrium, Kornai has argued that the proﬁt maximization rule has more drawbacks than advantages for economic theorizing and that the mainstream view is untenable. Later, he went further and shortly before his death he summarized his arguments in Dynamism, Rivalry, and the Surplus Economy, for the market economy as a surplus economy. He refrained from using the pair excess supply economy – excess demand economy, because he maintained that the aggregation of ex-ante plans in real terms is void of real meaning. We have argued that in the context of an excess supply – excess demand economy, when only the possible sign of the total aggregate excess supply in nominal terms is at issue, the concerns raised by Kornai can be avoided. Namely, the lower bound of this total aggregate excess supply in nominal terms is the difference between the lower bound of total aggregate supply in nominal terms and the upper bound of total aggregate demand in nominal terms. The ﬁrst item is simply the planned total income – present and future – in nominal terms; the second is the possible total expenditures. We have defined business plans not with the goal of profit maximization, but with the net retained profit rule, which implies that business plans always aim to realize more income than expenses in nominal terms. Consequently, this difference between planned income and expenses is necessarily always strictly positive for an aggregation of all ongoing business plans. Thus, a surplus economy is an economy in which there is a realization problem of business plans in the sense that new business plans must always be started to allow the completion of ongoing business plans. That is, there is a need for growth in nominal terms. These phenomena are absent in the equilibrium economies. Therefore, the static setting is inappropriate to fully understand the functioning of the surplus economies: “Dynamism” is a primordial element of the analytical framework. Unauthenticated \| Downloaded 05/21/24 01:56 AM UTC 126 Acta Oeconomica 73 (2023) S1, 119–127 We cannot neglect the usual question of what is new in this work. If Kornai was so modest as to say in Dynamism, Rivalry, and the Surplus Economy that “There is hardly a sentence in the essay that has no antecedent in the literature.” (2013: 54), then we are really in a difﬁcult situation. Our core hypothesis, which implies a surplus economy with the realization problem and the property of the growth imperative, goes back at least to Aristotle’s concept of chrematistics; the realization problem is already a central problem for Marx (1867), although for him it is a problem of the realization of intrinsic value. In recent literature, as we have already referred to, Smithin (2016) discusses it in detail. The growth imperative has a relatively recent but rich literature related to the over-indebtedness (e.g., Rosier 1991, 1992) on the one hand and sustainability (e.g., Binswanger H. C. 2006; Binswanger, M. 2009; Gilányi 2015; Richters – Siemoneit 2017) on the other. The restriction of the analysis of excess supply economies to monetary production economies allowed us to define the excess supply exclusively in nominal terms. This restriction made it possible to use the post-Keynesian tools (the logic of the monetary circuit and accounting) and to combine the seemingly disparate results from different areas of the economic literature. To our knowledge, these concepts, namely surplus/excess supply economy – nominal growth imperative – realization problem have never been linked. ACKNOWLEDGEMENT The author is grateful for comments on an earlier version of this paper presented at the Kornai 95 Conference, Corvinus University of Budapest, May 2023. REFERENCES Binswanger, H. C. (2006): Die Wachstumspirale. Marburg: Metropolis Verlag. Binswanger, M. (2009): Is there a Growth Imperative in Capitalist Economies? A Circular Flow Perspective. Journal of Post Keynesian Economics, 31(4): 707–727. Gilányi, Zs. (2015): A Brief Note on Mathias Binswanger’s Model. Journal of Post Keynesian Economics, 37(4): 590–596. Kornai, J. (1971): Anti-Equilibrium. Budapest: Közgazdasági és Jogi Könyvkiadó. Kornai, J. (1980): A hiány (The Shortage). Budapest: Közgazdasági és Jogi Könyvkiadó. Kornai, J. (1992): The Socialist System: The Political Economy of Communism. Princeton University Press. Kornai, J. (2013): Dynamism, Rivalry, and the Surplus Economy. Oxford University Press. Marx, K. (1867): Das Kapital. Kritik der politischen Oekonomie. Hamburg: Otto Meissner. Richters, O. – Siemoneit, A. (2017): Consistency and Stability Analysis of Models of a Monetary Growth Imperative. Ecological Economics, 136: 114–125. Rosier, M. (1991): Eléments d’une approche théorique et comptable du phénomène d’endettement (Elements of a Theoretical and Accounting Approach to the Phenomenon of Indebtedness). Cahier monnaie et ﬁnancement, la monnaie entre formel et informel, 20: 176–185. Unauthenticated \| Downloaded 05/21/24 01:56 AM UTC Acta Oeconomica 73 (2023) S1, 119–127 127 Rosier, M. (1992): Des causes et des remèdes aux surendettements nationaux et internationaux (Causes and Remedies for National and International Over-Indebtedness). Université de Paris, Cahier du Gratice: 9202: 85–114. Smithin, J. (2016): Some Puzzles About Money, Finance and the Monetary Circuit. Cambridge Journal of Economics, 40(5): 1259–1274. Open Access statement. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the CC License is provided, and changes – if any – are indicated. (SID_1) Unauthenticated \| Downloaded 05/21/24 01:56 AM UTC
https://openalex.org/W4379374819	https://zenodo.org/records/8004038/files/LESLLASP_2009_VinogradovFinal.pdf	English	null	Balancing Top and Bottom: Learner-Generated Texts for Teaching Phonics	Zenodo (CERN European Organization for Nuclear Research)	2,010	cc-by	5,473	Recommended citation of this article Recommended citation of this article Vinogradov, P. (2010). Balancing Top and Bottom: Learner-Generated Texts for Teaching Phonics. LESLLA Symposium Proceedings, 5(1), 3–14. https://doi.org/10.5281/zenodo.8004038 Vinogradov, P. (2010). Balancing Top and Bottom: Learner-Generated Texts for Teaching Phonics. LESLLA Symposium Proceedings, 5(1), 3–14. https://doi.org/10.5281/zenodo.8004038 Citation for LESLLA Symposium Proceedings This article is part of a collection of articles based on presentations from the 2009 Symposium held at Bow Valley College in Banff, Alberta, Canada. Please note that the year of publication is often different than the year the symposium was held. We recommend the following citation when referencing the edited collection. Wall, T., & Leong, M. (Eds.) (2010). Low-educated adult second language and literacy Acquisition (LESLLA): Proceedings of the 5th symposium. Bow Valley College. https://lesllasp.journals.publicknowledgeproject.org/index.php/lesllasp/issue/view/470 LESLLA Symposium Proceedings Recommended citation of this article Vinogradov, P. (2010). Balancing Top and Bottom: Learner-Generated Texts for Teaching Phonics. LESLLA Symposium Proceedings, 5(1), 3–14. https://doi.org/10.5281/zenodo.8004038 LESLLA Symposium Proceedings Recommended citation of this article Vinogradov, P. (2010). Balancing Top and Bottom: Learner-Generated Texts for Teaching Phonics. LESLLA Symposium Proceedings, 5(1), 3–14. https://doi.org/10.5281/zenodo.8004038 About the Organization LESLLA aims to support adults who are learning to read and write for the first time in their lives in a new language. We promote, on a worldwide, multidisciplinary basis, the sharing of research findings, effective pedagogical practices, and information on policy. LESLLA Symposium Proceedings LESLLA Symposium Proceedings https://lesllasp.journals.publicknowledgeproject.org Website https://www.leslla.org/ https://lesllasp.journals.publicknowledgeproject.org Website https://www.leslla.org/ https://www.leslla.org/ 3 1 “LESLLA learners” refers to adult language learners with little or no literacy in their first languages. The acronym is derived from the LESLLA: Low-Educated Second Language and Literacy Acquisition, www.leslla.org, which is specifically focused on such learners. 1 Introduction Adult learners learn best when instruction is consistently grounded in meaningful, relevant contexts and is closely connected to learners‟ daily lives and needs (Auerbach, 1992; Imel, 1998; Knowles, 1973). LESLLA learners1 are no exception. At the same time, reading research places a high value on explicit phonics instruction for emergent readers: the direct teaching of “alphabetic skills” such as phonological awareness, decoding, and encoding. While these two needs may appear to pull instructors in different directions, this report offers practical options for balanced instruction that addresses both priorities. After briefly exploring the tenets of balanced literacy and the value of phonics instruction for adult emergent readers, classroom practices are illustrated that use learner-generated texts as a basis for developing alphabetic print literacy. Patsy Vinogradov, University of Minnesota Patsy Vinogradov, University of Minnesota 2 Balanced literacy: Weaving top and bottom The “whole language vs. phonics great debate” among reading experts is largely a thing of the past (see Chall, 1967). Reading is viewed as an interactive, meaning- making endeavor that includes both top-down and bottom-up processes (Birch, 2007; Campbell, 2004). While adult language classrooms vary greatly, generally instructors include both bottom-up and top-down reading instruction in their classrooms, as each approach develops different skills that strong readers need (Campbell 2004; Parrish, 2004; Vinogradov, 2008). LESLLA learners, as adult emergent readers new to alphabetic print literacy, need lessons that both focus on meaning and also bring attention to the building blocks of literacy. Effective LESLLA literacy lessons are balanced: grounded in interesting, relevant contexts that emphasize meaning, while also explicitly teaching patterns of sounds, syllables, and word families (Fish, Knell & Buchanan, 2007; Vinogradov, 2008). As Michael Pressley writes, “balanced-literacy teachers combine the strengths of whole language and skills instruction, and in doing so, create instruction that is more than the sum of its parts” (1998, p. 1). One method that integrates such explicit phonics instruction into meaningful, theme-based lessons is termed Whole-Part-Whole (WPW). Here teachers begin with a topic that is interesting, important, and familiar to learners. They elicit words, phrases, and stories from students, and they strengthen their vocabularies surrounding the given topic. Then, once learners are engaged in the topic, they examine particular words to present and practice alphabetics (phonics and 4 Patsy Vinogradov Patsy Vinogradov phonemic awareness skills). Later, they return these words to the larger context to continue reading and oral language practice (Haverson & Haynes, 1982; Trupke- Bastidas & Poulos, 2007). Instead of presenting phonics in a decontextualized way with nonsense words and endless worksheets on word families, WPW strives to provide a balance: on one side of the coin is meaningful language, and on the other side are the building blocks that combine to create this language. When students are familiar with a given topic and have a bank of words, teachers can then spend time on sound-symbol correspondence, and learners can discover how letters and sounds are related (Brod, 1999, p.16). This creates a much needed connection between the larger topic at hand and the emergent reading activities. 3 Explicit phonics instruction Adults who lack print literacy generally also lack critical pre-literacy skills such as phonemic awareness and phonics skills (Gombert, 1994; Kurvers & van de Craats, 2007; Young-Scholten & Strom, 2006). The LESLLA research study by Young- Scholten and Strom shows that while all the study‟s participants “demonstrated solid knowledge of the alphabet in their ability to read letters in different fonts and out of order…many demonstrated no phonemic awareness and no decoding ability” (Young-Scholten & Strom, 2006, p. 63). Such findings indicate that teachers should include explicit instruction of phonemic awareness and decoding in their classrooms, since such skills do not develop with alphabet knowledge alone. Anecdotally, many of my own LESLLA learners have shown similar patterns; when shown a word, they may be able to name the letters, but they are often not able to assign sounds to the letters or to combine them to sound-out the word or to derive meaning from it. There is much more to reading than simply knowing the alphabet, a fact that decades of reading research have proven. LESLLA instructors struggle with exactly what should be taught and how. Can these literacy skills be taught to LESLLA learners? And how should precious instructional time be spent to develop such skills? While the research surrounding literacy instruction for low-literate adult second language learners is growing, it is still quite limited. The field of LESLLA can draw from the large reading research base done with children both in first and second language literacy and also from research on adult first language literacy development. A remarkable number of studies have been done that focus on reading development in children in the early elementary years. The National Reading Panel (2000) reviewed relevant research, and their conclusions support explicit and systematic literacy instruction. While these conclusions were based on research with native speaking children, another important research review by Kruidenier (2002) focused on adult literacy development in native speaking adults, and it also supports explicit and systematic instruction. A few small practitioner research projects have demonstrated the positive impact on reading ability when specific, concentrated phonics instruction is implemented into instruction of LESLLA learners (Evans, 2008; Trupke-Bastidas & Poulos, 2007). While further research is needed in this area, evidence points to the high value of such interventions. 5 Balancing top and bottom What exactly is meant by explicit and systematic? 3 Explicit phonics instruction Kruidenier‟s review states that such instruction should focus on both phonemic awareness, the ability to manipulate sounds within a spoken word, and phonics, the knowledge of sound- symbol relationships. Specifically, he suggests that teachers build phonemic awareness by (1) focusing on one or two types of phonemic awareness tasks at a time; (2) focusing on segmenting and blending, which may be most useful to learners; and (3) using letters as well as sounds for instruction (Kruidenier, 2002, p. 50). “Decoding,” or mastering how sounds relate to the alphabet, requires that learners 1) recognize letters, 2) identify and produce the sounds represented by the letters, 3) blend the individual sounds in sequence, and finally 4) recognize the word (Kruidenier, 2002). Research on the language and literacy development of children who are non-native speakers of English can also inform LESLLA practice. The American Education Research Association advocates explicit phonics as one of four critical components of reading instruction to help English language learners (ELLs) catch up to their native speaking peers (AERA, 2004, p.3). Interestingly, the other three recommendations include intensive vocabulary instruction, a focus on reading comprehension strategies, and oral language development. This package of critical components for reading constitutes a balanced literacy approach. Additionally, research studies focused on English language learning children were reviewed by the National Literacy Panel (Grant & Wong, 2003) and indicate a strong relationship between oral proficiency and literacy skills. The LESLLA research done by Tarone, Bigelow, and Hansen (2009) further explores this connection between oracy and literacy, and they too conclude that practitioners must connect oral language and the written word, and they must do so in a balanced, engaging, multi-faceted way. LESLLA instructors need a toolbox full of effective balanced literacy activities, and the use of learner-generated texts is one of these tools. 4 The value of learner-generated texts As stated at the beginning of this report, decades of adult learning research demonstrates the importance of meaningful, engaging lessons for adults (Knowles, 1973; Imel, 1998; Weinstein, 1999; Wrigley & Guth, 1992). Learners‟ lives must be central to instructional approaches and materials (Auerbach & Burgess, 1985; Lado, 1991; Williams & Chapman, 2007). As Gail Weinstein writes, “ESL and literacy classrooms can and should be settings where adults find opportunities to develop language and literacy skills while reflecting, as individuals and in collaboration, on their changing lives” (1999, p. 6). Learning should be contextualized, relevant, and lessons should draw upon the actual experiences and concerns of learners (Auerbach, 1992). Adult learners have pressing needs and interests in their communities, and they must manage a great deal of environmental print. And yet when teachers reach for reading development texts and materials to build literacy in their LESLLA learners, they may find very little that is fitting for adults, and even less that combines learners needs and interests with level-appropriate literacy work (see CAELA, 2009). Much of the emergent reading curricula available and created by teachers is childish in nature, and tends to replicate the literacy work done in early childhood classrooms and primary grades. While reading-level appropriate, 6 Patsy Vinogradov Patsy Vinogradov such material is not engaging for adult learners as it is neither age-appropriate nor relevant to the daily needs of LESLLA learners. Creating and capitalizing on learner-generated texts is one classroom practice that exemplifies balanced literacy instruction. Learner-generated texts immediately provide relevant, meaningful, level and age-appropriate reading material. In creating learner-generated texts, teachers tap into learners‟ often more developed listening and speaking skills to build literacy (Geva & Zadeh, 2006). Oral processing skills and print literacy skills are interconnected and interdependent (Tarone, Bigelow & Hansen, 2009), and as learners practice and build their oral abilities, learner- generated texts provide a means to connect these skills and to present oral language on paper. The result is an array of rich and interesting readings for students. The traditional method of producing learner-generated texts is the Language Experience Approach (LEA). In LEA, students first share a common experience, perhaps a field trip or an experience like making a sandwich in the classroom. Then, the teacher guides them to re-tell the experience aloud. Students recall what happened to a teacher or another scribe who writes down their words. 4 The value of learner-generated texts Later, these words are then used as reading texts. From here, a number of bottom-up focused techniques can be used to focus on word analysis and particular sounds and structures. Then, students revisit the entire text they have created, and perhaps add to it (see Whole Part Whole description above). LEA is an efficient technique in working with emergent readers as it connects what they are able to communicate orally to what they are learning to do in print (Crandall & Peyton, 1993). Figure 1: Ways to Generate Learner Texts • Shared experience • Students‟ newsletters • Picture stories • Responding to a photograph or visual • Transcribed taped conversations • Journal entries • Texts for wordless books • Photo books • Class posters • Overheard student stories (Liden, Poulos, & Vinogradov, 2008) Figure 1: Ways to Generate Learner Texts The Language Experience Approach is one way to create learner-generated texts, but texts can be created less formally as well (see Croydan, 2005). For example, a teacher and students can look at a photograph together and write about what they see and think. Journaling, free-writing, or a teacher simply transcribing a story a student shared during break-time all create learner-generated texts. Figure 1 above lists more ways to generate learner texts. Such stories provide abundant opportunities for looking at bottom-up strategies. The advantage of using learner- generated texts is that the text is already comprehensible, meaningful, and interesting to the learner. Since the learner created it, he/she has ownership over those words and that story. By using these texts within the Whole-Part-Whole The Language Experience Approach is one way to create learner-generated texts, but texts can be created less formally as well (see Croydan, 2005). For example, a teacher and students can look at a photograph together and write about what they see and think. Journaling, free-writing, or a teacher simply transcribing a story a student shared during break-time all create learner-generated texts. Figure 1 above lists more ways to generate learner texts. Such stories provide abundant opportunities for looking at bottom-up strategies. The advantage of using learner- generated texts is that the text is already comprehensible, meaningful, and interesting to the learner. Since the learner created it, he/she has ownership over those words and that story. 4 The value of learner-generated texts By using these texts within the Whole-Part-Whole 7 Balancing top and bottom method, teachers can then focus on particular sounds, word families, or other reading skills within content that the student created him/herself. This creates an engaging and memorable lesson for learners. 5 Examples of learner generated texts and phonics activities Mix up the words the next day, or after break, and have pairs or individuals repeat ordering the story. Order the Story (individually). Type up the story for the next day, and have students cut the story into sentences, words or phrases and re-order. Order the Story (individually). Type up the story for the next day, and have students cut the story into sentences, words or phrases and re-order. Word Recognition Tasks Circle the Word in the Text. Have students circle all the times a certain word is repeated in the story. For example, following “Hot Day,” students can circle “today” each time it appears, either on the board or on their own copy of the story. Repeat with the word “is.” Choose the Word You Hear. Create a worksheet that lists words from the story alongside other words. Students must circle the word that is called out. For example: Teacher: In number one, circle the word “hot.” (Learners choose from a worksheet; the options are hot/sunny/we). Teacher: Number two, circle the word “we.” (Learners choose from the words wear/we/water. Choose the Word You Hear. Create a worksheet that lists words from the story alongside other words. Students must circle the word that is called out. For example: p cher: In number one, circle the word “hot.” (Learners choose from a worksheet; the options are hot/sunny/we). Teacher: Number two, circle the word “we.” (Learners choose from the words wear/we/water. Flyswatter Game. Tape each card (with a word or phrase from the story) on the wall or board and have two learners sit in front of the board, each with a flyswatter. As the teacher or a fellow student calls out a word, the two learners race to hit it first. Nine-patch. Each student receives a piece of paper that has been marked to create 9 squares (3 X 3). Have small pieces of paper with words from the story ready for each student. As the teacher calls out a word from the story, the learner must find the slip of paper with that word and place on the grid. More advanced students can write the words on the grid. 1. summer 2. sunny 3. sweating 4. wear 5. we 6. water 7. today 8. is 9. are Same or Different? This activity helps build the „automaticity‟ that fluent readers use when reading. 5 Examples of learner generated texts and phonics activities Once a learner-generated text has been created, teachers can draw on the text in many ways to practice bottom-up reading skills. To explore some of these activities, we turn to a sample of LESLLA learner writing. This text (Figure 2 below) was generated in an adult English as a Second Language class for low-literate adults in Minnesota, USA. This particular class consists mostly of Southeast Asian learners, refugees from Burma, Laos, and Thailand. A summer project for this LESLLA class involved creating a community garden near the school. As a shared experience that allowed students to share their farming expertise, the gardening project provided a rich resource for language and balanced literacy lessons. As they worked in the garden throughout the summer months, their instructor elicited and helped learners write a number of stories. During classroom time, she capitalized on these stories to widen their vocabularies, build their conversation skills, and develop their emerging literacy skills. Figure 2: Hot Day Hot Day Today is summer. Today is sunny. Today is hot. Today we are sweating. Today drink water. Today wear t-shirt. Figure 2: Hot Day Once meaning is well established (“whole” in WPW method), the teacher can focus on pieces of language (“parts” in WPW). A number of active, engaging tasks can help students build word recognition and sound-symbol correspondence skills with this text. While by no means a comprehensive list, a sample of such tasks is listed below. Sequencing tasks q g Order the Story (with group). Write each word or phrase of the story on cards and place one full set of cards on each table. In small groups, learners need to re-create the story on their tables and practice reading it to each other. Stand Up and Sequence. Hand out a card with one word or phrase from the story to each student. Have students stand up and come to the front of the room to re-order the story, saying their words aloud as they read together. Pocket Chart. Write the story on index cards, one word on each card. Hand out the cards and have student re-create the story in order in the pocket 8 Patsy Vinogradov Patsy Vinogradov chart. Mix up the words the next day, or after break, and have pairs or individuals repeat ordering the story. chart. 5 Examples of learner generated texts and phonics activities Create a pile of paper strips with a line in the center, and write two words on each paper strip that differ only by one or two letters. Students turn over a strip very quickly, just for a moment, and they must quickly determine whether the two words are the same or different. Then turn the paper over to check. shirt skirt 9 Balancing top and bottom Phonemic Awareness Tasks Phonemic Awareness Tasks Sound Chain. Choose a word from the story and say it aloud. Students must listen for the final sound of that word and say a word that begins with that sound. Continue listening for the final sound and stating a word that uses that sound initially. For example: Teacher: Today. What‟s the last sound in “today?” Learners: /eI/. Teacher: Right! What‟s a word that starts with /eI/? Learners: Age. Teacher: Good! And what‟s the last sound in „age‟? Teacher: Good! And what‟s the last sound in „age‟? Learners: /dz/ Teacher: Yes. What‟s a word that starts with /dz/? Teacher: Yes. What‟s a word that starts with /dz/? Learners: Jump. Etc. Where‟s the Sound? Give each student or pair of students 3 paper cups, one for the first sound, one for a middle sound, and one for the final sound. Have student label the cups appropriately 1, 2, and 3. Say a word, and then ask students where one of the sounds in the word occurs. They must drop a bean or penny into the appropriate cup. For example, if the word is “hot,” and you ask, “Where is the /t/?” Students drop a bean into the final cup, or number 3. You can then ask, “Where is the /h/ sound?” and students will drop a bean into the first cup. Where‟s the Sound? Give each student or pair of students 3 paper cups, one for the first sound, one for a middle sound, and one for the final sound. Have student label the cups appropriately 1, 2, and 3. Say a word, and then ask students where one of the sounds in the word occurs. They must drop a bean or penny into the appropriate cup. For example, if the word is “hot,” and you ask, “Where is the /t/?” Students drop a bean into the final cup, or number 3. 5 Examples of learner generated texts and phonics activities You can then ask, “Where is the /h/ sound?” and students will drop a bean into the first cup. Blend the Word. Say a word from the story and have students say the word back in segments, emphasizing each sound. For example: Teacher: Summer. Learners: /s/, /u/, /m/, /er/ After several words, repeat the process. Teacher says a word‟s phonemes, and learners must blend them mentally and say the word. For example: Teacher: /t/, /iy/, /sh/, /ir/, /t/ Learners: T-shirt! Does it Rhyme? Emphasize through examples that rhyme concerns the ends of words. Have students listen to three words (at least one from the learner-generated story) and identify the one that doesn‟t belong. For example: hot, pot, pat. Alternatively, students can identify rhyming words in a story or the teacher can say a word (for example, pot) and students must find a word in the story that rhymes (hot). Phonics Tasks Large Cards. Put letters or letter combinations on large cards, hand to each student, and have students “spell out” words, starting with one from the story, at the front of the room. Teacher: D-R-I-N-K, now D and R sit down, S come up. What word do we have now? (sink) Now I sit down, A come up. What now? (sank) Take time to point out similarities and differences among the words and to have students 10 Patsy Vinogradov Patsy Vinogradov physically „blend‟ the cards together in front of the room to demonstrate the combining of sounds. Sort the Words. Sort pictures or words by the letter sound. This can be done with the word cards from the story (see sequencing tasks above). Or, give students a worksheet with empty boxes for each sound, and they sort the pictures under the sound or words under the sound. W wear, we, water S sunny, summer T today Sh shirt Fill in the Missing Letters. To review the vocabulary words for your unit, give students a list of words that have one or more letters missing (try to choose ones that they could easily hear the sound of). Have students write the missing letters (without dictation). su___ ___y su___ ___er ___ ___irt Dictation. The literacy task of assigning symbols to sounds is a major undertaking, and students will need a great deal of practice. Dictation is also a good progress-checking activity. 5 Examples of learner generated texts and phonics activities Connect oral and written language by having students try to write the sounds or words you (or fellow students) call out. Encourage new readers to write only the first sound they hear, or the final sound, and later the entire word if possible. Encourage “inventive” spelling. Dictation. The literacy task of assigning symbols to sounds is a major undertaking, and students will need a great deal of practice. Dictation is also a good progress-checking activity. Connect oral and written language by having students try to write the sounds or words you (or fellow students) call out. Encourage new readers to write only the first sound they hear, or the final sound, and later the entire word if possible. Encourage “inventive” spelling. Word Families. Using a starter-word from the learner-generated text, elicit or introduce more words that fall into this word family. For example: hot (pot, rot, shot, dot, got, not), or wear (pear, bear), or drink (sink, think, blink). Students can sort words on cards or slips of paper into particular word families, or you can do a number of the other phonics activities listed above working not on specific sounds, but on this specific word family pattern. Letter Tiles. Put a handful of letter tiles (similar to Scrabble® tiles) on each table for use by 2-4 students. You (or a student) call out words that the learners must spell with their tiles. They can work individually, in pairs or groups. Make it into a game: Assign teams and award points to the team who can spell the word correctly first. Clearly, once a text has been generated by LESLLA learners, teachers need only read it carefully to find a treasure chest of bottom-up tasks that can build emergent literacy skills. Some of these tasks are described above, but innovative teachers can no doubt create many, many more. The final step in the Whole-Part-Whole method is to then return to the whole text and again focus on meaning, perhaps expanding the original text in the process. By taking the time to work on the pieces of 11 Balancing top and bottom language, including word recognition, phonemic awareness, and phonics, students then return to the whole text with deeper understanding and increased attention to how print functions. 6 Conclusion LESLLA learners face a unique set of challenges: acquiring literacy as adults while learning a new language and resettling in a new culture. While tackling these incredible tasks, learners bring with them remarkable resources for learning that can be capitalized on by skilled teachers to build literacy. In this report, learner- generated texts are presented as one route to effective, balanced literacy instruction that addresses the top-down needs of adult emergent readers while also nurturing bottom-up skills. LESLLA teachers can capitalize on students‟ oral language abilities to build literacy by recording their spoken stories and using their words as reading texts. Using the Whole-Part-Whole method, teachers can focus first on meaning and comprehension, and then they can turn attention to specific readings skills such as sequencing, word recognition, phonemic awareness, and phonics. Then returning to the „whole‟ text, students gain new understanding of the building blocks of language within their own stories. Such practice uses language that is familiar and engaging to learners to nurture their emergent reading skills. In this way, teachers and learners work together to create motivating, achievable lessons that gently move LESLLA learners to becoming proficient readers and writers. For discussion Below is another text gathered from a LESLLA class at the Minnesota Literacy Council. What kinds of emergent literacy activities could be done with learners based on this text? Figure 3: Beautiful Garden Beautiful Garden Mr. J tiller on the dirt. Many students clean the stones and grass. Dirt in the pot and plant tomatoes, chilies, cilantro, eggplants. Put in the stakes and put in the fence. Plant broccoli, onions, green beans, flowers in the garden. Water the plants everyday. We like cucumbers, carrots, broccoli, onions, radishes. We feel happy. Figure 3: Beautiful Garden Beautiful Garden Mr. J tiller on the dirt. Many students clean the stones and grass. Dirt in the pot and plant tomatoes, chilies, cilantro, eggplants. Put in the stakes and put in the fence. Plant broccoli, onions, green beans, flowers in the garden. Water the plants everyday. We like cucumbers, carrots, broccoli, onions, radishes. We feel happy. 12 Patsy Vinogradov Acknowledgments Acknowledgments The presentation at the LESLLA conference was adapted from a presentation at TESOL in 2008 with Astrid Liden and Andrea Poulos. As always, I am grateful for their support and encouragement. 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https://openalex.org/W2096208867	https://npg.copernicus.org/articles/20/1031/2013/npg-20-1031-2013.pdf	English	null	The local ensemble transform Kalman filter and the running-in-place algorithm applied to a global ocean general circulation model	Nonlinear processes in geophysics	2,013	cc-by	13,641	Correspondence to: S. G. Penny (steve.penny@noaa.gov) Correspondence to: S. G. Penny (steve.penny@noaa.gov) Received: 20 February 2013 – Revised: 21 June 2013 – Accepted: 20 September 2013 – Published: 26 November 2013 Received: 20 February 2013 – Revised: 21 June 2013 – Accepted: 20 September 2013 – Published: 26 November 2013 Received: 20 February 2013 – Revised: 21 June 2013 – Accepted: 20 September 2013 – Publishe Abstract. The most widely used methods of data assim- ilation in large-scale oceanography, such as the Simple Ocean Data Assimilation (SODA) algorithm, specify the background error covariances and thus are unable to reﬁne the weights in the assimilation as the circulation changes. In contrast, the more computationally expensive Ensemble Kalman Filters (EnKF) such as the Local Ensemble Trans- form Kalman Filter (LETKF) use an ensemble of model fore- casts to predict changes in the background error covariances and thus should produce more accurate analyses. The EnKFs are based on the approximation that ensemble members re- ﬂect a Gaussian probability distribution that is transformed linearly during the forecast and analysis cycle. In the pres- ence of nonlinearity, EnKFs can gain from replacing each analysis increment by a sequence of smaller increments ob- tained by recursively applying the forecast model and data assimilation procedure over a single analysis cycle. This has led to the development of the “running in place” (RIP) algo- rithm by Kalnay and Yang (2010) and Yang et al. (2012a,b) in which the weights computed at the end of each analysis cycle are used recursively to reﬁne the ensemble at the beginning of the analysis cycle. To date, no studies have been carried out with RIP in a global domain with real observations. This paper provides a comparison of the aforementioned assimilation methods in a set of experiments spanning seven years (1997–2003) using identical forecast models, initial conditions, and observation data. While the emphasis is on understanding the similarities and differences between the assimilation methods, comparisons are also made to indepen- dent ocean station temperature, salinity, and velocity time se- ries, as well as ocean transports, providing information about the absolute error of each. Comparisons to independent ob- servations are similar for the assimilation methods but the observation-minus-background temperature differences are distinctly lower for LETKF and RIP. Correspondence to: S. G. Penny (steve.penny@noaa.gov) The results support the potential for LETKF to improve the quality of ocean analyses on the space and timescales of interest for seasonal prediction and for RIP to accelerate the spin up of the system. Nonlinear Processes in Geophysics Open Access Nonlinear Processes in Geophysics Open Access 1 Introduction Seasonal and longer climate predictions depend on accu- rate estimates of the initial state of the ocean. Most fore- cast centers base their ocean state estimates on data assim- ilation methods such as Optimal Interpolation or 3D-Var, in which the calculation is simpliﬁed by the assumption of pre- speciﬁed statistics of the model errors (Carton and Santorelli, The local ensemble transform Kalman ﬁlter and the running-in-place algorithm applied to a global ocean general circulation model S. G. Penny1,2,7, E. Kalnay2,4, J. A. Carton2, B. R. Hunt3,4, K. Ide1,2,4,5, T. Miyoshi2,6, and G. A. Chepurin2 1Applied Mathematics and Scientiﬁc Computation, University of Maryland, College Park, Maryland, USA 2Department of Atmospheric and Oceanic Science, University of Maryland, College Park, Maryland, USA 3Department of Mathematics, College Park, Maryland, USA 4Institute for Physical Science and Technology, University of Maryland, College Park, Maryland, USA 5Center for Scientiﬁc Computation and Mathematical Modeling, University of Maryland, College Park, Maryland, USA 6RIKEN Advanced Institute for Computational Science Kobe Japan S. G. Penny1,2,7, E. Kalnay2,4, J. A. Carton2, B. R. Hunt3,4, K. Ide1,2,4,5, T. Miyoshi2,6, and G. A. Chepurin2 1Applied Mathematics and Scientiﬁc Computation, University of Maryland, College Park, Maryland, USA 2Department of Atmospheric and Oceanic Science, University of Maryland, College Park, Maryland, USA 3Department of Mathematics, College Park, Maryland, USA S. G. Penny , E. Kalnay , J. A. Carton , B. R. Hunt , K. Ide , T. Miyoshi , and G. A. Chepurin 1Applied Mathematics and Scientiﬁc Computation, University of Maryland, College Park, Maryland, USA 2Department of Atmospheric and Oceanic Science, University of Maryland, College Park, Maryland, USA 3Department of Mathematics, College Park, Maryland, USA 4Institute for Physical Science and Technology, University of Maryland, College Park, Maryland, USA 5Center for Scientiﬁc Computation and Mathematical Modeling, University of Maryland, College Park, Maryland, USA 6RIKEN Advanced Institute for Computational Science, Kobe, Japan 7National Centers for Environmental Prediction (NCEP), NOAA Center for Weather and Climate Prediction, College Park, Maryland, USA 4Institute for Physical Science and Technology, University of Maryland, College Park, Maryland, USA 5Center for Scientiﬁc Computation and Mathematical Modeling, University of Maryland, College Park, Maryland, USA 6RIKEN Advanced Institute for Computational Science, Kobe, Japan 7National Centers for Environmental Prediction (NCEP), NOAA Center for Weather and Climate Prediction, College Park, M l d USA S. G. Penny et al.: The local ensemble transform Kalman ﬁlter 2008; Cummings et al., 2009; Xue et al., 2012). In reality these errors evolve with the changing circulation. Ensemble Kalman Filters (EnKF) have been developed as a computa- tionally efﬁcient way to estimate these evolving errors. How- ever, to date there are few direct comparisons of the results of these different approaches in a realistic situation to eval- uate the improvement in state estimates. This study attempts to help ﬁll this void by comparing the widely used Simple Ocean Data Assimilation (SODA) system against the Local Ensemble Kalman Filter (LETKF) of Hunt et al. (2007), and the LETKF with RIP (Kalnay and Yang, 2010; Yang et al., 2012a,b). Here we compare these methods as adapted for the ocean by Penny 2011) in a set of seven- year-long exper- iments (1997–2003) using identical forecast models, initial conditions, and observation data. Gaussian probability distribution that is transformed linearly during the forecast and analysis cycle. In the presence of non- linearity, LETKF can be improved by replacing each analysis increment by a sequence of smaller increments over a single analysis cycle. This has led to the development of the “run- ning in place” (RIP) algorithm by Kalnay and Yang (2010) in which the weights computed at the end of each analysis cycle are used to adapt the distribution of ensemble mem- bers at the beginning of the analysis cycle. This procedure may be repeated multiple times per analysis cycle, but in this study it is only applied once per cycle. The RIP algorithm has been applied successfully in a simple Lorenz system, a quasi-geostrophic model (Kalnay and Yang, 2010; Yang et al., 2012a), observing system simulation experiments for ty- phoon prediction (Yang et al., 2012b) and the forecast of Ty- phoon Sinlaku (Yang et al., 2013). RIP is implemented here for the ﬁrst time in the global domain with historical obser- vation data. Data assimilation blends a weighted combination of ob- servations and model forecasts to improve their agreement while remaining consistent with the estimated uncertainties in each. If the forecast error is unbiased, stationary, Gaus- sian distributed, homogeneous, and isotropic then the op- timal weights for the observations in a least squares sense are given by Optimal Interpolation or equivalently, 3D-Var (Kalnay, 2003). However, we know that errors are more cor- related in the direction of wave and ﬂuid ﬂows. S. G. Penny et al.: The local ensemble transform Kalman ﬁlter For example, the errors have greater correlation scales in the zonal direc- tion than in the meridional direction in the tropics. As another example, error correlations decrease rapidly across oceanic fronts such as the Gulf Stream. We compare the results of the LETKF and LETKF-RIP ﬁl- ters against an implementation of SODA in the same quasi- global model for the seven-year period (1997–2003). The pe- riod was chosen because it contains strong climate signals beginning with the massive 1997/1998 El Niño, the La Niña of 1998 and 1999, and the return of El Niño in 2002/2003. This period is also of interest because of the rich array of ocean observations available, including temperature, salinity, and currents from the tropical mooring arrays in the Atlantic and Paciﬁc, satellite SST, satellite altimetry, and the com- mencement of the extensive Argo ﬂoat observing system in 1999. In the 1990s, variants of the Kalman ﬁlter were devel- oped for ocean data assimilation. These methods used adap- tive approximations of the background error covariance, but were computationally expensive. Fukumori et al. (1993) pro- posed reducing the cost of sequential ﬁltering by using the asymptotic steady-state form of the Kalman ﬁlter. Pham et al. (1998) proposed a modiﬁed form of the Extended Kalman Filter that approximated the error covariance with a singular low rank matrix to control only the most unstable dimensions of error growth. The ensemble Kalman Filter was introduced by Evensen (1994) as a computationally efﬁcient way to pre- dict key aspects of the nonstationary, inhomogeneous error statistics – those associated with the most unstable dimen- sions of error growth. The ensemble Kalman Filter estimates these error statistics from examination of the correlations of O(10–100) ensemble members, each consisting of a model forecast. The computational cost of the ﬁlter is proportional to the number of ensemble members. The original formula- tion of Evensen had some limitations, including a tendency to become unstable, but it led to many alternative implemen- tations to address these limitations (e.g. Brusdal et al., 2003; Zhang et al., 2007; Keppenne et al., 2008; Wan, 2008, 2010; Zhang and Rosati, 2010). Published by Copernicus Publications on behalf of the European Geosciences Union & the American Geophysical Union. Published by Copernicus Publications on behalf of the European Geosciences Union & the American Geophysical Union. 1032 S. G. Penny et al.: The local ensemble transform Kalman ﬁlter Thus τ i(tk) is the weighted combination at time tk, Figure 1. Temperature and Salinity super-observation counts from Jan 1996 to Jan 2004 data point represents a single day’s Argo and XBT profile data used for data assimilatio after horizontal binning and interpolation to the model’s vertical levels. Fig. 1. Temperature and Salinity super-observation counts from Jan- uary 1996 to January 2004. Each data point represents a single day’s Argo and XBT proﬁle data used for data assimilation, after horizon- tal binning and interpolation to the model’s vertical levels. Figure 2. 12-month moving average of global temperature (ºC) RMSD O-B and O-A in top 500 m for Free-Run, SODA, Standard-LETKF1, and LETKF-RIP1. The background 1996 1997 1998 1999 2000 2001 2002 2003 2004 0 0.5 1 1.5 2 Date Temperature RMSD (ºC) Mean Obs Error 0ï500m Free Run SODA B SODA A LETKF1 B LETKF1 A RIP1 B0 RIP1 B1 RIP1 A1 quasi-uniform 18 ◦C mode water (Joyce and Robins, 1996; Phillips and Joyce, 2007). The upper layer experiences shal- low depths, warming and reduced salinity in boreal summer and fall, and corresponding cooling, saliniﬁcation, and deep- ening in late winter and spring (annual ranges of temper- ature and salinity near-surface are 6 ◦C and 0.15 psu). Su- perimposed on this seasonal cycle temperature and salinity of the upper 200 m undergo year-to-year ﬂuctuations on the order of 0.5 ◦C and 0.05 psu, with cool temperatures in the mid-1990s followed by a warming trend, and substantially reduced salinities for several years in the late 1990s. Below this layer the mode water thickness and salinity varies as a function of the intensity of winter storm activity, which was reduced in the late 1990s and early 2000s. tk, (1) 23 τ i(tk) = (1 −αwτ(tk) + αwτ i(tk + m · 12 mo) (1) For observed zonal velocities, we use the Acoustic Doppler Current Proﬁle (ADCP) data that are part of the TAO/TRITON array located in the equatorial Paciﬁc. The available data during the experiment period from 1997–2003 are located on the equator at 165◦E, 170◦W, 140◦W, and 110◦W. Volume transports were estimated in the model as the integral of the velocity over a vertical cross section of each current, from the surface to 700 m depth for the Gulf Stream and Kuroshio Current, and from the surface to 2400 m depth for the Agulhas Current. S. G. Penny et al.: The local ensemble transform Kalman ﬁlter S. G. Penny et al.: The local ensemble transform Kalman ﬁ Figure 1. Temperature and Salinity super-observation counts from Jan 1996 to Jan 2004. data point represents a single day’s Argo and XBT profile data used for data assimilation after horizontal binning and interpolation to the model’s vertical levels. !" #!!" $!!!" $#!!" %!!!" %#!!" &!!!" $'$'()" $'$'(" $'$'(+" $'$'((" $'$'!!" $'$'!$" $'$'!%" $'$'!&" ,-./-01230-" 41567628" Fig. 1. Temperature and Salinity super-observation counts from Jan- uary 1996 to January 2004. Each data point represents a single day’s Argo and XBT proﬁle data used for data assimilation, after horizon- tal binning and interpolation to the model’s vertical levels. relaxation to Levitus and Boyer (1994) climatological tem- perature and salinity at higher latitudes. We make no attempt to model cryospheric or deep-water formation processes ex- plicitly. A weak relaxation of the global temperature and salinity ﬁelds to climatological values with a 5 yr timescale is included in order to reduce weak forecast bias in deep water masses. Horizontal friction and diffusion coefﬁcients have a constant value of 6 × 107 cm s−2. Vertical friction and dif- fusion are Richardson number dependent with a maximum value of 3 cm s−2. No explicit treatment of model bias was performed. ch p Surface momentum and thermal forcing is provided by the NCEP/NCAR reanalysis of Kalnay et al. (1996) while surface salinity is represented with a monthly climatology from Levitus and Boyer (1994). Surface forcing ﬁelds are lin- early interpolated from monthly averages. The Free Run was started from a state of rest with climatological temperature and salinity and run from years 1970 to 2004. Differences between the observations and the Free Run were calculated for 1996–2003. These give a point of reference for the back- ground and analysis errors in the experiments. We assume the main source of forecast error in the ocean model comes from the surface forcing. For this study, we focus on wind stress error, though we acknowledge additional sources of surface forcing error are important as well. In order to intro- duce wind error with realistic spatial scales and seasonality into the stress of the ith ensemble member, τ i, perturbations were constructed by selecting stresses from the same month in different, randomly selected years from the entire NCEP reanalysis period (a similar approach was used by Zhang et al., 2007). 2 Data and methods A set of 4.3 million temperature proﬁles and moored time series and 1.9 million salinity proﬁles spanning 1979–2003 was obtained from the World Ocean Database 2005 (Boyer et al., 2006). This proﬁle data coverage is concentrated along shipping routes in the Northern Hemisphere and in some coastal regions. Temperature observations are also concen- trated in the tropical Paciﬁc while salinity observations are most prevalent in the North Atlantic and Indian Oceans. Salinity observation coverage increases rapidly throughout the experiment period due to the introduction of the Argo ﬂoat network. This data underwent minor additional qual- ity control and was then binned into daily 1◦× 1◦horizontal bins and linearly interpolated onto the model vertical levels (Fig. 1). We use validation data from two stations: the ALOHA sta- tion of the Hawaiian Ocean Time-series (Karl and Lukas, 1996) and Station S of the Bermuda Atlantic Time-series Study (Steinberg et al., 2001). The hydrography at Station S consists of a seasonally varying, up to 200 m-thick sur- face layer whose properties are controlled by local condi- tions superimposed on a roughly 200–400 m depth layer of In this paper we explore a formulation known as LETKF that was introduced by Hunt et al. (2007) and has found wide application in meteorology. As with other EnKFs, LETKF is based on the approximation that ensemble members reﬂect a Nonlin. Processes Geophys., 20, 1031–1046, 2013 www.nonlin-processes-geophys.net/20/1031/2013/ 1033 S. G. Penny et al.: The local ensemble transform Kalman ﬁlter S. G. Penny et al.: The local ensemble transform Kalman ﬁlter The model estimates are compared to observations of volume transport as reported by Richardson (1985), Imawaki et al. (2001), and Bryden et al. (2005). While the major western boundary currents are not well resolved in this model, the relative impacts of the different assimilation methods on the general circulation are noted. for some random integer n ̸= 0. αw is the weighting that de- termines the proportion of the historical perturbation used. For the experiments reported here we used αw = 0.1. A simi- lar approach was used to generate the initial ensemble, using the historical Free Run data set as a basis with a weighting coefﬁcient equal to 0.5. Our benchmark data assimilation experiment uses SODA (Carton et al., 2000a, b; Carton and Giese, 2008). SODA aggregates an extended (±45 day) window of observations, with observations weighted by a Gaussian taper function centered at the analysis time. SODA speciﬁes an empirical anisotropic background error covariance that depends on lat- itude, depth and dynamic height (see Carton et al., 2000a for details). The vertical background error covariance is assumed to be large within the mixed layer, and decreases at deeper levels where the ocean is well mixed. Observation error is assumed to be uncorrelated white noise with an amplitude equal to 20 % of the zero lag model background error vari- ance for temperature (T ), and 40 % for salinity (S), as spec- iﬁed by Carton et al. (2000a). The salinity observation error Forecasts are made using an ocean model based on the Geophysical Fluid Dynamics Laboratory (GFDL) Modular Ocean Model 2.2 (Pacanowski, 1996). The model resolution is 1◦×0.58◦×20 levels in the tropics, expanding to a uniform 1◦× 1◦× 20 levels in mid-latitude for a total of 936 000 grid points. The basin domain extends from 62◦S–62◦N with a Nonlin. Processes Geophys., 20, 1031–1046, 2013 www.nonlin-processes-geophys.net/20/1031/2013/ S. G. Penny et al.: The local ensemble transform Kalman ﬁlter LETKF implements localization by choosing observations around each model grid point subject to a cutoff radius. It then applies a Gaussian localization function to the esti- mated observation errors in this radius. The localization is parameterized by the σ-radius, which is the distance cor- responding to one standard deviation of the Gaussian lo- calization function. We report results from two parameter settings in LETKF, indicated by superscripts (if no super- script is used the statements apply to both). The ﬁrst uses a horizontal σ-radius at the equator of about 300 km with a cutoff at 1100 km (denoted LETKF1 and LETKF-RIP1), and the second uses a σ-radius of 1100 km with a cutoff at about 4000 km (denoted LETKF2 and LETKF-RIP2). Both decrease linearly to an 80 km σ-radius with cutoff at 300 km at the ±60◦latitude. The change in localization exhibited a nonlinear effect on the inﬂation values computed with the adaptive inﬂation algorithm, thus a smaller time-scaling fac- tor σb = 0.0001 was used with the larger localization radius, versus σb = 0.001 for the smaller radius. In the vertical, a σ- radius of about 80 m with a cutoff of 300 m was used. We will focus primarily on the results from LETKF1 and LETKF- RIP1, but include LETKF2 and LETKF-RIP2 for compari- son. The separation of ensemble members in the state space should occur because of a combination of internal ﬂuid in- stabilities and variations in surface forcing. The impacts of internal instabilities are greatly reduced because of our choice of non-eddy resolving resolution and associated phys- ical parameterization errors. Additionally, limitations on the size of the ensemble further reduce the ensemble variance. We adjust for this missing error variance (1) in the model by adding perturbations to the wind ﬁeld (as described above), and (2) in the data assimilation procedure through an adaptive error covariance inﬂation scheme developed by Miyoshi (2011). Miyoshi’s scheme has been applied to adjust inﬂation to automatically balance the background error with the estimated observation error. Occasionally, the ensemble spread becomes under-dispersive, meaning that the back- ground error covariance estimate is small, while the mean state is “far” from the observations (with respect to the ob- servation error). In these cases the inﬂation is automatically increased and the ensemble spread increases as a direct re- sult. S. G. Penny et al.: The local ensemble transform Kalman ﬁlter 1034 values are effectively taken from the climatological T /S rela- tionship to be consistent with the estimated temperature ob- servation error values. SODA uses the two-stage Incremental Analysis Update (IAU) time ﬁlter of Bloom et al. (1996) to avoid generation of spurious gravity waves. After perform- ing the analysis on the 5-day forecast, a 10-day forecast is performed in which the analysis increment is gradually in- troduced as a restoring force into the predictive equations for temperature and salinity. to the initial conditions in order to improve its performance for the sparse observation network. For the experiments de- scribed here, this relaxation was only applied in areas where the analysis spread for temperature was greater than 2 ◦C. The ﬁnal data assimilation algorithm is LETKF-RIP, uti- lizing the iterative dual-pass RIP procedure introduced by Kalnay and Yang (2010) and Yang et al. (2012a). RIP ap- plies the weights computed for the end of the LETKF anal- ysis cycle to the ensemble members at the beginning of the cycle, and the forecast and analysis steps are subsequently re- peated. The result is a gradual correction to the background ensemble mean. Each repetition is based on the same set of observations, but initiates the dynamical model from differ- ent initial conditions. While this procedure could be repeated several times, it is only repeated once per analysis cycle in this study. Both the standard LETKF and LETKF-RIP use a 5 day analysis cycle so the background and analysis times correspond with SODA. Both ensemble methods use a 40- member ensemble in all experiments reported here. p y The second data assimilation algorithm we use is LETKF, a computationally efﬁcient ensemble Kalman ﬁlter designed by Hunt et al. (2007) that uses the localization method of Ott et al. (2004) and a transformation between background and analysis ensemble perturbations similar to Bishop et al. (2001), Wang et al. (2003) and Ott et al. (2004). The al- gorithm uses an ensemble of numerical forecast model runs to estimate the background error covariance and assimilates observations at the time they occur rather than aggregating them at a ﬁxed analysis time. A variable localization radius is used in LETKF to model the reduction of the radius of deformation with increasing latitude. LETKF’s estimates of observation error are taken from the SODA assimilation sys- tem. S. G. Penny et al.: The local ensemble transform Kalman ﬁlter The ensemble spread continues to increase over multi- ple analysis cycles until the background error covariance is large enough that the observations begin to impact the anal- ysis again. A parameter for adaptive inﬂation σb allows the inﬂation to be smoothed in time to achieve a larger effective sample size of observations. 3 Results The following experiments are compared: A Free Run of the model with no data assimilation applied, SODA, LETKF1,2 and LETKF-RIP1,2. We assess the performance of these data assimilation systems by ﬁrst examining the consistency of statistical assumptions used in formulating the assimila- tion algorithms. In particular we focus on the root mean square deviation (RMSD) statistic of observation-minus- background (O–B) and observation-minus-analysis (O–A) differences evaluated at the observation locations and times. Miyoshi’s original method assumed that the spatial struc- ture of the observing system remains constant in time. When applied to the oceanographic problem for which the observ- ing system is constantly changing, Miyoshi’s approach has a tendency to increase background covariance in areas where observational coverage declines over time. Here, whenever observation coverage declines we relax inﬂation values back www.nonlin-processes-geophys.net/20/1031/2013/ Nonlin. Processes Geophys., 20, 1031–1046, 2013 1996 1997 1998 1999 2000 2001 2002 2003 2004 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 Date Salinity RMSD (psu) Mean Obs Error 0ï500m Free Run SODA B SODA A LETKF1 B LETKF1 A RIP1 B0 RIP1 B1 RIP1 A1 1996 1997 1998 1999 2000 2001 2002 2003 2004 0 0.5 1 1.5 2 Date Temperature RMSD (ºC) Mean Obs Error 0ï500m Free Run SODA B SODA A LETKF1 B LETKF1 A RIP1 B0 RIP1 B1 RIP1 A1 9 Figure 2. 12-month moving average of global temperature (ºC) RMSD O-B and O-A Fig. 2. A 12-month moving average of global temperature (◦C)10 Figure 2. 12-month moving average of global temperature (ºC) RMSD O-B and O-A top 500 m for Free-Run, SODA, Standard-LETKF1, and LETKF-RIP1. The backgrou Fig. 2. A 12-month moving average of global temperature (◦C) RMSD O–B and O–A in the top 500 m for Free Run, SODA, Standard-LETKF1, and LETKF-RIP1. The background data are shown with dashed lines, and the analysis data with solid lines. In the legend, the sufﬁx “B” signiﬁes background, while “A” signiﬁes analysis. For the RIP methods, “B0” is the background of the ini- tial iteration of LETKF while “B1” and “A1” are the background and analysis after the 1st iteration of RIP. The mean temperature observation error in the top 500 m is shown with a dashed gray line. As cases LETKF-B and RIP-B0 have not used “future” observa- tions, they are directly comparable and show improvement by RIP. Because SODA uses a long window of observations, it is more ap- propriate to compare the background SODA-B versus RIP-B1 and the analysis SODA-A versus LETKF-A and RIP-A1. 10 1 12 13 14 15 16 23 he ata Figure 3. 12-month moving average of global salinity (psu) RMSD O-B and O-A in th 500 m for Free-Run, SODA, Standard-LETKF1, and LETKF-RIP1. The background d shown with dashed lines, and the analysis data with solid lines. In the legend, the suffi signifies background, while ‘A’ signifies analysis. For the RIP methods, ‘B0’ is the background of the initial iteration of LETKF while ‘B1’ and ‘A1’ are the background analysis after the 1st iteration of RIP. The mean salinity observation error in the top 50 shown with a dashed gray line. As with the temperature RMSD in Figure 2, cases LET Fig. 3. Nonlin. Processes Geophys., 20, 1031–1046, 2013 The mean salinity observation error in the top 500 m is shown with a dashed gray line. Comparisons between the background SODA-B versus RIP-B1 and the analysis SODA-A versus LETKF-A and RIP-A1 show improvement by RIP. 3.1 RMSD S. G. Penny et al.: The local ensemble transform Kalman ﬁlter after horizontal binning and interpolation to the model’s vertical levels. SOD 8 1035 IP-A1. 23 the data Figure 3. 12-month moving average of global salinity (psu) RMSD O-B and O-A in th 500 m for Free-Run, SODA, Standard-LETKF1, and LETKF-RIP1. The background da shown with dashed lines, and the analysis data with solid lines. In the legend, the suffi signifies background, while ‘A’ signifies analysis. For the RIP methods, ‘B0’ is the background of the initial iteration of LETKF while ‘B1’ and ‘A1’ are the background analysis after the 1st iteration of RIP. The mean salinity observation error in the top 50 shown with a dashed gray line. As with the temperature RMSD in Figure 2, cases LET 1996 1997 1998 1999 2000 2001 2002 2003 2004 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 Date Salinity RMSD (psu) Mean Obs Error 0ï500m Free Run SODA B SODA A LETKF1 B LETKF1 A RIP1 B0 RIP1 B1 RIP1 A1 Fig. 3. A 12-month moving average of global salinity (psu) RMSD O–B and O–A in the top 500 m for Free Run, SODA, Standard- LETKF1, and LETKF-RIP1. The background data are shown with dashed lines, and the analysis data with solid lines. In the legend, the sufﬁx “B” signiﬁes background, while “A” signiﬁes analysis. For the RIP methods, “B0” is the background of the initial iteration of LETKF while “B1” and “A1” are the background and analysis after the 1st iteration of RIP. The mean salinity observation error in the top 500 m is shown with a dashed gray line. Comparisons between the background SODA-B versus RIP-B1 and the analysis SODA-A versus LETKF-A and RIP-A1 show improvement by RIP. Nonlin. Processes Geophys., 20, 1031–1046, 2013 A 12-month moving average of global salinity (psu) RMSD O–B and O–A in the top 500 m for Free Run, SODA, Standard- LETKF1, and LETKF-RIP1. The background data are shown with dashed lines, and the analysis data with solid lines. In the legend, the sufﬁx “B” signiﬁes background, while “A” signiﬁes analysis. For the RIP methods, “B0” is the background of the initial iteration of LETKF while “B1” and “A1” are the background and analysis after the 1st iteration of RIP. The mean salinity observation error in the top 500 m is shown with a dashed gray line. Comparisons between the background SODA-B versus RIP-B1 and the analysis SODA-A versus LETKF-A and RIP-A1 show improvement by RIP. 3.1 RMSD The aggregate RMSD for the ﬁnal three years of the experi- ment period are shown for temperature (Table 1) and salinity (Table 2). Globally, LETKF and LETKF-RIP substantially improve the temperature ﬁelds over the SODA baseline. As can be seen in Fig. 2, the (O–B) temperature RMSD for LETKF1 and LETKF-RIP1 both fall below the SODA (O–B) baseline. The LETKF-RIP1 temperature (O–A) RMSD falls below the estimated observation error over a year before the standard LETKF analysis, while the SODA baseline never reaches this level. For all methods other than SODA, the (O–B) RMSD are computed with observations that have not been used at all by the assimilation up to that time, thus these observations may be considered withheld for the purpose of comparison. The (O–A) RMSD at the corresponding time indicates the relative impact of the assimilation method. Adaptive inﬂation values are provided for additional perspective on the performance of the LETKF-based approaches. An increase in RMSD was noted as the ensemble size was reduced from 40 to 20 mem- bers, though results were qualitatively similar. Throughout the experiment period, across all regions, the temperature ﬁelds for LETKF-RIP are as good or better than LETKF. LETKF-RIP takes advantage of the large esti- mated observation error (e.g. with a mean 0.67 psu in the top 500 m, as speciﬁed by SODA) and increases salinity RMSD in some regions to facilitate greater improvements in tem- perature. However, as shown in Fig. 3, by the end of the ex- periment period the salinity (O–B) RMSD for both LETKF1 and LETKF-RIP1 have fallen below the SODA baseline. The LETKF-RIP1 salinity (O–A) RMSD falls below the SODA baseline early in the experiment period, and this occurs over a year before the crossover by the standard LETKF1 analysis. Recall from Fig. 1 that there is an order of magnitude fewer salinity observations than temperature observations. For the salinity RMSD calculations, a small number of out- lier observations from the Gulf Stream that caused a spike in RMSD (for both the Free Run and SODA) in the ﬁrst two years of the experiment period were removed, though they Throughout the experiment period, across all regions, the temperature ﬁelds for LETKF-RIP are as good or better than LETKF. LETKF-RIP takes advantage of the large esti- mated observation error (e.g. Nonlin. Processes Geophys., 20, 1031–1046, 2013 Nonlin. Processes Geophys., 20, 1031–1046, 2013 S. G. Penny et al.: The local ensemble transform Kalman ﬁl data point represents a single day’s Argo and XBT profile data used for data assimilatio after horizontal binning and interpolation to the model’s vertical levels. Figure 2. 12-month moving average of global temperature (ºC) RMSD O-B and O-A in top 500 m for Free-Run, SODA, Standard-LETKF1, and LETKF-RIP1. The background 1996 1997 1998 1999 2000 2001 2002 2003 2004 0 0.5 1 1.5 2 Date Temperature RMSD (ºC) Mean Obs Error 0ï500m Free Run SODA B SODA A LETKF1 B LETKF1 A RIP1 B0 RIP1 B1 RIP1 A1 Fig. 2. A 12-month moving average of global temperature (◦C) RMSD O–B and O–A in the top 500 m for Free Run, SODA, Standard-LETKF1, and LETKF-RIP1. The background data are shown with dashed lines, and the analysis data with solid lines. In the legend, the sufﬁx “B” signiﬁes background, while “A” signiﬁes analysis. For the RIP methods, “B0” is the background of the ini- tial iteration of LETKF while “B1” and “A1” are the background and analysis after the 1st iteration of RIP. The mean temperature observation error in the top 500 m is shown with a dashed gray line. As cases LETKF-B and RIP-B0 have not used “future” observa- tions, they are directly comparable and show improvement by RIP. Because SODA uses a long window of observations, it is more ap- propriate to compare the background SODA-B versus RIP-B1 and the analysis SODA-A versus LETKF-A and RIP-A1. 7 8 9 10 11 12 13 14 15 16 S. G. Penny et al.: The local ensemble transform Kalman ﬁlter 1035 23 data point represents a single day s Argo and XBT profile data used for data assimilation, after horizontal binning and interpolation to the model’s vertical levels. Figure 2. 12-month moving average of global temperature (ºC) RMSD O-B and O-A in the top 500 m for Free-Run, SODA, Standard-LETKF1, and LETKF-RIP1. The background data 1996 1997 1998 1999 2000 2001 2002 2003 2004 0 0.5 1 1.5 2 Date Temperature RMSD (ºC) Mean Obs Error 0ï500m Free Run SODA B SODA A LETKF1 B LETKF1 A RIP1 B0 RIP1 B1 RIP1 A1 Fig. 2. A 12-month moving average of global temperature (◦C) RMSD O–B and O–A in the top 500 m for Free Run, SODA, Standard-LETKF1, and LETKF-RIP1. Nonlin. Processes Geophys., 20, 1031–1046, 2013 The background data are shown with dashed lines, and the analysis data with solid lines. In the legend, the sufﬁx “B” signiﬁes background, while “A” signiﬁes analysis. For the RIP methods, “B0” is the background of the ini- tial iteration of LETKF while “B1” and “A1” are the background and analysis after the 1st iteration of RIP. The mean temperature observation error in the top 500 m is shown with a dashed gray line. As cases LETKF-B and RIP-B0 have not used “future” observa- tions, they are directly comparable and show improvement by RIP. Because SODA uses a long window of observations, it is more ap- propriate to compare the background SODA-B versus RIP-B1 and the analysis SODA-A versus LETKF-A and RIP-A1. uses a long window of observations, it is more appropriate to compare the background 7 SODA-B versus RIP-B1 and the analysis SODA-A versus LETKF-A and RIP-A1. 8 9 Figure 3. 12-month moving average of global salinity (psu) RMSD O-B and O-A in the to 10 500 m for Free-Run, SODA, Standard-LETKF1, and LETKF-RIP1. The background data 11 shown with dashed lines, and the analysis data with solid lines. In the legend, the suffix ‘B 12 signifies background, while ‘A’ signifies analysis. For the RIP methods, ‘B0’ is the 13 background of the initial iteration of LETKF while ‘B1’ and ‘A1’ are the background an 14 analysis after the 1st iteration of RIP. The mean salinity observation error in the top 500 m 15 shown with a dashed gray line. As with the temperature RMSD in Figure 2, cases LETKF 16 1996 1997 1998 1999 2000 2001 2002 2003 2004 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 Date Salinity RMSD (psu) Mean Obs Error 0ï500m Free Run SODA B SODA A LETKF1 B LETKF1 A RIP1 B0 RIP1 B1 RIP1 A1 Fig. 3. A 12-month moving average of global salinity (psu) RMSD O–B and O–A in the top 500 m for Free Run, SODA, Standard- LETKF1, and LETKF-RIP1. The background data are shown with dashed lines, and the analysis data with solid lines. In the legend, the sufﬁx “B” signiﬁes background, while “A” signiﬁes analysis. For the RIP methods, “B0” is the background of the initial iteration of LETKF while “B1” and “A1” are the background and analysis after the 1st iteration of RIP. Nonlin. Processes Geophys., 20, 1031–1046, 2013 S. G. Penny et al.: The local ensemble transform Kalman ﬁlter 1036 Table 1. Mean (observation-background) temperature RMS deviations (◦C) for each method from January 2001–December 2003 listed globally, regionally, and by vertical level. ackground) temperature RMS deviations (◦C) for each method from January 2001–December 2003 listed ical level. Table 1. Mean (observation background) temperature RMS deviations ( C) for each method from January 2001 December 2003 listed globally, regionally, and by vertical level. Free Run SODA LETKF1 LETKF-RIP1 LETKF2 LETKF-RIP2 Global 2.03 1.23 1.03 1.01 1.23 1.17 N Paciﬁc 2.17 1.47 1.35 1.30 1.57 1.50 Eq Paciﬁc 2.19 1.18 0.89 0.89 1.05 1.00 S Paciﬁc 1.43 0.98 0.96 0.94 1.21 1.12 N Atlantic 1.63 1.12 0.99 0.96 1.28 1.19 Eq Atlantic 1.94 1.18 0.94 0.92 1.15 1.09 S Atlantic 1.86 1.66 1.06 0.81 1.39 1.36 Indian 1.61 1.17 1.12 1.09 1.32 1.27 Global SFC 1.91 1.03 0.95 0.91 1.20 1.13 Global 100 m 2.19 1.37 1.12 1.12 1.26 1.10 Global 500 m 1.15 0.68 0.67 0.63 0.88 0.82 Table 2. Mean (observation-background) salinity RMS deviations (psu) or each method from January 2001–December 2003 listed globally, regionally, and by vertical level. ckground) salinity RMS deviations (psu) or each method from January 2001–December 2003 listed globally, Table 2. Mean (observation-background) salinity RMS deviations (psu) or each method from January 2001–December 2003 listed globally, regionally, and by vertical level. tion-background) salinity RMS deviations (psu) or each method from January 2001–December 2003 listed glo al level. Free Run SODA LETKF1 LETKF-RIP1 LETKF2 LETKF-RIP2 Global 0.34 0.26 0.26 0.26 0.31 0.31 N Paciﬁc 0.29 0.14 0.12 0.19 0.36 0.31 Eq Paciﬁc 0.25 0.20 0.24 0.26 0.29 0.30 S Paciﬁc 0.19 0.18 0.20 0.24 0.16 0.17 N Atlantic 0.34 0.30 0.25 0.25 0.32 0.31 Eq Atlantic 0.35 0.29 0.25 0.24 0.30 0.30 S Atlantic 0.23 0.11 0.21 0.18 0.20 0.19 Indian 0.30 0.18 0.16 0.15 0.30 0.25 Global SFC 0.34 0.27 0.31 0.30 0.33 0.34 Global 100 m 0.34 0.27 0.24 0.26 0.31 0.30 Global 500 m 0.23 0.21 0.14 0.13 0.20 0.20 RIP-B0 RMSD with the temperature observations is signiﬁ- cantly smaller than the corresponding LETKF-B during the spin up of the ﬁrst four years, indicating that, in agreement with Kalnay and Yang (2010), RIP is particularly helpful dur- ing the spin up. During the last 3 yr included in Table 1, the advantage of RIP over the standard LETKF for temperatures is smaller but still positive. were used in the analyses. 3.1 RMSD with a mean 0.67 psu in the top 500 m, as speciﬁed by SODA) and increases salinity RMSD in some regions to facilitate greater improvements in tem- perature. However, as shown in Fig. 3, by the end of the ex- periment period the salinity (O–B) RMSD for both LETKF1 and LETKF-RIP1 have fallen below the SODA baseline. The LETKF-RIP1 salinity (O–A) RMSD falls below the SODA baseline early in the experiment period, and this occurs over a year before the crossover by the standard LETKF1 analysis. To evaluate the analyses, we present comparisons to inde- pendent observations. We examine the relationship between observed and analysis temperature and salinity in the north- ern subtropical gyres through comparison to observed time series at Station S (30.6◦N, 63.5◦W) in the western sub- tropical North Atlantic and the Hawaii Ocean Time-series site (24.8◦N, 158.0◦W) near Oahu, Hawaii in the subtrop- ical North Paciﬁc. We examine zonal velocity anomalies us- ing Tropical Atmosphere Ocean (TAO/TRITON) moorings in the equatorial Paciﬁc (at 165◦E, 170◦W, 140◦W and 110◦W). Both observation and model data for the stations and moorings have been smoothed with a monthly average prior to this comparison. We also compare volume transport in selected major western boundary currents to observed val- ues. Recall from Fig. 1 that there is an order of magnitude fewer salinity observations than temperature observations. For the salinity RMSD calculations, a small number of out- lier observations from the Gulf Stream that caused a spike in RMSD (for both the Free Run and SODA) in the ﬁrst two years of the experiment period were removed, though they www.nonlin-processes-geophys.net/20/1031/2013/ Nonlin. Processes Geophys., 20, 1031–1046, 2013 3.2 Adaptive inﬂation parameter by region to increase the effect of speciﬁc observations on the analysis. While adaptive inﬂation builds a long-term “memory” of ge- ographic areas where the model consistently disagrees with observations, RIP reforms the initial ensemble to immedi- ately increase the impact of observations in areas where the model uncertainty is largest. As discussed in Sect. 2, inﬂation is needed to artiﬁcially en- hance growth of the ensemble spread due to the viscous na- ture of the 1 × 1◦model grid resolution. The adaptive in- ﬂation procedure increases inﬂation for areas in which the model error is estimated to be too small based on comparison with local observations. In a well-observed region, smaller values of inﬂation indicate a more appropriate ensemble in approximating the background covariance given the obser- vation errors, as described by the diagnostic equations of Desroziers et al. (2005) and Li et al. (2009). Average inﬂation values for LETKF and LETKF-RIP are given for each region in Table 3. In general, the inﬂation values are larger in the western boundary currents and in the equatorial regions of the Atlantic and Paciﬁc oceans. However, because the adap- tive inﬂation calculation can only be performed when ob- servations are present, it is noted that adaptive inﬂation is roughly proportional to the number of observations available in each region over the duration of the experiment period. The adaptive inﬂation values for LETKF-RIP are less than that for LETKF. This indicates a reduced need for inﬂation with the RIP method. Both inﬂation and RIP provide a means S. G. Penny et al.: The local ensemble transform Kalman ﬁlter Table 3. Adaptive inﬂation values averaged per region. Values are reported at the end of the experiment period (approximately 1 Jan- uary 2004). Larger adaptive inﬂation indicates model deﬁciencies in representing observed features. Adaptive inﬂation values are small in the southern oceans due to low data availability. Percent Inﬂation LETKF1 LETKF-RIP1 LETKF2 LETKF-RIP2 N Paciﬁc 14.1 9.1 5.7 4.2 Eq Paciﬁc 36.6 24.2 16.4 12.6 S Paciﬁc 4.2 2.6 1.1 0.9 N Atlantic 19.3 12.2 6.7 4.6 Eq Atlantic 28.3 18.1 11.2 8.0 S Atlantic 2.8 1.8 0.4 0.3 Indian 9.5 6.6 3.2 2.5 Gulf Stream 24.6 13.6 9.5 6.8 Kuroshio 38.6 22.0 20.6 14.8 Percent Inﬂation LETKF1 LETKF-RIP1 LETKF2 LETKF-RIP2 N Paciﬁc 14.1 9.1 5.7 4.2 Eq Paciﬁc 36.6 24.2 16.4 12.6 S Paciﬁc 4.2 2.6 1.1 0.9 N Atlantic 19.3 12.2 6.7 4.6 Eq Atlantic 28.3 18.1 11.2 8.0 S Atlantic 2.8 1.8 0.4 0.3 Indian 9.5 6.6 3.2 2.5 Gulf Stream 24.6 13.6 9.5 6.8 Kuroshio 38.6 22.0 20.6 14.8 Table 4. Mean and standard deviation of volume transports for the Gulf Stream (off Cape Hatteras, 35◦N, 0–700 m), Kuroshio Current (off Shikoku Island, ASUKA Line, 0–700 m), and Agulhas Current (off Port Edward, 33◦S, 0–2400 m) from 1997 to 2003. Mean Volume Transport in Sverdrups (Sv), and (standard deviation) Free Run SODA LETKF1 LETKF- RIP1 LETKF2 LETKF- RIP2 Observed Gulf Stream 28.8 (1.7) 46.2 (21.1) 38.0 (7.4) 41.5 (10.1) 32.4 (2.0) 34.0 (2.5) 60 (55) Kuroshio 34.1 (1.9) 56.7 (16.7) 44.7 (11.0) 46.5 (14.0) 40.1 (3.0) 42.1 (3.9) 42 (1.3) Agulhas 77.5 (22.7) 78.3 (23.3) 76.8 (22.7) 76.4 (22.6) 77.4 (22.7) 77.5 (22.8) 69.7* (21.5) * Richardson (1985); Imawaki et al. (2001); * Bryden et al. (2005). Mean Volume Transport in Sverdrups (Sv), and (standard deviation) S. G. Penny et al.: The local ensemble transform Kalman ﬁlter While the estimated observation error proﬁles were used from published results derived for the second half of the 20th century, the results in Fig. 3 in- dicate that a more sophisticated estimation of observation er- rors, varying not only by depth but also geographically and temporally, may further improve the analyses, particularly with the increase in quantity and accuracy of in situ salin- ity observations due to the Argo system. Both for salinity and for temperatures, the forecast from RIP-B1, started from the previous analysis updated with RIP is about as accurate as the analysis of the standard LETKF, and the LETKF-RIP analysis is signiﬁcantly more accurate in RMSD, indicating that RIP succeeded in improving the quality of the previous analysis. We note that for reanalysis, the improvement of the previous analysis can be performed with the “no-cost smoother” of Kalnay et al., 2007; Yang et al., 2009, without incurring in the main computational cost of RIP, which is the re-forecasting of the ensemble. y g y In Figs. 2 and 3 we show RMSD for both the background and analysis ﬁelds for SODA and LETKF1. Because RIP is an iterative method, we show the background for the ﬁrst (B0) and last (B1) iterations of LETKF-RIP1, as well as the ﬁnal analysis (A1). We note that the background ﬁelds for LETKF1-B and RIP1-B0 can be fairly compared since they do not use future observations, relative to the analysis time. Because SODA uses a long window of observations extending both into the past and future relative to the anal- ysis time, it is more appropriate to compare the background ﬁeld SODA-B with RIP1-B1 and the analysis ﬁeld SODA- A with LETKF1-A and RIP1-A1. Figure 2 shows that the www.nonlin-processes-geophys.net/20/1031/2013/ Nonlin. Processes Geophys., 20, 1031–1046, 2013 1037 Nonlin. Processes Geophys., 20, 1031–1046, 2013 3.3 Comparisons with independent observations at station S and Aloha Salinity analysis minus bottle measurement (psu) at Station S (Bermuda; (30.55,- 2 63 5)) from Jan 1997 to Dec 2003 for the top 500 m From top to bottom: Free Run SODA 3 Free Run SODA LETKF1 LETKF-RIP1 LETKF2 LETKF-RIP2 Fig. 5. Salinity analysis minus bottle measurement (psu) at Station S (Bermuda; (30.55–63.5)) from January 1997 to December 2003 for the top 500 m. From top to bottom: Free Run, SODA, LETKF1, LETKF-RIP1, LETKF2, and LETKF-RIP2. 4 Figure 4. Temperature analysis minus bottle temperature measurement (ºC) at Station S 5 Fig. 4. Temperature analysis minus bottle temperature measurement (◦C) at Station S (Bermuda; (30.55–63.5)) from January 1997 to De- cember 2003 for the top 500 m. From top to bottom: Free Run, SODA, LETKF1, LETKF-RIP1, LETKF2, and LETKF-RIP2. Red shows that the analysis is too warm, and blue too cold, compared to the independent observation. White gaps indicate missing observation data. 25 (Bermuda; (30.55, 63.5)) from Jan. 1997 to Dec. 2003 for the top 500 m. From top to bottom: 6 Free Run, SODA, LETKF1, LETKF-RIP1, LETKF2, and LETKF-RIP2. Red shows that the 7 analysis is too warm, and blue too cold, compared to the independent observation. White gaps 8 indicate missing observation data. 9 1 Figure 5. Salinity analysis minus bottle measurement (psu) at Station S (Bermuda; (30.55,- 2 Free Run SODA LETKF1 LETKF-RIP1 LETKF2 LETKF-RIP2 Fig. 5. Salinity analysis minus bottle measurement (psu) at Station S (Bermuda; (30.55–63.5)) from January 1997 to December 2003 for the top 500 m. From top to bottom: Free Run, SODA, LETKF1, LETKF-RIP1, LETKF2, and LETKF-RIP2. 1 Figure 5. Salinity analysis minus bottle measurement (psu) at Station S (Bermuda; (30.55,- 2 Fig. 5. Salinity analysis minus bottle measurement (psu) at Station S (Bermuda; (30.55–63.5)) from January 1997 to December 2003 for the top 500 m. From top to bottom: Free Run, SODA, LETKF1, LETKF-RIP1, LETKF2, and LETKF-RIP2. LETKF1, LETKF-RIP1, LETKF2, and LETKF-RIP2. 4 Nonlin. Processes Geophys., 20, 1031–1046, 2013 www.nonlin-processes-geophys.net/20/1031/2013/ LETKF1, LETKF-RIP1, LETKF2, and 4 Nonlin. Processes Geophys., 20, 1031–1046, 2013 3.3 Comparisons with independent observations at station S and Aloha Next we present comparisons of the analyses against inde- pendent time series of temperature and salinity at two sub- tropical locations in the North Atlantic and North Paciﬁc. Our comparison of the Free Run and assimilation experi- ments at Station S shows that the Free Run has a difﬁcult time representing the mean vertical structure of temperature (Fig. 4) or salinity (Fig. 5). In particular, it is over 3 ◦C too warm in the upper 200 m and 3 ◦C too cold in the depth range 200–400 m, implying that it is too strongly stratiﬁed, and is also too fresh by up to 0.5 psu. All of the assimilation methods reduce this mean stratiﬁ- cation error. After assimilation the salinity analyses are no longer uniformly fresh, but develop a pattern of salty and www.nonlin-processes-geophys.net/20/1031/2013/ Nonlin. Processes Geophys., 20, 1031–1046, 2013 S. G. Penny et al.: The local ensemble transform Kalman ﬁlter 1038 1038 S. G. Penny et al.: The local ensemble transform Kalman ﬁlter 4 Figure 4. Temperature analysis minus bottle temperature measurement (ºC) at Station S 5 Free Run SODA LETKF1 LETKF-RIP1 LETKF2 LETKF-RIP2 Fig. 4. Temperature analysis minus bottle temperature measurement (◦C) at Station S (Bermuda; (30.55–63.5)) from January 1997 to De- cember 2003 for the top 500 m. From top to bottom: Free Run, SODA, LETKF1, LETKF-RIP1, LETKF2, and LETKF-RIP2. Red shows that the analysis is too warm, and blue too cold, compared to the independent observation. White gaps indicate missing observation data. 25 4 Figure 4. Temperature analysis minus bottle temperature measurement (ºC) at Station S 5 (Bermuda; (30.55,-63.5)) from Jan. 1997 to Dec. 2003 for the top 500 m. From top to bottom: 6 Free Run, SODA, LETKF1, LETKF-RIP1, LETKF2, and LETKF-RIP2. Red shows that the 7 analysis is too warm, and blue too cold, compared to the independent observation. White gaps 8 indicate missing observation data. 9 Free Run SODA LETKF1 LETKF-RIP1 LETKF2 LETKF-RIP2 Fig. 4. Temperature analysis minus bottle temperature measurement (◦C) at Station S (Bermuda; (30.55–63.5)) from January 1997 to De- cember 2003 for the top 500 m. From top to bottom: Free Run, SODA, LETKF1, LETKF-RIP1, LETKF2, and LETKF-RIP2. Red shows that the analysis is too warm, and blue too cold, compared to the independent observation. White gaps indicate missing observation data. 1 Figure 5. LETKF1, LETKF-RIP1, LETKF2, and 4 Nonlin. Processes Geophys., 20, 1031–1046, 2013 www.nonlin-processes-geophys.net/20/1031/2013/ www.nonlin-processes-geophys.net/20/1031/2013/ S. G. Penny et al.: The local ensemble transform Kalman ﬁlter S. G. Penny et al.: The local ensemble transform Kalman ﬁlter 1039 1 Figure 6. Temperature analysis minus bottle measurement (ºC) at Aloha Station (24.75,- 2 Free Run SODA LETKF1 LETKF-RIP1 LETKF2 LETKF-RIP2 Fig. 6. Temperature analysis minus bottle measurement (◦C) at Aloha Station (24.75–158.0) from January 1997 to December 2003 for the top 500 m. From top to bottom: Free Run, SODA, LETKF1, LETKF-RIP1, LETKF2, and LETKF-RIP2. 1 Figure 6 Temperature analysis minus bottle measurement (ºC) at Aloha Station (24 75 - 2 Fig. 6. Temperature analysis minus bottle measurement (◦C) at Aloha Station (24.75–158.0) from January 1997 to December 2003 for the top 500 m. From top to bottom: Free Run, SODA, LETKF1, LETKF-RIP1, LETKF2, and LETKF-RIP2. 27 158.0) from Jan. 1997 to Dec. 2003 for the top 500 m. From top to bottom: Free Run, SODA, 3 LETKF1, LETKF-RIP1, LETKF2, and LETKF-RIP2. 4 1 Figure 7. Salinity analysis minus bottle measurement (psu) at Aloha Station (24.75,-158.0) 2 Free Run SODA LETKF1 LETKF-RIP1 LETKF2 LETKF-RIP2 Fig. 7. Salinity analysis minus bottle measurement (psu) at Aloha Station (24.75–158.0) from Januar 1997 to December 2003 for the top 500 m. From top to bottom: Free Run, SODA, LETKF1, LETKF-RIP1, LETKF2, LETKF-RIP2. 1 Figure 7. Salinity analysis minus bottle measurement (psu) at Aloha Station (24.75,-158.0) 2 Fig. 7. Salinity analysis minus bottle measurement (psu) at Aloha Station (24.75–158.0) from Januar 1997 to December 2003 for the top 500 m. From top to bottom: Free Run, SODA, LETKF1, LETKF-RIP1, LETKF2, LETKF-RIP2. Nonlin. Processes Geophys., 20, 1031–1046, 2013 S. G. Penny et al.: The local ensemble transform Kalman ﬁlter The summer of 2002 is unusual in all three experiments in showing anomalously cool wa- ter throughout the upper 400 m relative to observed condi- tions, which were themselves anomalously cool with deep penetration of the winter mixed layer. During the anoma- lous conditions of summer 2002, all three assimilation ex- periments exhibit elevated surface salinities, while SODA has anomalously low salinity below 200 m. In some cases, LETKF-RIP reduces the salinity errors exhibited by LETKF, e.g. below 200 m in 1997–1998, throughout 0–400 m from 1999–2001. The anomalously high surface salinities in sum- mer 2002 are reduced by LETKF-RIP, though this correction appears to have caused fresh anomalies in the subsequent winter months for LETKF-RIP1. In other cases, LETKF-RIP increases the salinity errors, e.g. the autumn months of 1997, 2000, 2002 and 2003. In winter 2002, errors are improved by 29 At Station Aloha in the subtropical North Paciﬁc, the up- per 400 m contains a warm salty layer at 0–200 m with tem- peratures of 18◦−23 ◦C and salinities 34.9–35.2 psu, drop- ping to 14 ◦C and 34.1 psu by 400 m depth. The seasonal cy- cle at this location is relatively weak. Salinity in this upper layer typically exhibits a subsurface maximum reﬂecting its origin through subduction along the subtropical front. Dur- ing our period of interest conditions were anomalously dry, leading to heavier than normal surface water and more exten- sive exchanges throughout the upper 200 m (Lukas, 2001). The Free Run at this location has temperatures that are too cool by several degrees and salinities that are too fresh by 0.3 psu (more consistent with salinities earlier in the decade). Again, the analyses all show a reduction in the temperature (Fig. 6) and salinity deviations (Fig. 7) from observations. In all experiments, temperatures remain too cold in the upper 300 m. SODA, LETKF2 and LETKF-RIP2 remain too fresh in the upper 200–300 m. This fresh bias is corrected starting in 2000 by LETKF1, and to a greater degree LETKF-RIP1, with some slight overshooting. However, as in the case for Station S, LETKF1 and LETKF-RIP1 analysis errors have more variation in time than the other methods. This is ex- pected, as SODA uses a rolling window of observations that extends well beyond the analysis cycle and is implemented via a forcing term (via IAU). S. G. Penny et al.: The local ensemble transform Kalman ﬁlter observation error profile shown for refere 6 7 8 fresh anomalies that are trapped near the surface. LETKF seems to still be adjusting mean temperatures throughout the early years of the experiment and thus are still too cold between 200–400 m, whereas in SODA they are too warm. This corrective warming occurs more gradually in LETKF but is accelerated by LETKF-RIP. All analyses show erro- neously warm surface temperatures in winter. In most cases, LETKF-RIP reduces the error compared to LETKF, though there is a slight increase in errors in the summer of 2000 by LETKF-RIP1. Both methods seem to improve the warm win- ter anomalies versus SODA. While LETKF1 does this by allowing freshwater anomalies at the surface, the effect is mitigated by LETKF-RIP. The summer of 2002 is unusual in all three experiments in showing anomalously cool wa- ter throughout the upper 400 m relative to observed condi- tions, which were themselves anomalously cool with deep penetration of the winter mixed layer. During the anoma- lous conditions of summer 2002, all three assimilation ex- periments exhibit elevated surface salinities, while SODA has anomalously low salinity below 200 m. In some cases, LETKF-RIP reduces the salinity errors exhibited by LETKF, e.g. below 200 m in 1997–1998, throughout 0–400 m from 1999–2001. The anomalously high surface salinities in sum- mer 2002 are reduced by LETKF-RIP, though this correction appears to have caused fresh anomalies in the subsequent winter months for LETKF-RIP1. In other cases, LETKF-RIP increases the salinity errors, e.g. the autumn months of 1997, 2000, 2002 and 2003. In winter 2002, errors are improved by observation error profile shown for refere 6 7 8 fresh anomalies that are trapped near the surface. LETKF seems to still be adjusting mean temperatures throughout the early years of the experiment and thus are still too cold between 200–400 m, whereas in SODA they are too warm. This corrective warming occurs more gradually in LETKF but is accelerated by LETKF-RIP. All analyses show erro- neously warm surface temperatures in winter. In most cases, LETKF-RIP reduces the error compared to LETKF, though there is a slight increase in errors in the summer of 2000 by LETKF-RIP1. Both methods seem to improve the warm win- ter anomalies versus SODA. While LETKF1 does this by allowing freshwater anomalies at the surface, the effect is mitigated by LETKF-RIP. S. G. Penny et al.: The local ensemble transform Kalman ﬁlter S. G. Penny et al.: The local ensemble transform Kalman ﬁlter 1040 S. G. Penny et al.: The local ensemble transform Kalman ﬁlter 1 2 Figure 8. Mean temperature and salinity (analysis minus independent bottle measurements) 3 RMSD at Station S and Aloha Station corresponding to Figures 4-7. Results are shown for the 4 1 1 0 0.5 1 1.5 2 2.5 0 100 200 300 400 500 RMSD (ºC) Station S Mean Temperature RMSD Depth (m) 0 0.2 0.4 0.6 0.8 1 0 100 200 300 400 500 RMSD (psu) Station S Mean Salinity RMSD Depth (m) 0 0.5 1 1.5 2 2.5 0 100 200 300 400 500 RMSD (ºC) ALOHA Station Mean Temperature RMSD Depth (m) 0 0.2 0.4 0.6 0.8 1 0 100 200 300 400 500 RMSD (psu) ALOHA Station Mean Salinity RMSD Depth (m) Fig. 8. Mean temperature and salinity (analysis minus independent bottle measurements) RMSD at Station S and Aloha Station corresponding to Figs. 4–7. Results are shown for the Free Run (solid grey), SODA (green), LETKF1 (blue), and LETKF-RIP1 (purple), with the observation error proﬁle shown for reference (dashed grey). .5 0 0.2 0.4 0.6 0.8 1 0 100 200 300 400 500 RMSD (psu) Station S Mean Salinity RMSD Depth (m) 0 0.5 1 1.5 2 2.5 0 100 200 300 400 500 RMSD (ºC) Station S Mean Temperature RMSD Depth (m) 0 0.2 0.4 0.6 0.8 1 0 100 200 300 400 500 RMSD (psu) Station S Mean Salinity RMSD Depth (m) 0 0.2 0.4 0.6 0.8 1 0 100 200 300 400 500 RMSD (psu) ALOHA Station Mean Salinity RMSD Depth (m) 0 0.5 1 1.5 2 2.5 0 100 200 300 400 500 RMSD (ºC) ALOHA Station Mean Temperature RMSD Depth (m) ALOHA Station Mean Temperature RMSD Figure 8. Mean temperature and salinity (analysis minus independent bottle measurements) 3 RMSD at Station S and Aloha Station corresponding to Figures 4-7. Results are shown for the 4 Fig. 8. Mean temperature and salinity (analysis minus independent bottle measurements) RMSD at Station S and Aloha Station corresponding to Figs. 4–7. Results are shown for the Free Run (solid grey), SODA (green), LETKF1 (blue), and LETKF-RIP1 (purple), with the observation error proﬁle shown for reference (dashed grey). (dashed grey). LETKF-RIP1 over LETKF1, but worsened by LETKF-RIP2 over LETKF2. LETKF , LETKF RIP , LETKF , LE 4 www.nonlin-processes-geophys.net/20/1031/2013/ LETKF , LETKF RIP , LETKF , LE 4 www.nonlin-processes-geophys.net/20/1031/2013/ LETKF , LETKF RIP , LETKF , LETKF RIP . 4 www.nonlin-processes-geophys.net/20/1031/2013/ Nonlin. Processes Geophys., 20, 1031–1046, 2013 Nonlin. Processes Geophys., 20, 1031–1046, 2013 Nonlin. Processes Geophys., 20, 1031–1046, 2013 1040 S. G. Penny et al.: The local ensemble transform Kalman ﬁlter S. G. Penny et al.: The local ensemble transform Kalman ﬁlter 1041 1 Figure 9. Monthly averaged zonal velocity at (0N, 165E) for observations and analyses from 2 Observed Free Run SODA LETKF1 LETKF-RIP1 LETKF2 LETKF-RIP2 Fig. 9. Monthly averaged zonal velocity at (0◦N, 165◦E) for observations and analyses from January 1997 to December 2003 from 30 to 270 m, with time mean removed. From top to bottom: Observed ADCP data, Free Run, SODA, LETKF1, LETKF-RIP1, LETKF2, LETKF- RIP2. 1 Fi 9 M thl d l l it t (0N 165E) f b ti d l f 2 Observed Free Run SODA LETKF1 LETKF-RIP1 LETKF2 LETKF-RIP2 Fig. 9. Monthly averaged zonal velocity at (0◦N, 165◦E) for observations and analyses from January 1997 to December 2003 from 30 to 270 m, with time mean removed. From top to bottom: Observed ADCP data, Free Run, SODA, LETKF1, LETKF-RIP1, LETKF2, LETKF- 2 1 Figure 9. Monthly averaged zonal velocity at (0N, 165E) for observations and analyses from 2 Fig. 9. Monthly averaged zonal velocity at (0◦N, 165◦E) for observations and analyses from January 1997 to December 2003 from 30 to 270 m, with time mean removed. From top to bottom: Observed ADCP data, Free Run, SODA, LETKF1, LETKF-RIP1, LETKF2, LETKF- RIP2. Observed ADCP data, Free Run, SODA, LET 4 spatial localization and the more gradual time smoothing pa- rameter used for adaptive inﬂation. F1, LETKF-RIP1, LETKF2, LETKF-RIP2. model, all of the methods underestimate the intensity of the observed zonal velocity. Therefore, we remove the time mean of each ﬁeld and examine the monthly mean observed and analyzed zonal velocity anomalies. Due to the large obser- vation window used in its analysis cycle, SODA appears to average out much of the small-scale temporal variability. In contrast, the ensemble methods exhibit some features on smaller temporal scales. The time mean (A–O) RMSD are shown for the Free Run, SODA, LETKF1 and LETKF-RIP1, relative to the observa- tion error proﬁle. At both stations, the mean temperature de- viations are reduced by all methods. For Station S, LETKF- RIP1 gives the greatest improvement in the top 200 m, fol- lowed by LETKF1 and SODA. From 200 to 500 m SODA give results closest to the observation error proﬁle, followed by LETKF-RIP1 and LETKF1. LETKF-RIP1 and LETKF1 have larger salinity RMSD at Station S in the upper 270 m, closer to the estimated observation error proﬁle. S. G. Penny et al.: The local ensemble transform Kalman ﬁlter While be- low 270 m, the RMSD is smallest with LETKF-RIP1, then LETKF1 and SODA, the latter being closest to the estimated observation error. At Aloha Station all methods give approx- imately equal RMSD, though LETKF-RIP1 is lower overall. The salinity RMSD at this station are lowest with SODA and closest to the estimated observation error with LETKF-RIP1. 30 At 165◦E (Fig. 8), a strong positive anomaly occurs throughout much of 1998. This feature is captured in part by all methods, but is strongest in LETKF2 and LETKF-RIP2. A negative anomaly that exists weakly in the Free Run in early 2002 appears to be represented most accurately by LETKF- RIP1,2. At 170◦W (Fig. 9), the overall anomaly pattern that alternates between positive and negative appears to be represented most accurately by LETKF-RIP1. The positive anomaly of 1998 is best captured by LETKF2 and LETKF- RIP2. At 140◦W (Fig. 10), SODA best represents the strong positive anomalies, though all of the methods strengthen the signal compared to the Free Run. LETKF2 has a slight rep- resentation of the positive anomaly that occurs just after the strong negative anomaly at the start of 1998. While absent in the SODA result, some of the weaker signals throughout 3.4 Comparison with independent observations of velocity We next compare to the observed zonal velocity proﬁles in the equatorial Paciﬁc. Due to the coarse resolution of the Nonlin. Processes Geophys., 20, 1031–1046, 2013 www.nonlin-processes-geophys.net/20/1031/2013/ S. G. Penny et al.: The local ensemble transform Kalman ﬁlter Both LETKF2 and LETKF- RIP2 exhibit a more gradual adjustment due to the larger www.nonlin-processes-geophys.net/20/1031/2013/ Nonlin. Processes Geophys., 20, 1031–1046, 2013 S. G. Penny et al.: The local ensemble transform Kalman ﬁlter 4 Conclusions 31 We have performed a comparison of the Local Ensemble Transform Kalman Filter (LETKF) to a benchmark ocean as- similation system (SODA) using an identical global ocean model with identical observed temperature and salinity pro- ﬁle data set, initial conditions, and surface forcing. Two ver- sions of LETKF were examined: the standard LETKF and LETKF-RIP, which uses the “running-in-place” (RIP) algo- rithm of Kalnay and Yang (2010) and Yang et al. (2012a,b). The results from each experiment were evaluated through two approaches: (1) through comparison of the background ﬁeld to the (not yet assimilated) observations of tempera- ture and salinity, and (2) through comparisons of the anal- yses to independent observations that included ocean station time series in the North Paciﬁc and North Atlantic subtropi- cal gyres, ADCP zonal velocity in the equatorial Paciﬁc, and major western boundary transports. S. G. Penny et al.: The local ensemble transform Kalman ﬁlter 1 Figure 10. Monthly averaged zonal velocity at (0N, 170W) for observations and analyses 2 Observed Free Run SODA LETKF1 LETKF-RIP1 LETKF2 LETKF-RIP2 Fig. 10. Monthly averaged zonal velocity at (0◦N, 170◦W) for observations and analyses from January 1997 to December 2003 from 30 to 270 m, with time mean removed. From top to bottom: observed ADCP data, Free Run, SODA, LETKF1, LETKF-RIP1, LETKF2, LETKF-RIP2. 1 Figure 10. Monthly averaged zonal velocity at (0N, 170W) for observations and analyses 2 Fig. 10. Monthly averaged zonal velocity at (0◦N, 170◦W) for observations and analyses from January 1997 to December 2003 from 30 to 270 m, with time mean removed. From top to bottom: observed ADCP data, Free Run, SODA, LETKF1, LETKF-RIP1, LETKF2, LETKF-RIP2. p 1, LETKF-RIP1, LETKF2, LETKF-RIP2. level and heat content. The transports in the Gulf Stream and Kuroshio are increased slightly toward the observed values. The transport in the Agulhas is relatively unchanged com- pared to the observed and modeled variance. Observed ADCP data, Free Run, SODA, LET 4 the experiment seem to be present in LETKF1 and LETKF- RIP2. At 110◦W (Fig. 11), the analysis signal corresponding most to the observed signal appears to come from LETKF1. All of the methods have a negative anomaly at the surface that conﬂict with the observed signal, though SODA has the largest error. A number of observed positive anomalies are present in the Free Run and strengthened in the LETKF1 and LETKF-RIP1 analyses. SODA generates some spurious positive anomalies throughout 2001 and 2002. LETKF1 and LETKF-RIP1 best capture the negative anomy at the end of 2002, while none of the methods adequately capture the al- ternating positive and negative anomalies of 2003. www.nonlin-processes-geophys.net/20/1031/2013/ 1042 S. G. Penny et al.: The local ensemble transform Kalman ﬁlter 3.5 Volume transport in select western boundary currents Volume transports for the Gulf Stream, Kuroshio Current, and Agulhas Current are given in Table 4 as compared with published observed values. All assimilation methods im- proved transport toward observed levels when compared to the Free Run baseline. The increased transport in the ma- jor ocean basins, combined with a geostrophic balance as imposed by the model, implies an increased density gradi- ent within the basin and thus an increased anomaly of sea www.nonlin-processes-geophys.net/20/1031/2013/ Nonlin. Processes Geophys., 20, 1031–1046, 2013 S. G. Penny et al.: The local ensemble transform Kalman ﬁlter S. G. Penny et al.: The local ensemble transform Kalman ﬁlter 1043 S. G. Penny et al.: The local ensemble transform Kalman ﬁlter 1043 1 Figure 11. Monthly averaged zonal velocity at (0N, 140W) for observations and analyses 2 Observed Free Run SODA LETKF1 LETKF-RIP1 LETKF2 LETKF-RIP2 Fig. 11. Monthly averaged zonal velocity at (0◦N, 140◦W) for observations and analyses from January 1997 to December 2003 from 30 to 270 m, with time mean removed. From top to bottom: observed ADCP data, Free Run, SODA, LETKF1, LETKF-RIP1, LETKF2, LETKF-RIP2. 1 Figure 11. Monthly averaged zonal velocity at (0N, 140W) for observations and analyses 2 Fig. 11. Monthly averaged zonal velocity at (0◦N, 140◦W) for observations and analyses from January 1997 to December 2003 from 30 to 270 m, with time mean removed. From top to bottom: observed ADCP data, Free Run, SODA, LETKF1, LETKF-RIP1, LETKF2, LETKF-RIP2. F1, LETKF-RIP1, LETKF2, LETKF-RIP2. substantial biases of both temperature and salinity. These bi- ases were reduced by all assimilation methods. At Aloha a cool, fresh bias remains in the high salinity upper 200 m, while a slight warm bias with elevated salinity is generally present between 200–400 m. The time series at Aloha is no- table for cool anomalies in 1998–1999, and for a period of enhanced salinity beginning in the late 1990s where salin- ities exceeded 35.1 psu. These anomalies were weak in the Free Run, but better reproduced in the assimilation experi- ments. During the years of the high salinity anomaly SODA has salinity errors below 0.4 psu. LETKF-RIP shows much smaller average salinity errors, but with stronger year-to-year variability. Observed ADCP data, Free Run, SODA, LET 4 Examination of temperature observation-minus- background differences shows a reduction of errors compared to the SODA baseline when using both LETKF and LETKF-RIP, with the latter showing the greatest impact especially during the ﬁrst four years of spin up. Globally, temperature observation-minus-background differences were smaller for LETKF-RIP than for SODA while salinity remained relatively unchanged compared the magnitude of estimated observation errors. We emphasize that both the LETKF and LETKF-RIP background ﬁelds, which did not use future data relative to the analysis time and whose RMS deviations were computed against observations that were not yet assimilated, achieved lower RMSD than SODA which did use future data. S. G. Penny et al.: The local ensemble transform Kalman ﬁlter Regionally the same results were found for temperature, while salinity RMSD was typically smaller in well-observed areas and larger in poorly observed areas. The reduction in background error implies a corresponding improvement of the prior analysis, however additional comparisons were made to independent observed data to assess the analyses themselves. 32 At Station S the Free Run also exhibits large biases in tem- perature and salinity, with too warm temperatures in the up- per 200 m, too cold temperatures below, and too fresh every- where. The three assimilation experiments all reduced these mean biases, though all three have a residual seasonal bias in near-surface temperature (too warm in winter, too cold in summer) likely reﬂecting errors in surface heating. As- similation substantially reduced the fresh bias in all three experiments. Both LETKF1 and LETKF-RIP1 exhibit more year-to-year variations in salinity error than SODA, while LETKF2 and LETKF-RIP2 exhibit somewhat less, indicating In order to evaluate the performance of the experiments in the subtropical gyres the analyses were compared against withheld ocean station time series at Aloha near Hawaii, and Station S near Bermuda. At these stations the Free Run has www.nonlin-processes-geophys.net/20/1031/2013/ Nonlin. Processes Geophys., 20, 1031–1046, 2013 www.nonlin-processes-geophys.net/20/1031/2013/ 1044 S. G. Penny et al.: The local ensemble transform Kalman ﬁlter 1 Figure 12. Monthly averaged zonal velocity at (0N, 110W) for observations and analyses 2 Observed Free Run SODA LETKF1 LETKF-RIP1 LETKF2 LETKF-RIP2 Fig. 12. Monthly averaged zonal velocity at (0◦N, 110◦W) for observations and analyses from January 1997 to December 2003 from 30 to 270 m, with time mean removed. From top to bottom: observed ADCP data, Free Run, SODA, LETKF1, LETKF-RIP1, LETKF2, LETKF-RIP2. 1 Figure 12. Monthly averaged zonal velocity at (0N, 110W) for observations and analyses 2 Fig. 12. Monthly averaged zonal velocity at (0◦N, 110◦W) for observations and analyses from January 1997 to December 2003 from 30 to 270 m, with time mean removed. From top to bottom: observed ADCP data, Free Run, SODA, LETKF1, LETKF-RIP1, LETKF2, LETKF-RIP2. 33 p 1, LETKF-RIP1, LETKF2, LETKF-RIP2. This LETKF system has been implemented at the National Centers for Environmental Prediction (NCEP) with the op- erational GFDL ocean model that is currently used by the Global Ocean Data Assimilation System (GODAS), which is a component of NCEP’s Coupled Forecast System (CFS). The LETKF algorithm will form the ensemble component of a hybrid ocean data assimilation system that is being de- veloped for use by NCEP. For the purpose of reanalysis, the practically cost-free RIP step applying the LETKF weights obtained at the end of each assimilation window to the en- semble at the beginning of the window may be used to im- prove the previous analysis and thus smooth the overall time evolution of the analyzed ﬁelds. In addition, this LETKF system will be used to assimilate the ocean component of a strongly coupled data assimilation system that is currently being designed by the University of Maryland for India’s Na- tional Monsoon Mission. Observed ADCP data, Free Run, SODA, LET 4 that the localization and inﬂation parameters can be tuned to control how rapidly LETKF adjusts to available observa- tions. 5 Due to model resolution, the modeled zonal velocities at the equator were much weaker than observed. After remov- ing the time mean, comparisons to observed zonal velocities in the equatorial Paciﬁc still showed that many of the ob- served anomalies were either weak or not present in the Free Run. www.nonlin-processes-geophys.net/20/1031/2013/ However, all of the assimilation methods improved the pattern of anomalies, typically strengthening both the posi- tive and negative anomalies to better coincide with the ob- served anomalies. This study has a number of limitations. The model, cho- sen for its computational efﬁciency, does not resolve the ocean mesoscale. The lack of mesoscale processes likely contributes to the presence of large mean biases. The ex- periments are limited to a seven-year period, which is insuf- ﬁcient to examine the impact of differences in assimilation on longer time-scale processes. While we made great efforts to make a fair comparison between methods, differences in practical implementation made a truly exact comparison im- possible. Acknowledgements. We gratefully acknowledge: the Laboratory for Microbial Oceanography within the School of Ocean and Earth Science and Technology at the University of Hawaii at Manoa which maintains the Aloha time series, the Bermuda Biological Station for Research which maintains Station S, and the National Oceanographic Data Center which maintains global hydrographic Nonlin. Processes Geophys., 20, 1031–1046, 2013 References Lukas, R.: Freshening of the upper thermocline in the North Pa- ciﬁc subtropical gyre associated with decadal changes of rainfall, Geophys. Res. Lett., 28, 3485–3488, 2001. Carton, J. A. and Santorelli, A.: Global Decadal Upper-Ocean Heat Content as Viewed in Nine Analyses, J. Climate, 21, 6015–6035, 2008. Miyoshi, T.: The Gaussian Approach to Adaptive Covariance Inﬂa- tion and its Implementation with the Local Ensemble Transform Kalman Filter, Mon. Weather Rev., 139, 1519–1535, 2011. Carton, J. 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https://openalex.org/W2566537557	https://europepmc.org/articles/pmc5186779?pdf=render	English	null	Comparison of Local Information Indices Applied in Resting State Functional Brain Network Connectivity Prediction	Frontiers in neuroscience	2,016	cc-by	13,166	Comparison of Local Information Indices Applied in Resting State Functional Brain Network Connectivity Prediction Chen Cheng 1, 2, Junjie Chen 1, Xiaohua Cao 3 and Hao Guo 1, 2* 1 Department of Computer Science and Technology, Taiyuan University of Technology, Taiyuan, China, 2 National Laboratory of Pattern Recognition, Institute of Automation, Chinese Academy of Sciences, Beijing, China, 3 Department of Psychiatry, First Hospital of Shanxi Medical University, Taiyuan, China 1 Department of Computer Science and Technology, Taiyuan University of Technology, Taiyuan, China, 2 National Laboratory of Pattern Recognition, Institute of Automation, Chinese Academy of Sciences, Beijing, China, 3 Department of Psychiatry, First Hospital of Shanxi Medical University, Taiyuan, China Anatomical distance has been widely used to predict functional connectivity because of the potential relationship between structural connectivity and functional connectivity. The basic implicit assumption of this method is “distance penalization.” But studies have shown that one-parameter model (anatomical distance) cannot account for the small-worldness, modularity, and degree distribution of normal human brain functional networks. Two local information indices–common neighbor (CN) and preferential attachment index (PA), are introduced into the prediction model as another parameter to emulate many key topological of brain functional networks in the previous study. In addition to these two indices, many other local information indices can be chosen for investigation. Different indices evaluate local similarity from different perspectives. Currently, we still have no idea about how to select local information indices to achieve higher predicted accuracy of functional connectivity. Here, seven local information indices are chosen, including CN, hub depressed index (HDI), hub promoted index (HPI), Leicht-Holme-Newman index (LHN-I), Sørensen index (SI), PA, and resource allocation index (RA). Statistical analyses were performed on eight network topological properties to evaluate the predictions. Analysis shows that different prediction models have different performances in terms of simulating topological properties and most of the predicted network properties are close to the real data. There are four topological properties whose average relative error is less than 5%, including characteristic path length, clustering coefﬁcient, global efﬁciency, and local efﬁciency. CN model shows the most accurate predictions. Statistical analysis reveals that ﬁve properties within the CN-predicted network do not differ signiﬁcantly from the real data (P > 0.05, false-discovery rate method corrected for seven comparisons). PA model shows the worst prediction performance which was ﬁrst applied in models of growth networks. Our results suggest that PA is not suitable for predicting connectivity in a small-world network. Furthermore, in order to evaluate the predictions rapidly, prediction power was proposed as an evaluation metric. The current study compares the predictions of functional connectivity with seven local information indices and provides a reference of method selection for construction of prediction models. Keywords: functional connectivity, local information, link prediction, brain network, graph theory ORIGINAL RESEARCH published: 27 December 2016 doi: 10.3389/fnins.2016.00585 Edited by: Jorge Bosch-Bayard, UNAM, Campus Juriquilla, Mexico Reviewed by: Roberto C. Sotero, University of Calgary, Canada Hui-Jie Li, Institute of Psychology (CAS), China Courtney Gallen, University of California, Berkeley, USA *Correspondence: Hao Guo feiyu_guo@sina.com Reviewed by: Roberto C. Sotero, University of Calgary, Canada Hui-Jie Li, Institute of Psychology (CAS), China Courtney Gallen, University of California, Berkeley, USA Specialty section: This article was submitted to Brain Imaging Methods, a section of the journal Frontiers in Neuroscience Specialty section: This article was submitted to Brain Imaging Methods, a section of the journal Frontiers in Neuroscience Received: 11 January 2016 Accepted: 07 December 2016 Published: 27 December 2016 Received: 11 January 2016 Accepted: 07 December 2016 Published: 27 December 2016 INTRODUCTION On the contrary, some studies have shown that strong functional connectivity may exist among regions lacking structural connectivity (Honey et al., 2009). This suggests that information in the network is not only transmitted directly through structural paths, but may also be aﬀected by network topology (Adachi et al., 2012). Additionally, analyses of the anatomical distance (Euclidean distance) between brain regions have shown that functional connectivity can be interpreted as a “distance penalty.” That means the closer two brain regions are, the stronger the functional connectivity is (Alexander-Bloch et al., 2013a,b). Although neuroscience investigations that apply local information methods have been conducted at the micro-scale, few have done macro-scale analyses. Vértes applied local information methods to connectivity prediction in resting-state functional brain networks and showed that the best predictions came from the model that combined anatomical distance with the indices—“common neighbor” (CN) (Vértes et al., 2012) among a dozen models. Common neighbor is one of local information indices whose mathematical deﬁnition is the number of neighbors that two locations x and y have in common. However, long-distance functional interactions cannot be explained by the distance penalty (Vértes et al., 2012), implying that relying solely on anatomical distance is not enough and we need to combine other factors to achieve better predictions of resting-state brain functional connectivity. Some researchers regard neuronal activity as a bridge between structural and functional connectivity. Several models of neural activity have been proposed, including neural mass models (Ponten et al., 2010), neural ﬁeld models (Power et al., 2013), the Kuramoto model (Cabral et al., 2011), and spiking models (Nakagawa et al., 2013). Other researchers have applied brain network topological information as parameters to model functional connectivity. Nodal degree is a commonly used attribute based on the basic assumptions of random models. The probability of a connection existing between the two regions is proportional to the product of their degrees (Newman, 2010). Several diﬀerent network topological properties have been proposed to predict the existence of connectivity in resting-state functional brain networks, including structural degree (Tewarie et al., 2014), degree distribution (Friedman et al., 2014), network communication measure (Goñi et al., 2014), and local information (Vértes et al., 2012). Local information reveals the topological similarity of nodes and reﬂects local topological coherence in networks (Lü et al., 2015). INTRODUCTION Local information is the simplest direct method in link- prediction research, which utilizes relevant network topological information to predict the possibility of an edge between two given nodes in a network (Getoor and Diehl, 2005). Link prediction reﬂects the eﬀect of inherent network topology characteristics during the process of network evolution (Wang et al., 2012). As a measurement of topological similarity between two given nodes, local information is the most commonly used method to predict the probability of connections between them (Lü and Zhou, 2011). Because of its signiﬁcant practical value, local information has been widely used in several scientiﬁc ﬁelds including information research (Popescul and Ungar, 2003), biomedical research (Stumpf et al., 2008), mobile communications (Dasgupta et al., 2008), and social networks (Kossinets, 2006; Kumar et al., 2010). In neuroscience, methods that use local information have been applied to simulate neural remodeling that occurs during the learning and memorizing tasks (Ziv and Ahissar, 2009), predicting connectivity of neuronal synapses in the rat primary visual cortex (Bock et al., 2011), optimizing component rearrangements to reduce total wiring length in the macaque nervous system (Kaiser and Hilgetag, 2006), analyzing network properties in the Caenorhabditis.elegans neuronal network (Varshney et al., 2011), and constructing local neuronal circuits in patients with autism (Markram et al., 2007). As a combination of non-invasive brain imaging techniques and complex network theory, brain networks have been widely used to characterize the morphology (Hagmann et al., 2008) and functional characteristics (Buldyrev et al., 2010) of human brain. Recently, increasing number of researchers have focused on the relationship between structural and functional networks. Structural connectivity has been deﬁned with diﬀusion imaging and tractography, while functional connectivity has been deﬁned as a time-series correlation between regions of interest (ROIs). Studies have found out structural connectivity is closely related to resting-state functional connectivity at both macro- (Honey et al., 2009; Hermundst et al., 2013) and micro- scales (Wang et al., 2013). Related studies have demonstrated that regions with structural connectivity also exhibit a strong functional connectivity. This ﬁnding suggests that functional connectivity might be predicted by characteristics of brain topology (Alexander-Bloch et al., 2013b; Ercsey-Ravasz et al., 2013). Here, “predict” means to deduce the strength or existence of functional connectivity between two ROIs in a resting-state network. The prediction model is mathematical expression of the prediction method. Citation: Citation: Cheng C, Chen J, Cao X and Guo H (2016) Comparison of Local Information Indices Applied in Resting State Functional Brain Network Connectivity Prediction. Front. Neurosci. 10:585. doi: 10.3389/fnins.2016.00585 December 2016 \| Volume 10 \| Article 585 Frontiers in Neuroscience \| www.frontiersin.org 1 Prediction Method for Brain Functional Connectivity Cheng et al. INTRODUCTION The basic implicit assumption of local information is that the more similar the topology between two given nodes, the higher the probability of an edge existing between them (Lü and Zhou, 2011). This method has been validated by the research in which two local information indices—“common neighbor” and “preferential attachment” (PA)—were introduced with the mathematical deﬁnition of the models for predicting resting-state functional connectivity (Vértes et al., 2012). In addition to these two indices, many other local information indices can be chosen for investigation. Diﬀerent indices evaluate nodal similarity from diﬀerent perspectives. Currently, we still have no idea about how to select a local information index to achieve higher predicted accuracy of functional connectivity. To address this issue, we performed a similar experiment mentioned above with two main diﬀerences. Firstly, we separately evaluated the inclusion of seven local information indices into the model and compared the prediction accuracy among indices. Secondly, prediction assessment was performed with a reliable and rapid method that avoided vast amounts of calculation and contrastive analysis of network topological December 2016 \| Volume 10 \| Article 585 Frontiers in Neuroscience \| www.frontiersin.org 2 Prediction Method for Brain Functional Connectivity Cheng et al. properties. The results showed that adding local information to the model allowed good simulations of functional brain network, which reﬂected its basic characteristics, such as high clustering coeﬃcient, high local eﬃciency, hub nodes, and small- worldness. Among the local information indices that were tested, “common neighbor” resulted in the best predictions. These results were consistent with the previous research (Vértes et al., 2012), despite using diﬀerent mathematical models, methods for evaluating topological properties and indices for evaluating network similarity. The current study compares the predictions of functional connectivity with seven local information indices and provides a reference of method selection for construction of prediction models. as well as the six parameters from motion correction. The residuals of these regressions constituted the set of regional mean time-series used for undirected graph analysis. Pearson correlation coeﬃcients among all node pairs in the network were calculated to generate a 90 × 90 correlation matrix. According to predeﬁned thresholds, the correlation matrix was converted into a binary matrix. See supplemental Text S2 for a detailed mathematical deﬁnition of the Pearson correlation coeﬃcient. In the contrast analysis of the complex networks, the compared networks must have the same number of nodes and edges (Bollobás, 2001). Network Construction An automated anatomical labeling atlas was used to deﬁne network nodes (Tzourio-Mazoyer et al., 2002). The whole brain was divided into 90 regions (45 in each hemisphere) and each region was deﬁned as a node in the network. Each regional mean time-series was regressed against the average cerebral spinal ﬂuid (CSF) and white matter signals INTRODUCTION Because the quantitative values of topological metrics will depend on size and connection density of the graphs. In order to identify topological diﬀerences between graphs pointed to the diﬀerence between groups, it is important to control these general eﬀects before making any quantitative comparisons. Sparsity (S) was chosen as the threshold to control the number of edges in the networks. S was deﬁned as the ratio of real existing edges to the maximum possible number of existing edges. We set the threshold space to be Sǫ[5%, 40%] because this is the standard used in similar studies (Bullmore and Bassett, 2011) and assures the small-worldness of network, which is one of the most important features in human functional brain networks (Bullmore and Sporns, 2009). Considering the high computing costs, we set the interval in threshold space to 5%. Supplemental Figure S2 illustrates the small-world scalar as a function of sparsity. Prediction Model Mathematic Deﬁnition Prediction Model Mathematic Deﬁnition Anatomical distance and nodal local information indices were chosen as parameters to deﬁne the mathematical model. Euclidean distance was chosen to deﬁne the distance between two given ROIs. Although Euclidean distance between nodal centroids is an imperfect approximation of the anatomical distance between the regions, it has previously been shown to be comparable to more reﬁned diﬀusion imaging-based measures of connection distance (Supekar et al., 2009). Considering the positive inﬂuence that similar topology between nodes has on connections, the introduced nodal local information indices were those used to measure similarity between nodes in complex networks (Lü and Zhou, 2011). The mathematical deﬁnition of the prediction model was: Data preprocessing was performed with SPM8 (http://www.ﬁl.ion.ucl.ac.uk/spm). First, slice-timing correction and head-movement correction were carried out. Two samples exhibiting more than 3.0 mm of translation and 3.0◦of rotation were discarded which were not included in the ﬁnal 28 samples. The corrected images were optimized with a 12-dimensional aﬃne transformation and normalized to 3 × 3 × 3 mm voxel in the Montreal Neurological Institute (MNI) standard space. Finally, linearly detrending and band-pass ﬁltering (0.01– 0.10 Hz) were performed to reduce the eﬀects of low-frequency drift and high frequency physiological noise. Pi, j = di, j si, j γ , where Pi, j is the probability that a connection between node i and node j exists, di, j is the anatomical distance (Euclidean distance) between node i and node j, si, j represents the local information indices (seven of which were chosen in the current study), and γ is a constant parameter. Considering computing costs, γ was set to [0, 3], with a step length of 0.1. The modeling process is illustrated in Figure 1. Data Acquisition and Preprocessing q p g This study was carried out in accordance with the recommendations of the medical ethics committee of Shanxi Province (reference number: 2012013) with written informed consent from all subjects. All subjects have been given written informed consent in accordance with the Declaration of Helsinki. Twenty-eight healthy right-handed volunteers (13 male; mean age: 26.6 ± 9.4 years, range: 17–51 years) underwent resting-state functional magnetic resonance imaging (fMRI) in a 3T MR scanner (Siemens Trio 3-Tesla scanner, Siemens, Erlangen, Germany). Data collection was completed at the First Hospital of Shanxi Medical University. All scans were performed by radiologists who were familiar with magnetic resonance. During the scan, participants were asked to relax with their eyes closed but not to fall asleep. Each scan consisted of 248 contiguous EPI functional volumes (33 axial slices, repetition time (TR) = 2000 ms, echo time (TE) = 30 ms, thickness/skip = 4/0 mm, ﬁeld of view (FOV) = 192 × 192 mm, matrix = 64 × 64 mm, ﬂip angle = 90◦) and the ﬁrst 10 volumes of time series were discarded regarding magnetization stabilization. See Supplemental Text S1 for detail scanning parameters. Local Information Indices We chose to assess seven indices for their usefulness in functional connectivity prediction, including common neighbors (CN) (Liben-Nowell and Kleinberg, 2007), hub depressed index (HDI) (Ravasz et al., 2002), hub promoted index (HPI) (Ravasz et al., 2002), Leicht-Holme-Newman index (LHN-I) (Leicht et al., 2006), Sørensen index (SI) (Sørensen, 1948), preferential attachment index (PA) (Barabasi and Albert, 1999) and resource allocation index (RA) (Zhou et al., 2009). These indices were incorporated into the model’s mathematical formula to generate seven functional connectivity prediction models (Table 1). See previous study (Vértes et al., 2012). “Common neighbors” is deﬁned as the number of common direct neighbors (nodes that have edges with both nodes x and y) between the given nodes x and y. The underlying assumption is that the more common their direct neighbors are, the more similar the topology of a node pair will be and the more likely an edge will be between them. In addition to “common neighbors,” there are many other local information indices. Diﬀerent indices describe similarity in network nodal topology with diﬀerent perspectives. We chose to assess seven indices for their usefulness in functional connectivity prediction, including common neighbors (CN) (Liben-Nowell and Kleinberg, 2007), hub depressed index (HDI) (Ravasz et al., 2002), hub promoted index (HPI) (Ravasz et al., 2002), Leicht-Holme-Newman index (LHN-I) (Leicht et al., 2006), Sørensen index (SI) (Sørensen, 1948), preferential attachment index (PA) (Barabasi and Albert, 1999) and resource allocation index (RA) (Zhou et al., 2009). These indices were incorporated into the model’s mathematical formula to generate seven functional connectivity prediction models (Table 1). See Local Information Indices The local information index termed “common neighbors” was used for resting-state functional connectivity prediction in a December 2016 \| Volume 10 \| Article 585 Frontiers in Neuroscience \| www.frontiersin.org 3 Cheng et al. Prediction Method for Brain Functional Connectivity FIGURE 1 \| Illustration of network modeling process with local information indices. For a given real network, we could calculate the local information between all of the node pairs and generate the corresponding local information matrix (e.g., common neighbor in the illustration), which could be plugged into the model’s mathematical deﬁnition to generate a predicted network. Compared with real data, some missing and spurious edges might exist, which could lead to some changes in the network’s topological properties. In previous research, differences in the topological properties between real data and the predicted networks are always used to quantitatively evaluate the model-predicted effect. FIGURE 1 \| Illustration of network modeling process with local information indices. For a given real network, we could calculate the local information between all of the node pairs and generate the corresponding local information matrix (e.g., common neighbor in the illustration), which could be plugged into the model’s mathematical deﬁnition to generate a predicted network. Compared with real data, some missing and spurious edges might exist, which could lead to some changes in the network’s topological properties. In previous research, differences in the topological properties between real data and the predicted networks are always used to quantitatively evaluate the model-predicted effect. Supplemental Text S3 for detailed mathematical deﬁnitions of the indices. An illustration of prediction networks based on diﬀerent local information indices is shown in Supplemental Figure S1. previous study (Vértes et al., 2012). “Common neighbors” is deﬁned as the number of common direct neighbors (nodes that have edges with both nodes x and y) between the given nodes x and y. The underlying assumption is that the more common their direct neighbors are, the more similar the topology of a node pair will be and the more likely an edge will be between them. In addition to “common neighbors,” there are many other local information indices. Diﬀerent indices describe similarity in network nodal topology with diﬀerent perspectives. Network Topology Properties In the above formulas, Si, j is the local information between node i and j, Γi is the direct neighbors of node i, ki is the degree of node i, and node z is the common neighbor of node i and j. See Supplemental Text S3 for a detailed mathematical explanation of the above indices. TABLE 1 \| Local information indices in the current study. Deformation indices of CN. This index is used mainly for ecological community data. In the above formulas, Si, j is the local information between node i and j, Γi is the direct neighbors of node i, ki is the degree of node i, and node z is the common neighbor of node i and j. See Supplemental Text S3 for a detailed mathematical explanation of the above indices. TABLE 2 \| Network topological properties in the current study. Property Symbol Description Assortativity R A measure of the correlation between the degree of a node and the mean degree of its nearest neighbors (Newman, 2002). Clustering Coefﬁcient C The ratio of the actual number of edges between direct neighbor nodes of given nodes to the number of maximum possible edges for those nodes (Newman, 2003). Characteristic Path Length L The average of the shortest path lengths from a given node to other nodes in the network (Newman, 2003). Degree Distribution P(k) Frequency distribution of nodal degree in the network (Amaral et al., 2000). Global Efﬁciency Eglob The efﬁciency of information transmission in the whole network (Latora and Marchiori, 2001). Local Efﬁciency Eloc The efﬁciency of information transmission from each node to the adjacent nodes (Latora and Marchiori, 2001). Modularity Q The degree to which the network may be subdivided into clearly delineated and non-overlapping groups (Newman and Girvan, 2004). Transitivity T A variant of the clustering coefﬁcient (Newman, 2003). TABLE 2 \| Network topological properties in the current study. The average of the shortest path lengths from a given node to other nodes in the network (Newman, 2003). The efﬁciency of information transmission in the whole network (Latora and Marchiori, 2001). The efﬁciency of information transmission from each node to the adjacent nodes (Latora and Marchiori, 2001). The degree to which the network may be subdivided into clearly delineated and non-overlapping groups (Newman and Girvan, 2004). re = (pd −pm) pd × 100%, dependent variables: age, gender, and educational attainments). Network Topology Properties p gy p Eight common global topological properties were chosen for prediction: assortativity, clustering coeﬃcient, characteristic path length, degree distribution, global eﬃciency, local eﬃciency, modularity, and transitivity (Table 2). Each property was plotted and the area under the curve (AUC) was calculated. This provided a summarized scalar for the selected threshold space and was a widely used technique in similar studies (Achard and Bullmore, 2007). Multiple linear regression analyses were applied to remove the confounding eﬀects of age, gender and educational attainments for each network properties excluded degree distribution (diﬀerent from other topological properties, degree distribution shows a distribution function) (independent variable: the AUC of each network properties; December 2016 \| Volume 10 \| Article 585 Frontiers in Neuroscience \| www.frontiersin.org 4 Prediction Method for Brain Functional Connectivity Cheng et al. TABLE 1 \| Local information indices in the current study. Local information index Index abbreviation Mathematical deﬁnition Description Common Neighbors (Liben-Nowell and Kleinberg, 2007) CN si, j = Γi ∩Γj The number of neighbors that x and y have in common. Hub Depressed Index (Ravasz et al., 2002) HDI si, j = Γi ∩Γj max[ki, kj] Deformation indices of CN. The links adjacent to hubs are probably assigned high scores since the denominator is determined by the higher degree only. Hub Promoted Index (Ravasz et al., 2002) HPI si, j = Γi ∩Γj min[ki, kj] Deformation indices of CN. An opposite index to HDI. Leicht-Holme-Newman Index (Leicht et al., 2006) LHN-I si, j = Γi ∩Γj ki × kj Deformation indices of CN. This index gives high similarity to node pairs that have many common neighbors compared not to the possible maximum, but to the expected number of such neighbors. Preferential Attachment (Sørensen, 1948) PA si, j = ki × kj An index can be used to generate evolving scale-free networks. The probability this new link is connecting x and y is proportional to ki × kj. Resource Allocation (Barabasi and Albert, 1999) RA si, j = P z∈Γi ∩Γj 1 kz This index is motivated by the resource allocation dynamics on complex networks. The similarity between x and y can be deﬁned as the amount of resource y received from x. Sørensen Index (Zhou et al., 2009) SI si, j = 2 Γi ∩Γj ki + kj Deformation indices of CN. This index is used mainly for ecological community data. Network Topology Properties The result showed the signiﬁcant correlation had not been found between network properties and confounding variables (Table 3). where pd is the property value of the real network and pm is the property value of the predicted network. The evaluation of degree distribution is special. Diﬀerent from other topological properties, degree distribution shows a distribution function. In the current study, the predicted networks and the real data exhibited an exponential truncated power- law distribution, but the diﬀerences were reﬂected in two parameters: the estimated exponent and the cutoﬀdegree. To quantitatively evaluate the diﬀerences between degree distribution functions, the average relative error of the two parameters was computed. The average relative error was deﬁned as: where pd is the property value of the real network and pm is the property value of the predicted network. The evaluation of degree distribution is special. Diﬀerent from other topological properties, degree distribution shows a distribution function. In the current study, the predicted networks and the real data exhibited an exponential truncated power- law distribution, but the diﬀerences were reﬂected in two parameters: the estimated exponent and the cutoﬀdegree. To quantitatively evaluate the diﬀerences between degree distribution functions, the average relative error of the two parameters was computed. The average relative error was deﬁned as: Frontiers in Neuroscience \| www.frontiersin.org In the above formulas, Si, j is the local information between node i and j, Γi is the direct neighbors of node i, ki is the degree of node i, and node z is the common neighbor of node i and j. See Supplemental Text S3 for a detailed mathematical explanation of the above indices. December 2016 \| Volume 10 \| Article 585 Evaluation of the Prediction Model To measure the statistical signiﬁcance of the prediction, we compared the predicted topological properties with the real data using a two-sample paired nonparametric test, which was corrected with Benjamini and Hochberg false-discovery rate (FDR) method (q = 0.05) (Benjamini and Hochberg, 1995). False-discovery rate method, retaining strong control over type 1 error in the context of multiple comparisons, was considered appropriate to correct the small number of comparisons entailed by testing the whole 28 subjects. As has been done in similar EEG (Astolﬁet al., 2004), MEG (Fasoula et al., 2013), and structural MRI (Jovicich et al., 2006) studies, we computed the relative error (Guimerà and Sales-Pardo, 2009) to quantitatively evaluate the between-group diﬀerences. Relative error was deﬁned as: MRI (Jovicich et al., 2006) studies, we computed the relative error (Guimerà and Sales-Pardo, 2009) to quantitatively evaluate the between-group diﬀerences. Relative error was deﬁned as: reP(k) = reα + rekc 2 × 100%, Frontiers in Neuroscience \| www.frontiersin.org 5 December 2016 \| Volume 10 \| Article 585 uted the relative error titatively evaluate the was deﬁned as: reP(k) = reα + rekc 2 × 100%, 5 December 2016 \| Volume 10 \| Article 585 reP(k) = reα + rekc 2 × 100%, December 2016 \| Volume 10 \| Article 585 Frontiers in Neuroscience \| www.frontiersin.org 5 Prediction Method for Brain Functional Connectivity Cheng et al. TABLE 3 \| Results of multiple linear regression analysis between network properties and confounding variables. Confounding variables Coefﬁcients Std. Error T Stat. P Lower 95% Upper 95% ASSORTATIVITY (Adj. Rsqr = −0.579, P = 0.681) Intercept 0.100 0.087 1.145 0.263 −0.080 0.281 Gender 0.026 0.029 0.876 0.389 −0.035 0.088 Age 0.000 0.001 0.466 0.645 −0.002 0.004 Educational Attainments 0.005 0.013 0.383 0.704 −0.022 0.032 CLUSTERING COEFFICIENT (Adj. Rsqr = 0.004, P = 0.391) Intercept 0.419 0.014 29.243 <0.001 0.389 0.448 Gender 0.007 0.004 1.472 0.153 −0.002 0.017 Age 0.000 0.000 −1.119 0.274 0.000 0.000 Educational Attainments 0.001 0.002 0.532 0.599 −0.003 0.005 CHARACTERISTIC PATH LENGTH (Adj. Rsqr = 0.064, P = 0.629) Intercept 0.647 0.065 25.331 <0.001 0.513 0.781 Gender 0.001 0.022 1.890 0.196 −0.003 0.087 Age 0.026 0.029 0.876 0.389 −0.035 0.088 Educational Attainments 0.000 0.009 −0.049 0.961 −0.021 0.020 GLOBAL EFFICIENCY (Adj. Evaluation of the Prediction Model Rsqr = 0.074, P = 0.187) Intercept 0.383 0.015 24.332 <0.001 0.351 0.416 Gender −0.008 0.005 −1.651 0.111 −0.019 0.002 Age 0.000 0.000 −0.915 0.369 0.000 0.000 Educational Attainments −0.001 0.002 −0.518 0.608 −0.006 0.003 LOCAL EFFICIENCY (Adj. Rsqr = −0.046, P = 0.623) Intercept 0.535 0.018 28.857 <0.001 0.497 0.574 Gender 0.001 0.006 0.213 0.832 −0.011 0.014 Age 0.000 0.000 1.890 0.196 −0.001 0.000 Educational Attainments 0.000 0.002 −0.062 0.951 −0.006 0.005 MODULARITY (Adj. Rsqr = −0.052, P = 0.650) Intercept 0.279 0.038 7.341 <0.001 0.201 0.358 Gender −0.004 0.013 −0.323 0.749 −0.031 0.022 Age 0.000 0.000 −0.388 0.701 −0.001 0.001 Educational Attainments −0.006 0.005 −1.10 0.278 −0.018 0.005 TRANSITIVITY (Adj. Rsqr = 0.022, P = 0.327) Intercept 0.393 0.033 11.672 <0.001 0.323 0.463 Gender 0.018 0.011 1.592 0.124 0.000 0.042 Age 0.000 0.000 0.549 0.587 0.000 0.001 Educational Attainments 0.000 0.005 0.087 0.930 −0.010 0.011 The range of age is 17–51 years. Optional values of gender are male and female. Optional values of educational attainments are illiteracy, primary school, junior high school, senior high school, junior college, college, graduate degree, and above. Adj. Rsqr, adjusted R square; Coefﬁcients, regression coefﬁcient; Std. Error, standard error; T stat., T statistic; Lower 95%, low bound of 95% conﬁdence limits. Upper 95%, upper bound of 95% conﬁdence limits. TABLE 3 \| Results of multiple linear regression analysis between network properties and confounding variables. ltiple linear regression analysis between network properties and confounding variables. The range of age is 17–51 years. Optional values of gender are male and female. Optional values of educational attainments are illiteracy, primary school, junior high school, senior high school, junior college, college, graduate degree, and above. Adj. Rsqr, adjusted R square; Coefﬁcients, regression coefﬁcient; Std. Error, standard error; T stat., T statistic; Lower 95%, low bound of 95% conﬁdence limits. Upper 95%, upper bound of 95% conﬁdence limits. where reα is the relative error of the estimated exponent and rekc is the relative error of the cutoﬀ degree. where reR is the relative error in assortativity, reC is the relative error in the clustering coeﬃcient, reL is the relative error in the characteristic path length, reEloc is the relative error in local eﬃciency, reEglob is the relative error in global eﬃciency, reQ is the relative error in modularity, reT is the relative error in transitivity, and reP(k) is the relative error in degree distribution. Frontiers in Neuroscience \| www.frontiersin.org Evaluation of the Prediction Model To measure the similarity between two networks, we comprehensively considered several network topological properties and deﬁned the network of similar indices, energy E, to evaluate the outcome of connectivity prediction. The deﬁnition of the E-value used here did not consider the weight of the properties. Thus, all of the network’s topology properties had equal importance in the model. Energy was deﬁned as: Faced with numerous local information indices, we needed a reliable and rapid prediction evaluation method in order to avoid a large amount of calculation and contrastive analyses of the topological properties. We hypothesized that the more covered the edges were between predicted networks and real data, the better the prediction would be. To test the hypothesis, prediction power was proposed as a metric and a correlation 1 E = reR + reC + reL + reEloc + reEglob + reQ + reT + reP(k) , E = reR + reC + reL + reEloc + reEglob + reQ + reT + reP(k) , December 2016 \| Volume 10 \| Article 585 Frontiers in Neuroscience \| www.frontiersin.org 6 Prediction Method for Brain Functional Connectivity Cheng et al. FIGURE 2 \| Average relative error of seven selected local information indices for different topological properties. The mathematical deﬁnition of relative error is (pd−pm) pd × 100%. Topological properties were sorted by average relative error. The results showed that the average relative errors of four properties were around or below 5%. CN, common neighbor; HDI, hub depressed index; HDI, hub promoted index; LHN-I, Leicht-Holme-Newman index; SI, Sørensen index; PA, preferential attachment index; RA, resource allocation index. analysis was performed between the E-value and the prediction power. In similar studies, prediction power is often used to evaluate link-prediction eﬀects (Cannistraci et al., 2013). Higher prediction power indicates a better prediction, while the closer the prediction power is to 0, the more random the prediction is. Prediction power is deﬁned as: Prediction Power = 10 × log10 PreM PreR , Prediction Power = 10 × log10 PreM PreR , where PreM is the ratio of the number of correct edges in a prediction network model to the number of existing edges in real data, and PreR is the ratio of the number of correct edges in a network model using random prediction methods to the number of existing edges in real data. RESULTS The average relative error for each network topological property was used to evaluate the predictions. Analysis showed that the diﬀerent prediction models had diﬀerent performances in terms of simulating topological properties and most of the predicted network properties were close to the real data (Figure 2). There are four topological properties whose average relative error is less than 5%, including characteristic path length, clustering coeﬃcient, global eﬃciency, and local eﬃciency. Modularity and transitivity had relative errors that ranged from 5 to 10%, while assortativity and degree distribution had average relative errors around 40%. Thus, most of the predicted global network properties were close to the real data, except for assortativity and degree distribution. See Supplemental Figure S3 for the detailed information about the distribution of relative errors for each topological property. To comprehensively evaluate the predictions of all the network topological properties in the seven models, we deﬁned a uniﬁed measurement metric termed energy (E). The E-value considered all eight topological property diﬀerences. The higher the E-value is, the more similar the predicted networks and real data are. The result of ANOVA analysis showed that there were signiﬁcant diﬀerences among seven models (F = 30.529, P < 0.0001, uncorrected). The results showed that the performance for the CN model was the best among the seven models, with the RA model being second best, and the PA model being the worst (Figure 4A). Relative error can be used to quantitatively measure how diﬀerent predicted networks are from real data. To determine if there were any statistically signiﬁcant between-group diﬀerences, we performed a two-sample paired, nonparametric test with false-discovery rate correction (q = 0.05; degree distribution was not statistically analyzed because of its speciﬁcity). The results showed that properties with high relative error were always signiﬁcantly diﬀerent and low relative error did not necessarily indicate a lack of signiﬁcant diﬀerences. Properties with high relative error, such as assortativity (Figure 3A) and modularity (Figure 3G), showed signiﬁcant diﬀerences in most of the models (P < 0.05, FDR corrected for 7 comparisons), except for the CN and RA models. Properties with low relative error, such as characteristic path length (Figure 3B), clustering coeﬃcient (Figure 3C), global eﬃciency (Figure 3E), local global (Figure 3F), and transitivity (Figure 3H), showed signiﬁcant diﬀerences in some of the models. Degree distribution was not analyzed because of its particularity (Figure 3D). Evaluation of the Prediction Model See Supplemental Text S4 for a detailed mathematical deﬁnition and explanation of prediction power. FIGURE 2 \| Average relative error of seven selected local information indices for different topological properties. The mathematical deﬁnition of (p p ) FIGURE 2 \| Average relative error of seven selected local information indices for different topological properties. The mathematical deﬁnition of relative error is (pd−pm) pd × 100%. Topological properties were sorted by average relative error. The results showed that the average relative errors of four properties were around or below 5%. CN, common neighbor; HDI, hub depressed index; HDI, hub promoted index; LHN-I, Leicht-Holme-Newman index; SI, Sørensen index; PA, preferential attachment index; RA, resource allocation index. FIGURE 2 \| Average relative error of seven selected local information indices for different topological properties. The mathematical deﬁnition of relative error is (pd−pm) pd × 100%. Topological properties were sorted by average relative error. The results showed that the average relative errors of four properties were around or below 5%. CN, common neighbor; HDI, hub depressed index; HDI, hub promoted index; LHN-I, Leicht-Holme-Newman index; SI, Sørensen index; PA, preferential attachment index; RA, resource allocation index. RESULTS These results suggest that although the property values of the predicted networks were close to those of the real data, the tiny diﬀerences were signiﬁcant. Here, we proposed that prediction power could be used to rapidly evaluate the predictions immediately after model generation instead of requiring large computing costs and contrast analysis of model properties. We compared the predictions among seven local information indices by prediction power as well. Signiﬁcant diﬀerences also has been found among seven models (F = 100.36, P < 0.0001, uncorrected) after ANOVA analysis. The result was very similar to the E value. Both the evaluation metrics showed the similar results. CN showed the best predicted eﬀect and PA showed the worst (Figure 4B). The only change is that SI showed better performance than HDI by prediction power. We performed a correlation analysis between prediction power and E value and corrected using the Benjamini & Hochberg false-discovery rate method (q = 0.05) (Figure 5; Benjamini and Hochberg, 1995). The four models that showed a signiﬁcant positive correlation (p < 0.05, FDR corrected for 7 comparisons) were HDI, HPI, and LHN-I. The CN and Sorensen models showed a marginally signiﬁcant correlation (0.05 < p < 0.10, FDR corrected for 7 comparisons). Notably, completely December 2016 \| Volume 10 \| Article 585 Frontiers in Neuroscience \| www.frontiersin.org 7 Prediction Method for Brain Functional Connectivity Cheng et al. et al. Prediction Method for Brain Functional Connect RE 3 \| Topological properties of real data and predicted networks. Error bars show standard deviation. Asterisks indicate a signiﬁcant difference betwe ata and the predicted networks (p < 0.05, FDR corrected for 7 comparisons). The statistical test method was a two-sample paired nonparametric test and the cted method was Benjamini and Hochberg false-discovery rate method (q = 0.05). The illustration of degree distribution was on the sparsity of 15%. CN, mon neighbor; HDI, hub depressed index; HDI, hub promoted index; LHN-I, Leicht-Holme-Newman index; SI, Sørensen index; PA, preferential attachment inde esource allocation index. FIGURE 3 \| Topological properties of real data and predicted networks. Error bars show st FIGURE 3 \| Topological properties of real data and predicted networks. Error bars show standard deviation. Asterisks indicate a signiﬁcant difference between real data and the predicted networks (p < 0.05, FDR corrected for 7 comparisons). RESULTS The statistical test method was a two-sample paired nonparametric test and the corrected method was Benjamini and Hochberg false-discovery rate method (q = 0.05). The illustration of degree distribution was on the sparsity of 15%. CN, common neighbor; HDI, hub depressed index; HDI, hub promoted index; LHN-I, Leicht-Holme-Newman index; SI, Sørensen index; PA, preferential attachment index; RA, resource allocation index. December 2016 \| Volume 10 \| Article 585 8 Frontiers in Neuroscience \| www.frontiersin.org Prediction Method for Brain Functional Connectivity Cheng et al. FIGURE 4 \| E value and prediction power comparison among local information indices. (A) The E-value was used to evaluate the predictions comprehensively. Local information indices were sorted by E-value. (B) Prediction power was a rapid evaluation metric. Local information indices were sorted by prediction power. Both the evaluation methods showed the similar results that common neighbor showed the best predicted effect and preferential attachment index showed the worst in seven selected local information indexes. Error bars show standard deviation. The F-value and P-value was the result of one-way ANOVA analysis (uncorrected). The right illustration was the P-value of two-sample paired T-test between any two indices (FDR corrected for 21 comparisons, q = 0.05). CN, common neighbor; HDI, hub depressed index; HDI, hub promoted index; LHN-I, Leicht-Holme-Newman index; SI, Sørensen index; PA, preferential attachment index; RA, resource allocation index. FIGURE 4 \| E value and prediction power comparison among local information indices. (A) The E-value was used to evaluate the predictions comprehensively. Local information indices were sorted by E-value. (B) Prediction power was a rapid evaluation metric. Local information indices were sorted by prediction power. Both the evaluation methods showed the similar results that common neighbor showed the best predicted effect and preferential attachment index showed the worst in seven selected local information indexes. Error bars show standard deviation. The F-value and P-value was the result of one-way ANOVA analysis (uncorrected). The right illustration was the P-value of two-sample paired T-test between any two indices (FDR corrected for 21 comparisons, q = 0.05). CN, common neighbor; HDI, hub depressed index; HDI, hub promoted index; LHN-I, Leicht-Holme-Newman index; SI, Sørensen index; PA, preferential attachment index; RA, resource allocation index. diﬀerent from the other models, the PA model showed a signiﬁcantly negative correlation (p < 0.0001, FDR corrected for 7 comparisons). RESULTS organization as well as a new viewpoint on the understanding and explanation of network organization (Wang et al., 2012; Zhang et al., 2013). The results of analysis of E value were consistent with that of another study (Vértes et al., 2012), even though the mathematical model, method of topological property evaluation and indices for evaluating network similarity were diﬀerent. Similar to our results, predictions from that study were best for the CN model and worst for the PA model (see the detailed comparisons in Table 4). The previous study focused on diﬀerent mathematical deﬁnitions of the prediction model, while our current study focused on comparing prediction ability of a single mathematical model that incorporated diﬀering local information indexes. Frontiers in Neuroscience \| www.frontiersin.org DISCUSSION This means that a signiﬁcant triadic closure structure (Liben-Nowell and Kleinberg, 2007; Zhou et al., 2009) exists in resting-state functional brain networks, which might cause the high clustering coeﬃcient and local eﬃciency that have been found in resting-state functional brain networks in previous studies (see Bullmore and Bassett, 2011 for a review). This conclusion has been demonstrated in network models with similar properties in the real world, including protein–protein interaction networks (Von Mering et al., 2002), US political blog networks (Ackland, 2005), US air-transportation system networks (Batageli and Pajek, 2006) and social collaboration networks (Newman, 2001). On the contrary, two properties are signiﬁcantly diﬀerent between real data and CN model, including characteristic path length and global eﬃciency. Both properties are related to the long-range links in the network. Compared with regular network, the long-range links in a small-world network ensure the lower characteristic path length, the higher global eﬃciency and the higher information transferring eﬃciency. CN evaluated the local similarity but was not sensitive to the long-range links. neighborhood-based measures. In the current study, signiﬁcant diﬀerences in prediction accuracy were found between CN and other neighborhood-based measures, which suggest a high degree of heterogeneity in functional brain networks. The high degree of heterogeneity is an important characteristic of power- law degree distribution (Espinosa et al., 2012), which has been found in the resting-state functional brain network in many studies (see Bullmore and Bassett, 2011 for a review). Preferential attachment (PA) index was calculated with the least information (only the nodal degree)and resulted in the least accurate predictions. PA was ﬁrst applied to models of growth networks (Barabasi and Albert, 1999; Mitzenmacher, 2004). The basic premise was that the probability that an edge has node x as an endpoint is proportional to the current number of neighbors of x. Similar mechanisms could also lead to scale- free networks without growth (Xie et al., 2008). Therefore, PA resulted in accurate predictions in a scale-free network. However, PA performance was disappointing, compared with other indices in small-world networks, such as human functional brain networks (Vértes et al., 2012) and nervous system networks for other species (Cannistraci et al., 2013). Studies have shown that PA performs badly in a rich-club network consisting of many components (Zhou et al., 2009). The same properties exist in human functional brain networks (van den Heuvel and Sporns, 2011). DISCUSSION As a characteristic of network topology, we proposed local information as a ﬁtting parameter for the predicted models. Local information characterizes network topology and reﬂects network’s local similarity. Our research was able to predict the existence of connections in the brain functional network with local information. The results showed that local information improved the accuracy of predictions. Among the eight network topology properties, most showed good ﬁtting: the relative errors of six properties (characteristic path length, clustering coeﬃcient, global eﬃciency, local eﬃciency, modularity, and transitivity) were less than 10%. Additionally, as an eﬃcient type of information within network topology, local information might provide strong evidence regarding the mechanisms of network The mathematical deﬁnition of CN is the number of neighbors that two locations x and y have in common. Among the seven models, CN model showed the most accurate predictions. Statistical analysis revealed that ﬁve properties (assortativity, clustering coeﬃcient, local eﬃciency, modularity, and transitivity) within the CN-predicted network did not diﬀer signiﬁcantly from the real data (P > 0.05, FDR corrected for December 2016 \| Volume 10 \| Article 585 9 Prediction Method for Brain Functional Connectivity Cheng et al. FIGURE 5 \| The correlation analysis between E-value and prediction power. Prediction power was used to evaluate predicted effect rapidly, whose mathematical deﬁnition was 10 × log10 PreM PreR . The results showed that there was a strong correlation between E value and prediction power. Adj_Rsqr, adjusted R square; CN, common neighbor; HDI, hub depressed index; HDI, hub promoted index; LHN-I, Leicht-Holme-Newman index; SI, Sørensen index; PA, preferential attachment index; RA, resource allocation index. FIGURE 5 \| The correlation analysis between E-value and prediction power. Prediction power was used to evaluate predicted effect rapidly, whose mathematical deﬁnition was 10 × log10 PreM PreR . The results showed that there was a strong correlation between E value and prediction power. Adj_Rsqr, adjusted R square; CN, common neighbor; HDI, hub depressed index; HDI, hub promoted index; LHN-I, Leicht-Holme-Newman index; SI, Sørensen index; PA, preferential attachment index; RA, resource allocation index. seven comparisons). Meanwhile, the E-value for CN model was the highest among all models. As a measurement of network local connectivity, better predictions by CN model implies a higher local connected density in the network. Frontiers in Neuroscience \| www.frontiersin.org DISCUSSION Results are sorted by energy value in descending order. Topological properties evaluation method relative error, whose mathematical deﬁnition is re = (pd−pm) pd × 100% . Lower value indicates the closer properties between real data and simulated networks. Network similarity indices, energy, was deﬁned as = 1 reR + reC + reL + reEloc + reEglob + reQ + reT + reP(k) . Higher value indicates the more similar between real data and simulated networks. In the previous similar research (Vértes et al., 2012), results are sorted by energy value in cending order. Topological properties evaluation method is the P-value associated with the t-test for a difference. P-value is higher than 0.05 indicates no signiﬁcant difference between real data and stimulated networks. Network milar indices is deﬁned as E = 1 PC × PEglob × PQ×Pp(k) . Lower value indicates the more similar between real data and simulated networks. P(k), Degree Distribution; R, Assortativity; C, Clustering Coefﬁcient; L, Characteristic Path ngth; Eglob, Global Efﬁciency; Eloc, Local Efﬁciency; Q, Modularity; T, Transitivity; CN, common neighbor; HDI, hub depressed index; HDI, hub promoted index; LHN-I, Leicht-Holme-Newman index; SI, Sørensen index; PA, preferential TABLE 4 \| Comparison of simulated effect evaluation between the current research and the previous similar research. Model Local information Abbr. DISCUSSION Mathematical deﬁnition Topological properties comparison Energy P(k) R Q T L C Eglob Eloc THE CURRENT RESEARCH Pi, j = di, j si, j γ Common Neighbors CN si, j = Γi ∩Γj 0.353 0.318 0.075 0.038 0.048 0.029 0.041 0.025 1.839 Resource Allocation RA si, j = P z∈Γi ∩Γj 1 kz 0.645 0.354 0.085 0.069 0.043 0.049 0.041 0.022 1.212 Hub Depressed Index HDI si, j = Γi ∩Γj max[ki, kj] 0.767 0.410 0.122 0.052 0.042 0.037 0.027 0.020 1.034 Sørensen Index SI si, j = 2 Γi ∩Γj ki + kj 0.670 0.396 0.118 0.048 0.041 0.036 0.025 0.021 1.026 Hub Promoted Index HPI si, j = Γi ∩Γj min[ki, kj] 1.066 0.435 0.143 0.062 0.050 0.044 0.024 0.023 0.729 Leicht-Holme-Newman Index LHN-I si, j = Γi ∩Γj ki × kj 1.235 0.439 0.162 0.064 0.047 0.051 0.022 0.028 0.628 Preferential Attachment PA si, j = ki × kj 1.540 0.414 0.221 0.056 0.047 0.037 0.042 0.037 0.534 THE PREVIOUS SIMILAR RESEARCH (Vé t t l 2012) HE PREVIOUS SIMILAR RESEARCH (Vértes et al., 2012) j = exp si, j γ (di, j)−η Common Neighbors CN si, j = Γi ∩Γj 2 × 10−4 – 0.10 – – 0.79 0.74 – 105 j = si, j γ exp(−ηdi, j) Common Neighbors CN si, j = Γi ∩Γj 10−3 – 0.24 – – 0.85 0.02 – 2 × 105 j = si, j γ (di, j)−η Variant of Hub Promoted Index Variant of HPI si, j = max[ki, kj] 5.5 × 10−4 – 0.05 – – 0.95 0.08 – 5 × 105 j = si, j γ exp(−ηdi, j) Variant of Hub Promoted Index Variant of HPI si, j = max[ki, kj] 1.5 × 10−5 – 0.33 – – 0.41 0.88 – 5 × 105 j = si, j γ (di, j)−η Variant of Sørensen Index Variant of SI si, j = ki + kj 7 × 10−6 – 0.12 – – 0.27 0.78 – 6 × 106 j = si, j γ exp(−ηdi, j) Variant of Sørensen Index Variant of SI si,j = ki + kj 10−4 – 0.008 – – 0.007 0.95 – 2 × 108 j = si, j γ exp(di, j)−η Variant of Hub Depressed Index Variant of HDI si, j = min[ki, kj] 9 × 10−6 – 0.0003 – – 0.05 0.34 – 2 × 1010 j = si, j γ (di, j)−η Preferential Attachment PA si, j = ki × kj 3 × 10−9 – 0.001 – – 0.01 0.80 – 3 × 1013 j = si, j γ exp(−ηdi, j) Variant of Hub Depressed Index Variant of HDI si, j = min[ki, kj] 10−9 – 0.004 – – 0.001 0.65 – 2 × 1014 j = si, j γ exp(−ηdi, j) Preferential Attachment PA si, j = ki × kj 1.5 × 10−11 – 0.0001 – – 0.005 0.51 – 2 × 1017 e above two researches deﬁne different topological properties evaluation method and network similarity evaluation indices. DISCUSSION P(k), Degree Distribution; R, Assortativity; C, Clustering Coefﬁcient; L, Characteristic Path Length; Eglob, Global Efﬁciency; Eloc, Local Efﬁciency; Q, Modularity; T, Transitivity; CN, common neighbor; HDI, hub depressed index; HDI, hub promoted index; LHN-I, Leicht-Holme-Newman index; SI, Sørensen index; PA, preferential attachment index; RA, resource allocation index. December 2016 \| Volume 1 ABLE 4 \| Comparison of simulated effect evaluation between the current research and the previous similar research. odel Local information Abbr. DISCUSSION Mathematical deﬁnition Topological properties comparison Energy P(k) R Q T L C Eglob Eloc HE CURRENT RESEARCH j = di, j si, j γ Common Neighbors CN si, j = Γi ∩Γj 0.353 0.318 0.075 0.038 0.048 0.029 0.041 0.025 1.839 Resource Allocation RA si, j = P z∈Γi ∩Γj 1 kz 0.645 0.354 0.085 0.069 0.043 0.049 0.041 0.022 1.212 Hub Depressed Index HDI si, j = Γi ∩Γj max[ki, kj] 0.767 0.410 0.122 0.052 0.042 0.037 0.027 0.020 1.034 Sørensen Index SI si, j = 2 Γi ∩Γj ki + kj 0.670 0.396 0.118 0.048 0.041 0.036 0.025 0.021 1.026 Hub Promoted Index HPI si, j = Γi ∩Γj min[ki, kj] 1.066 0.435 0.143 0.062 0.050 0.044 0.024 0.023 0.729 Leicht-Holme-Newman Index LHN-I si, j = Γi ∩Γj ki × kj 1.235 0.439 0.162 0.064 0.047 0.051 0.022 0.028 0.628 Preferential Attachment PA si, j = ki × kj 1.540 0.414 0.221 0.056 0.047 0.037 0.042 0.037 0.534 HE PREVIOUS SIMILAR RESEARCH (Vértes et al., 2012) j = exp si, j γ (di, j)−η Common Neighbors CN si, j = Γi ∩Γj 2 × 10−4 – 0.10 – – 0.79 0.74 – 105 j = si, j γ exp(−ηdi, j) Common Neighbors CN si, j = Γi ∩Γj 10−3 – 0.24 – – 0.85 0.02 – 2 × 105 j = si, j γ (di, j)−η Variant of Hub Promoted Index Variant of HPI si, j = max[ki, kj] 5.5 × 10−4 – 0.05 – – 0.95 0.08 – 5 × 105 j = si, j γ exp(−ηdi, j) Variant of Hub Promoted Index Variant of HPI si, j = max[ki, kj] 1.5 × 10−5 – 0.33 – – 0.41 0.88 – 5 × 105 j = si, j γ (di, j)−η Variant of Sørensen Index Variant of SI si, j = ki + kj 7 × 10−6 – 0.12 – – 0.27 0.78 – 6 × 106 j = si, j γ exp(−ηdi, j) Variant of Sørensen Index Variant of SI si,j = ki + kj 10−4 – 0.008 – – 0.007 0.95 – 2 × 108 j = si, j γ exp(di, j)−η Variant of Hub Depressed Index Variant of HDI si, j = min[ki, kj] 9 × 10−6 – 0.0003 – – 0.05 0.34 – 2 × 1010 j = si, j γ (di, j)−η Preferential Attachment PA si, j = ki × kj 3 × 10−9 – 0.001 – – 0.01 0.80 – 3 × 1013 j = si, j γ exp(−ηdi, j) Variant of Hub Depressed Index Variant of HDI si, j = min[ki, kj] 10−9 – 0.004 – – 0.001 0.65 – 2 × 1014 j = si, j γ exp(−ηdi, j) Preferential Attachment PA si, j = ki × kj 1.5 × 10−11 – 0.0001 – – 0.005 0.51 – 2 × 1017 e above two researches deﬁne different topological properties evaluation method and network similarity evaluation indices. DISCUSSION Results are sorted by energy value in descending order. Topological properties evaluation method relative error, whose mathematical deﬁnition is re = (pd−pm) pd × 100% . Lower value indicates the closer properties between real data and simulated networks. Network similarity indices, energy, was deﬁned as = 1 reR + reC + reL + reEloc + reEglob + reQ + reT + reP(k) . Higher value indicates the more similar between real data and simulated networks. In the previous similar research (Vértes et al., 2012), results are sorted by energy value in ending order. Topological properties evaluation method is the P-value associated with the t-test for a difference. P-value is higher than 0.05 indicates no signiﬁcant difference between real data and stimulated networks. Network ilar indices is deﬁned as E = 1 PC × PEglob × PQ×Pp(k) . Lower value indicates the more similar between real data and simulated networks. P(k), Degree Distribution; R, Assortativity; C, Clustering Coefﬁcient; L, Characteristic Path ngth; Eglob, Global Efﬁciency; Eloc, Local Efﬁciency; Q, Modularity; T, Transitivity; CN, common neighbor; HDI, hub depressed index; HDI, hub promoted index; LHN-I, Leicht-Holme-Newman index; SI, Sørensen index; PA, preferential achment index; RA, resource allocation index. The above two researches deﬁne different topological properties evaluation method and network similarity evaluation indices. Results are sorted by energy value in descending order. Topological properties evaluation method is relative error, whose mathematical deﬁnition is re = (pd−pm) pd × 100% . Lower value indicates the closer properties between real data and simulated networks. Network similarity indices, energy, was deﬁned as E = 1 reR + reC + reL + reEloc + reEglob + reQ + reT + reP(k) . Higher value indicates the more similar between real data and simulated networks. In the previous similar research (Vértes et al., 2012), results are sorted by energy value in ascending order. Topological properties evaluation method is the P-value associated with the t-test for a difference. P-value is higher than 0.05 indicates no signiﬁcant difference between real data and stimulated networks. Network similar indices is deﬁned as E = 1 PC × PEglob × PQ×Pp(k) . Lower value indicates the more similar between real data and simulated networks. December 2016 \| Volume 10 \| Article 585 DISCUSSION Our results show that PA is not suitable for predicting functional connectivity. Aside from the basic CN model, we also tested HDI, HPI, LHN-I, and SI models, which are deformation indices of CN model. These indices and CN were mentioned as neighborhood- based measures. These deformation indices were subjected to the inﬂuence of nodal degree (see the mathematical formula in Table 1). Degree heterogeneity was a measure used to quantify the amount of variation or dispersion of degree of all the nodes in a network (Barabasi and Albert, 1999). If nodal degrees tended to be the same, degree heterogeneity would be very small and there would be no obvious diﬀerence between these Unlike other indices, RA focuses on the degree of direct neighbor. Consider a pair of nodes, x and y, which are not directly connected. Node x can send resources to y, with their common neighbors playing the role of transmitters. In the simplest case, we assume that each transmitter has one unit of resource and will distribute it evenly to all its neighbors. The similarity between x and y can be deﬁned as the amount of December 2016 \| Volume 10 \| Article 585 10 Prediction Method for Brain Functional Connectivity Cheng et al. The above two researches deﬁne different topological properties evaluation method and network similarity evaluation indices. Results are sorted by energy value in descending order. Topological properties evaluation method is relative error, whose mathematical deﬁnition is re = (pd−pm) pd × 100% . Lower value indicates the closer properties between real data and simulated networks. Network similarity indices, energy, was deﬁned as E = 1 reR + reC + reL + reEloc + reEglob + reQ + reT + reP(k) . Higher value indicates the more similar between real data and simulated networks. In the previous similar research (Vértes et al., 2012), results are sorted by energy value in ascending order. Topological properties evaluation method is the P-value associated with the t-test for a difference. P-value is higher than 0.05 indicates no signiﬁcant difference between real data and stimulated networks. Network similar indices is deﬁned as E = 1 PC × PEglob × PQ×Pp(k) . Lower value indicates the more similar between real data and simulated networks. Frontiers in Neuroscience \| www.frontiersin.org DISCUSSION P(k), Degree Distribution; R, Assortativity; C, Clustering Coefﬁcient; L, Characteristic Path Length; Eglob, Global Efﬁciency; Eloc, Local Efﬁciency; Q, Modularity; T, Transitivity; CN, common neighbor; HDI, hub depressed index; HDI, hub promoted index; LHN-I, Leicht-Holme-Newman index; SI, Sørensen index; PA, preferential attachment index; RA, resource allocation index. , j , j , j , j j Pi, j = si, j γ exp(−ηdi, j) Variant of Hub Depressed Index Variant of HDI si, j = min[ki, kj] 10−9 – 0.004 – – 0.001 0.65 – 2 × 1014 Pi, j = si, j γ exp(−ηdi, j) Preferential Attachment PA si, j = ki × kj 1.5 × 10−11 – 0.0001 – – 0.005 0.51 – 2 × 1017 The above two researches deﬁne different topological properties evaluation method and network similarity evaluation indices. Results are sorted by energy value in descending order. Topological properties evaluation method is relative error, whose mathematical deﬁnition is re = (pd−pm) pd × 100% . Lower value indicates the closer properties between real data and simulated networks. Network similarity indices, energy, was deﬁned as E = 1 reR + reC + reL + reEloc + reEglob + reQ + reT + reP(k) . Higher value indicates the more similar between real data and simulated networks. In the previous similar research (Vértes et al., 2012), results are sorted by energy value in ascending order. Topological properties evaluation method is the P-value associated with the t-test for a difference. P-value is higher than 0.05 indicates no signiﬁcant difference between real data and stimulated networks. Network similar indices is deﬁned as E = 1 PC × PEglob × PQ×Pp(k) . Lower value indicates the more similar between real data and simulated networks. P(k), Degree Distribution; R, Assortativity; C, Clustering Coefﬁcient; L, Characteristic Path Length; Eglob, Global Efﬁciency; Eloc, Local Efﬁciency; Q, Modularity; T, Transitivity; CN, common neighbor; HDI, hub depressed index; HDI, hub promoted index; LHN-I, Leicht-Holme-Newman index; SI, Sørensen index; PA, preferential attachment index; RA, resource allocation index. Preferential Attachment December 2016 \| Volume 10 \| Article 585 11 Prediction Method for Brain Functional Connectivity Cheng et al. resource y received from x. Like CN, RA is suitable for networks with a large clustering coeﬃcient, a high degree of heterogeneity (Zhou et al., 2009), in which resources tend to ﬂow to high- degree nodes rather than low-degree nodes (Ou et al., 2007). Functional brain networks have been demonstrated to organize intrinsically as highly modular small-world architectures, capable of transferring information at a low wiring cost eﬃciently as well as formatting highly connected hub nodes. Hub node is usually deﬁned as the node with a degree greater than the mean degree plus the standard deviation (He et al., 2009). Previous research has shown that brain networks are vulnerable to a targeted attack on hub nodes—expressed in the signiﬁcant reduction of connectivity and eﬃciency—regardless of whether the network is structural (He et al., 2007) or functional (Crossley et al., 2014). must be paid to construct a complete network because the numbers of nodes and connections are enormous. In this case, link prediction can solve this problem to some extent, so long as it can provide satisfactory accuracy. Generally, application of link prediction in network construction decreases computation costs at the risk of increasing the error rate. Apart from predicting missing connections, link prediction can also be used to evaluate the reliability of existing connections in an unreliable network, namely the possibility of pseudo connections. The connections in brain networks also need to be veriﬁed reliably, regardless of whether the networks are structural or functional. Previous research has lacked methods of quality control when constructing brain networks, which are disturbed by many factors. How do we know that the connections we obtained through correlation analysis actually exist in the brain? How can we to verify the reliably of the connections in network? There are two key points to solving this problem—a dependable method for quantiﬁable evaluation and a comparable golden rule. Link prediction is a choice that can satisfy the ﬁrst point. For a given network, we can evaluate the possibility of a pseudo connection with the link-prediction method. For the latter point, unlike in functional networks, tract-tracing measures provide an importance reference for reconstructing pathways in DTI structural networks (non- human species). Preferential Attachment Faced with numerous local information indices, we needed a reliable and rapid method to evaluate the prediction accuracy of the models. As a widely used measurement in link-prediction research, prediction power was introduced to evaluate the predictions. Correlation analysis with the E value revealed a strong positive correlation. Six of the local information indices showed signiﬁcant or marginally signiﬁcant correlations, while PA index appeared more suitable for scale-free networks than for small-world networks. The result fully veriﬁed our hypothesis and implied that we could transform the problem of prediction evaluation into a problem of link prediction, which avoids vast amounts of calculation and contrastive analysis of network topological properties. CONCLUSION Anatomical distance has widely been used to predict functional connectivity because of the potential relationship between structural connectivity and functional connectivity. But studies have shown that one-parameter model (anatomical distance) cannot account for the small-worldness, modularity, and degree distribution of normal human brain functional networks. Frontiers in Neuroscience \| www.frontiersin.org METHODOLOGY Diﬀerent from other methods, local information is a topological property of functional network itself. This means that the topological properties of the network itself are used to predict its own connections. This is circular. The precondition for this thinking is that we have a complete network (all connections in the given network are known). In contrast, if we have an incomplete network because the connection data are diﬃcult to collect or the connection computation costs are high, we can predict the missing connections with the known connections. That is the value of the method. Local information is the simplest and direct method in link-prediction research, which utilizes relevant network topological information to predict the possibility of an edge between two given nodes in a network. The underlying basic assumption was that the higher similarity between the nodes in network, the higher probability of an edge existing between them. Based on previous researches, the current study separately evaluated the inclusion of seven local information indices into the model and compared the prediction accuracy among indices. Results showed that the simulated networks reﬂected the basic characteristics of brain networks, such as high clustering coeﬃcient, high local eﬃciency, hub nods, and small-worldness. But when it comes to some properties related with long-range links, the simulated result is disillusionary. It reﬂected the limitation of local information method. Local information method evaluated the local similarity well and but it was not sensitive to the long-range links. Local information is a common method of link prediction, which is among the most important research ﬁelds in network science. The aim of link prediction is to predict the existence of potentially missing connections and to evaluate the reliability of the existing connections according to the available incomplete or unreliable network. The same problems occur in brain network research. We thus applied link-prediction methods to this ﬁeld and hoped it could be the part of solution. Large-scale brain network construction is an intractable and urgent problem. Beneﬁting from the promotion of hardware performance and advancement of computation frames, brain networks can be constructed at the voxel level. However, at larger scales (e.g., the neuron level), constructing a complete network is diﬃcult. A huge computational cost As is mentioned in Methodology, the main application of local information is in incomplete or unreliable network. REFERENCES Bullmore, E., and Sporns, O. (2009). Complex brain networks: graph theoretical analysis of structural and functional systems. Nat. Rev. Neurosci. 10, 186–198. doi: 10.1038/nrn2575 Achard, S., and Bullmore, E. (2007). Eﬃciency and cost of economical brain functional networks. PLoS Comput. Biol. 3:e17. doi: 10.1371/journal.pcbi.0030017 Bullmore, E. T., and Bassett, D. S. (2011). Brain graphs: graphical models of the human brain connectome. Annu. Rev. Clin. Psychol. 7, 113–140. doi: 10.1146/annurev-clinpsy-040510-143934 Ackland, R. (2005). “Mapping the US political blogosphere: Are conservative bloggers more prominent,” in Presentation to BlogTalk Downunder, (Sydney, NSW: Conference of BlogTalk Downunder), 19–22. Cabral, J., Hugues, E., Sporns, O., and Deco, G. (2011). 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Neuron 80, 184–197. doi: 10.1016/j.neuron.2013.07.036 Amaral, L. A. N., Scala, A., Barthelemy, M., and Stanley, H. E. (2000). Classes of small-world networks. Proc. Natl. Acad. Sci. U.S.A. 97, 11149–11152. ACKNOWLEDGMENTS connections is unknown, local information can predict the missing connections. For an unreliable network, in which some existing connections might not be real, local information can evaluate the reliability of connections. Both of them are intractable problems in brain network construction, especially the latter. We think local information method has practical applicability in brain network research as a feasible and eﬀective tool. We thank Tianzi Jiang and Yong Liu from the Institute of Automation, Chinese Academy of Sciences for valuable suggestions. This study was supported by research grants from the National Natural Science Foundation of China (61373101, 61472270, and 61402318), Natural Science Foundation of Shanxi Province (201601D021073) and Scientiﬁc and Technological Innovation Programs of Higher Education Institutions in Shanxi (2016139). SUPPLEMENTARY MATERIAL CC was responsible for the study design and writing the manuscript. JC performed data analysis and statistical processing. XC provided and integrated experimental data. HG was the heads of the funds and supervised the paper. All authors approved the ﬁnal version of the manuscript. The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fnins. 2016.00585/full#supplementary-material METHODOLOGY For an incomplete network, in which the existence of some December 2016 \| Volume 10 \| Article 585 12 Prediction Method for Brain Functional Connectivity Cheng et al. 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https://openalex.org/W3165360498	https://www.ajol.info/index.php/tjpr/article/download/207252/195366	English	null	Development of reduced-salt gel of silver carp meat batter using low frequency ultrasound: Effect on color, texture, cooking loss and microstructure	Tropical journal of pharmaceutical research	2,021	cc-by	5,153	Development of reduced-salt gel of silver carp meat batter using low frequency ultrasound: Effect on color, texture, cooking loss and microstructure Riya Liuhartana Nasyiruddin , Willard Burton Navicha , Abuubakar Hassan Ramadhan1, Fang Yang1, Qixing Jiang1, Yanshun Xu1, Peipei Yu1, Wenshui Xia1* 1State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Collaborative Innovation Center of Food Safety and Quality Control in Jiangsu Province, Jiangnan University, Wuxi, Jiangsu 214122, China, 2The Fishery Faculty, University of PGRI Palembang, Palembang, South Sumatera, 30263, Indonesia, 3Department of Human Ecology, Domasi College of Education, PO Box 49, Domasi, Zomba, Malawi Xia1* 1State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Collaborative Innovation Center of Food Safety and Quality Control in Jiangsu Province, Jiangnan University, Wuxi, Jiangsu 214122, China, 2The Fishery Faculty, University of PGRI Palembang, Palembang, South Sumatera, 30263, Indonesia, 3Department of Human Ecology, Domasi College of Education, PO Box 49, Domasi, Zomba, Malawi For correspondence: Email: xiaws@jiangnan.edu.cn; Tel: +86 510 85919121 For correspondence: Email: xiaws@jiangnan.edu.cn; Tel: +86 510 85919121 Revised accepted: 17 March 2019 Sent for review: 12 August 2018 Abstract Purpose: To develop reduced-salt silver carp meat gels using low frequency ultrasound. Purpose: To develop reduced-salt silver carp meat gels using low frequency ultrasound. Methods: Silver carp meat batters were prepared with 0 5 1 (reduced-salt) and 2 % (regular s p p p g g q y Methods: Silver carp meat batters were prepared with 0.5, 1 (reduced-salt) and 2 % (regular salt), and sonicated (20 KHz, 500 W) for 30 and 40 min, or unsonicated (control). Changes in gel properties were evaluated in terms of color, texture, cooking loss and microstructure using color measurement, puncture test, cooking loss and scanning electron microscopic (SEM) analysis, respectively. , g g p ( ) y , p y Results: Ultrasound and salt exposure led to marked effects on color, texture and cooking loss in fish meat gels (p < 0.05). Reduction in salt content increased the lightness (L) and cooking loss; and also decreased the sample values of greenness (-a), breaking force, rupture distance and gel strength. Scanning electron microscopy (SEM) on regular-salt level samples showed that ultrasonic exposure decreased dense aggregates and increased the number and distribution of small cavity samples. Reduced-salt samples (1 % salt) subjected to 30 min sonication had better color (lighter) than control (0 min sonication), better texture (higher gel strength) and cooking loss comparable to that of regular-salt level sample subjected to 30 min sonication, and similar to microstructures from normal salt samples without ultrasound exposure. p Conclusion: Low frequency ultrasound is suitable for preparing reduced-salt fish meat gels under suitable ultrasonic conditions. Keywords: Silver carp, Fish meat batter, Gel, Reduced salt, Low frequency ultrasound Keywords: Silver carp, Fish meat batter, Gel, Reduced salt, Low frequency ultrasound This is an Open Access article that uses a fund-ing model which does not charge readers or their institutions for access and distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0) and the Budapest Open Access Initiative (http://www.budapestopenaccessinitiative.org/read), which permit unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. Tropical Journal of Pharmaceutical Research is indexed by Science Citation Index (SciSearch), Scopus, International Pharmaceutical Abstract, Chemical Abstracts, Embase, Index Copernicus, EBSCO, African Index Medicus, JournalSeek, Journal Citation Reports/Science Edition, Directory of Open Access Journals (DOAJ), African Journal Online, Bioline International, Open-J-Gate and Pharmacy Abstracts Tropical Journal of Pharmaceutical Research December 2017; 18 (4): 773-780 ISSN: 1596-5996 (print); 1596-9827 (electronic) © Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin City, 300001 Nigeria. Tropical Journal of Pharmaceutical Research December 2017; 18 (4): 773-780 ISSN: 1596-5996 (print); 1596-9827 (electronic) © Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin City, 300001 Nigeria. Tropical Journal of Pharmaceutical Research December 2017; 18 (4): 773-780 ISSN: 1596-5996 (print); 1596-9827 (electronic) © Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin City, 300001 Nigeria. Tropical Journal of Pharmaceutical Research December 2017; 18 (4): 773-780 ISSN: 1596-5996 (print); 1596-9827 (electronic) © Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin City, 300001 Nigeria. Available online at http://www.tjpr.org http://dx.doi.org/10.4314/tjpr.v18i4.14 Original Research Article INTRODUCTION harvested commercially in China [1]. In recent years, food products such as fermented sausages [2], surimi [3-5] and restructured fish [1] have been developed from silver carp. Silver carp (Hypophthalmichthys molitrix) is one of the major inexpensive freshwater fish species Trop J Pharm Res, April 2019; 18(4): 7 19 The authors. This work is licensed under the Creative Commons Attribution 4.0 International License Nasyiruddin et al Nasyiruddin et al polyethylene bags and kept at 4 oC prior to preparation of meat batter. All of the chemicals and reagents used in this study were of analytical grade (Sinopharm Chemical Reagent, Shanghai, China). Common salt or sodium chloride is a vital additive in the processing of many foods. However, excessive sodium intake is associated with hypertension [6]. Aligned with that, there is a great interest in reduced-salt products. However, reduction in salt content could influence the characteristics of meat products. Reduced salt content impairs the complete solubilization or dissociation of myofibrillar proteins, thereby contributing to coarse product structure [4]. Moreover, it has been shown that salt concentrations below 2 % negatively affect the mechanical and functional attributes of fish products [7]. Ultrasound treatment and meat gel preparation The aim of the present study was to develop reduced-salt gels of silver carp meat batters using low frequency ultrasound. Available literature shows that there is limited information on this area of research. This was performed using a probe system (Intelligent Ultrasonicator Generator USC201L, 500 W and 20 kHz, Hangzhou Biao, Hangzhou, China) equipped with horn of 10 mm diameter. EXPERIMENTAL The sample was immersed in 1500 mL iced-cold water in a 2000-mL glass beaker. The beaker was placed in ice bath. During the ultrasound application, the temperature of the iced-cold water was maintained below 15 oC. The distance between sample and tip of the probe was established at 2 cm. the meat batters were sonicated for periods of 30 and 40 min. Measurements were taken in triplicate. Meat batter not subjected to sonication served as control at the same temperature (15 oC). Samples were stored at 4 oC prior to analysis. Meat batter preparation The meat batter samples were prepared with three levels of salt (NaCl) namely 0.5, 1 % (reduced-salt concentrations) and 2 % (regular- salt concentration); and three levels of ultrasound treatment, namely 0 min (unsonicated sample used as control), 30 min and 40 min. The meat batters were prepared in line with a modified version of the procedure described earlier [14]. The preparation procedure was as follows: ground meat was homogenized in a food processor (JYL-D025, Joyoung, Shandong, China) with ice-cold water for 30 s, and salt was added at different concentrations as indicated in Table 1, followed with chopping for 30 sec. Approximately 60 g of each meat batter was placed in a plastic bag, spread out to a thickness of about 2 mm, and vacuum-sealed. Ultrasound is a novel, harmless and eco- compatible technique employed in food industries for production of food products. It involves the use of mechanical waves with frequencies beyond the auditory threshold of humans. In the food processing industries, ultrasound waves of low frequency are employed for altering the physical and chemical properties of foods [8], while those of high frequencies are useful for assessing the physicochemical attributes foods. Some studies have reported the enhancing effect of low frequency ultrasound on the properties of meat protein [8-11]. According to Li et al [12], ultrasound waves are capable of improving the properties of low-salt gels. In addition, it has been suggested that ultrasound parameters such as the type of equipment, frequency, and time and power of sonication should be considered in research studies [8,13]. Table 1: Composition of the fish meat batters Table 1: Composition of the fish meat batters Ingredient Salt levels (%) 0.5 1 2 Fish meat 80.0 80.0 80.0 Salt 0.5 1.0 2.0 Ice water 19.5 19.0 18.0 Total 100.0 100.0 100.0 Ultrasound treatment and meat gel preparation Ultrasound treatment and meat gel preparation Color measurement The meat gels were sliced into cylinder-shaped patterns of diameter 2.5 cm and length 2.0 cm. The color of the sample surfaces was determined with a color spectrophotometer (UltraScan, USA), after prior calibration using a white and black standard plate. Color was measured at four different points on a sample to get the magnitudes of lightness (L), redness (+a), greenness (-a), yellowness (+b) or blueness (- b) of the gels. The measurements were carried out in triplicate. Cooking loss analysis This was determined using a modified version of the procedure outlined previously [15]. Portions of each meat batter (approximately 10 g) were processed to meat gel using meat gel preparation as described above. The tubes were kept in inverted position for 1 h, and filter paper (Whatman no. 1 with diameter 90 mm) was put on the open tubes to absorb separated fluid. Cooking loss was estimated in terms of post- cooking weight decrease (%) relative to original weight. Measurements were done in triplicate. With decreased NaCl content, the b level was enhanced by the two ultrasound exposure periods, while in samples with normal NaCl content, b* was reduced as a function of duration of ultrasound exposure. However, in samples containing 1 % NaCl, ultrasound exposure had no marked effect on b* value. Color The values of L, a and b* for fish meat gels are shown in Figure 1. The L* value varied from 78.31 to 81.27; a* value varied from -1.07 to - 1.83, while b* value varied from 7.20 to 9.74. There were marked increases in L* values of fish meat gels subjected to ultrasound for 30 min and 40 min, with decreasing NaCl content (p < 0.05). In addition, the L* values of regular-salt fish meat gels were reduced by ultrasound exposure. However, the -a* values at decreasing NaCl levels were reduced by ultrasound exposure for the two periods of time. Furthermore, at normal NaCl level, there were increases in -a* value as a result of ultrasound exposure. Materials Fresh live silver carp fish (mean weight = 2.5 ± 0.2 kg) were purchased from a local supermarket (Wuxi, Jiangsu, China). Trained store personnel sacrificed the fish using percussive stunning. Then the fish were scaled, eviscerated, decapitated and washed in running tap water. Thereafter, they were covered with crushed ice, and transported to Food Processing Technology Laboratory within 20 min. Then, the fish were manually skinned and filleted. Only the white muscles were used: the red muscles were manually removed with a knife. The muscles were passed through a grinder using a 7-mm plate (MGJ-090, Foshan Shunde Mingjian, Guangdong, China). Samples were placed in Meat gel was prepared as follows: after the ultrasound treatment, meat batter portions (about 35 g) were placed in plastic centrifuge tubes of inner diameter 2.5 cm, and height 10 cm. The tubes were hermetically sealed. The sealed Trop J Pharm Res, April 2019; 18(4): 774 Nasyiruddin et al tubes were centrifuged at 2500 g at 3 oC for 15 min using Sigma Laboratory Centrifuges 4K15, Germany. Thereafter, the tubes were heated in a water bath at 40 oC for 1 h, and at 90 oC for 15 min. Then, the meat gels were immediately cooled by immersing the tubes in ice-cold water at 4 oC for 30 min. The meat gels were stored in a refrigerator at 4 oC prior to further analysis. Japan) which was set at the operating voltage of 5 kV and at a magnification of ×3k. Prior to SEM, the 4 samples were processed with a modified version of an earlier method [12]. The samples were subjected to fixation for 48 h using phosphate buffered solution of 2.5 % glutaraldehyde, pH 7.0. Following fixation, the samples were rinsed thrice with 0.1 M phosphate buffer at the same pH, and then dehydrated through increasing ethanol concentrations (30 – 100 %). Finally, the dehydrated samples were dried in a freeze drier dryer (Alpha 1-2 LDplus, Germany). Statistical analysis Data were subjected to statistical analysis using ANOVA. Groups were compared with Duncan’s multiple range tests. All analyses were carried out with SPSS 20.0 software. Statistical significance of difference was assumed at p < 0.05. Puncture test Puncture test was performed in line with a modified version of the procedure reported earlier [12]. The test was done on cylinder- shaped slices of meat gels (same dimensions as in color measurement) with TA.XT Plus texture analyzer bearing a 5-mm diameter plunger operating at a velocity of 1 mm/sec. The parameters measured were distance to rupture (cm) and breaking force (g). Gel strength, expressed in the unit of g.cm, was obtained from the product of breaking force and distance to rupture. The test was repeated six times for each sample at room temperature. SEM of meat gels The breaking force values of fish meat gels varied from 186.55 to 291.66 g. The distance to rupture values varied from 0.56 to 0.86 cm, while the gel strength varied from 107.15 to 251.12 g.cm (Figure 2). The breaking force decreased in decreased NaCl content as a function of duration of ultrasound exposure, while it was enhanced as The ultra-structures of the 4 meat gel samples (sample containing 1 % salt with 30 min ultrasound, and samples containing 2 % salt subjected to 0, 30 and 40 min ultrasound) were examined under SEM (SU1510, Hitachi, Tokyo, Trop J Pharm Res, April 2019; 18(4): 775 Nasyiruddin et al Nasyir Figure 1: Color of fish meat gels. Different letters (a, b and c) indicate significant differences (p < 0.05) among different salt levels at the same sonication time. The letters x and y indicate significant differences (p < 0.05) between the sonication times at the same salt level a function of ultrasound exposure duration in increase in ultrasound exposure time, but increased with ultrasound exposure period at normal NaCl content. Figure 1: Color of fish meat gels. Different letters (a, b and c) indicate significant differences (p < 0.05) among different salt levels at the same sonication time. The letters x and y indicate significant differences (p < 0.05) between the sonication times at the same salt level a function of ultrasound exposure duration in samples with normal NaCl content. The distance of rupture of samples decreased with reducing salt levels at the two ultrasound treatments. Moreover, the distance of rupture increased significantly with ultrasound treatments in samples with normal salt levels such that a p p normal NaCl content. Figure 2: Distance to rupture, breaking force, and gel strength of the fish meat gels. Different letters (a, b and c) reflect significant differences (p < 0.05) among samples with different salt contents at the same sonication time. The letters x and y indicate significant differences (p < 0.05) between the two sonication periods at the same salt level Cooking loss The results of cooking losses in fish meat gels Figure 2: Distance to rupture, breaking force, and gel strength of the fish meat gels. Different letters (a, b and c) reflect significant differences (p < 0.05) among samples with different salt contents at the same sonication time. SEM of meat gels The letters x and y indicate significant differences (p < 0.05) between the two sonication periods at the same salt level Figure 1: Color of fish meat gels. Different letters (a, b and c) indicate significant differences (p < 0.05) among different salt levels at the same sonication time. The letters x and y indicate significant differences (p < 0.05) between the sonication times at the same salt level Figure 2: Distance to rupture, breaking force, and gel strength of the fish meat gels. Different letters (a, b and c) reflect significant differences (p < 0.05) among samples with different salt contents at the same sonication time. The letters x and y indicate significant differences (p < 0.05) between the two sonication periods at the same salt level a function of ultrasound exposure duration in samples with normal NaCl content. a function of ultrasound exposure duration in samples with normal NaCl content. The distance of rupture of samples decreased with reducing salt levels at the two ultrasound treatments. Moreover, the distance of rupture increased significantly with ultrasound treatments in samples with normal salt levels, such that a sample with 40 min ultrasound had higher distance of rupture than the control. Structural features The results also showed that with reduced salt levels, ultrasound treatment decreased the greenness (-a) of fish meat gels, but increased the greenness in samples with regular salt levels. In addition, ultrasound treatment of samples with 1 % salt content had no significant effect on yellowness (b) of fish meat gels. Color is an important factor which determines the quality and acceptability of fish processing products such as surimi gels. Surimi gels with high lightness and low yellowness are generally in high demand [20]. The microstructures of the four kinds of fish meat gels are presented in Figure 4. These are: sample containing 1 % salt exposed to 30 min ultrasound, and sample containing 2 % salt exposed to 0, 30 and 40 min of ultrasound. Figure 4: SEM images of ultrasound-treated fish meat gel with different salt level: (A) 1 % salt exposed to 30- min ultrasound; (B) 2 % salt unexposed to ultrasound; (C) 2 % salt exposed to 30-min ultrasound; (D) 2 % salt exposed to 40-min ultrasound (x 3000; bar = 10.0 µm) (ag = aggregate; ca = cavity) In texture observations, the results showed that breaking force, distance to rupture and gel strength values of samples were affected by salt content and ultrasound treatments. In texture observations, the results showed that breaking force, distance to rupture and gel strength values of samples were affected by salt content and ultrasound treatments. Ultrasound treatment in reduced salt samples decreased the breaking force and distance of rupture values of the fish meat gels, whereas, the ultrasound treatment gave the opposite effect on the breaking force values at regular-salt level and 1% salt of samples, and on the distance of rupture values of fish meat gels with normal salt content. Figure 4: SEM images of ultrasound-treated fish meat gel with different salt level: (A) 1 % salt exposed to 30- min ultrasound; (B) 2 % salt unexposed to ultrasound; (C) 2 % salt exposed to 30-min ultrasound; (D) 2 % salt exposed to 40-min ultrasound (x 3000; bar = 10.0 µm) (ag = aggregate; ca = cavity) Some studies have also showed that salt had a positive effect on the mechanical properties of fish gel [4,21-24]. These studies showed that the breaking force, distance to rupture and gel strength of fish gel decreased at decreased salt concentration. Salt significantly influences the extraction and mechanical attributes of proteins Cooking loss The results of cooking losses in fish meat gels are presented in Figure 3. The values ranged from 10.76 to 16.16 %. Cooking loss was enhanced with reduction in NaCl content of fish meat gels as a function of duration of ultrasound exposure. In samples with normal NaCl contents, The gel strength value of samples decreased in sample with decreased NaCl content and with Trop J Pharm Res, April 2019; 18(4): 776 Nasyiruddin et al cooking loss was markedly enhanced by ultrasound exposure. treatment at decreasing salt levels increased the lightness (L) value of fish meat gels. However, ultrasound treatment of samples at regular salt level produced the opposite effect. These results are supported by the findings of Hu et al [16], who reported that the whiteness values of squid muscle decreased on exposure to ultrasound treatment due to ultrasound-induced enhancement of browning reaction. On the other hand, reduction of salt level increased the L value of samples, which is consistent with other studies [15,17,18]. This was attributed to the differences in levels of protein abstracted during chopping, and differences in salt content. It has been shown that increases in protein content decreased the L* value of beef meat batters [19]. This means that reduction of salt level might increase the L* value of samples. In the present study, cooking loss increased with reduction in salt content. Studies have shown that L* value is positively correlated with cooking loss [15]. This phenomenon might be due to heat-induced loss of water from the samples, resulting in accentuated light-scattering capacity of the meat batters. Figure 3: Cooking loss of fish meat gels. The letters a, b and c reflect significant differences (p < 0.05) among samples with different salt levels at the same sonication time. The letters x and y indicate significant differences (p < 0.05) between the two sonication times at the same salt level Figure 3: Cooking loss of fish meat gels. The letters a, b and c reflect significant differences (p < 0.05) among samples with different salt levels at the same sonication time. The letters x and y indicate significant differences (p < 0.05) between the two sonication times at the same salt level CONCLUSION The results obtained in this study demonstrate that it is feasible to obtain reduced-salt silver carp meat gels using low frequency ultrasound (20 KHz, 500 W). The findings show that reduced-salt fish meat gels (1% salt) subjected to 30 min of sonication have better color (lighter) than control (0 min sonication), better texture (higher gel strength), and minimal changes in cooking loss, relative to samples containing regular salt level (2% salt). These products also have microstructures similar to regular-salt content sample without sonication. Thus, the findings suggest that low frequency ultrasound may be a useful technique for preparing reduced- salt fish meat gels using suitable ultrasonic conditions. In this study, reduction in salt levels increased the cooking loss value of fish meat gels. This finding is consistent with previous reports [12,15,26]. When salt level is increased, the hydration and water-binding characteristics of processed meats are increased [26]. Thus, decreases in salt level increase the cooking loss of samples. Studies have shown that salt contents of 1.5 - 2 % result in solubilization of active myofibrillar proteins, thereby enhancing hydration and the binding capacity of the protein, resulting in better texture and less water loss during cooking [27]. As shown in the results of the present study, cooking losses of fish meat gels with 1 and 0.5% salt content were significantly higher than those of samples with regular salt level samples (2 %) at the same ultrasound treatment time. Ultrasound treatment significantly increased the cooking loss of fish meat gel samples with regular salt content, and samples with 1% salt content on exposure to 40 min of ultrasound. It has been reported that exposure of chicken meat gels to 20-min ultrasound reduced their water mobility and decreased their cooking loss at NaCl levels of 1 – 2 %, relative to cooking losses seen after their exposure to ultrasound for shorter or longer periods [12]. Contribution of authors The authors declare that this work was done by those mentioned in this manuscript. All liabilities in respect of claims associated with the contents of this manuscript will be shouldered by the authors. Results of SEM showed three dimensional networks in the sample of meat gels. The gel of regular-salt samples without sonication had visibly dense aggregates and low population of large cavities unevenly distributed. This sample presented a gel mesh of disorganized and dense aggregates, most likely as a result of disordered agglomeration prior to unfolding. When treated with ultrasound for 30 min and 40 min, there was a reduction in the population of dense aggregates and an elevation in the population of uniformly distributed small cavities. Similar results were also reported by other researchers Acknowledgement This research was financially supported by China Agriculture Research System (no. CARS-45-26) and Project from Jiangsu Fishery Administration (no. Y2017-30). The authors express their thanks to Associate Professor Xiaolin Qiu, Packaging Engineering, School of Mechanical Engineering, Jiangnan University for providing the ultrasonicator employed in the experiments. Conflict of interest This investigation is not linked to any conflict of interest. DISCUSSION In this study, low frequency ultrasound was used to develop reduced-salt gel of silver carp meat batter. The results showed that ultrasound In this study, low frequency ultrasound was used to develop reduced-salt gel of silver carp meat batter. The results showed that ultrasound Trop J Pharm Res, April 2019; 18(4): 777 Nasyiruddin et al [24]. Furthermore, other studies have revealed that the gel strength of chicken meat batters [25] and beef myofibrillar proteins [8] are positively influenced by ultrasound exposure. These effects are due to the fact that ultrasound exposure improves gel strength due to its effect on the degree of acidity of proteins, which in turn decreases sample particle size thereby resulting in increased solubility. However, prolonged ultrasound treatment causes denaturation of proteins and adversely influences the texture of products [12]. It has been reported that the gel strength of chicken breast meat batter with salt contents of 1, 1.5 and 2 % tended to increase with 10 and 20 min ultrasound treatment, but decreased after 30 and 40 min sonication (40 kHz, 300 W) [12]. [9,25]. Furthermore, SEM of reduced-salt sample (1 % salt content, 30 min ultrasound) showed a structure similar to that of non-sonicated, regular salt-containing sample. REFERENCES 15. Shao JH, Deng YM, Jia N, Li RR, Cao JX, Liu DY, Li JR. Low-field NMR determination of water distribution in meat batters with NaCl and polyphosphate addition. Food Chem 2016, 200, 308-314. 1. Wang Y, Zhang M, Mujumdar AS, Mothibe KJ. Quality changes of dehydrated restructured fish product from silver Carp (Hypophthalmichthys molitrix) as affected by arying methods. Food Bioproc Tech 2012; 6: 1664- 1680. 16. Hu Y, Yu H, Dong K, Yang S, Ye X, Chen S. 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https://openalex.org/W2037015222	http://old.scielo.br/pdf/rbpm/v14n4/14.pdf	Portuguese	null	Avaliação das atividades antioxidante, anti e pró-hemolítica do extrato etanólico das folhas de Pterogyne nitens Tul. (Fabaceae-Caesalpinioideae)	Revista brasileira de plantas medicinais	2,012	cc-by	4,478	Recebido para publicação em 19/05/2011 Aceito para publicação em 12/03/2011 Palavras-chave: Pterogyne nitens, dano oxidativo, radicais livres Palavras-chave: Pterogyne nitens, dano oxidativo, radicais livres ABSTRACT: Evaluation of antioxidant, anti- and pro-hemolytic activities of ethanol extract from the leaves of Pterogynenitens Tul. (Fabaceae-Caesalpinioideae). The search for natural products as a widespread practice enables the discovery of new active principles, or the discovery of new activities for plant extracts (extensively used by the population) and natural active principles already known. Pterogynenitensis is a plant whose descriptions of activities are relatively recent and therefore has in its crude extract a good source for research in the field of natural products. Thus, the aim of this study was to evaluate the antioxidant profile of crude ethanol extract from P. nitens leaves and a possible influence on the hemolysis caused by AAPH radical. For the studied oxidant species, ABTS+, DPPH, HOCl and H2O2, the IC50 values were found of 5.0 g mL-1, 17 g mL-1, no action at all, and 3.9 g mL-1, respectively, relatively low values, indicating a good antioxidant potential. Pro- and anti-hemolytic activities were found for the extract in a concentration-dependent way. The studied extract showed to be a good source of natural molecules with potential biological action. Key words: Pterogynenitens, oxidative damage, free radicals 666 666 Avaliação das atividades antioxidante, anti e pró-hemolítica do extrato etanólico das folhas de Pterogyne nitens Tul. (Fabaceae-Caesalpinioideae) PASQUINI-NETTO, H.1; MANENTE, F.A.1; MOURA, E.L.1; REGASINI, L.O.2; PINTO, M.E.F.2; BOLZANI, V.S.2; OLIVEIRA, O.M.M.F.2; VELLOSA, J.C.R.1* 1Universidade Estadual de Ponta Grossa, Departamento de Análises Clínicas e Toxicológicas, Ponta Grossa- Brasil josevellosa@yahoo.com.br 2Universidade Estadual Paulista “Júlio de Mesquita Filho”, Instituto de Química de Araraquara, Departamento de Biotecnologia e Bioquímica e Departamento de Química Orgânica, Araraquara-Brasil 1Universidade Estadual de Ponta Grossa, Departamento de Análises Clínicas e Toxicológicas, Ponta Grossa- Brasil josevellosa@yahoo.com.br 2Universidade Estadual Paulista “Júlio de Mesquita Filho”, Instituto de Química de Araraquara, Departamento de Biotecnologia e Bioquímica e Departamento de Química Orgânica, Araraquara-Brasil RESUMO: A pesquisa de produtos naturais permite a descoberta de novos princípios ativos, ou ainda, a descoberta de novas atividades para extratos de plantas (amplamente utilizados pela população brasileira) e princípios ativos naturais já conhecidos. Pterogyne nitens é uma planta cuja descrição das atividades é relativamente recente e, portanto, tem no extrato bruto boa fonte para pesquisas na área de produtos naturais. Desta forma, o objetivo deste trabalho foi estudar o perfil antioxidante do extrato bruto etanólico das folhas de P. nitens e possível interferência sobre a hemólise provocada pelo radical AAPH. No estudo da ação antioxidante das espécies estudadas, ABTS+, DPPH, H2O2 e HOCl, encontrou-se os valores de IC50 de 5,0 g mL-1, 17 g mL-1, sem ação e 3,9 g mL-1, respectivamente, valores relativamente baixos e que indicam bom potencial antioxidante. Foram encontradas atividades pró-hemolítica e anti-hemolítica para o extrato de forma concentração-dependente. O extrato estudado mostro boa fonte de moléculas naturais com potencial de ação biológica. Material Vegetal e Extração As folhas de P. nitens foram coletadas no Instituto Botânico, São Paulo-SP, Brasil, entre os meses de abril e maio de 2003, pela Dra Maria Cláudia Marx Young e identificadas como Pterogyne nitens Tul. (Fabaceae-Caesalpinioideae) pela Dra Inês Cordeiro do mesmo instituto. Uma exsicata (SP204319) encontra-se depositada no herbário “Maria Eneida P. Kaufmann” do Instituto Botânico, São Paulo-SP, Brasil. Contudo, existem relatos limitados das aplicações medicinais e da composição química de P. nitens. Segundo os levantamentos etnofarmacológicos de Crivos et al. (2007), em comunidades guaranis do nordeste da Argentina, as cascas do caule possuem ação anti-helmíntica contra o “tacho”, nome popular de Ascaris lumbricoides. As folhas foram secas ao abrigo da luz por quatro semanas, sendo trituradas em moinho de facas. O pó seco e pulverizado (2,7 kg) foi submetido à maceração em hexano (cinco etapas com 2,0 L de solvente cada) durante cinco semanas. Posteriormente, a torta foi submetida à maceração em etanol (cinco etapas com 4,0 L de solvente cada). As soluções extrativas hexânicas e etanólicas, obtidas dos processos extrativos foram concentradas separadamente em evaporador rotatório, obtendo-se 3,6 g e 12,7 g dos extratos hexânico e etanólico, respectivamente. O extrato etanólico foi utilizado nos ensaios de atividade antioxidante, anti- e pró-hemolítica. Interessante estratégia na pesquisa de produtos naturais é direcionar o estudo do isolamento de substâncias por meio da atividade. Para tanto, os extratos vegetais são fracionados e as frações são testadas em ensaios biológicos. Aquelas tidas como positivas são, então, encaminhadas para o processo de isolamento e purificação. Essa investigação evidencia estudos que se dizem biodirigidos ou, também denominados, bioguiados. g Os estudos fitoquímicos biodirigidos/ bioguiados com P. nitens tem indicado a presença de alcalóides guanidínicos com atividade citotóxica frente a inúmeras linhagens celulares de mama, melanoma e leucemia (Bolzani et al., 1995; Regasini et al., 2007, 2009a). Outros alcalóides de estrutura química similar a esses foram isolados de P. nitens e demonstraram atividade antifúngica contra fungos oportunistas humanos (Regasini et al., 2010). Compostos fenólicos, incluindo flavonóides (Regasini et al., 2008a) e derivados de ácido gálico (Regasini et al., 2008b), foram descritos como constituintes de P. nitens, bem como se mostraram inibidores potentes de mieloperoxidase (MPO), uma enzima pró- inflamatória (Fernandes et al., 2008; Regasini et al., 2008c). Esteróides e triterpenos também foram identificados em inúmeras partes de P. nitens, empregando Cromatografia Gasosa de Alta Resolução (Regasini et al., 2009b). INTRODUÇÃO Pterogyne nitens Tul. (Fabaceae- Caesalpinioideae) é uma espécie vegetal que habita o Cerrado, Caatinga e Mata Atlântica e se encontra com risco de extinção, fazendo parte da lista de espécies arbóreas recomendadas para conservação genética no Estado de São Paulo (Carvalho, 1994). Esta espécie pode ser encontrada também nas regiões Nordeste (Ceará) e Sul (Paraná) do Brasil, além de ocorrer em outros países da América do Sul, tais como a Argentina e o Paraguai (Nassif, 2000). Esta é a única espécie do gênero, sendo conhecida popularmente como “amendoim-do-campo”, “bálsamo”, “vi-raró”, “cocal”, “tipa” ou “amendoim-bravo”. A madeira de P. nitens apresenta-se moderadamente densa e com relativa resistência física, sendo amplamente empregada na construção civil (Carvalho, 1994). Rev. Bras. Pl. Med., Botucatu, v.14, n.4, p.666-672, 2012. 667 MATERIAL E MÉTODO P. nitens possui valor ornamental, devido à beleza e odor das flores, com folhagem brilhante e frutificação que varia de tons à medida que amadurecem, são recomendadas para arborização de vias urbanas e rodovias e na reposição de mata ciliar em locais com inundações periódicas (Lorenzi, 1998). O lenho de P. nitens apresenta seiva rósea e o preparado aquoso do “pó-de-serra” tem coloração roxa, sendo muito utilizado na tinturaria (Carvalho, 1994). Ação scavenger sobre o radical ABTS·+ Ação scavenger sobre o radical ABTS·+ Este ensaio foi realizado de acordo com Pellegrini et al. (1999), com pequenas modificações. O radical ABTS+ é gerado a partir da reação do sal 2,22 -Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium (ABTS) com o persulfato de potássio (K2S2O8). A atividade sequestradora de radicais pelas amostras foi realizada em tampão fosfato de sódio 10 mM (pH 7,0) e foi avaliada por meio do decréscimo da absorbância em 734 nm, comprimento em que se encontra o pico de absorção do radical (Vellosa et al., 2007a). Material Vegetal e Extração Os estudos de bioprospecção investigaram o potencial biológico dos extratos de P. nitens, evidenciando inúmeras outras bioatividades, tais como, antidiabética (Souza et al., 2009, 2010), mutagênica (Oliveira et al., 2007) e antimutagênica (Ferreira et al., 2009). Ação scavenger sobre espécies reativas Nos ensaios de ação direta sobre as espécies reativas foram construídas curvas concentração-resposta a partir das quais foram obtidos os valores de concentração das amostras (extrato e padrão/rutina) que promoveram inibição de 50% do sistema estudado (IC50). Ação scavenger sobre o ácido hipocloroso As reações foram feitas de acordo com Costa et al. (2004), com algumas modificações (Costa et al., 2004; Vellosa et al., 2009). As reações foram realizadas em tampão fosfato de sódio 50 mM, pH 7,4. Procedeu-se incubação da amostra com ácido hipocloroso 30 M (HOCl) durante 10 minutos e logo em seguida foi adicionada tetrametilbenzidina (TMB; 2,8 mM), revelador do HOCl remanescente em solução. Após 5 minutos, realizou-se a leitura da absorbância em 652 nm. Ação scavenger sobre espécies reativas Investigar a ação scavenger de espécies reativas por meio dos ensaios com ABTS+ e DPPH traz vantagens por estes serem métodos rápidos e economicamente acessíveis. Os resultados da ação das amostras sobre ABTS+ e DPPH estão apresentados nas Figuras 1 e 2 respectivamente, bem como, os valores de IC50 obtidos, são apresentados na Tabela 1. Os extratos apresentaram IC50 de 5 e 17 g mL-1 contra o ABTS·+ e DPPH·, porém, para a rutina estes valores foram menores (0,98 e 1,55 g mL-1, respectivamente), indicando o padrão como agente mais eficiente. Esses resultados são estabelecidos considerando que, quanto menores forem as concentrações das amostras necessárias para promoverem menores medidas de absorbâncias nos comprimentos de onda específicos, maior potencial scavenger de radicais livres é observado. Ação scavenger sobre o peróxido de hidrogênio dos eritrócitos e a hemólise promovida foi avaliada pela liberação de hemoglobina pelas células (suspensão de eritrócitos 5%) após 2 horas e meia de incubação em 37ºC. O grau de hemólise é proporcional à leitura da absorbância em 414 nm (Vissers et al., 1998; Yang et al., 2006). A ação sequestradora de peróxido de hidrogênio (H2O2) pelas amostras foi avaliada através da variação da absorbância em 412 nm, como resultado da oxidação do ácido 5-tio-2-nitrobenzóico (TNB) em ácido 5-5’-ditio-2-nitrobenzóico (DTNB), na presença de peróxido 100 M (Vellosa et al., 2007b). Ação scavenger sobre o radical DPPH· A atividade sequestradora de radicais livres do extrato foi avaliada em etanol, segundo Soares et al. (1997). A ação das amostras é evidenciada por meio do decréscimo da absorbância em 531 nm da solução de radical 2,2-Diphenyl-1- picrylhydrazyl 60 M (DPPH). Considerando o potencial biológico dos extratos e das substâncias isoladas de P. nitens, incluindo os flavonóides, compostos com ação antioxidante (Pietta, 2000), o presente estudo objetivou a avaliação das atividades antioxidante, anti- e pró-hemolítica do extrato etanólico obtido das folhas de P. nitens. Rev. Bras. Pl. Med., Botucatu, v.14, n.4, p.666-672, 2012. 668 Ação scavenger sobre o peróxido de hidrogênio Dano provocado pelo radical AAPH em eritrócitos Os testes foram realizados conforme protocolo de pesquisa 08766/10 aprovado pela Comissão de Ética em Pesquisa da UEPG (parecer 68/2010). As hemácias utilizadas foram colhidas do sangue de voluntários sadios por punção venosa, separadas por centrifugação e lavadas sucessivamente com solução tampão fosfato de sódio 10 mM, pH7,4 contendo NaCl 150 mM. A ação dos compostos sobre os eritrócitos foi avaliada tanto na ausência de radicais livres como na presença do radical 2,22 -Azobis(2-methylpropionamidine) dihydrochloride 25 mM (AAPH). Os ensaios foram realizados no mesmo tampão utilizado para lavagem O ABTS+ e o DPPH são radicais modelo amplamente utilizados para a avaliação das atividades de diferentes compostos ou extratos frente a radicais livres. Petacci et al. (2010) estudaram a ação da astilbina, um flavonóide isolado do barbatimão, sobre o radical ABTS+, encontrando IC50 de aproximadamente 9 g mL-1 (20 M), consideravelmente maior aos Rev. Bras. Pl. Med., Botucatu, v.14, n.4, p.666-672, 2012. 0 10 20 30 40 50 60 0,0 0,2 0,4 0,6 0,8 0 10 20 30 40 50 60 0,0 0,2 0,4 0,6 0,8 A734nm Concentração (µg mL-1) Extrato etanólico das folhas de P. nitens Rutina FIGURA 1. Ação scavenger sobre o radical ABTS+ 55 M. FIGURA 1. Ação scavenger sobre o radical ABTS+ 55 M. Rev. Bras. Pl. Med., Botucatu, v.14, n.4, p.666-672, 2012. 669 0 5 10 15 20 0,0 0,1 0,2 0,3 0,4 0,5 0,6 0 5 10 15 20 0,0 0,1 0,2 0,3 0,4 0,5 0,6 A531nm Concentração (µg mL-1) Extrato etanólico das folhas de P. nitens Rutina FIGURA 2. Ação scavenger sobre o radical DPPH 60 M. 0 5 10 15 20 0,0 0,1 0,2 0,3 0,4 0,5 0,6 0 5 10 15 20 0,0 0,1 0,2 0,3 0,4 0,5 0,6 A531nm Concentração (µg mL-1) Extrato etanólico das folhas de P. nitens Rutina FIGURA 2. Ação scavenger sobre o radical DPPH 60 M. Amostra Espécie reativa Extrato bruto etanólico das folhas de P. nitens Rutina ABTS 55 M 5,0 g mL-1 0,98 g mL-1 DPPH 60 M 17,0 g mL -1 1,55 g mL -1 HOCl 30 M 3,9 g mL-1 0,90 g mL-1 TABELA 1. Valores de concentrações das amostras testadas inibitórias em 50% (IC50) frente ao ABTS+, DPPH e HOCl, obtidos a partir dos gráficos concentração-resposta. TABELA 1. Valores de concentrações das amostras testadas inibitórias em 50% (IC50) frente ao ABTS+, DPPH e HOCl, obtidos a partir dos gráficos concentração-resposta. Dano provocado pelo radical AAPH em eritrócitos e concentrações das amostras testadas inibitórias em 50% (IC50) frente ao ABTS+, DPPH e dos gráficos concentração-resposta. valores obtidos pelo extrato e rutina estudados no presente trabalho. Tal dado indica que as amostras ora avaliadas apresentam maior potencial de ação frente a radicais livres do que a astilbina. A espécie Dimorphandra mollis Benth. (Fabaceae, Caesalpinioideae), é uma árvore comum na região Sudeste do Brazil (Petacci et al., 2010) e o potencial antioxidante também foi estudado por Hubinger et al. (2010), que obtiveram IC50 de 3,5 g mL-1 e 38 g mL-1 frente aos radicais ABTS·+ e DPPH, respectivamente, para o extrato etanólico dos frutos desta planta. Tal resultado indica a especificidade de ação frente ao radical testado, pois o extrato das folhas de P. nitens teve ação inferior frente ao ABTS+ e superior frente ao DPPH, quando comparado ao extrato etanólico dos frutos do barbatimão. O peróxido de hidrogênio é uma molécula importante para a ação da mieloperoxidase, enzima que catalisa a formação de ácido hipocloroso in vivo (Lapenna & Cuccurollo, 1996) e, portanto, um alvo farmacológico importante. O ensaio utilizando peróxido de hidrogênio evidenciou que nem o extrato, nem o padrão (Rutina), foram capazes de agir sobre o H2O2 (Figura 3). 2 2 O extrato etanólico das folhas de P. Nitens também apresentou grande eficiência frente ao ácido hipocloroso (Figura 4), com IC50 de 3,9 g mL-1, enquanto a rutina apresentou-se com IC50 de 0,9 g mL-1 (Tabela 1). Em trabalho anterior, Vellosa et al. (2006) observaram que o extrato bruto etanólico da casca das raízes da espinheira santa apresentou IC50 de 2,0 g mL-1 e 1,9 g mL-1 frente ao ABTS+ e HOCl, respectivamente. Apesar de o extrato etanolico das folhas de P. Nitens apresentar potencial de ação inferior ao observado para a espinheira santa, frente ao ácido hipocloroso e ABTS+, ainda assim, os dados obtidos no presente trabalho (baixas concentrações para exercer a ação) são bastante relevantes, pois, em sistemas biológicos, o ácido hipocloroso é o agente mais tóxico e abundante dentre os oxidantes As folhas de P. Nitens mostraram-se excelente fonte de agentes contra espécies oxidantes e radicalares na medida que apresentaram potencial de ação frente aos radicais ABTS+ e DPPH superior aos observados para os extratos etanólicos das raízes, frutos, ramos e cascas do caule desta mesma planta, obtidos por Regasini et al. (2008a). Rev. Bras. Pl. Med., Botucatu, v.14, n.4, p.666-672, 2012. Dano provocado pelo radical AAPH em eritrócitos 670 FIGURA 3. Ação scavenger sobre o peróxido de hidrogênio 100 M. 0 10 20 30 40 50 60 0,50 0,55 0,60 0,65 0,70 0,75 0,80 0,85 0,90 0 10 20 30 40 50 60 0,50 0,55 0,60 0,65 0,70 0,75 0,80 0,85 0,90 A412nm Concentração (µg mL-1) Extrato etanólico das folhas de P. nitens Rutina 0 10 20 30 40 50 60 0,50 0,55 0,60 0,65 0,70 0,75 0,80 0,85 0,90 0 10 20 30 40 50 60 0,50 0,55 0,60 0,65 0,70 0,75 0,80 0,85 0,90 A412nm Concentração (µg mL-1) Extrato etanólico das folhas de P. nitens Rutina Extrato etanólico das folhas de P. nitens Concentração (µg mL-1) FIGURA 3. Ação scavenger sobre o peróxido de hidrogênio 100 M. indiscriminadamente pela construção civil e, por tratar- se de um gênero monoespecífico, a torna seriamente ameaçada de extinção (Carvalho, 1994). Dessa forma, os estudos químicos e biológicos (de quaisquer partes do vegetal) tornam-se prementes para o conhecimento de uma espécie encontrada exclusivamente na América do Sul e impactada pelo extrativismo. Com os dados de bioprospecção apresentados busca-se agregar valor à biodiversidade brasileira. produzidos pelos leucócitos polimorfonucleares (Lapenna & Cuccurollo, 1996; Vellosa et al., 2007b) e pode atacar moléculas importantes, além de gerar outras espécies reativas nocivas (Weiss, 1989; Eaton, 1993). Desta forma, apesar de valores menores que o padrão, o extrato de P. nitens mostrou-se com promissora ação contra espécies reativas e, portanto, boa fonte de moléculas com potencial ação farmacológica. A descrição destas atividades de metabólitos presentes nas folhas de P. nitens, por meio do estudo do extrato etanólico, é, ainda, de grande valia, pois, mesmo não sendo considerada madeira nobree vem sendo empregada Ação sobre o dano provocado pelo radical AAPH em eritrócitos Ação sobre o dano provocado pelo radical AAPH em eritrócitos No estudo da ação do extrato bruto das folhas de P. nitens frente ao radical AAPH torna-se Rev. Bras. Pl. Med., Botucatu, v.14, n.4, p.666-672, 2012. FIGURA 4. Ação scavenger sobre o ácido hipocloroso 30 M. 0 5 10 15 20 -0,1 0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0 5 10 15 20 -0,1 0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 A652nm Concentração (µg mL-1) Extrato etanólico das folhas de P. nitens Rutina FIGURA 4. Ação scavenger sobre o ácido hipocloroso 30 M. Rev. Bras. Pl. Med., Botucatu, v.14, n.4, p.666-672, 2012. 671 ação. A rutina, entretanto, nas menores concentrações, inibe a hemólise provocada pelo radical AAPH e, em concentrações maiores, a ação hemolítica volta a elevar-se de forma concentração-dependente. Isso pode ser explicado pela formação de um produto com maior potencial lesivo, cuja concentração se eleva à medida que as amostras têm aumentadas suas concentrações frente ao AAPH. necessário avaliar, primariamente, se o próprio extrato e o padrão não apresentam potenciais hemolíticos. Por meio da Figura 5, observa-se que o extrato apresenta baixa ação hemolítica enquanto o padrão (rutina) mostrou grande potencial de dano aos eritrócitos. O resultado observado pode ter sido ocasionado por distúrbio de membrana, provocado pelas moléculas presentes no extrato, incluindo a própria rutina, em decorrência de interações com lipídios, proteínas e/ou carboidratos da superfície celular, ou então, um possível distúrbio eletrostático promovido pelo potencial redox destas moléculas. Perfil semelhante já havia sido observado por Vellosa et al. (2011) para os flavonóides kaempferol, quercetina e isoquercetrina, isolados de P. nitens. Estes três flavonóides apresentaram ação scavenger sobre o ABTS+, DPPH e HOCl, dentre outros. Entretanto, apresentaram elevado Observa-se ainda na Figura 5, que a ação hemolítica das amostras é alterada na presença do radical livre, o que potencializa ligeiramente esta FIGURA 5. Ação hemolítica das amostras e interferência sobre a hemólise provocada pelo radical AAPH 25 mM. 0,00 0,01 0,02 0,03 0,04 0,05 0,00 0,08 0,16 0,24 0,32 0,40 0,48 0,56 0,64 0,72 0,80 0,00 0,01 0,02 0,03 0,04 0,05 0,00 0,08 0,16 0,24 0,32 0,40 0,48 0,56 0,64 0,72 0,80 0 10 20 30 40 50 0,0 0,2 0,4 0,6 0,8 1,0 0 10 20 30 40 50 0,0 0,2 0,4 0,6 0,8 1,0 A414nm Concentração (µg mL-1) A414nm Concentração (µg mL-1) Extrato etanólico das folhas de P. AGRADECIMENTO À À Fundação Araucária e FAPESP pela bolsa concedida e apoio financeiro respectivamente. Ação sobre o dano provocado pelo radical AAPH em eritrócitos nitens Rutina Com radical AAPH Sem radical AAPH Com radical AAPH Sem radical AAPH 0 10 20 30 40 50 0,0 0,2 0,4 0,6 0,8 1,0 0 10 20 30 40 50 0,0 0,2 0,4 0,6 0,8 1,0 A414nm Concentração (µg mL-1) Rutina Com radical AAPH Sem radical AAPH Rutina Com radical AAPH Sem radical AAPH 0,00 0,01 0,02 0,03 0,04 0,05 0,00 0,08 0,16 0,24 0,32 0,40 0,48 0,56 0,64 0,72 0,80 0,00 0,01 0,02 0,03 0,04 0,05 0,00 0,08 0,16 0,24 0,32 0,40 0,48 0,56 0,64 0,72 0,80 Concentração (µg mL-1) A414nm Extrato etanólico das folhas de P. nitens Com radical AAPH Sem radical AAPH Com radical AAPH Sem radical AAPH Concentração (µg mL-1) Concentração (µg mL-1) FIGURA 5. Ação hemolítica das amostras e interferência sobre a hemólise provocada pelo radical AAPH 25 mM. efeitos observados, mecanismos de ação e produtos formados nas reações. potencial hemolítico ou oxidante na ausência de radical. Na presença de HOCl, estes flavonóides haviam inibido a hemólise em baixas concentrações, mas, em elevadas concentrações, observou-se aumento da liberação de hemoglobina. Além disso, frente ao radical AAPH, os flavonóides puderam reduzir a hemólise nas concentrações avaliadas. REFERENCIA Frente aos dados obtidos, observa-se que o extrato bruto etanólico de P. nitens apresenta importante potencial de ação contra espécies reativas oxidativas, notadamente, frente a radicais livres e HOCl. Entretanto, a despeito desta atividade, também foi constatado um potencial lesivo às células, revelado pela lise de eritrócitos, seja pelo extrato, ou, até mesmo pela rutina, um antioxidante reconhecido e utilizado como padrão positivo para as atividades antioxidantes estudadas neste trabalho. Novos ensaios aprofundados são necessários para maiores esclarecimentos dos agentes responsáveis pelos BOLZANI, V.S.; GUNATILAKA, A.A.L.; KINGSTON, D.G.I. Bioactive guanidine alkalaoids from Pterogyne nitens. 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https://openalex.org/W2038731675	https://europepmc.org/articles/pmc4094074?pdf=render	English	null	Calcium Entry in Toxoplasma gondii and Its Enhancing Effect of Invasion-linked Traits	Journal of biological chemistry/The Journal of biological chemistry	2,014	cc-by	12,017	* Thisworkwassupported,inwholeorinpart,byNationalInstitutesofHealth Grants AI096836 (to S. N. J. M.) and AI101167 (to V. J.) and National Insti- tutes of Health T32 Training Grant AI060546 (to D. A. P. and the Center for Tropical and Emerging Global Diseases). □ S This article contains supplemental Videos S1 and S2. 1 Present address: California State University, Long Beach, CA 90840. 2 Both authors contributed equally to this work. 3 Present address: California State University, Fullerton, CA 92831 4 To whom correspondence should be addressed: Dept. of Cellular Biology and Center for Tropical and Emerging Global Disease, 350A Paul D. Cover- dell Center, University of Georgia, Athens, GA 30602. Tel.: 706-542-4736; Fax: 706-542-9493; E-mail: smoreno@uga.edu. 5 The abbreviations used are: ORAI, store-operated channel; STIM, stromal interaction molecule; EB, extracellular buffer; IB, intracellular buffer; SOCE, Calcium Entry in Toxoplasma gondii and Its Enhancing Effect of Invasion-linked Traits□ S Received for publication,March 20, 2014, and in revised form, May 19, 2014 Published, JBC Papers in Press,May 27, 2014, DOI 10.1074/jbc.M114.565390 Douglas A. Pace‡1, Ciara A. McKnight‡§2, Jing Liu‡¶2, Veronica Jimenez‡3, and Silvia N. J. Moreno‡§4 From the ‡Center for Tropical and Emerging Global Diseases and the §Department of Cellular Biology, University of Georgia, Athens, Georgia 30602 and the ¶College of Veterinary Medicine, China Agricultural University, Beijing 100193, China Background: Toxoplasma gondii is exposed to large Ca2 gradients during its lytic cycle. Results: Ca2 entry in T. gondii is a source of Ca2 increase and is mediated by a nifedipine-sensitive pathway and not by a canonical store-operated Ca2 entry (SOCE) pathway. Conclusion: Ca2 entry enhances parasite virulence traits. Significance: This is the first study linking regulation of Ca2 entry and virulence traits of T. gondii. Background: Toxoplasma gondii is exposed to large Ca2 gradients during its lytic cycle. Background: Toxoplasma gondii is exposed to large Ca2 gradients during its lytic cycle. Results: Ca2 entry in T. gondii is a source of Ca2 increase and is mediated by a nifedipine-sensitive pathway and not by a canonical store-operated Ca2 entry (SOCE) pathway. Background: Toxoplasma gondii is exposed to large Ca gradients during its lytic cycle. Results: Ca2 entry in T. gondii is a source of Ca2 increase and is mediated by a nifedipine-sensitiv canonical store-operated Ca2 entry (SOCE) pathway. y y Conclusion: Ca2 entry enhances parasite virulence traits. y Significance: This is the first study linking regulation of Ca2 entry and virulence traits of T. gondii. During invasion and egress from their host cells, Apicomplexan parasites face sharp changes in the surrounding calcium ion (Ca2) concentration. Our work with Toxoplasma gondii pro- vides evidence for Ca2 influx from the extracellular milieu leading to cytosolic Ca2 increase and enhancement of viru- lence traits, such as gliding motility, conoid extrusion, microneme secretion, and host cell invasion. Assays of Mn2 and Ba2 uptake do not support a canonical store-regulated Ca2 entry mechanism. Ca2 entry was blocked by the L-type Ca2 channel inhibitor nifedipine and stimulated by the increase in cytosolic Ca2 and by the specific L-type Ca2 chan- nel agonist Bay K-8644. Our results demonstrate that Ca2 entry is critical for parasite virulence. We propose a regulated Ca2 entry mechanism activated by cytosolic Ca2 that has an enhancing effect on invasion-linked traits. Calcium Entry in Toxoplasma gondii and Its Enhancing Effect of Invasion-linked Traits□ S endoplasmic reticulum sensor protein stromal interaction mol- ecule (STIM), voltage-operated channels, ligand-operated channels, transient receptor potential (TRP) channels, and sec- ond messenger-operated channels (3), but little is known about the mechanisms involved in Ca2 entry in the Apicomplexan parasite Toxoplasma gondii. During its lytic cycle, T. gondii actively invades host cells, creating a parasitophorous vacuole, where it divides to finally exit in search of a new host cell. Parasite invasion is a highly coordinated and active process involving several discrete steps (4). Gliding motility, conoid extrusion, secretion of specific proteins, attachment to the host cell, and active invasion are all critical steps for invasion. Previous studies have shown that intracellular Ca2 stores are important for initiation of gliding motility (5), microneme secretion (6), conoid extrusion (7), active parasite invasion (8–10), and egress (8) (reviewed in Ref. 11), but the role of extracellular Ca2 in these processes was not studied in detail or was considered minor. Elevation in cytoplasmic Ca2 mediates a plethora of cellular responses in all cells. Protein function is dependent on shape and charge, and both of these features are affected by Ca2 binding. Cells contain a sophisticated set of mechanisms to bal- ance the cytosolic Ca2 concentration, and the signals that increase Ca2 in the cytosol are compensated by mechanisms that decrease it (1). Considering the enormous Ca2 concentration gradient (20,000-fold), to which tachyzoites are exposed upon egress (100 nM in the host cytosol, taking into account that the para- sitophorous vacuole functions as a molecular sieve (12) versus 1.8 mM in the extracellular space), it seems a feasible hypoth- esis that the extracellular stage represents an opportunity to use extracellular Ca2 as a mechanism to enhance Ca2-dependent invasion processes as well as to replenish intracellular Ca2 stores that are likely used during the process of parasite egress. In this work, we explore the mechanism of Ca2 entry in extra- cellular tachyzoites and its role during the lytic cycle of the parasite. We present evidence for a regulated Ca2 entry pro- cess and its enhancing effect on invasion-related traits. This is the first detailed study linking Ca2 entry and its regulation directly to virulence traits of an eukaryotic microbe. Ca2 entry plays an important role in replenishing intracel- lular organelles and in activating signaling pathways that respond to elevated cytosolic Ca2 (2). store-operated Ca2 entry; TG, thapsigargin; SERCA, sarcoplasmic/endo- plasmic reticulum Ca2-ATPase; 2-APB, 2-aminomethoxydiphenyl borate; Bay K, Bay K-8644; ANOVA, analysis of variance; IO, ionomycin; VGCC, volt- age-gated calcium Ca2 channel. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 28, pp. 19637–19647, July 11, 2014 © 2014 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 28, pp. 19637–19647, July 11, 2014 © 2014 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 28, pp. 19637–19647, July 11, 2014 © 2014 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. Calcium Entry in Toxoplasma gondii and Its Enhancing Effect of Invasion-linked Traits□ S Received for publication,March 20, 2014, and in revised form, May 19, 2014 Published, JBC Papers in Press,May 27, 2014, DOI 10.1074/jbc.M114.565390 Douglas A. Pace‡1, Ciara A. McKnight‡§2, Jing Liu‡¶2, Veronica Jimenez‡3, and Silvia N. J. Moreno‡§4 From the ‡Center for Tropical and Emerging Global Diseases and the §Department of Cellular Biology, University of Georgia, Athens, Georgia 30602 and the ¶College of Veterinary Medicine, China Agricultural University, Beijing 100193, China Background: Toxoplasma gondii is exposed to large Ca2 gradients during its lytic cycle. Results: Ca2 entry in T. gondii is a source of Ca2 increase and is mediated by a nifedipine-sensitive pathway and not by a canonical store-operated Ca2 entry (SOCE) pathway. Conclusion: Ca2 entry enhances parasite virulence traits. Significance: This is the first study linking regulation of Ca2 entry and virulence traits of T. gondii. Calcium Entry in Toxoplasma gondii and Its Enhancing Effect of Invasion-linked Traits□ S Five types of plasma membrane Ca2 channels are present in vertebrate cells such as the store-operated channel (ORAI),5 which is linked to the Calcium Entry in T. gondii The cells were resuspended to a final density of 1 l09 cells/ml in loading buffer (similar to the iso- lation buffer plus 1.5% sucrose and 5 M Fura-2/AM). The sus- pensions were incubated for 26 min in a 26 °C water bath with mild agitation. Subsequently, the cells were washed twice to remove extracellular dye. Cells were resuspended to a final den- sity of 1 109 cells/ml in the indicated buffer and kept in ice. Parasites were viable for several hours under these conditions. For fluorescence measurements, a 50-l portion of cell suspen- sion was diluted into 2.5 ml of the indicated buffer (final den- sity, 2 l07 cells/ml) in a cuvette placed in a Hitachi F-4500 spectrofluorometer. Excitation was at 340 and 380 nm, and emission was at 510 nm. The Fura-2 fluorescence response to intracellular Ca2 concentration ([Ca2]i) was calibrated from the ratio of 340/380-nm fluorescence values after subtraction of the background fluorescence of the cells at 340 and 380 nm as described by Grynkiewicz et al. (15). Ca2 influx (Ca2/s) was evaluated by measuring the rate of [Ca2]i change during the first 20 s after its addition (linear rate) when the mechanisms of compensation are not yet at full capacity. Manganese quench- ing experiments were conducted on Fura-2/AM-loaded para- sites, and Fura-2 fluorescence was monitored at the isosbestic wavelength (excitation 360 nm, emission 510 nm). MnCl2 was added to a final concentration of 2 mM, and the rate of Fura-2 fluorescence quenching was recorded. FU was calcu- lated by measuring the rate of change in fluorescence during the first 20 s after adding manganese (when the rate is linear). No Fura-2 leakage into the extracellular media was detected (data not shown). Microneme Secretion—Purified intracellular parasites were collected in IB and resuspended in the same buffer with varying amounts of free calcium. Microneme secretion was measured at 37 °C for 15 min following published protocols (6). Rabbit anti-GRA1 at 1:10,000 was used as secretion control. Host Cell Invasion—Red-Green assay was modified from the assay described previously (16). Intracellular RH tachyzoites were collected in IB, centrifuged, and resuspended in the fol- lowing concentrations of free calcium: 160 nM, 99.3 M, 1 mM, and 2 mM at a parasite concentration of 1 108 cells/ml. Calcium Entry in T. gondii DMEM with 1% fetal bovine serum. Intracellular tachyzoites were isolated under intracellular conditions by scraping off infected fibroblasts and passing them through a 27-gauge nee- dle. In this way, parasites were released from host cells while still at intracellular ionic conditions. Extracellular tachyzoites were collected from cultures with 50–75% of egressed para- sites. Parasites (both intracellular and extracellular) were puri- fied as described (13) and washed in either low Ca2 extracel- lular buffer (EB) or intracellular buffer (IB) (see below). using an Olympus IX-71 inverted microscope with a Photome- trix CoolSnap CCD camera operated by DeltaVision image reconstruction software (Applied Precision, Seattle, WA). Glid- ing trail length was quantified with ImageJ software (National Institutes of Health) by measuring trails (positively stained with SAG1 antibody) that were directly associated with a parasite. For all treatments, at least 50 parasite trails were measured within each experiment. Conoid Extrusion—Conoid extrusion was tested following the protocol of Mondragon and Frixione (7). Parasites were kept in IB at 110 nM free Ca2. Parasites were spun down and resuspended in IB or EB at different conditions (e.g. Ca2 or in the presence of the calcium ionophore ionomycin). Experi- ments were initiated by placing the parasites at 37 °C and ter- minated by the addition of 4% formaldehyde. The percentage of parasites with their conoids extruded, reflecting the initiation of the invasion process, was assessed using an Olympus BX60 microscope. At least 100 parasites were evaluated for treatment within each independent experiment. Experiments testing Ca2 entry were conducted using EB (116 mM NaCl, 5.4 mM KCl, 1 mM MgSO4, 5.5 mM glucose, 20 mM Hepes, pH 7.4), IB (10 mM NaCl, 130 mM KCl, 10 mM glu- cose, 0.8 mM MgCl2, 1 mM EGTA, 0.62 mM CaCl2, 20 mM Hepes, pH 7.4) (final free Ca2, 117 nM) or buffer A (116 mM NaCl, 5.4 mM KCl, 0.8 mM MgSO4, 5.5 mM D-glucose, and 50 mM Hepes, pH 7.4). For low Ca2 EB, 1 mM EGTA and 0.62 mM CaCl2 were added to bring the free Ca2 to 110 nM. The final Ca2 concen- trations were determined by using CaCl2-EGTA combinations and calculated using Maxchelator software. Cytosolic Ca2 Measurements—Parasites (intracellular or extracellular) were loaded with Fura-2/AM as described (14). Briefly, after harvesting the cells, they were washed twice at 500 g for 10 min at room temperature in the buffer indicated in each figure legend. EXPERIMENTAL PROCEDURES Cell Culture and Preparation—T. gondii tachyzoites (RH strain) were maintained in hTERT human fibroblasts using JOURNAL OF BIOLOGICAL CHEMISTRY 19637 JULY 11, 2014•VOLUME 289•NUMBER 28 19637 Calcium Entry in T. gondii 2.5 107 tachyzoites were added to subconfluent hTERT monolay- ers, allowed to settle for 15 min on ice, and then incubated for 2 min at 37 °C. Subsequently, the medium was aspirated and fixed with 2.5% formaldehyde for 20 min. Subsequent steps were done as published (16). Cell counting data from three inde- pendent experiments were collected; each one by triplicate, counting eight randomly selected fields per individual cover- slip. The number of invaded parasites was calculated as the difference between the total number of parasites (green) and the number of attached parasites (red). Nifedipine inhibition of invasion was tested in invasion media (DMEM, 10 mM Hepes, pH 7.4, and 3% fetal bovine serum). RESULTS At 100 s, varying concentrations of Ca2 were added to the extracellular buffer: 2 mM (blue), 1.5 mM (red), 1 mM (green), and no FIGURE 1. Ca2 entry in extracellular tachyzoites. Measurements were made in low Ca2 EB (see “Experimental Procedures”). A, addition of Ca2 (1 mM) to Fura-2/AM-loaded tachyzoites leads to an increase in cytosolic Ca2. Inset, no addition. B, addition of various extracellular Ca2 concentrations leads to higher intracellular Ca2 increase. At 100 s, varying concentrations of Ca2 were added to the extracellular buffer: 2 mM (blue), 1.5 mM (red), 1 mM (green), and no addition (black). C, increase in extracellular Ca2 concentration resulted in a large increase in cytosolic Ca2 followed by a plateau at 200 nM. D, amplification of the boxed area shown in C. The error bars in all cases indicate means S.E. (n 3). This phenomenon of saturable [Ca2]i increase with increas- ing extracellular Ca2, and the maintenance of cytosolic Ca2 levels orders of magnitude below that of the extracellular Ca2 concentration, support the conclusion that entry of extracellu- lar Ca2 is regulated by the parasite and probably occurs through the coordination of specific entry channels (probably more than one) and compensatory mechanisms operated by Ca2 pumps. centrations (1.5 and 2 mM) led to higher cytosolic Ca2 (Fig. 1B). We measured [Ca2]i over a wide range of extracellular Ca2 concentrations (Fig. 1C), consistently observing an increase in final [Ca2]i (after return to basal levels) up to 0.6 M (Fig. 1D). Higher extracellular Ca2 concentrations (1.5–2 mM) led to minor changes in intracellular levels (Fig. 1C). Although there was significant entry of Ca2, its final concen- tration was orders of magnitude lower than the extracellular concentration (Fig. 1C). The curve in Fig. 1C shows a biphasic phenomenon, which could be due to the participation of more than one Ca2 permeation pathway. Tachyzoites isolated under extracellular conditions (see “Experimental Procedures”) also showed this phenomenon of Ca2 entry upon addition of Ca2 (data not shown) (see “Experimental Procedures” for buffer compositions). centrations (1.5 and 2 mM) led to higher cytosolic Ca2 (Fig. 1B). We measured [Ca2]i over a wide range of extracellular Ca2 concentrations (Fig. 1C), consistently observing an increase in final [Ca2]i (after return to basal levels) up to 0.6 M (Fig. 1D). Higher extracellular Ca2 concentrations (1.5–2 mM) led to minor changes in intracellular levels (Fig. 1C). RESULTS Evidence for a Regulated Ca2 Entry Pathway in T. gondii Tachyzoites—Upon egress from their host cell, T. gondii tachyzoites face a large change in extracellular Ca2 concentra- tion from nanomolar levels, when in contact with the host cyto- plasm through the parasitophorous vacuole, to 1.8 mM, in the extracellular milieu. Our hypothesis is that tachyzoites allow for some of this Ca2 to enter but in a regulated manner. To simulate conditions present upon exit of tachyzoites from the host cells, we suspended parasites obtained under intracellular conditions (see “Experimental Procedures”) and loaded them with Fura-2/AM at a Ca2 concentration usually present in the cytosol (110 nM; low Ca2 EB; see “Experimental Procedures”). The addition of extracellular Ca2 (1 mM) elicited an increase in intracellular Ca2 concentration ([Ca2]i) (Fig. 1A) that slowly returns to the basal level. Higher extracellular Ca2 con- Gliding Assays—Purified parasites were lightly adhered to polylysine-coated coverslips by placing them (2 107 cells/ml) on a coverslip on ice for 4 min. Non-adhered cells were washed away, and coverslips were incubated at different conditions at 37 °C for 10 min, and immunofluorescence images were acquired as described previously (5). Images were acquired 19638 19638 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289•NUMBER 28•JULY 11, 2014 GURE 1. Ca2 entry in extracellular tachyzoites. Measurements were made in low Ca2 EB (see “Experimental Procedures”). A, addition of Ca2 (1 mM) to ura-2/AM-loaded tachyzoites leads to an increase in cytosolic Ca2. Inset, no addition. B, addition of various extracellular Ca2 concentrations leads to higher tracellular Ca2 increase. At 100 s, varying concentrations of Ca2 were added to the extracellular buffer: 2 mM (blue), 1.5 mM (red), 1 mM (green), and no ddition (black). C, increase in extracellular Ca2 concentration resulted in a large increase in cytosolic Ca2 followed by a plateau at 200 nM. D, amplification f the boxed area shown in C. The error bars in all cases indicate means S.E. (n 3). Calcium Entry in T. gondii Calcium Entry in T. gondii y g FIGURE 1. Ca2 entry in extracellular tachyzoites. Measurements were made in low Ca2 EB (see “Experimental Procedures”). A, addition of Ca2 (1 mM) to Fura-2/AM-loaded tachyzoites leads to an increase in cytosolic Ca2. Inset, no addition. B, addition of various extracellular Ca2 concentrations leads to higher intracellular Ca2 increase. RESULTS Although there was significant entry of Ca2, its final concen- tration was orders of magnitude lower than the extracellular concentration (Fig. 1C). The curve in Fig. 1C shows a biphasic phenomenon, which could be due to the participation of more than one Ca2 permeation pathway. Tachyzoites isolated under extracellular conditions (see “Experimental Procedures”) also showed this phenomenon of Ca2 entry upon addition of Ca2 (data not shown) (see “Experimental Procedures” for buffer compositions). Calcium Entry Is Not Stimulated by Store Depletion but Blocking the SERCA-type Ca2 ATPase Leads to Higher Cytoso- lic Ca2 Levels—We next investigated whether Ca2 entry could be further enhanced by depletion of Ca2 from intracel- lular stores (store-operated Ca2 entry, or SOCE), a mecha- nism proposed to operate in the related Apicomplexan Plasmo- dium falciparum (17). To test for SOCE, we used thapsigargin JULY 11, 2014•VOLUME 289•NUMBER 28 19639 JOURNAL OF BIOLOGICAL CHEMISTRY Calcium Entry in T. gondii FIGURE2.Ca2entryintachyzoitesisenhancedinthepresenceofthapsigarginbutentryisnotthroughacanonicalstore-regulatedchannel.Parasites (5 107 cells) were loaded with Fura-2/AM and resuspended in 2.5 ml of EB in the presence of 100 M EGTA. A, cytosolic Ca2 changes upon addition of 1 M TG at 100 s and then 1 mM CaCl2 at 400 s (blue line). The red line represents the cytosolic Ca2 changes after Ca2 addition alone. The inset shows quantification of the initial rate of Ca2 entry after adding Ca2. At least three independent experiments were used for the quantification. B, Ca2 entry measured by Mn2 quenching. Fura-2/AM-loaded parasites were monitored at the Fura-2 isosbestic fluorescence point (excitation 360 nm). The blue tracing is the tracing obtainedafteradding1MTG.TheinsetshowsquantificationoftherateofMnCl2uptakefromthreeormoreindependentexperiments.2mMMnCl2wasadded where indicated. C, Ba2 entry and binding of Fura-2. The blue line shows the increase in Fura-2 fluorescence after store depletion with 1 M TG. The red line indicatesincreaseinfluorescencewithoutdepletionofintracellularstores.ThereisnosignificantdifferenceintheinitialrateoffluorescenceincreaseafterBa2 addition (1 mM) under both conditions (inset). The inset shows the quantification of the initial rate of BaCl2 uptake from a minimum of three independent experiments. D, Gd3 inhibition. The blue line shows preincubation with 5 M Gd3 and addition of 1 M TG and then 1 mM Ca2. The red line shows increased Fura-2 fluorescence after store depletion with 1 M TG alone. The inset shows quantification of initial rates of Ca2 uptake. A minimum of three independent experiments was used for the quantification. E, Ca2 entry enhanced by TG is not affected by 2-APB. 2-APB (30 M) was added where indicated. RESULTS The error bars in all cases indicate means S.E. (n 3). Calcium Entry in T. gondii FIGURE2.Ca2entryintachyzoitesisenhancedinthepresenceofthapsigarginbutentryisnotthroughacanonicalstore-regulatedchannel.Parasites (5 107 cells) were loaded with Fura-2/AM and resuspended in 2.5 ml of EB in the presence of 100 M EGTA. A, cytosolic Ca2 changes upon addition of 1 M TG at 100 s and then 1 mM CaCl2 at 400 s (blue line). The red line represents the cytosolic Ca2 changes after Ca2 addition alone. The inset shows quantification of the initial rate of Ca2 entry after adding Ca2. At least three independent experiments were used for the quantification. B, Ca2 entry measured by Mn2 quenching. Fura-2/AM-loaded parasites were monitored at the Fura-2 isosbestic fluorescence point (excitation 360 nm). The blue tracing is the tracing obtainedafteradding1MTG.TheinsetshowsquantificationoftherateofMnCl2uptakefromthreeormoreindependentexperiments.2mMMnCl2wasadded where indicated. C, Ba2 entry and binding of Fura-2. The blue line shows the increase in Fura-2 fluorescence after store depletion with 1 M TG. The red line indicatesincreaseinfluorescencewithoutdepletionofintracellularstores.ThereisnosignificantdifferenceintheinitialrateoffluorescenceincreaseafterBa2 addition (1 mM) under both conditions (inset). The inset shows the quantification of the initial rate of BaCl2 uptake from a minimum of three independent experiments. D, Gd3 inhibition. The blue line shows preincubation with 5 M Gd3 and addition of 1 M TG and then 1 mM Ca2. The red line shows increased Fura-2 fluorescence after store depletion with 1 M TG alone. The inset shows quantification of initial rates of Ca2 uptake. A minimum of three independent experiments was used for the quantification. E, Ca2 entry enhanced by TG is not affected by 2-APB. 2-APB (30 M) was added where indicated. The error bars in all cases indicate means S.E. (n 3). (TG), an inhibitor of the SERCA-type Ca2-ATPase, which results in leakage of Ca2 from the endoplasmic reticulum as a consequence of inhibiting the uptake pathway. The addition of TG 300 s prior to adding CaCl2 resulted in a reproducible higher influx, which can be observed by an increase in [Ca2]i as compared with the control in the absence of TG (Fig. 2A). We measured the initial rate of entry under identical conditions used to measure Ca2 entry after thapsigargin (Ca2 “add back”), and rates were similar in the presence and absence of thapsigargin (Fig. 2C, inset). This is in agreement with the Mn2 quenching experiment and indicates that the phenome- non of Ca2 entry is likely not store-regulated. RESULTS We also tested two inhibitors of SOCE channels, gadolinium (Gd3) at 5 M (Fig. 2D) and 2-aminomethoxydiphenyl borate (2-APB) at 30 M (Fig. 2E). We did not observe any effect on Ca2 entry with any of these inhibitors. Although it appears as if Gd3 inhibits the amount of Ca2 entry, initial rates (see “Experimental Pro- cedures” for explanation of calculations) were not significantly different to the initial rate of Ca2 entry after adding Ca2 alone (Fig. 2D, inset). It is possible that Gd3 could partially block a plasma membrane voltage-gated calcium Ca2 channel (VGCC) as reported previously (19), limiting the amount of Ca2 entering, but the initial rate is not affected. The results described above (Fig. 2A) seemed to indicate the existence of SOCE in T. gondii. However, the increases in [Ca2]i evoked by restoring Ca2 with and without prior TG treatment differed only in that the former had a transient over- shoot, but thereafter the “sustained” phases were similar (after the initial peak). These results seem most easily explained by a larger increase in [Ca2]i because of the inhibition of SERCA, which can no longer contribute to sequester incoming Ca2. We next tested this possibility by different strategies. The sur- rogate ion Mn2 is known to permeate through Ca2 channels, effectively quenching Fura-2 fluorescence. In addition, Mn2 is a poor substrate for most Ca2-dependent enzymes/processes so that any quenching observed in Fura-2/AM-loaded parasites would be due to Mn2 entry. An abrupt quenching of cytosolic Fura-2 fluorescence was observed at the Ca2-independent excitation wavelength of 360 nm (Fig. 2B). However, the rate of quenching was not increased by thapsigargin, suggesting that this flux of Mn2 was not stimulated through store depletion (Fig. 2B, inset). To further investigate this phenomenon, we used Ba2, which enters cells through Ca2 channels and binds Fura-2 modifying its fluorescence in a similar way to Ca2 (18). In summary, our results do not support the presence of a Ca2 entry channel regulated by store depletion. The absence of store regulation of Ca2 entry is supported by the lack of genomic evidence for the presence of SOCE molecular players (STIM and ORAI) in any Apicomplexan parasite (EuPath.DB). The Influence of Cytosolic Ca2 on Ca2 Entry—The in- creased Ca2 accumulation after TG addition as a result of SERCA inhibition could result in opening of a plasma mem- brane Ca2 channel through a Ca2 induced Ca2 uptake mechanism. RESULTS To study whether this was the case, we investi- In summary, our results do not support the presence of a Ca2 entry channel regulated by store depletion. The absence of store regulation of Ca2 entry is supported by the lack of genomic evidence for the presence of SOCE molecular players (STIM and ORAI) in any Apicomplexan parasite (EuPath.DB). The Influence of Cytosolic Ca2 on Ca2 Entry—The in- creased Ca2 accumulation after TG addition as a result of SERCA inhibition could result in opening of a plasma mem- brane Ca2 channel through a Ca2 induced Ca2 uptake mechanism. To study whether this was the case, we investi- VOLUME 289•NUMBER 28•JULY 11, 2014 19640 JOURNAL OF BIOLOGICAL CHEMISTRY 19640 FIGURE 3. Role of [Ca2]i in Ca2 entry as detected by varying the time interval between TG and Ca2 additions. Fura-2/AM loaded parasites were treated with 1 M TG and Ca2 added back at different times. A, the red line shows a representative tracing where Ca2 (1 mM) was added 50 s after TG (at maximum TG response, red arrow). Green line shows the response to addition of Ca2 100 s after TG (green arrow). The blue line represents a time interval of 150 s (after TG response, blue arrow). Inset, quantified responses for four inde- pendent experiments measuring Ca2 entry for Ca2 alone (Ca2) and Ca2 addition at different time points (in seconds) after TG addition. Rates of Ca2 entry were measured in the first 20 s. Rate of Ca2 entry was significantly higher at 50 s when the cytosolic Ca2 was highest (ANOVA, p 0.05). B, Ca2 entry in T. gondii after the addition of TG. Fura-2/AM loaded parasites in EB were treated with 1 M TG at 50 s, and then the concentrations of Ca2 indi- cated were added at 100 s. For reference, the increase in cytosolic Ca2 upon Calcium Entry in T. gondii Calcium Entry in T. gondii Calcium Entry in T. gondi gated whether there was a correlation between the [Ca2]i lev- els and the rate of Ca2 entry. The addition of TG leads to an increase in cytosolic Ca2 and a decrease after reaching a max- imum caused by reuptake or efflux of released Ca2. We added Ca2 back at different times after the addition of TG (50, 100, and 150 s) at points when the cell reached different levels of cytosolic Ca2 (Fig. RESULTS 3A, inset). If Ca2 entry was modulated by cytosolic Ca2, then as [Ca2]i decays, so too would the rate of influx of extracellular Ca2. Calcium “add back” at different times after TG showed that there is a significant difference between the rate of Ca2 entry at 50 s versus 100 and 150 s after TG addition (Fig. 3A, inset). Ca2 entry rate at 100 and 150 s was 30% lower than the rate at 50 s, which is when [Ca2]i is highest. We also observed that the rate of Ca2 entry after addition of 1, 1.5, and 2.0 mM Ca2 after TG addition increased signifi- cantly when compared with the rates of entry with the same Ca2 concentrations without TG (Fig. 3B) (compare gray and color bars of Fig. 3B, inset). We added Ca2 back 50 s after the addition of TG, and we observed that the rate of Ca2 entry was consistently 2.5 times higher in the presence of TG for all the treatments (Fig. 3B, inset). This enhancing effect of TG was independent of the ionic conditions used for purification of parasites and measurements (data not shown). In summary, Ca2 release from intracellular stores results in an increase in cytosolic Ca2, and this elevated Ca2 could directly modulate a Ca2 entry channel. This result agrees with the results shown in Fig. 2A where a higher level of Ca2 entry was observed after blocking the SERCA with TG. g Inhibition of Ca2 Entry—To investigate the mechanism of Ca2 entry we tested a range of channel blockers and inhibitors like 2-APB (30 M) (20), nifedipine (1–10 M), verapamil (1–100 M), and diltiazem (10–100 M) (21). Only nifedipine, an inhibitor of L-type voltage-activated Ca2 channels, was effective in blocking Ca2 entry (Fig. 4A). 2-APB at 30 M had no effect on Ca2 entry enhanced by thapsigargin, supporting the lack of participation of a SOCE Ca2 channel (Fig. 2E). Nifedipine also decreased Mn2 quenching in a dose-depen- dent manner (Fig. 4B), suggesting that entry of extracellular Ca2 is through a channel-mediated process and that nifedi- pine is an effective blocker of such Ca2 entry. FIGURE 3. Role of [Ca2]i in Ca2 entry as detected by varying the time interval between TG and Ca2 additions. Fura-2/AM loaded parasites were treated with 1 M TG and Ca2 added back at different times. RESULTS A, the red line shows a representative tracing where Ca2 (1 mM) was added 50 s after TG (at maximum TG response, red arrow). Green line shows the response to addition of Ca2 100 s after TG (green arrow). The blue line represents a time interval of 150 s (after TG response, blue arrow). Inset, quantified responses for four inde- pendent experiments measuring Ca2 entry for Ca2 alone (Ca2) and Ca2 addition at different time points (in seconds) after TG addition. Rates of Ca2 entry were measured in the first 20 s. Rate of Ca2 entry was significantly higher at 50 s when the cytosolic Ca2 was highest (ANOVA, p 0.05). B, Ca2 entry in T. gondii after the addition of TG. Fura-2/AM loaded parasites in EB were treated with 1 M TG at 50 s, and then the concentrations of Ca2 indi- cated were added at 100 s. For reference, the increase in cytosolic Ca2 upon addition of TG alone is also shown (gray line). Inset, quantification of the initial Ca2 entry rates of three or more independent experiments. Gray bars repre- sent increase in cytosolic Ca2 after adding only extracellular Ca2, colored bars represent increase when the same amount of Ca2 addition is preceded by 1 M TG. Error bars in all cases indicate means S.E. (n 3). FIGURE 3. Role of [Ca2]i in Ca2 entry as detected by varying the time interval between TG and Ca2 additions. Fura-2/AM loaded parasites were treated with 1 M TG and Ca2 added back at different times. A, the red line shows a representative tracing where Ca2 (1 mM) was added 50 s after TG (at maximum TG response, red arrow). Green line shows the response to addition of Ca2 100 s after TG (green arrow). The blue line represents a time interval of 150 s (after TG response, blue arrow). Inset, quantified responses for four inde- pendent experiments measuring Ca2 entry for Ca2 alone (Ca2) and Ca2 addition at different time points (in seconds) after TG addition. Rates of Ca2 entry were measured in the first 20 s. Rate of Ca2 entry was significantly higher at 50 s when the cytosolic Ca2 was highest (ANOVA, p 0.05). B, Ca2 entry in T. gondii after the addition of TG. RESULTS Fura-2/AM loaded parasites in EB were treated with 1 M TG at 50 s, and then the concentrations of Ca2 indi- cated were added at 100 s. For reference, the increase in cytosolic Ca2 upon addition of TG alone is also shown (gray line). Inset, quantification of the initial Ca2 entry rates of three or more independent experiments. Gray bars repre- sent increase in cytosolic Ca2 after adding only extracellular Ca2, colored bars represent increase when the same amount of Ca2 addition is preceded by 1 M TG. Error bars in all cases indicate means S.E. (n 3). Taking into account the inhibition of Ca2 entry by nifedi- pine, we tested Bay K-8644 (Bay K), a Ca2 channel agonist that acts by opening L-type channels (CaV1 family), increasing the time the channel is open and causing a large increase in the channel macroscopic response (22). Bay K (2.5–5 M) stimu- lated Ca2 entry when tested in Fura-2-loaded cells (Fig. 4C, inset). Using the Mn2-quenching technique (Fig. 4D), we found that Ca2 entry in extracellular tachyzoites was signifi- cantly enhanced when parasites were treated with 5 M Bay K (Fig. 4D). The inset in Fig. 4D shows the quantification of rates obtained after adding 1, 2.5, and 5 M Bay K. We also tested nifedipine and Bay K after blocking the SERCA with TG to obtain higher cytosolic Ca2 levels (Fig. 5). We observed a significant inhibition by nifedipine of Ca2 entry measuring the initial rate (Fig. 5A, inset). The addition of TG followed by the simultaneous addition of CaCl2 and 5 M Bay K (blue trace) elicited a greater accumulation of Ca2 over that of TG and Ca2 alone (Fig. 5B, red trace). The quantifica- tion of these relative differences in these measurements dem- onstrated that Bay K addition resulted in a significant increase JULY 11, 2014•VOLUME 289•NUMBER 28 19641 JOURNAL OF BIOLOGICAL CHEMISTRY Calcium Entry in T. gondii n T. gondii FIGURE 4. Ca2 entry inhibition and stimulation. Conditions as in Fig. 2. A, Ca2 entry (1 mM) is inhibited in parasites preincubated for 2 min with nifedipine (Nif) ranging from 1 to 10 M. Inset, quantification of initial rates measure from at least three independent experiments. B, Mn2 quenching inhibition by nifedipine. Fura-2/AM-loaded parasites were monitored at the Fura-2 isosbestic fluorescence point (excitation 360 nm). 2 mM MnCl2 was added where indicated. RESULTS Inset, rate of Mn2 quenching was significantly decreased (ANOVA of slope: n 4, p 0.05) by nifedipine. C, Bay K-8644 enhances Ca2 entry. Fura-2/AM-loaded parasites in EB were analyzed in the presence of 100 M EGTA, and 1 mM Ca2 was added at the indicated time. Inset, quantification of initial rates S.E. after adding Ca2 of at least three independent experiments. D, Mn2 entry in tachyzoites in Fura-2/AM-loaded parasites (5 107 cells in EB). Quenching by Mn2 was measured in the presence of Bay K (5 M) (red line) or 10 M nifedipine (maroon line). Mn2 (2 mM) was added where indicated (100 s).Theconditionswere2mMMnCl2DMSO(controlentry,grayline),2mMMnCl25MBayK(redline),and2mMMnCl210Mnifedipine(maroonline).Inset, rates of change in Fura-2 fluorescence at the isosbestic point S.E. (n 4) during the first 20 s after addition of manganese alone or plus 1, 2.5, and 5 M Bay K. , p 0.01 (one-way ANOVA). Calcium Entry in T. gondii FIGURE 4. Ca2 entry inhibition and stimulation. Conditions as in Fig. 2. A, Ca2 entry (1 mM) is inhibited in parasites preincubated for 2 min with nifedipine (Nif) ranging from 1 to 10 M. Inset, quantification of initial rates measure from at least three independent experiments. B, Mn2 quenching inhibition by nifedipine. Fura-2/AM-loaded parasites were monitored at the Fura-2 isosbestic fluorescence point (excitation 360 nm). 2 mM MnCl2 was added where indicated. Inset, rate of Mn2 quenching was significantly decreased (ANOVA of slope: n 4, p 0.05) by nifedipine. C, Bay K-8644 enhances Ca2 entry. Fura-2/AM-loaded parasites in EB were analyzed in the presence of 100 M EGTA, and 1 mM Ca2 was added at the indicated time. Inset, quantification of initial rates S.E. after adding Ca2 of at least three independent experiments. D, Mn2 entry in tachyzoites in Fura-2/AM-loaded parasites (5 107 cells in EB). Quenching by Mn2 was measured in the presence of Bay K (5 M) (red line) or 10 M nifedipine (maroon line). Mn2 (2 mM) was added where indicated (100 s).Theconditionswere2mMMnCl2DMSO(controlentry,grayline),2mMMnCl25MBayK(redline),and2mMMnCl210Mnifedipine(maroonline).Inset, rates of change in Fura-2 fluorescence at the isosbestic point S.E. (n 4) during the first 20 s after addition of manganese alone or plus 1, 2.5, and 5 M Bay K. , p 0.01 (one-way ANOVA). (ANOVA comparison of slopes: p 0.001) in Ca2 accumula- tion (Fig. 5B, inset). RESULTS physiological context of Ca2 entry by addition of extracellular Ca2 or that had an increase in Ca2 accumulation by addition of TG and extracellular Ca2 (Fig. 6A, TG Ca2) or ionomy- cin (Fig. 6A, IO) showed an increase in both the percentage of gliding parasites and the length that the parasites moved (Fig. 6, B and C). Fig. 6A shows representative images used to obtain the quantification shown in Fig. 6 (B and C), which show the percentage of motile parasites and the trail length, respectively. Ionomycin (IO, 1 M) was used as a positive control because this reagent releases all Ca2 stored in neutral compartments and also increases Ca2 entry evoking a maximal response. Importantly, the addition of nifedipine (10 M) (in the presence of TG Ca2) caused a significant decrease in gliding response (Fig. 6, B and C, maroon bars). Interestingly, motility, as expressed as the percentage of parasites associated with a trail, did not get reduced beyond the levels of Ca2 alone (Fig. 6B), whereas gliding motility, expressed as average trail length, did (Fig. 6C), suggesting that nifedipine is inhibiting gliding motil- ity rather than other types of motility. The Role of Ca2 Entry in Virulence-related Traits: Gliding Motility, Conoid Extrusion, Microneme Secretion, and Host Cell Invasion—Previous work (6–10) has shown that [Ca2]i increase is involved during all these steps of the tachyzoite lytic cycle, but the source of this Ca2 increase has been proposed to be mostly Ca2 release from intracellular Ca2 stores on the basis of experiments using high concentrations of extracellular EGTA to prevent Ca2 entry. Our hypothesis is that upon egress, the parasite encounters a 20,000-fold higher concen- tration of Ca2, and this gradient should result in a strong elec- trochemical driving force favoring Ca2 influx. We therefore investigated whether this Ca2 influx plays a role in gliding motility, conoid extrusion, microneme secretion, and host cell invasion. We isolated parasites using both intra- and extracel- lular ionic concentrations and exposed them to higher, physio- logically relevant Ca2 concentrations to quantify these processes. Parasites treated with different reagents to promote high cytosolic Ca2 concentration were stained to determine whether they possessed a SAG1 positive gliding trail and the length of the trail. Parasites kept in standard buffer conditions showed only minimal movement and relatively small gliding trails (Fig. 6A, no additions). RESULTS TG (1 M), Ca2 (2 mM) and Ca2 Bay K (5 M) were added where indicated. Blue line, 2 mM Ca2 TG BayK; red line, Ca2 TG; light blue line, baseline Ca2 addition, green line is the response to TG (1 M) alone; black line, cytosolic Ca2 with no additions. Inset shows the quantification of the rate of Ca2 entry for multiple replicate measurements of TG Ca2 (red bar) and TG Ca2 Bay K (blue bar). *, p 0.01 (one-way ANOVA). Conditions as in Fig. 3. addition of both caused a substantial increase in motility (sup- plemental Videos S1 and S2). extracellular environment to an intracellular environment resulted in similar patterns of conoid extrusion (data not shown). Before invasion, tachyzoites extend their conoids (conoid extrusion), a highly dynamic organelle situated at the apical end of the parasite, which has been shown to be stimulated by an increase in [Ca2]i (7). We measured the ability of parasites to extrude their conoid over a relevant period of time (Fig. 6, D–F). Parasites kept in extracellular buffer conditions but at low Ca2 concentration (Ca2 110 nM) for the 5-min experiment showed no significant increase in conoid extrusion (Fig. 6E, EB). The addition of IO (1 M), which has been used previously to stimulate conoid extrusion (7), increased conoid extrusion when compared with control parasites. Extracellular Ca2 (1 mM) produced a minor increase in conoid extrusion. A high percentage of parasites treated with both IO and extracellular Ca2 extruded their conoids. Importantly, this increase in conoid response was maintained throughout the 5-min exper- iment, whereas the IO-treated parasites exhibited a higher ini- tial increase in conoid extrusion followed by a significant reduc- tion for the remainder of the experiment (Fig. 6E, inset). The difference obtained between IO treatment alone and in the presence of extracellular Ca2 shows the importance of extra- cellular calcium in enhancing conoid extrusion and also that in the absence of extracellular Ca2 (Fig. 6E, inset), the response is not sustained. In agreement with the enhancing effect of Ca2 entry, nifedipine inhibited conoid extrusion (Fig. 6F) in the presence of extracellular Ca2. We did not find a significant difference on the percentage of parasites with their conoids extruded when preincubated in the presence of Bay K. This could be because compensatory mechanisms in the parasite (i.e. RESULTS Parasites that were placed in the Experiments were also conducted to directly visualize the role of Ca2 accumulation caused by Ca2 entry in tachyzoite motility. Using the same conditions as seen in Fig. 1C parasites were treated with Ca2 alone, thapsigargin alone, or with both. Time lapse microscopy showed that although the addition of Ca2 or thapsigargin alone had a small effect on motility, the VOLUME 289•NUMBER 28•JULY 11, 2014 19642 19642 JOURNAL OF BIOLOGICAL CHEMISTRY Calcium Entry in T. gondii FIGURE 5. Nifedipine inhibits Ca2 entry enhanced by thapsigargin. A, Ca2 accumulation enhanced by TG (1 M), which was added at 50 s followed by 2 mM Ca2 at 100 s (red line), is inhibited by preincubating parasites with 10 M nifedipine (blue line). The black line is the baseline cytosolic Ca2 level, the gray line shows addition of TG, and the light blue line shows the response to Ca2 addition alone. Inset, quantification of rate of Ca2 entry after nifedipine inhibition. One-way ANOVA of regression model yielded a significant relationship between inhibition of Ca2 entry and nifedipine concentration: p 0.001. B, Bay K enhances Ca2 entry. TG (1 M), Ca2 (2 mM) and Ca2 Bay K (5 M) were added where indicated. Blue line, 2 mM Ca2 TG BayK; red line, Ca2 TG; light blue line, baseline Ca2 addition, green line is the response to TG (1 M) alone; black line, cytosolic Ca2 with no additions. Inset shows the quantification of the rate of Ca2 entry for multiple replicate measurements of TG Ca2 (red bar) and TG Ca2 Bay K (blue bar). , p 0.01 (one-way ANOVA). Conditions as in Fig. 3. Calcium Entry in T. gondii FIGURE 5. Nifedipine inhibits Ca2 entry enhanced by thapsigargin. A, Ca2 accumulation enhanced by TG (1 M), which was added at 50 s followed by 2 mM Ca2 at 100 s (red line), is inhibited by preincubating parasites with 10 M nifedipine (blue line). The black line is the baseline cytosolic Ca2 level, the gray line shows addition of TG, and the light blue line shows the response to Ca2 addition alone. Inset, quantification of rate of Ca2 entry after nifedipine inhibition. One-way ANOVA of regression model yielded a significant relationship between inhibition of Ca2 entry and nifedipine concentration: p 0.001. B, Bay K enhances Ca2 entry. JULY 11, 2014•VOLUME 289•NUMBER 28 RESULTS TG, TG (2 M); Ca2 1.6 mM free Ca2; TG Ca2, TG and 1.6 mM free Ca2; Nifedipine, same as TG Ca2 with a 60-s preincubation with 10 M nifedipine; IO 1 M ionomycin. D, role of extracellular Ca2 conoid extrusion. Representative images obtained after the treatments: extracellular parasites were collected and exposed to Ca2 (2 mM, EB), ionomycin ( IO), or both (IO Ca2 ) for 5 min at 37 °C. E, quantification of conoid extrusion response. Parasites were collected at specified times, fixed, and assessed fo percentage of parasites with an extruded conoid. Inset, kinetic analysis of conoid extrusion in extracellular parasites. Kinetic monitoring of conoid extru shows that when ionomycin-treated parasites were also given extracellular Ca2 (2 mM), they responded with a greater percentage of extruded conoids for extended amount of time (red symbols) as evidenced by a statistically nonsignificant slope value (p 0.05). Parasites treated with ionomycin in buffer cytosolic levels of Ca2 (110 nM) responded with an initial increase in conoid extrusion that displayed exponential decay kinetics (green symbols). F, effe nifedipine (10 M, Nif) and Bay K (2.5 M) on conoid extrusion. Parasites were treated as described under “Experimental Procedures,” and the percenta parasites with their conoid extruded was evaluated at 300 s. The average S.E. of three experiments of parasites with their conoids extruded under extr lular buffer with Ca2 (1 mM) was 21.3%. Incubation with nifedipine reduced this number to 12% (p 0.0062) and in the presence of Bay K; 23.7% o i h d h i id d d M d i i d d i ll l b ff Calcium Entry in T. gondii FIGURE 6. Ca2 entry enhances gliding motility and conoid extrusion. A, gliding trails visualization by SAG1 immunolocalization. Representative micro- graphs showing the SAG1 signal after different treatments (see label details below). These micrographs were used for the quantification number of motile parasitesinBandtheaveragetraillengthinC.Scalebar,5m.B,averagepercentage(n5S.E.)ofextracellulartachyzoitesthatweredirectlyassociatedwith a gliding trail. C, the average length (n 100 S.E.) of parasites directly associated with a gliding trail. TG, TG (2 M); Ca2 1.6 mM free Ca2; TG Ca2, 2 M TG and 1.6 mM free Ca2; Nifedipine, same as TG Ca2 with a 60-s preincubation with 10 M nifedipine; IO 1 M ionomycin. D, role of extracellular Ca2 on conoid extrusion. RESULTS Representative images obtained after the treatments: extracellular parasites were collected and exposed to Ca2 (2 mM, EB), ionomycin (1 M IO), or both (IO Ca2 ) for 5 min at 37 °C. E, quantification of conoid extrusion response. Parasites were collected at specified times, fixed, and assessed for the percentage of parasites with an extruded conoid. Inset, kinetic analysis of conoid extrusion in extracellular parasites. Kinetic monitoring of conoid extrusion shows that when ionomycin-treated parasites were also given extracellular Ca2 (2 mM), they responded with a greater percentage of extruded conoids and for extended amount of time (red symbols) as evidenced by a statistically nonsignificant slope value (p 0.05). Parasites treated with ionomycin in buffer with cytosolic levels of Ca2 (110 nM) responded with an initial increase in conoid extrusion that displayed exponential decay kinetics (green symbols). F, effect of nifedipine (10 M, Nif) and Bay K (2.5 M) on conoid extrusion. Parasites were treated as described under “Experimental Procedures,” and the percentage of parasites with their conoid extruded was evaluated at 300 s. The average S.E. of three experiments of parasites with their conoids extruded under extracel- lular buffer with Ca2 (1 mM) was 21.3%. Incubation with nifedipine reduced this number to 12% (p 0.0062) and in the presence of Bay K; 23.7% of the parasites had their conoids extruded. Measurements were done using parasites suspended in extracellular buffer. cellular Ca2 (Fig. 7, C and D). Additionally, when access to extracellular Ca2 was blocked through the use of nifedipine (10 M), there was a significant decrease in parasite attachment and invasion in host cells relative to DMSO vehicle controls (Fig. 7D). not support the activation of a SOCE channel. We assayed the rate of Mn2 entry and its response to the degree of store filling (2). The rate of Mn2 quenching was not stimulated by the preaddition of TG, indicating that store depletion did not acti- vate Ca2 entry in tachyzoites. We also could not demonstrate store-operated Ba2 influx. The higher accumulation of cyto- solic Ca2 in the presence of TG, could be the result of the SERCA pump inhibition rather than of direct stimulation of the entry channel by store depletion. RESULTS plasma membrane Ca2-ATPase) would not allow cytosolic Ca2 to stay elevated for extended lengths of time. As with other experiments, switching the ionic environment from an T. gondii tachyzoites contain micronemes, which are secre- tory organelles involved in host cell invasion. Microneme secre- tion is stimulated naturally upon host cell attachment. Although microneme proteins are secreted at a low rate in extracellular tachyzoites (23), the process can be accelerated 10–100-fold by elevating the intracellular Ca2 concentration (6). We studied the secretion of MIC2, a protein used as a marker to study microneme secretion (24). Parasites were exposed to increasing amounts of extracellular calcium (0–2.4 mM) after a short preincubation in a Ca2-free buffer to mea- sure secretion of MIC2 (Fig. 7A). This treatment resulted in a significant increase in the amount of MIC2 protein secreted from the parasite into the medium, as detected by Western blot analysis (Fig. 7A). The amount of MIC2 secretion in the same cohort of parasites when treated with TG (1 M) followed by the same Ca2 concentrations resulted in MIC2 secretion that was significantly higher than after the addition of Ca2 alone (Fig. 7A, compare TG). Fig. 7B is the quantification of three inde- pendent experiments and displays the consistency of this phe- nomenon. Importantly, MIC2 secretion was significantly higher when parasites were exposed to TG (blue bars). These results show the previously documented importance of intra- cellular stored Ca2 while demonstrating the significant enhancement that occurs in microneme secretion when Ca2 accumulates under Ca2 entry conditions. The cumulative effect of Ca2 on invasion-linked traits was assessed by the direct measurement of host cell attachment and invasion of parasites. Red-green assays demonstrated that par- asites exhibited higher frequencies of host cell attachment and invasion when incubated in increasing concentrations of extra- JULY 11, 2014•VOLUME 289•NUMBER 28 19643 JOURNAL OF BIOLOGICAL CHEMISTRY Calcium Entry in T. gondii FIGURE 6. Ca2 entry enhances gliding motility and conoid extrusion. A, gliding trails visualization by SAG1 immunolocalization. Representative m graphs showing the SAG1 signal after different treatments (see label details below). These micrographs were used for the quantification number of m parasitesinBandtheaveragetraillengthinC.Scalebar,5m.B,averagepercentage(n5S.E.)ofextracellulartachyzoitesthatweredirectlyassociated a gliding trail. C, the average length (n 100 S.E.) of parasites directly associated with a gliding trail. DISCUSSION This study provides evidence for a highly regulated mecha- nism of Ca2 entry in T. gondii extracellular tachyzoites. Ca2 levels were enhanced after depleting intracellular Ca2 stores with TG, a SERCA-type Ca2-ATPase inhibitor. Our results do This highly regulated Ca2 entry was stimulated by [Ca2]i, inhibited by the L-type Ca2 channel blocker nifedipine, and VOLUME 289•NUMBER 28•JULY 11, 2014 VOLUME 289•NUMBER 28•JULY 11, 2014 19644 JOURNAL OF BIOLOGICAL CHEMISTRY 19644 JOURNAL OF BIOLOGICAL CHEMISTRY Calcium Entry in T. gondii FIGURE 7. Extracellular Ca2 entry enhances microneme secretion and host cell invasion. A, Western blot analysis of a representative MIC2 secretion experiment (upper panel) under different extracellular Ca2 concentrations. Lower panel is secretion control with anti-GRA1. Where indicated, 1 M TG was added. MIC2 signal was below the level of detection at 0 mM Ca2. B, quantification of gel band intensity of MIC2 secretion ( S.E.) from three independent experiments (one of which is shown in A) with (blue bars) and without (red bars) TG addition. Experiments were standardized to loading controls and quantified using ImageJ (National Institutes of Health). C, relationship of extracellular Ca2 concentration and number of parasites attached to or invaded into host cells as determined by a red-green invasion assays. Both attachment and invasion showed significant relationships with Ca2 concentration (p 0.01 for both attachment and invasion). D, inhibition of invasion and attachment by nifedipine. IM, invasion medium; DMSO, vehicle control; Nifedipine, 10 M nifedipine. , p 0.01. Calcium Entry in T. gondii FIGURE 7. Extracellular Ca2 entry enhances microneme secretion and host cell invasion. A, Western blot analysis of a representative MIC2 secretion experiment (upper panel) under different extracellular Ca2 concentrations. Lower panel is secretion control with anti-GRA1. Where indicated, 1 M TG was added. MIC2 signal was below the level of detection at 0 mM Ca2. B, quantification of gel band intensity of MIC2 secretion ( S.E.) from three independent experiments (one of which is shown in A) with (blue bars) and without (red bars) TG addition. Experiments were standardized to loading controls and quantified using ImageJ (National Institutes of Health). C, relationship of extracellular Ca2 concentration and number of parasites attached to or invaded into host cells as determined by a red-green invasion assays. Both attachment and invasion showed significant relationships with Ca2 concentration (p 0.01 for both attachment and invasion). D, inhibition of invasion and attachment by nifedipine. IM, invasion medium; DMSO, vehicle control; Nifedipine, 10 M nifedipine. *, p 0.01. stimulated by the L-type Ca2 channel agonist Bay K. Fura-2 fluorescence quenching by Mn2 entry was also inhibited by nifedipine and significantly stimulated by Bay K, suggesting a similar mechanism of entry. 19644 JOURNAL OF BIOLOGICAL CHEMISTRY ORAI) involved in SOCE in some eukaryotes were discovered only a few years ago, almost 20 years after the phenomenon was described in mammalian cells (26). Specific searches in the T. gondii genome, as well as in other Apicomplexan genomes, have revealed no evidence for the existence of STIM or ORAI (27, 28), in agreement with our results on the absence of SOCE in T. gondii. It is interesting to note, however, that evidence for the presence of STIM and ORAI orthologs has been reported in other unicellular protists, like the choanoflagellate Monosiga brevicollis, which belongs to the supergroup Ophisotkonta, which also includes animals and fungi (11, 29). Our data support the presence of a Ca2 channel in tachyzoites probably voltage-gated, modulated by Ca2 itself. Certain voltage-gated Ca2 channels (e.g. L-type channels) upon becoming permeable to Ca2 (i.e. gate opening) will respond to cytosolic Ca2 by becoming more permeable, a process called Ca2-dependent facilitation (25). We tested this possibility by exploring the correlation between cytosolic Ca2 concentration and extent of Ca2 influx. We manipulated the intracellular Ca2 concentration using TG to block uptake by the ER and allow other uptake mechanisms to operate for dif- ferent lengths of times, resulting in different cytosolic Ca2 concentrations prior to the addition of extracellular Ca2. Under these conditions, the rate of Ca2 entry was significantly higher at higher cytosolic Ca2 concentration, indicating a pos- sible enhancement effect of cytosolic Ca2 itself on the putative Ca2 entry channel. A critical question emerging from our data is the molecular identity of the Ca2 permeation pathways. The dihydropyri- dine analog, nifedipine, an L-type calcium channel inhibitor, showed consistent inhibition of Ca2 entry, whereas the use of an agonist of L-type Ca2 channels, Bay K, demonstrated the ability to enhance Ca2 and Mn2 entry. Other members of the VGCC group (N, P/Q, and R) are insensitive to the effects of these modulators (30). The genome of T. gondii predicts the presence of a putative VGCC (31) (TGME49_205265), the type of channel expected to be nifedipine-sensitive. In addition, a recent evaluation by Prole and Taylor (27) found a second gene with homology to VGCCs (Cav), TgME49_267720, which may encode a subunit of a Ca2 channel as the sequence predicts for the presence of six transmembrane domains. An attractive Calcium ions impact nearly every aspect of cellular life. Calcium Entry in T. gondii Model for the role of Ca2 entry in the lytic cycle of T. gondii. The proposed model shows Ca2 changes in the cytosol and intracellular stores (IS) of the parasite when exposed to changes in extraparasite Ca2 concentrations during the lytic cycle of T. gondii. Step 1, cytoplasmic Ca2 [Ca2]i increases in the tachyzoite (darker orange) before egress because of Ca2 release from intracellular stores. Step 2, once outside the parasite, [Ca2]i returns to nanomolar levels (lighter orange). Step 3, because of large electrochemical gradient, extracellular Ca2 (represented by darker blue) enters parasite cytosol through the regulated mechanisms observed in this study (darker orange in tachyzoite). Step 4, according to our data, release of Ca2 from intracellular stores could contribute to this increase by an unknown mechanism. Step 5, an increase in cytosolic Ca2 would lead to initiation of invasion. Step 6, gliding, conoid extrusion, microneme secretion, and invasion. Step 7, the parasite [Ca2]i (lighter orange) decreases by the action of Ca2 pumps at the plasma membrane and the SERCA at the ER. This last event would result in replenishment of the intracellular stores. Step 8, tachyzoite invades its host cell and forms a parasitophorous vacuole where it resides and replicates exposed to host [Ca2]i (50–100 nM). The blue color barrepresentsrelativeextraparasiteCa2concentration.Theorangecolorbar represents parasite Ca2 concentration. The arrows show Ca2 fluxes. The black lines and dots on invading parasites represent conoid extrusion and microneme secretion, respectively. (Christina A. Moore drew the model.) lost, and then egress and begin the process anew. In fact, studies show that parasites commonly invade and exit with little or no replication (35, 36). Such complex invasion behavior, all of which is dependent on Ca2, likely requires the replenishing of intracellular stored Ca2 with extracellular Ca2. Similarly, the process of egress is dependent on Ca2 (8). A parsimonious feature for the parasites is to utilize the extracellular Ca2 avail- able to supplement invasion-dependent processes and to also resupply intracellular stores to use during reinvasion or to ini- tiate egress. Other studies have found some evidence for the role of extra- cellular Ca2 in the lytic cycle of T. gondii. Lovett and Sibley (33) demonstrated that release of Ca2 via ryanodine-sensitive stores was enhanced in the presence of extracellular Ca2. Importantly, without extracellular Ca2, ryanodine was incapa- ble of stimulating microneme secretion. Treatment of parasites with caffeine (an agonist of ryanodine receptors) also elicited release of stored Ca2. 19644 JOURNAL OF BIOLOGICAL CHEMISTRY SOCE mechanisms are activated specifically in response to depletion of Ca2 from intracellular organelles. This Ca2 is used to replenish those organelles and to activate Ca2-sensi- tive signaling pathways. The molecular players (STIM1 and JULY 11, 2014•VOLUME 289•NUMBER 28 19645 JOURNAL OF BIOLOGICAL CHEMISTRY Calcium Entry in T. gondii Step 7, the parasite [Ca2]i (lighter orange) decreases by the action of Ca2 pumps at the plasma membrane and the SERCA at the ER. This last event would result in replenishment of the intracellular stores. Step 8, tachyzoite invades its host cell and forms a parasitophorous vacuole where it resides and replicates exposed to host [Ca2]i (50–100 nM). The blue color barrepresentsrelativeextraparasiteCa2concentration.Theorangecolorbar represents parasite Ca2 concentration. The arrows show Ca2 fluxes. The black lines and dots on invading parasites represent conoid extrusion and microneme secretion, respectively. (Christina A. Moore drew the model.) hypothesis is that the Ca2 entry observed in this study involves one or more of these Ca2 channels. Interestingly, Ca2 entry in response to various extracellular Ca2 concentrations showed a biphasic mode in support of the participation of more than one channel or permeation pathway. The use of T. gondii as a model basal eukaryote may provide very useful information for understanding the evolution of Ca2 regulation and signal- ing. Further exploration of these genes is warranted given the supporting evidence in ToxoDB.org. Interestingly, Pezzella et al. (32) reported an effect on invasion when using dihydropyri- dine Ca2 channel inhibitors similar to nifedipine, the reagent used in this study to inhibit Ca2 entry and invasion-linked responses. In this study, we quantitatively correlated Ca2 entry with invasion-linked behavior. This strategy proved to be very useful in highlighting how extracellular Ca2 enhances invasion- linked traits. We designed our experiments so that free Ca2 concentrations were always defined and tightly controlled (110 nM range). We looked at motility, conoid extrusion, microneme secretion, and invasion, and Ca2 entry enhanced all of these traits. The enhancement was more dramatic when cytosolic levels were elevated with ionophores or with the SERCA inhibitor TG. The use of ionophores in the presence of extracellular Ca2 led to higher intracellular Ca2 levels, sug- gesting a potential association. This can be appreciated in fur- ther examination of the conoid extrusion data. Release of stored Ca2 by ionophore treatment results in a large response in conoid extrusion. However, the combined use of ionophore with extracellular Ca2 results not only in a higher proportion of cells extruding their conoids, but the population also main- tains extruded conoids for longer times, an advantageous trait for increasing invasion efficiency. The inhibition of conoid extrusion by nifedipine agrees with the Ca2 entry enhance- ment effect on this trait. FIGURE 8. Calcium Entry in T. gondii FIGURE 8. Model for the role of Ca2 entry in the lytic cycle of T. gondii. The proposed model shows Ca2 changes in the cytosol and intracellular stores (IS) of the parasite when exposed to changes in extraparasite Ca2 concentrations during the lytic cycle of T. gondii. Step 1, cytoplasmic Ca2 [Ca2]i increases in the tachyzoite (darker orange) before egress because of Ca2 release from intracellular stores. Step 2, once outside the parasite, [Ca2]i returns to nanomolar levels (lighter orange). Step 3, because of large electrochemical gradient, extracellular Ca2 (represented by darker blue) enters parasite cytosol through the regulated mechanisms observed in this study (darker orange in tachyzoite). Step 4, according to our data, release of Ca2 from intracellular stores could contribute to this increase by an unknown mechanism. Step 5, an increase in cytosolic Ca2 would lead to initiation of invasion. Step 6, gliding, conoid extrusion, microneme secretion, and invasion. Step 7, the parasite [Ca2]i (lighter orange) decreases by the action of Ca2 pumps at the plasma membrane and the SERCA at the ER. This last event would result in replenishment of the intracellular stores. Step 8, tachyzoite invades its host cell and forms a parasitophorous vacuole where it resides and replicates exposed to host [Ca2]i (50–100 nM). The blue color barrepresentsrelativeextraparasiteCa2concentration.Theorangecolorbar represents parasite Ca2 concentration. The arrows show Ca2 fluxes. The black lines and dots on invading parasites represent conoid extrusion and microneme secretion, respectively. (Christina A. Moore drew the model.) FIGURE 8. Model for the role of Ca2 entry in the lytic cycle of T. gondii. The proposed model shows Ca2 changes in the cytosol and intracellular stores (IS) of the parasite when exposed to changes in extraparasite Ca2 concentrations during the lytic cycle of T. gondii. Step 1, cytoplasmic Ca2 [Ca2]i increases in the tachyzoite (darker orange) before egress because of Ca2 release from intracellular stores. Step 2, once outside the parasite, [Ca2]i returns to nanomolar levels (lighter orange). Step 3, because of large electrochemical gradient, extracellular Ca2 (represented by darker blue) enters parasite cytosol through the regulated mechanisms observed in this study (darker orange in tachyzoite). Step 4, according to our data, release of Ca2 from intracellular stores could contribute to this increase by an unknown mechanism. Step 5, an increase in cytosolic Ca2 would lead to initiation of invasion. Step 6, gliding, conoid extrusion, microneme secretion, and invasion. REFERENCES 22. Yamamoto, H., Hwang, O., and Van Breemen, C. (1984) Bay K8644 dif- ferentiates between potential and receptor operated Ca2 channels. Eur. J. Pharmacol. 102, 555–557 1. Berridge, M. J., Bootman, M. D., and Roderick, H. L. (2003) Calcium sig- nalling: dynamics, homeostasis and remodelling. Nat. Rev. Mol. Cell Biol. 4, 517–529 23. Wan, K. L., Carruthers, V. B., Sibley, L. D., and Ajioka, J. W. 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Pezzella, N., Bouchot, A., Bonhomme, A., Pingret, L., Klein, C., Burlet, H., Balossier, G., Bonhomme, P., and Pinon, J. M. (1997) Involvement of cal- cium and calmodulin in Toxoplasma gondii tachyzoite invasion. Eur. J. Cell Biol. 74, 92–101 14. Moreno, S. N., and Zhong, L. (1996) Acidocalcisomes in Toxoplasma gon- dii tachyzoites. Biochem. J. 313, 655–659 33. Lovett, J. L., Marchesini, N., Moreno, S. N., and Sibley, L. D. (2002) Toxo- plasma gondii microneme secretion involves intracellular Ca2 release from inositol 1,4,5-triphosphate (IP3)/ryanodine-sensitive stores. J. Biol. Chem. 277, 25870–25876 15. Grynkiewicz, G., Poenie, M., and Tsien, R. Y. (1985) A new generation of Ca2 indicators with greatly improved fluorescence properties. J. Biol. Chem. Calcium Entry in T. gondii Although this release was enhanced in the presence of extracellular Ca2, it was not necessary for stimulating microneme secretion. These results support the assertion that extracellular Ca2 enhances virulence-related traits. Such enhancement was also observed in previous exper- iments on gliding motility (34) and conoid extrusion. Mon- dragon and Frixione (7) observed enhanced conoid extrusion in parasites that were treated with ionomycin. As observed in this study, ionomycin was able to initiate a significant response in conoid extrusion, but when combined with extracellular Ca2, the response was more robust. Likewise, previous publications have established that although Ca2 ionophores were sufficient to induce Ca2 mobilization, through their probable effect on intracellular stores, the presence of extracellular Ca2 resulted in greater microneme secretion (6). Our model of invasion events, timing of intracellular Ca2 release, and extracellular Ca2 entry is shown in Fig. 8. Intra- cellular stores are depleted upon egress, and the parasites need to replenish them when they come in contact with the high Ca2 of the extracellular environment. This wave of Ca2 influx enhances invasion-related traits. Active Ca2 pumping to intracellular compartments and to the extracellular space will allow control of the cytosolic Ca2 levels after signaling events. In conclusion, our data points toward a general mechanism of Ca2 entry, which may not be sensitive to the filling state of the ER but that could work in parallel to elevate intracellular Ca2 to activate downstream events essential for initiating bio- logical processes like invasion-linked traits. 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Acta 1191, 205–208 Acknowledgments—We thank Gary S. Bird and James W. Putney for suggestions and Drs. L. David Sibley, Vern Carruthers, and John Boothroyd for antibodies. We thank Samantha Lie Tjauw and Thayer King for excellent technical help. We also thank Christina Moore for the design of the model for Fig. 8. 20. Bootman, M. D., Collins, T. J., Mackenzie, L., Roderick, H. L., Berridge, M. J., and Peppiatt, C. M. (2002) 2-aminoethoxydiphenyl borate (2-APB) is a reliable blocker of store-operated Ca2 entry but an inconsistent inhib- itor of InsP3-induced Ca2 release. FASEB J. 16, 1145–1150 21. Capiod, T. (2011) Cell proliferation, calcium influx and calcium channels. Biochimie 93, 2075–2079 REFERENCES 260, 3440–3450 16. Kafsack, B. F., Beckers, C., and Carruthers, V. B. (2004) Synchronous in- vasion of host cells by Toxoplasma gondii. Mol. Biochem. Parasitol. 136, 309–311 34. Mondragon, R., Meza, I., and Frixione, E. 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https://openalex.org/W2064718480	https://europepmc.org/articles/pmc3731533?pdf=render	English	null	Learning and memory in zebrafish larvae	Frontiers in neural circuits	2,013	cc-by	13,250	Adam C. Roberts 1, Brent R. Bill 2,3 and David L. Glanzman1,4,5* 1 Department of Integrative Biology and Physiology, University of California at Los Angeles, Los Angeles, CA, USA 2 Center for Autism Research and Program in Neurobehavioral Genetics, David Geffen School of Medicine, Semel Institute for Neuroscience and Human Behavior, University of California at Los Angeles, Los Angeles, CA, USA 1 Department of Integrative Biology and Physiology, University of California at Los Angeles, Los Angeles, CA, USA 1 Department of Integrative Biology and Physiology, University of California at Los Angeles, Los A p g gy y gy, y g , g , , 2 Center for Autism Research and Program in Neurobehavioral Genetics, David Geffen School of Medicine, Semel Institute for Neuroscience and Human Behavior, University of California at Los Angeles, Los Angeles, CA, USA y g g 3 Department of Psychiatry, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA, USA 4 Department of Neurobiology David Geffen School of Medicine University of California at Los Angeles Los Angeles CA USA 3 Department of Psychiatry, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA, USA 4 Department of Neurobiology David Geffen School of Medicine University of California at Los Angeles Los Angeles CA USA 3 Department of Psychiatry, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA, USA 4 D t t f N bi l D id G ff S h l f M di i U i it f C lif i t L A l L A l CA USA 3 Department of Psychiatry, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA, USA 4 Department of Neurobiology, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA, USA p gy, , y g , g , , 5 Integrative Center for Learning and Memory, David Geffen School of Medicine, Brain Research Institute, University of California at Los Angeles, Los Angeles, CA, USA p gy, , y g , g , , 5 Integrative Center for Learning and Memory, David Geffen School of Medicine, Brain Research Institute, University of Ca CA, USA 5 Integrative Center for Learning and Memory, David Geffen School of Medicine, Brain Research Institute, University of California at Los Angeles, Los Angeles, CA, USA Larval zebraﬁsh possess several experimental advantages for investigating the molecular and neural bases of learning and memory. Edited by: German Sumbre, Ecole Normale Supérieure, France German Sumbre, Ecole Normale Supérieure, France German Sumbre, Ecole Normale Supérieure, France Reviewed by: Gonzalo G. De Polavieja, Instituto Cajal. CSIC, Spain Misha B. Ahrens, Howard Hughes Medical Institute, USA Correspondence: David L. Glanzman, Integrative Biology and Physiology, and Neurobiology, University of California, Los Angeles, 695 Charles E. Young Drive South, Los Angeles, CA 90035, USA e-mail: dglanzman@physci.ucla.edu German Sumbre, Ecole Normale Supérieure, France Reviewed by: Gonzalo G. De Polavieja, Instituto Cajal. CSIC, Spain Misha B. Ahrens, Howard Hughes Medical Institute, USA Correspondence: David L. Glanzman, Integrative Biology and Physiology, and Neurobiology, University of California, Los Angeles, 695 Charles E. Young Drive South, Los Angeles, CA 90035, USA e-mail: dglanzman@physci ucla edu Reviewed by: Gonzalo G. De Polavieja, Instituto Cajal. CSIC, Spain Misha B. Ahrens, Howard Hughes Medical Institute, USA Reviewed by: Gonzalo G. De Polavieja, Instituto Cajal. CSIC, Spain Misha B. Ahrens, Howard Hughes Medical Institute, USA Correspondence: David L. Glanzman, Integrative Biology and Physiology, and Neurobiology, University of California, Los Angeles, 695 Charles E. Young Drive South, Los Angeles, CA 90035, USA e-mail: dglanzman@physci.ucla.edu e-mail: dglanzman@physci.ucla.edu can be rapidly applied to zebraﬁsh simply by immersing the ﬁsh in drug-containing water, which greatly simpliﬁes pharma- cological manipulation (Goldsmith, 2004). Undoubtedly one of the most attractive properties of the zebraﬁsh as a model ver- tebrate organism for the study of behavior, however, is its ease of genetic manipulability. Indeed, the zebraﬁsh approaches such invertebrate models as Drosophila and C. elegans with respect to the number of forward (Gaiano et al., 1996; Haffter et al., 1996; Schier et al., 1996; Kotani et al., 2006; Sivasubbu et al., 2006) and reverse genetic approaches to which it is amenable (Nasevicius and Ekker, 2000; Wienholds et al., 2003; Guo, 2004; Doyon et al., 2008; Meng et al., 2008; Dong et al., 2009; Bedell et al., 2012; Cade et al., 2012; Dahlem et al., 2012; Hwang et al., 2013). A major advance in genetic manipulation in zebraﬁsh has been the recent development of an effective GAL4/Upstream Activating Sequence (GAL4/UAS) system for use in zebraﬁsh (Asakawa and Kawakami, 2008; Halpern et al., 2008). This system, described in more detail below, enables researchers to target the expres- sion of genes to speciﬁc cells. NEURAL CIRCUITS NEURAL CIRCUITS Adam C. Roberts 1, Brent R. Bill 2,3 and David L. Glanzman1,4,5 Despite this, neuroscientists have only recently begun to use these animals to study memory. However, in a relatively short period of time a number of forms of learning have been described in zebraﬁsh larvae, and signiﬁcant progress has been made toward their understanding. Here we provide a comprehensive review of this progress; we also describe several promising new experimental technologies currently being used in larval zebraﬁsh that are likely to contribute major insights into the processes that underlie learning and memory. Keywords: zebraﬁsh, learning, memory, habituation, NMDA receptor Edited by: In particular, the GAL4/UAS system has been used to express the genes, such as green ﬂuorescent protein (GFP), as well as of optical probes, such as channel- rhodopsin and halorhodopsin, in speciﬁc groups of neurons in the zebraﬁsh CNS (Scott et al., 2007; Scott, 2009; Wyart et al., 2009; Warp et al., 2012). These innovations allow neuroscien- tists to visually identify behaviorally relevant neural circuits in the zebraﬁsh brain and spinal cord, and to optically monitor the func- tional activity of these circuits. The technical challenges posed by such studies are greatly reduced in the zebraﬁsh by a remark- able feature of its larval form, namely that it is translucent; this feature permits optical investigations of neuronal structure and activity in the intact, and in some instances, behaving animal. Learning and memory in zebraﬁsh larvae Adam C. Roberts 1, Brent R. Bill 2,3 and David L. Glanzman1,4,5* REVIEW ARTICLE published: 02 August 2013 doi: 10.3389/fncir.2013.00126 REVIEW ARTICLE published: 02 August 2013 doi: 10.3389/fncir.2013.00126 INTRODUCTION Even relatively simple instances of learning in vertebrates can involve complex interactions of hundreds of molecules, each with distinct spatial and temporal kinetics, as well as neural circuits containing hundreds to thousands of neurons, and thousands to tens of thousands of synapses, which must ﬁrst be identiﬁed and then monitored over time. A proven strategy for reduc- ing this daunting complexity to a manageable level has been to study forms of learning and memory that involve restricted neu- ral circuits. The efﬁcacy of such a reductionist approach has been convincingly demonstrated by investigators of invertebrate learn- ing and memory during the past several decades (Byrne and Kandel, 1996; Dubnau and Tully, 1998; Rankin, 2002; Roberts and Glanzman, 2003; Menzel, 2012). Reductionist neurobiologi- cal approaches toward understanding learning in vertebrates have been generally impeded by the enormous size and complexity of the vertebrate brain, especially the mammalian brain. One verte- brate that possesses a nervous system that may be better suited to reductionist analyses of behavior, however, is the zebraﬁsh, Danio rerio. Zebraﬁsh display a rich repertoire of behaviors, includ- ing associative learning (Norton and Bally-Cuif, 2010; Sison and Gerlai, 2010; Aizenberg and Schuman, 2011; Valente et al., 2012), social learning (Zala and Määttänen, 2013), and shoaling, a type of group behavior (Engeszer et al., 2007). Importantly, they also exhibit simple behaviors that appear to be mediated by rela- tively simply neural circuits (Kimmel et al., 1974; O’Malley et al., 1996; Easter and Nicola, 1997; Liu and Fetcho, 1999; Eaton et al., 2001; Roeser and Baier, 2003; Gahtan et al., 2005; Burgess and Granato, 2007b; Orger et al., 2008). Furthermore, zebraﬁsh have other qualities that facilitate biological analyses of behavior. For example, because they readily absorb chemicals from water, drugs August 2013 \| Volume 7 \| Article 126 \| 1 Frontiers in Neural Circuits www.frontiersin.org Learning and memory in larval zebraﬁsh Roberts et al. Identiﬁed neurons can be photoactivated or inhibited in the intact zebraﬁsh larva, and the effect of this optical manipulation of neu- ronal activity on behavior examined (see Baier and Scott, 2009; Friedrich et al., 2010). Optical manipulation of neural activity is also presently feasible in mammals (e.g., Yizhar et al., 2011) of course; but the speciﬁcity of the resulting pattern of activity, as well as its behavioral consequences, is signiﬁcantly less restricted, and therefore less mechanistically informative, than in the larval zebraﬁsh. TYPES OF LEARNING AND MEMORY IN LARVAL ZEBRAFISH HABITUATION FIGURE 2 \| The C-start reﬂex in larval zebraﬁsh is mediated by the Mauthner neuron-mediated circuit. (A) An example of a larval zebraﬁsh C-start reﬂex in response to an auditory/vibrational stimulus. The initiation of the C-start reﬂex is marked by a white dot and images were recorded every 1 ms. Frames are shown every 2 ms for illustration purposes. (B) Model of the Mauthner neuron circuitry. Potential sites of NMDAR-dependent plasticity are indicated by asterisks. Adapted with permission from Roberts et al. (2011). Habituation is a non-associative form of learning during which the response of an animal to repeated presentations of a stimu- lus of ﬁxed intensity or strength gradually declines; furthermore, this decline is not due to sensory adaptation, motor fati, or injury (Thompson and Spencer, 1966; Rankin et al., 2009). Despite habituation’s simplicity and apparent ubiquity, at present we lack FIGURE 1 \| Repertoire of behavior and learning exhibited by zebraﬁsh during development together with major experimental techniques and their effective age of use. The timeline at the bottom of the ﬁgure indicates important developmental milestones in zebraﬁsh. Note that several forms of learning have been identiﬁed as early as 5 dpf, an age at which such powerful experimental techniques as optogenetics are still effective. Note, also, that there are many molecular tools (for example, the GAL4-UAS system; see Halpern et al., 2008) that are effective at all developmental stages in zebraﬁsh. FIGURE 1 \| Repertoire of behavior and learning exhibited by zebraﬁsh during development together with major experimental techniques and their effective age of use. The timeline at the bottom of the ﬁgure indicates important developmental milestones in zebraﬁsh. Note that several forms of learning have been identiﬁed as early as 5 dpf, an age at which such powerful experimental techniques as optogenetics are still effective. Note, also, that there are many molecular tools (for example, the GAL4-UAS system; see Halpern et al., 2008) that are effective at all developmental stages in zebraﬁsh. FIGURE 2 \| The C-start reﬂex in larval zebraﬁsh is mediated by the Mauthner neuron-mediated circuit. (A) An example of a larval zebraﬁsh FIGURE 2 \| The C-start reﬂex in larval zebraﬁsh is mediated by the Mauthner neuron-mediated circuit. (A) An example of a larval zebraﬁsh C-start reﬂex in response to an auditory/vibrational stimulus. The initiation of the C-start reﬂex is marked by a white dot and images were recorded every 1 ms. INTRODUCTION a comprehensive neurobiological understanding of this form of learning (Glanzman, 2009). Teleost ﬁsh, including zebraﬁsh, exhibit a simple startle response, the C-start, that is controlled by a bilateral pair of large command neurons, the Mauthner cells, in the ﬁsh’s hind- brain (Eaton et al., 2001) (Figure 2). The C-start is triggered by an abrupt sensory (auditory, visual, or tactile) stimulus (Eaton et al., 1984; Weiss et al., 2006); it ﬁrst appears in zebraﬁsh lar- vae in response to an auditory stimulus at 4 days postfertilization (dpf), and the response begins to exhibit habituation to a repeti- tious sensory stimulus at about the same time (Eaton et al., 1977). The onset latency of the C-start is rapid (∼6 ms) and the result- ing behavior of the ﬁsh—the bending of the ﬁsh into a C shape, from which the response gets its name—is highly stereotyped (Burgess and Granato, 2007b; Issa et al., 2011); its function is to rapidly propel the ﬁsh away from potential predators. Zebraﬁsh larvae also exhibit a related escape behavior that has a longer onset latency (∼30 ms), is less stereotyped than the C-start, and is medi- ated by the activity of non-Mauthner cell circuits rather than by the Mauthner cells (Burgess and Granato, 2007b; Issa et al., 2011) (but see Liu and Fetcho, 1999). The readiness with which zebraﬁsh larvae lend themselves to optogenetics and other molecular tools (each with its own distinct efﬁcacy across development), together with the general experimental advantages of zebraﬁsh for reductionist analyses of behavior possessing different ontogenies (Figure 1), might be expected to excite interest among neuroscientists focused on learning and memory. However, to date the cognitive capabilities of larval zebraﬁsh have been relatively unexplored. Here, we will review the various forms of learning and memory shown in these animals. We will also discuss what is currently known regarding the cellular and molecular mechanisms that underlie these forms of learning and memory. Finally, we will discuss potential future directions in learning and memory research in zebraﬁsh larvae. Three forms of habituation of the C-start have been described. These forms are induced by different training protocols and TYPES OF LEARNING AND MEMORY IN LARVAL ZEBRAFISH HABITUATION Frames are shown every 2 ms for illustration purposes. (B) Model of the Mauthner neuron circuitry. Potential sites of NMDAR-dependent plasticity are indicated by asterisks. Adapted with permission from Roberts et al. (2011). FIGURE 2 \| The C-start reﬂex in larval zebraﬁsh is mediated by the M h di d i i (A) A l f l l b ﬁh FIGURE 2 \| The C-start reﬂex in larval zebraﬁsh is mediated by the Mauthner neuron-mediated circuit. (A) An example of a larval zebraﬁsh C-start reﬂex in response to an auditory/vibrational stimulus. The initiation of the C-start reﬂex is marked by a white dot and images were recorded every 1 ms. Frames are shown every 2 ms for illustration purposes. (B) Model of the Mauthner neuron circuitry. Potential sites of NMDAR-dependent plasticity are indicated by asterisks. Adapted with permission from Roberts et al. (2011). August 2013 \| Volume 7 \| Article 126 \| 2 Frontiers in Neural Circuits www.frontiersin.org Learning and memory in larval zebraﬁsh Roberts et al. habituation as well. (The source of this discrepancy may be the speciﬁc NMDAR antagonist used by the two groups; Roberts and colleagues observed that MK801, a non-competitive NMDAR antagonist, used by Wolman and colleagues, did disrupt rapid habituation, whereas DL-2-amino-5-phosphonopentanoic acid (APV), a competitive NMDAR antagonist used in the experi- ments of Roberts et al., did not alter rapid habituation). Recently, we (Pearce et al., 2012) have shown that the C-start can also undergo long-term habituation (LTH) in larval zebraﬁsh. Here, the ﬁsh were stimulated with six spaced blocks of auditory pips; each block comprised spaced 8 trains of pips (120 pips at 1 Hz per train). The spaced training produced signiﬁcant habituation of the C-start in the larvae that persisted for at least 18 h. Like STH, the induction of LTH depended on the activity of NMDARs; unlike STH, however, LTH depended on macromolecular synthe- sis, because its induction was disrupted by cold shock, and gene transcription—bathing the ﬁsh in the transcriptional inhibitor 5,6-dichlorobenzimidazole 1-β-D-ribofuranoside (DRB) during training blocked LTH. LTH of the C-start in larval zebraﬁsh exhibits a striking mechanistic similarity to LTH of the gill- and siphon-withdrawal reﬂex in the marine snail Aplysia in its requirement for NMDAR activity, translation, and transcription (Ezzeddine and Glanzman, 2003; Esdin et al., 2010). are mechanistically distinct. TYPES OF LEARNING AND MEMORY IN LARVAL ZEBRAFISH HABITUATION There are two forms of relatively short-lived habituation that we have termed “rapid” and “short- term” (Roberts et al., 2011). Rapid habituation can be induced by massed presentation of 50–120 brief auditory pulses (1 ms in duration, 200 Hz ramp wave, 109 dB), or “pips,” at 1 Hz (Figure 3); the consequent habituation is signiﬁcant at 1 min after training but the response returns to its initial strength within 3–15 min post-training (Roberts et al., 2011; Wolman et al., 2011). Short-term habituation (STH), which persists for up to 1 h after training, is induced by spaced training, speciﬁcally, by 10 blocks of 900 pips (1 Hz) with a 5 min interblock interval (Roberts et al., 2011). Roberts et al. (2011) found that STH of the C-start requires N-methyl-d-aspartate receptor (NMDAR) activ- ity, whereas rapid habituation does not. Wolman et al. (2011), however, reported that NMDAR activity was required for rapid FIGURE 3 \| Rapid Habituation of the C-start (A) C-start responses to 120 pulses delivered at 1 Hz (binned into groups of 10). (B) Persistence of rapid habituation. The training protocol resulted in short-lived habituation of the C-start, which returned to its original (pretraining) level of responsiveness within 15 min. The asterisk indicates that the 10 s test is signiﬁcantly different from the pretest and 15 min test; the pound sign indicates that the 1 min test is signiﬁcantly different from the pretest and the 15 min test. Adapted with permission from Roberts et al. (2011). In response to the sudden extinction of light (“dark ﬂash”) larval zebraﬁsh show another form of escape behavior that dif- fers from the C-start; this response, the O-bend, is characterized by a signiﬁcantly larger amplitude bend of the ﬁsh’s body than occurs during the C-start (Burgess and Granato, 2007a). Unlike the C-start, the O-bend is not mediated by the Mauthner cells. Like the C-start, however, the O-bend can undergo LTH as a result of spaced training (120 min of exposure to dark ﬂashes using an interstimulus interval of 15–60 s) (Wolman et al., 2011). LTH of the O-bend persists for up to 24 h and requires protein synthe- sis, as indicated by its blockage when training is performed in cyclohexamide, a translational inhibitor. TYPES OF LEARNING AND MEMORY IN LARVAL ZEBRAFISH HABITUATION The experiments documenting LTH of escape behaviors in larval zebraﬁsh represent a major advance because they demon- strate, for the ﬁrst time to our knowledge, that immature zebraﬁsh possess the capacity for long-term memory. Similar to long-term memory for a wide variety of learning tasks in a broad range of organisms, long-term memory in zebraﬁsh larvae is more readily induced by spaced than by massed training (Ebbinghaus, 1964), training, and depends on protein synthesis and gene transcription (Davis and Squire, 1984; Goelet et al., 1986; Yin et al., 1994, 1995; Alberini, 2009; Ardiel and Rankin, 2010). DISHABITUATION AND SENSITIZATION Sensitization is a form of non-associative learning in which expo- sure to an arousing stimulus, commonly one that is painful or noxious, causes response enhancement (Groves and Thompson, 1970). The same stimulus that induces sensitization can typically be used to enhance a habituated response, a phenomenon known as dishabituation. Despite their phenomenological (Thompson and Spencer, 1966; Hawkins et al., 1998) and mechanistic (Antonov et al., 1999) similarities, sensitization and dishabitua- tion are now recognized to be distinct forms of learning (Hawkins et al., 2006; Antonov et al., 2010). FIGURE 3 \| Rapid Habituation of the C-start (A) C-start responses to 120 pulses delivered at 1 Hz (binned into groups of 10). (B) Persistence of rapid habituation. The training protocol resulted in short-lived habituation of the C-start, which returned to its original (pretraining) level of responsiveness within 15 min. The asterisk indicates that the 10 s test is signiﬁcantly different from the pretest and 15 min test; the pound sign indicates that the 1 min test is signiﬁcantly different from the pretest and the 15 min test. Adapted with permission from Roberts et al. (2011). FIGURE 3 \| Rapid Habituation of the C-start (A) C-start responses to 120 pulses delivered at 1 Hz (binned into groups of 10). (B) Persistence FIGURE 3 \| Rapid Habituation of the C-start (A) C-start responses to 120 pulses delivered at 1 Hz (binned into groups of 10). (B) Persistence of rapid habituation. The training protocol resulted in short-lived p g p habituation of the C-start, which returned to its original (pretraining) level of responsiveness within 15 min. The asterisk indicates that the 10 s test is signiﬁcantly different from the pretest and 15 min test; the pound sign indicates that the 1 min test is signiﬁcantly different from the pretest and the 15 min test. Adapted with permission from Roberts et al. (2011). August 2013 \| Volume 7 \| Article 126 \| 3 Frontiers in Neural Circuits www.frontiersin.org Roberts et al. Learning and memory in larval zebraﬁsh Dishabituation of the rapid escape response in zebraﬁsh lar- vae (5–7 dpf) has been observed by three laboratories. After habituating the escape response in larvae to auditory pips, Best et al. (2008) produced dishabituation by delivering a brief stim- ulus of a different sensory modality (a pulse of light) to the ﬁsh. Similarly, Wolman et al. DISHABITUATION AND SENSITIZATION (2011) ﬁrst habituated the C-start in lar- vae to acoustic stimuli, and then dishabituated it by applying a brief tactile stimulus to the larval head. Using similar methods we have recently succeeded in dishabituating the C-start following LTH of this response. The enhancing action of the ﬂash on the sound-elicited C- start in zebraﬁsh larvae appears to be quite brief. It remains to be determined whether more persistent enhancement could be induced in the larvae. Possibly, the briefness of the ﬂash-induced modulation of the escape response may reﬂects the developmen- tal immaturity of monoaminergic neurotransmission within the CNS of larval ﬁsh. In support of this idea, Buske and Gerlai (2012) report that levels of dopamine and serotonin increase dramatically in zebraﬁsh around 10–12 dpf (see below). y p Drug-induced sensitization of locomotor activity has been shown in both larval (Petzold et al., 2009) and adult (Blaser et al., 2010) zebraﬁsh. Petzold et al. (2009) observed that the locomotor activity of larval (5–6 dpf, but not 4 dpf) zebraﬁsh was enhanced by nicotine and that re-exposure to the drug sensitized the nico- tine response. Interestingly, administration of APV together with nicotine blocked the sensitization. Therefore, NMDAR activity appears to mediate at least some forms of habituation and sen- sitization. Blaser et al. (2010) examined the effects of repeated exposure to ethanol on locomotor activity in adult zebraﬁsh. They observed sensitization of ethanol-induced hyperactivity in the ﬁsh; furthermore, the sensitization was context-speciﬁc: ﬁsh given a second exposure to ethanol in the same context in which they received their ﬁrst exposure exhibited enhanced locomotor hyperactivity, whereas ﬁsh re-exposed to ethanol in a different context did not show sensitization. (Note that the ﬁsh did not classically condition to the context, because their locomotor activ- ity did not increase when they were re-exposed to the original context in the absence of ethanol.) Context speciﬁcity of drug- induced sensitization of locomotor activity remains to be shown in larval zebraﬁsh. p A short-lived form of cross-modal modulation of the C- start that resembles sensitization (the enhancement of a non- habituated response) has also recently been shown in larval zebraﬁsh. Mu et al. (2012) used auditory pips to evoke the C-start in 5–6 dpf zebraﬁsh. DISHABITUATION AND SENSITIZATION When the sound stimulus was preceded by about 500 ms by a brief (15 ms) pulse of white light (a “ﬂash”), the probability of a C-start being evoked by the subsequent sound stimulus was facilitated; by itself, the ﬂash did not evoke the escape response. Through whole-cell electrophysiological record- ings from the Mauthner cell in paralyzed ﬁsh embedded in agar, the investigators found that the preceding visual stimulus signiﬁ- cantly enhanced the compound synaptic current (CSC) evoked in the Mauthner cell by the auditory pips; the ﬂash alone, however, evoked only a very small CSC. Furthermore, a preceding ﬂash enhanced the biphasic excitatory postsynaptic current (EPSC) evoked in the Mauthner cell by extracellular stimulation of the VIIIth cranial nerve, which transmits auditory information to the brain. The biphasic EPSC contains an early electrical compo- nent and a later chemical component; the chemical component is mediated by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) and NMDARs; both the electrical and chemical components of the EPSC were enhanced by the pre- ceding ﬂash. Mu et al. showed that the enhancement of VIIIth nerve-Mauthner cell synaptic transmission produced by a pre- ceding visual stimulus was mimicked by exogenous application of dopamine and was blocked by antagonists of the D1 dopamine receptor. In support of the idea that the ﬂash causes release of dopamine within the C-start circuit, laser ablation of the GFP- expressing dopaminergic neurons in the brains of the larval ﬁsh, as well as down-regulation of dopamine synthesis in hypothala- mic dopaminergic neurons by knocking down tyrosine hydrox- ylase (the enzyme that converts L-tyrosine to L-DOPA) or two transcription factors required for the development of dopaminer- gic neurons with morpholino oligonucleotides (Mu et al., 2012) reduced the modulation of the auditory-evoked C-start by the preceding ﬂash. Finally, the investigators determined that the visual ﬂash induced bursting activity in dopaminergic neurons in the hypothalamus. It is interesting that the basis of cross-modal enhancement of the auditory-evoked C-start in larval zebraﬁsh is modulatory neuronal actions caused by the release of dopamine within the ﬁsh’s brain. This scheme is broadly consistent with that for sensitization of the defensive withdrawal reﬂex in Aplysia, which results from modulatory actions on sensorimotor pathways within the snail of another monoamine, serotonin; serotonin’s release, in turn, is triggered by noxious stimulation (Brunelli et al., 1976; Castellucci and Kandel, 1976; Kandel and Schwartz, 1982) CLASSICAL CONDITIONING Classical conditioning, ﬁrst described by Pavlov (1927), is the ability of an animal to associate a neutral stimulus (the condi- tioned stimulus or CS) with a reinforcing stimulus (the uncon- ditioned stimulus or US). As the result of the paired delivery of a CS and a US, the CS acquires the ability to predict the occurrence of the US and, consequently, the animal’s response to the CS (the conditioned response or CR) comes to resemble its response to the US (the unconditioned response or UCR). Classical conditioning is the most basic form of associative learn- ing; consequently, understanding its biological basis is a major goal of behavioral neuroscientists. Adult teleost ﬁsh classically condition readily (Agranoff and Davis, 1968; Flood et al., 1976; Amiro and Bitterman, 1980; Mattioli et al., 1998; Eisenberg et al., 2003; Salas et al., 2006; Yoshida and Kondo, 2012), and there have been several published reports of classical conditioning in adult zebraﬁsh (Braubach et al., 2009; Agetsuma et al., 2010; Karnik and Gerlai, 2012; Aoki et al., 2013). To date, there have been just two reports of suc- cessful classical conditioning in larval to juvenile zebraﬁsh. In one successful study, Aizenberg and Schuman (2011) trained 6- to-8-day-old larval zebraﬁsh to associate a moving spot of light (the CS) with a touch to the side of the body (the US). The ﬁsh were partially restrained in agarose during the experiments such that their tails were free to move. The CR was enhanced move- ment of the tail in response to the CS. Aizenberg and Schuman August 2013 \| Volume 7 \| Article 126 \| 4 Frontiers in Neural Circuits www.frontiersin.org www.frontiersin.org www.frontiersin.org Learning and memory in larval zebraﬁsh Roberts et al. recently, Bianco et al. (2012) also reported that larval zebraﬁsh possess a VOR. also measured changes in intracellular Ca2+ in cerebellar neu- rons in the restrained ﬁsh during the experiments. They observed that prior to training the CS and the US activated partially dis- tinct populations of cerebellar neurons prior to conditioning; as a consequence of learning, the number of CS-activated neurons in the cerebellum was increased. Laser-ablation of the cerebellum immediately after the ﬁrst training trial prevented conditioning, whereas cerebellar ablation after the last training trial impaired extinction of the CR. CLASSICAL CONDITIONING Interestingly, ablating the cerebellum after training, although it altered extinction, did not affect memory retention, which suggests that the memory for the CR is stored outside the cerebellum. A recent study used motor learning in paralyzed zebraﬁsh lar- vae, together with whole brain imaging of activity-dependent changes in intracellular calcium in individual neurons, to show the promise of larval zebraﬁsh as model organisms for cellu- lar investigations of learning (Ahrens et al., 2012). Ahrens and colleagues examined a type of motor adaptation (the optomo- tor response) related to the VOR. Here, paralyzed, restrained zebraﬁsh larvae were exposed to a moving whole-ﬁeld visual stim- ulus that simulated the visual effect in freely swimming ﬁsh of being swept backwards by the water ﬂow. In response, the ﬁsh initiated motor commands (“ﬁctive swims”) that would have— were they not paralyzed—moved them forward; the purpose of these ﬁctive swims was to stabilize the virtual location of the ﬁsh. The motor commands were recorded electrophysiologically from motor neurons in the ﬁsh, and these electrical signals were then translated into visual feedback that mimicked the optic ﬂow pro- duced in freely swimming ﬁsh by forward movement. The ﬁsh used in this study were transgenics that expressed the calcium sensor GCaMP2 (Akerboom et al., 2012) in almost all neurons. By means of two-photon microscopy the investigators were able to optically record neural activity throughout the brain at single- neuron resolution while the ﬁsh “behaved” in the virtual reality setup. They observed many neurons in the inferior olive and cerebellum whose ﬁring correlated with motor adaptation by the ﬁsh to the visual stimulation. That the activity of these neurons was somehow causally related to the ﬁsh’s behavior was indi- cated by the fact that lesioning the inferior olive post-training eliminated the motor adaptation. Although this study was unable to specify the actual cellular mechanism of motor learning, it nonetheless represents an impressive demonstration of the ana- lytic power of the combination of transgenic manipulation and optical recording in the living, intact brain that zebraﬁsh larvae enable. In the second demonstration of classical conditioning Valente et al. (2012) trained ﬁsh to associate a visual pattern projected onto an LCD screen below half of the tank of water in which the ﬁsh freely swam (the CS), to a whole-tank electric shock (the US). CLASSICAL CONDITIONING The experimenters measured the turns away from the side of the tank to which the CS was delivered as the CR. The zebraﬁsh did not exhibit signiﬁcant evidence of learning on this task until 4 weeks of age (∼late larval stage or early juvenile stage), after which their learning steadily improved, reaching an adult level at 6 weeks of age. Valente and colleagues also used a modiﬁcation of this learning task to attempt to train larval zebraﬁsh. In this mod- iﬁcation the CS was a visual stimulus projected from below the ﬁsh, which were restrained in agarose, and the US was either an electric shock applied to the head of the ﬁsh, or a tap delivered to the ﬁsh’s ear. However, the training did not produce evidence of learning in 7-day-old larvae. MOTOR LEARNING A type of vertebrate motor learning that has both formal and mechanistic similarities to classical conditioning is the vestibulo- ocular reﬂex (VOR) (Lac et al., 1995; Cohen et al., 2004). The VOR is a reﬂexive eye movement in which vestibular signals are used to generate compensatory eye movements in the oppo- site direction from head movements; its function is to stabilize retinal images. Calibration of the VOR requires motor learning; when head movements are consistently paired with the undesir- able motion of the retinal image, learning occurs and the gain of the reﬂex is changed to reduce the image motion. Learning in the VOR depends on the cerebellum; furthermore, the cellu- lar mechanisms that mediate this form of learning resemble those that mediate classical conditioning of the eyeblink response (Lac et al., 1995). Adult teleost ﬁsh, including adult zebraﬁsh, exhibit a robust VOR (Graf and Baker, 1983; Pastor et al., 1994; Marsh and Baker, 1997). Initially, it was reported that larval zebraﬁsh exhibit angular VORs (VORs evoked by stimulation of the semicircular canals) by 96 h postfertilization (hpf) (Easter and Nicola, 1997; Moorman et al., 2002); however, a later study did not ﬁnd angular VORs in zebraﬁsh until 35 dpf (Beck et al., 2004). Mo et al. (2010) reexamined this issue, and found evidence for an angular VOR in zebraﬁsh as early as 72 hpf; furthermore, this group showed that several lines of mutant ﬁsh with defects of the vestibular system exhibited either a loss of VOR or reduced VOR. Mo and col- leagues attributed the earlier failure to recognize the VOR in larval zebraﬁsh (Beck et al., 2004) to mistaken attribution of vestibularly mediated eye movements to visually mediated movements. More Frontiers in Neural Circuits OPERANT CONDITIONING Operant conditioning, another major form of associative learn- ing, differs from classical conditioning in that the consequences (outcomes) of an animal’s voluntary response to a reinforcing stimulus alters the future probability of the animal’s responses or behavior; in classical conditioning the animal’s (involuntary) responses to the training stimuli are not altered by the behav- ior’s outcomes (Gluck et al., 2014). One operant conditioning paradigm that has been used successfully with ﬁsh is avoid- ance conditioning. In a protocol originally developed for use with the goldﬁsh over 40 years ago (Agranoff and Davis, 1968; Agranoff, 1971), ﬁsh must learn to swim to the other side of a shuttle box at the onset of a light to avoid an electric shock. Adult zebraﬁsh condition readily in this protocol (Pradel et al., 1999, 2000; Xu et al., 2007) moreover, the learning depends on NMDARs (Blank et al., 2009). Two studies have used variants of the original shuttle box training protocol to show avoidance con- ditioning in larval to juvenile zebraﬁsh. Lee et al. (2010) trained three-to-ﬁve-week-old ﬁsh to avoid the side of a shuttle box illu- minated with a red light. They showed that the learning required the habenula—a diencephalic structure involved in the regulation August 2013 \| Volume 7 \| Article 126 \| 5 Frontiers in Neural Circuits www.frontiersin.org Learning and memory in larval zebraﬁsh Roberts et al. Two papers by Gerlai and colleagues provide some additional support for the notion that shoaling is a learned social behavior in zebraﬁsh. Al-Imari and Gerlai (2008) raised zebraﬁsh singly to adulthood (the experimental ﬁsh). Then the experimental ﬁsh were given 10 training trials in which they were placed in a four- arm aquatic maze. Beside each arm of the maze was a small tank—the contents of which were visible from the maze arm— one of which contained seven stimulus ﬁsh; a red plastic cue card was placed at the end of the maze arm next to the tank con- taining the stimulus ﬁsh. (The location within the maze of the stimulus ﬁsh and red cue card was changed from trial to trial.) Another group of ﬁsh (unpaired group) was given ten training trails in the maze, but the red cue card and the stimulus ﬁsh were placed separately in different arms. OPERANT CONDITIONING (This toxin prevents neurons from releasing transmitter by cleaving synaptobrevin.) Larvae with habenular expression of tetanus showed deﬁcits in avoidance conditioning, particularly in the later training trails. The study of Lee and colleagues nicely illustrates the potential of zebraﬁsh for investigations of learning and memory involving modern genetic tools. Valente et al. (2012) also used a shuttle box-type protocol to measure the ontogeny of operant conditioning in zebraﬁsh. The zebraﬁsh did not exhibit signiﬁcant conditioning until 3 weeks of age (∼late larval stage), reaching a maximal (adult) level by week 6. of dopaminergic and serotonergic pathways, and which medi- ates avoidance learning in mammals (Shumake et al., 2010)—by using genetic technologies to disrupt habenular circuits. Lee and colleagues used a mutant line that expresses the phototoxic ﬂuo- rescent protein, KillerRed, in forebrain afferents to the habenula; photobleaching KillerRed-expressing neurons by illuminating the larvae with green light damaged the afferents and, when per- formed prior to behavioral training, prevented acquisition of conditioned avoidance. Interestingly, photobleaching habenular afferents after training did not impair expression of the learning. Lee et al. also used the GAL4/UAS system to express tetanus toxin speciﬁcally in habenular neurons. (This toxin prevents neurons from releasing transmitter by cleaving synaptobrevin.) Larvae with habenular expression of tetanus showed deﬁcits in avoidance conditioning, particularly in the later training trails. The study of Lee and colleagues nicely illustrates the potential of zebraﬁsh for investigations of learning and memory involving modern genetic tools. Valente et al. (2012) also used a shuttle box-type protocol to measure the ontogeny of operant conditioning in zebraﬁsh. The zebraﬁsh did not exhibit signiﬁcant conditioning until 3 weeks of age (∼late larval stage), reaching a maximal (adult) level by week 6. OPERANT CONDITIONING (The locations of the cue card and the stimulus ﬁsh were also moved from trial to trial.) Following the training the ﬁsh were presented with the red cue card alone, and the amount of time the ﬁsh spent in proximity to the card was measured. Fish in the paired group spent signiﬁ- cantly more time near the red cue card than would be predicted by chance alone, whereas ﬁsh in the unpaired group preformed at chance level, i.e., the amount of time the unpaired ﬁsh spent in proximity to the card was no more than would predicted by chance given the total area of the maze. This result demonstrates that the zebraﬁsh found the presence of a shoal mate rewarding. A second paper from this laboratory, that of Buske and Gerlai (2012), used high-pressure liquid chromatographic (HPLC) anal- yses of whole-brain extracts and behavioral measurements of the tendency to shoal to gain insights into potential neurobiologi- cal processes underlying the ontogenesis of shoaling in zebraﬁsh. These investigators found, as had others (Engeszer et al., 2007; Buske and Gerlai, 2011), that shoaling-related behavior increased gradually in zebraﬁsh from 10 to 75 dpf. At the approximately the same time there were also signiﬁcant increases in the brain lev- els of both dopamine (DA) and serotonin (5-HT). Although this correlation does not prove that the increase in the brain levels of the monoamines underlay the increase in shoaling behavior, it is nonetheless suggestive; dopaminergic and serotonergic processes are known to play prominent roles in many forms of vertebrate and invertebrate learning and memory (e.g., Kandel, 2001; Wise, 2004; Riemensperger et al., 2005; Sitaraman et al., 2008; Hart et al., 2011; Johnson et al., 2011; Wood et al., 2011; Roberts and Hedlund, 2012). of dopaminergic and serotonergic pathways, and which medi- ates avoidance learning in mammals (Shumake et al., 2010)—by using genetic technologies to disrupt habenular circuits. Lee and colleagues used a mutant line that expresses the phototoxic ﬂuo- rescent protein, KillerRed, in forebrain afferents to the habenula; photobleaching KillerRed-expressing neurons by illuminating the larvae with green light damaged the afferents and, when per- formed prior to behavioral training, prevented acquisition of conditioned avoidance. Interestingly, photobleaching habenular afferents after training did not impair expression of the learning. Lee et al. also used the GAL4/UAS system to express tetanus toxin speciﬁcally in habenular neurons. SOCIAL LEARNING A shoal is a group of ﬁsh, typically of the same species and age, that swim together for social reasons. (Shoaling is distinguished from schooling in which ﬁsh swim together in tight, synchronized fashion.) It is believed that this social behavior serves, in part, as a protection against predation through increasing the detection of predators and decreasing the probability of individual capture (Peichel, 2004). As ﬁrst shown by McCann et al. (1971), shoal- ing preferences in zebraﬁsh have been shown to be at least partly learned. More recently, Engeszer et al. (2004) examined prefer- ences of zebraﬁsh who had been raised from hatching either in isolation, with siblings of the same phenotype, or with siblings of a different phenotype (cross-rearing). The ﬁsh in the study were either wild-type (normally striped) or mutant ﬁsh lack- ing melanophore stripes (nacre mutants). The ﬁsh were tested for social preference when they reached adulthood (≥90 dpf). Social isolates exhibited no preference for either the wild-type or nacre phenotypes. However, ﬁsh in the other groups preferred the phenotypes they had been raised with, e.g., wild-type ﬁsh raised with nacre ﬁsh from hatching preferred to shoal with nacre ﬁsh as adults. These results suggest that shoaling preferences are determined, at least partly, by early experience. In a later study (Engeszer et al., 2007) Engeszer and colleagues ascertained the onset of conspeciﬁc preferences in zebraﬁsh; they found that zebraﬁsh begin to show conspeciﬁc preferences at approximately the post-ﬂexion stage (∼12 dpf), and that zebraﬁsh ﬁrst exhibit signiﬁcant shoaling preferences as juveniles. Furthermore, shoal- ing preferences were not plastic; as adults zebraﬁsh preferred to shoal with the phenotypes they were reared with, even if given prolonged (30 days) exposure to the other phenotypes in adult- hood. In addition to visual features, olfactory cues have also been shown to be a signiﬁcant factor in determining shoaling pref- erences in zebraﬁsh (Gerlach and Lysiak, 2006; Gerlach et al., 2007). Frontiers in Neural Circuits FUTURE DIRECTIONS As the above review indicates, zebraﬁsh larvae possess a surpris- ingly rich repertoire of learning abilities, including not only non- associative, but also associative and even social learning. Given that at present neurobiologists lack a comprehensive under- standing of any form of learning in any organism (discussed in Glanzman, 2009), we should not underestimate the formidable analytic challenge posed by the types of learning and memory that zebraﬁsh larvae are known to exhibit. Moreover, the future is likely to bring an increased appreciation of the cognitive capa- bilities of these animals, which are almost certainly underrated at present. In addition to the development of new learning and mem- ory assays in larval zebraﬁsh, one can anticipate that the genetic Frontiers in Neural Circuits August 2013 \| Volume 7 \| Article 126 \| 6 www.frontiersin.org www.frontiersin.org www.frontiersin.org Learning and memory in larval zebraﬁsh Roberts et al. tools available for use in zebraﬁsh will steadily improve (see Figure 1). At present the most widely used method of altering gene function in larval zebraﬁsh is gene knockdown by mor- pholinos (Mullins et al., 1994) This method has been widely employed in standard studies of the zebraﬁsh developmental biology, but has had only partial success in behavioral studies due to the temporally restricted nature of the mRNA knock- down (Bill et al., 2009). Many behavioral assays (see, e.g., Valente et al., 2012) require ﬁsh to be 5 dpf or older, at which time the efﬁcacy of morpholino gene knockdown is questionable; this method is therefore of limited value in learning and memory research. A relatively new method that should ultimately prove more useful than morpholinos for molecular analyses of behav- ior is Targeting Induced Local Lesions IN Genomes, (TILLING); here, zebraﬁsh mutants are initially produced by exposure of embryos to the mutagen N-ethyl-N-nitrosourea (ENU), and then the DNA of these ﬁsh is screened and sequenced to identify muta- tions within speciﬁc genes (Wienholds et al., 2003). Currently, the Sanger Institute and the Zebraﬁsh TILLING Project have avail- able a large number of mutant ﬁsh with predicted mutations that should code for nonfunctional proteins (http://www.sanger.ac. uk/Projects/D_rerio/zmp/ and http://webapps.fhcrc.org/science/ tilling/index.php). This resource should lead to the identiﬁcation of novel molecular pathways involved in learning and mem- ory. FUTURE DIRECTIONS In addition, newer techniques, such as zinc ﬁnger nucleases, CRISPR, and TALENS, allow for site-directed mutagenesis of zebraﬁsh genes (Doyon et al., 2008; Meng et al., 2008; Bedell et al., 2012; Dahlem et al., 2012; Moore et al., 2012; Hwang et al., 2013). This ability, available in mice for decades through homologous recombination (Capecchi, 1989), has facilitated the identiﬁcation of many of the molecules important for learning and memory (Grant et al., 1992; Silva et al., 1992a,b). Site- directed mutagenesis promises to be equally useful in molecular analyses of zebraﬁsh learning and memory. Finally, a powerful genetic tool, the GAL4/UAS system (Scott, 2009, and above), has only just begun to be used in memory research in larval zebraﬁsh (Lee et al.), but is likely to become increasingly pop- ular. Scott et al. (2007) have developed several GAL4 combined enhancer trap zebraﬁsh lines; these will permit UAS-linked trans- genes to be targeted to speciﬁc regions or, in some instances, speciﬁc cell types of the larval brain. The GAL4/UAS system has been used to great effect in mechanistic studies of Drosophila memory (Joiner and Grifﬁth, 1999; Zars et al., 2000a,b; Akalal et al., 2006; Kasuya et al., 2009; Berry et al., 2012), and is likely to prove equally valuable in the analysis of memory in larval zebraﬁsh. Investigators have long taken advantage of the transparency of throughout the brain (Ahrens et al., 2012), or expressed in restricted regions of the brain (Del Bene et al., 2010; Muto et al., 2011). Furthermore, increasingly powerful genetically encoded calcium indicators are being developed (Akerboom et al., 2012) and this, together with improvements in optical techniques, such as two-photon microscopy and, more recently, light-sheet microscopy (Ahrens et al., 2013), make it now feasi- ble to record the activity of more than 80% of the neurons in the larval zebraﬁsh brain at the same time with single-cell res- olution (Ahrens et al., 2012). This is a remarkable advance, one that could revolutionize our understanding of the brain circuits generate behaviors and encode learned experiences. Moreover, zebraﬁsh larvae are uniquely suited to take advantage of this new technology. tools available for use in zebraﬁsh will steadily improve (see Figure 1). FUTURE DIRECTIONS At present the most widely used method of altering gene function in larval zebraﬁsh is gene knockdown by mor- pholinos (Mullins et al., 1994) This method has been widely employed in standard studies of the zebraﬁsh developmental biology, but has had only partial success in behavioral studies due to the temporally restricted nature of the mRNA knock- down (Bill et al., 2009). Many behavioral assays (see, e.g., Valente et al., 2012) require ﬁsh to be 5 dpf or older, at which time the efﬁcacy of morpholino gene knockdown is questionable; this method is therefore of limited value in learning and memory research. A relatively new method that should ultimately prove more useful than morpholinos for molecular analyses of behav- ior is Targeting Induced Local Lesions IN Genomes, (TILLING); here, zebraﬁsh mutants are initially produced by exposure of embryos to the mutagen N-ethyl-N-nitrosourea (ENU), and then the DNA of these ﬁsh is screened and sequenced to identify muta- tions within speciﬁc genes (Wienholds et al., 2003). Currently, the Sanger Institute and the Zebraﬁsh TILLING Project have avail- able a large number of mutant ﬁsh with predicted mutations that should code for nonfunctional proteins (http://www.sanger.ac. uk/Projects/D_rerio/zmp/ and http://webapps.fhcrc.org/science/ tilling/index.php). This resource should lead to the identiﬁcation of novel molecular pathways involved in learning and mem- ory. In addition, newer techniques, such as zinc ﬁnger nucleases, CRISPR, and TALENS, allow for site-directed mutagenesis of zebraﬁsh genes (Doyon et al., 2008; Meng et al., 2008; Bedell et al., 2012; Dahlem et al., 2012; Moore et al., 2012; Hwang et al., 2013). This ability, available in mice for decades through homologous recombination (Capecchi, 1989), has facilitated the identiﬁcation of many of the molecules important for learning and memory (Grant et al., 1992; Silva et al., 1992a,b). Site- directed mutagenesis promises to be equally useful in molecular analyses of zebraﬁsh learning and memory. Finally, a powerful genetic tool, the GAL4/UAS system (Scott, 2009, and above), has only just begun to be used in memory research in larval zebraﬁsh (Lee et al.), but is likely to become increasingly pop- ular. Scott et al. (2007) have developed several GAL4 combined enhancer trap zebraﬁsh lines; these will permit UAS-linked trans- genes to be targeted to speciﬁc regions or, in some instances, speciﬁc cell types of the larval brain. FUTURE DIRECTIONS The GAL4/UAS system has been used to great effect in mechanistic studies of Drosophila memory (Joiner and Grifﬁth, 1999; Zars et al., 2000a,b; Akalal et al., 2006; Kasuya et al., 2009; Berry et al., 2012), and is likely to prove equally valuable in the analysis of memory in larval zebraﬁsh. Another new optical method to which zebraﬁsh larvae are highly amenable is optogenetics. Through the use of light-gated glutamate receptors (Szobota et al., 2007), channelrhodopsin (Douglass et al., 2008; Bundschuh et al., 2012; Fajardo et al., 2013) and halorhodopsin (Arrenberg et al., 2009) one can either excite or inhibit neurons in the intact, behaving ﬁsh. Also, the speci- ﬁcity of optical manipulation of neuronal activity can be further reﬁned by means of genetic tools. This technology has already been used for mechanistic studies of behavior in zebraﬁsh larvae, although not yet for studies of learning and memory. However, optogenetic investigations of learning and memory have recently been carried out in mice (Alonso et al., 2012; Liu et al., 2012), and one can anticipate similar studies in zebraﬁsh larvae in the near future. Mention should also be made of other new methods for manipulating or monitoring neural function in the intact lar- val zebraﬁsh’s brain. For example, GAL4-UAS technology can be used to target the expression of tetanus toxin, which blocks neurotransmitter release, to speciﬁc neurons and thereby eliminate their contribution to behavior-related brain activ- ity (Asakawa et al., 2008; Wyart et al., 2009). 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For this reason, it is gratifying that these relatively simple animals possess signiﬁcant capabilities for learning. In particular, as we have discussed in this review, zebraﬁsh larvae exhibit not only short-term, but also long-term memory, as well as associative and social learn- ing. As neurobiologists increasingly recognize the advantages of zebraﬁsh larvae for investigations of learning and memory, our knowledge of the mnemonic repertoire of these animals will undoubtedly expand. Possibly, studies of this tiny, imma- ture, deceptively humble creature will one day unlock some of the most profound secrets about how brains acquire and store memories. Investigators have long taken advantage of the transparency of larvae to image optical activity in the brain of intact zebraﬁsh using calcium indicator dyes (Fetcho and O’Malley, 1995, 1997; Ritter et al., 2001; Higashijima et al., 2003). 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https://openalex.org/W3135006921	https://zenodo.org/records/4773966/files/Quality%20control%20and%20identification%20of%20organic%20biscuits.pdf	English	null	Quality control and identification of organic biscuits	Technology audit and production reserves	2,021	cc-by	5,787	1. Introduction It should be noted that the main trends in the global market for organic products have become an increase in demand for organic products in most countries. The situa tion in the global organic market is characterized by the concentration of demand in developed countries, an increase in demand in developing countries, consolidation of mar ket participants and the development of distribution [1]. However, the unresolved issue is the compliance of the quality of organic products, the falsification of organic products on the market, the study of quality and food safety indicators and their comparison with traditional products. In 2007, European Union Regulation (EC) 834/2007 introduced principles and criteria for the processing of or ganic food. This document sets out the goals and principles of organic production. One of the production goals is to create high quality organic products [2]. The main problem in the study of organic food products is the need to select methods for determining the quality, as well as determining the quality parameters of organic products [3]. It should be noted that national standards do not regulate the quality of organic products. Previous studies suggested the creation It should be noted that the main trends in the global market for organic products have become an increase in demand for organic products in most countries. The situa tion in the global organic market is characterized by the concentration of demand in developed countries, an increase in demand in developing countries, consolidation of mar ket participants and the development of distribution [1]. Certification of organic products is an effective tool for protecting consumers from counterfeiting. Organic certifica tion gives the manufacturer the right to label products using marks confirming their origin from organic farming [5, 6]. However, in this context, the issue of violation of labeling requirements and misleading consumers remains unresolved. Considering the above, quality control and identification of organic products is a timely and urgent task. However, the unresolved issue is the compliance of the quality of organic products, the falsification of organic products on the market, the study of quality and food safety indicators and their comparison with traditional products. QUALITY CONTROL AND IDENTIFICATION OF ORGANIC BISCUITS The object of research is children’s spelled biscuits Holle (Germany) and Belgian children’s biscuits with pieces of Fleur Alpine chocolate (Belgium). Consumers often buy organic-branded traditional food products through unfair advertising and labeling violations. Due to the fact that organic products are often the subject of counter- feiting, quality expertise is especially important. Also, thanks to the conformity assessment of the labeling given in the work, it is possible to establish whether the product is really organic. The labeling of biscuits was analyzed for compliance with the Law of Ukraine «On Organic Production» and the Law of Ukraine «On Information for Food Consumers». The study showed that the labeling complies with legal requirements in both samples. It was also found that both samples of biscuits meet the requirements of regulatory documents for organoleptic indicators. Organoleptic characteristics were tested: shape, surface, color, taste and smell, appearance in the fault. The tasting assessment was carried out according to a 45-point scale developed by the authors. The total number of points in the tasting score, taking into accounts the weight factor, for Fleur Alpine biscuits was 39.96, which corresponds to a quality level of 0.88; and Holle biscuits – 44, with a quality level of 0.97. Both samples meet the requirements of regulatory documents for such physicochemical indicators as humidity, alkalinity and moisture content. The moisture content of the biscuits was determined in an accelerated manner in an oven. Wetness was determined by the ratio of the weight of the items after wetting to the weight of the dry items. Alkalinity was determined by titration of the product filtrate. The moisture content for Fleur Alpine biscuits was 9.3 % and for Holle biscuits it was 8.0 %. The alkalinity was 1.6 and 1.4 degrees, respectively, and the wetness was also within the normal range with values of 120 and 110 %. The results indicate the quality of the studied samples in terms of organoleptic and physicochemical indicators. Keywords: organic products, organic production, organic biscuits, organoleptic indicators, biscuit labeling, physical and chemical indicators. Received date: 22.09.2020 Accepted date: 20.11.2020 Published date: 26.02.2021 © The Author(s) 2021 This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0) of a draft national standard «Organic production. General requirements» [4]. But this standard proposes to establish criteria for production, and not for quality parameters of finished products. UDC 664.681 DOI: 10.15587/2706-5448.2021.225531 Article type «Original research» UDC 664.681 DOI: 10.15587/2706-5448.2021.225531 Article type «Original research» TECHNOLOGY AUDIT AND PRODUCTION RESERVES — № 1/3(57), 2021 ISSN 2664-9969 ISSN 2664-9969 CHEMICAL ENGINEERING: FOOD PRODUCTION TECHNOLOGY © The Author(s) 2021 This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0) 3. The aim and objectives of research The aim of research is to study the quality of organic biscuits in terms of organoleptic and physicochemical in dicators and compliance with labeling. Given this aim, the objectives of research are: j 1. Analyze the labeling of organic biscuits. 1. Analyze the labeling of organic biscuits. 2. Conduct barcode identification of organic biscuits. 3. Investigate the organoleptic characteristics of the quality of organic biscuits. Fig. 1. Children’s spelled biscuits Holle 4. Determine the physical and chemical indicators of the quality of organic biscuits. Contains gluten, milk. May also contain traces of nuts, eggs, sesame seeds, soy. Energy value per 100 g: 1851 kJ/440 kcal. Nutritional value per 100 g: fat – 12.8 g, including satu rated – 8.5 g; carbohydrates – 70.1 g, of which sugar – 14.80 g, protein – 9.2 g, salt – 0.2 g. Method of preparation: open the package, stir before use and feed. After opening the package, store in the refrigerator and consume within 2 days. Storage conditions: store in a cool dry place. CHEMICAL ENGINEERING: FOOD PRODUCTION TECHNOLOGY – butter (dairy product); – banana powder; – natural vanilla extract; – antioxidant (tocopherol). – baking powder sodium bicarbonate; The packaging indicates that the product may contain traces of soybeans and nuts. The energy value of the biscuit is 1880 kJ/447 kcal. Nutritional value: fats – 18 g, includ ing saturated fatty acids – traces; carbohydrates – 60.8 g, including sucrose – 23 g; dietary fiber – 3.2 g; proteins – 7.5 g, salt – 0.3 g. Net weight: 150 g (6 servings in indi vidual packaging, 25 g each). Expiration date: 15 months. Storage conditions: store at a temperature not exceeding 25 °С and relative humidity not exceeding 75 %. Use an open sachet within 24 hours. – vitamin B1. Fig. 1. Children’s spelled biscuits Holle 4. Research of existing solutions of the problem Previous studies have established that the demand for sweets made from natural ingredients, in particular organic products, is growing now [7]. At the same time, there are many cases of food counterfeiting. A high level of standards and technical and other requirements can not only more reliably protect the market from low-quality goods, but also become an incentive for the growth of manufacturers’ competitiveness [8]. Certain countries have organic product standards. At the same time, many countries adhere to the Codex Alimentarius standards [9]. Fig. 2. Belgian children’s biscuits with pieces of Fleur Alpine chocolate The labeling of organic products deserves special attention during identification. A new EU regulation on the labeling of organic products is currently under discussion. It proposes a stricter policy on pesticide residues in organic products. In particular, if these are pesticide residues that are not permitted in organic agriculture, the sale of such products is prohibited for 2 months until the source of pollution is identified [10]. Many studies point to the great health benefits of or ganic foods. It has been found that organic food contains less harmful substances, and the risk of allergies decreases with their long-term consumption [11]. Available studies of comparative analysis of nutritional and biological value of organic and traditional food products. Thus, there are data on the comparative analysis of studies of the quality of organic and conventional food products – yogurt, cheese, green vegetables produced in Bulgaria, and a conceptual model for comparative testing of product quality has been proposed [12]. There is evidence that people who consume exclusively organic products and have a lower risk of obe sity [13]. Organic products are only gaining popularity in some countries [14]. Along with this, it should be noted that despite the benefits of organic products, a significant part of consumers remains aware of what kind of products are organic [15]. This is the reason for the small number of consumers of organic products. The survey [16], in which Fig. 2. Belgian children’s biscuits with pieces of Fleur Alpine chocolate Fig. TECHNOLOGY AUDIT AND PRODUCTION RESERVES — № 1/3(57), 2021 2. The object of research and its technological audit In 2007, European Union Regulation (EC) 834/2007 introduced principles and criteria for the processing of or ganic food. This document sets out the goals and principles of organic production. One of the production goals is to create high quality organic products [2]. The main problem in the study of organic food products is the need to select methods for determining the quality, as well as determining the quality parameters of organic products [3]. It should be noted that national standards do not regulate the quality of organic products. Previous studies suggested the creation The object of research is organic children’s spelled biscuits Hole, Germany (Fig. 1) and Belgian children’s biscuits with pieces of Fleur Alpine chocolate, Belgium (Fig. 2). Fig. 1 shows that the composition of the Children’s spelled biscuits Holle: – spelled flour 63 %; – spelled flour 63 %; – rice syrup; – rice syrup; – rice syrup; TECHNOLOGY AUDIT AND PRODUCTION RESERVES — № 1/3(57), 2021 39 ISSN 2664-9969 CHEMICAL ENGINEERING: FOOD PRODUCTION TECHNOLOGY 566 respondents took part, found that organic products are consumed mainly by people who adhere to a healthy lifestyle. At the same time, research data [17] show that the demo graphic profile of organic buyers is not related to income, age, or family size, but to educational level. A mandatory element for labeling organic products is the code number, which is placed under the state logo for organic products: 1. Acronym that identifies the state of origin. 2. The inscription «organic». The development of new recipes and the study of the quality of flour confectionery products are devoted to the previous works of the authors. These studies describe the search for new formulations from organic raw materials and proposed studies of quality, safety and biological value: 3. Registration code of the certification body, which has completed the certification of organic production [24]. In order to ensure that the printed acronyms and codes of the certification bodies are correct, a check was carried out on the Organic Farming Information System website [25]. An important requirement for the production of organic food is the identification of each batch of such products. Sam pling was carried out in accordance with DSTU 5904:2006 «Confectionery products. Acceptance rules, methods of sam pling and sample preparation». Organoleptic analysis was carried out according to the following parameters: appearance, taste, smell, and appearance in the fault [26]. An important requirement for the production of organic food is the identification of each batch of such products. Sam pling was carried out in accordance with DSTU 5904:2006 «Confectionery products. Acceptance rules, methods of sam pling and sample preparation». Organoleptic analysis was carried out according to the following parameters: appearance, taste, smell, and appearance in the fault [26]. – cakes [18]; – biscuits [19]; – cupcakes [20]. It has been established that organic products are dis tinguished by a lower content of toxic compounds and a high content of useful nutrients. However, the proposed studies did not address the quality problems of the exist ing range of organic products sold on the market. The moisture content of the biscuits is determined by drying to constant weight at a temperature of 130 ± 2 °C for 40 minutes according to DSTU 4910:2008 «Confec tionery products. Methods for Determination of Moisture and Dry Substances». 4. Research of existing solutions of the problem 2 it can be seen that the composition of the Belgian children’s biscuits with pieces of Fleur Alpine chocolate: h l i h t fl – whole grain wheat flour; – unrefined cane sugar; h d – non-hydrogenation vegetable oils (sunflower, cocoa butter); – 8 % chocolate chips (unrefined cane sugar, cocoa mass, cocoa butter); ) – rice flour; – rice flour; – egg; – skimmed milk powder; – baking powder (sodium bicarbonate); TECHNOLOGY AUDIT AND PRODUCTION RESERVES — № 1/3(57), 2021 40 ISSN 2664-9969 CHEMICAL ENGINEERING: FOOD PRODUCTION TECHNOLOGY The moisture content of the biscuits is determined by drying to constant weight at a temperature of 130 ± 2 °C for 40 minutes according to DSTU 4910:2008 «Confec tionery products. Methods for Determination of Moisture and Dry Substances». 5. Methods of research Organic products can be labeled on the packaging only if there is a corresponding certificate [21]. Organic products are those that are 95 % made from organic raw materials [22]. That is why, the first stage of the examina tion of organic biscuits was the analysis of the labeling for compliance with the requirements of Ukrainian legislation. The alkalinity of biscuits was determined according to DSTU 5024:2008 «Confectionery products. Methods for determination of acidity and alkalinity». The wetness of the biscuits was determined according to DSTU 5023:2008 «Confectionery products. Method for determining wetness». Labeling – words, descriptions, signs for goods and services (trade marks), graphics or symbols related to food, which are placed on any packaging. In the absence of packaging, in a document or message accompanying or referring to the food product. The conformity of the biscuit labeling has been twisted according to the requirements of the Law of Ukraine «On information for consumers regarding food products» [23]. 6. Research results Analysis of the labeling for compliance with the Law of Ukraine «On information for food consumers» and the Law of Ukraine «On organic production» are presented in Table 1. Table 1 Decoding the barcode of Fleur Alpine biscuits 1 2 3 11. For drinks with an ethyl alcohol content of more than 1.2 % by volume – the actual alcohol content in the drink Method of preparation: the product is ready to eat – 12. Information on the nutritional value of the food – Energy value 1894 kJ/450 kcal. Fat – 14.0 g, including Saturated fatty acids – 8.3 g. Car- bohydrates – 79.5 g, including Sucrose – 14.6 g. Dietary fiber – 3.5 g. Proteins – 8.8 g. Salt – 0.2 g. Thiamin (mg) – 66 Additional labeling requirements for organic products 1. Acronym that identifies the state of origin BE-BIO-01 DE- KO-001 2. The inscription «organic» + + 3. Registration code of the certification body, made the certification of organic production + + Continuation of Table 1 Continuation of Table 1 1 2 3 11. For drinks with an ethyl alcohol content of more than 1.2 % by volume – the actual alcohol content in the drink Method of preparation: the product is ready to eat – 12. Information on the nutritional value of the food – Energy value 1894 kJ/450 kcal. Fat – 14.0 g, including Saturated fatty acids – 8.3 g. Car- bohydrates – 79.5 g, including Sucrose – 14.6 g. Dietary fiber – 3.5 g. Proteins – 8.8 g. Salt – 0.2 g. Thiamin (mg) – 66 Additional labeling requirements for organic products 1. Acronym that identifies the state of origin BE-BIO-01 DE- KO-001 2. The inscription «organic» + + 3. Registration code of the certification body, made the certification of organic production + + Continuation of Table 1 So, product labeling complies with Ukrainian legislation. In order to make sure that the applied acronyms and codes of certification bodies are correct, a check was carried out on the site of the Organic Farming Information Sys tem [25] (Fig. 3, 4). It has been established (Fig. 3) that biscuits are pro duced in Belgium in Brussels, as indicated on the package. For Holle biscuits, the information is shown in Fig. 4. Fig. 3. Checking the Fleur Alpine biscuit information Fig. 3. Checking the Fleur Alpine biscuit information Fig. 4. Checking the acronym for Holle biscuits Fig. 5. Decoding the barcode of Holle biscuits Fig. 3. Checking the Fleur Alpine biscuit information Fig. 4. Checking the acronym for Holle biscuits Fig. 6. Table 1 41 TECHNOLOGY AUDIT AND PRODUCTION RESERVES №1/3(57) 2021 Table 1 Organic biscuit labeling analysis Mandatory information element on the label Belgian children’s biscuits with pieces of Fleur Alpine chocolate Children’s spelled biscuits Holle 1 2 3 1. Name of the food Belgian children’s biscuits with pieces of Fleur Alpine chocolate Children’s spelled biscuits Holle from 8 months 2. List of ingredients Whole wheat flour, unrefined cane sugar, non-hydrogenated vegetable oils (sunflower, cocoa butter), chocolate chips (8 %) (unrefined cane sugar, cocoa mass, cocoa butter), rice flour, egg, skimmed milk powder, sodium bicarbonate (bicarbonate), natural vanilla extract, antioxidant (tocopherol) [27] Spelled flour 63 %, rice syrup, butter (dairy product), banana powder, baking powder, so- dium bicarbonate, vitamin B1. Contains gluten, milk [27] 3. Any ingredients or processing aids used in the production or preparation of a food product and remain present in the finished product, even in a modified form May contain traces of soybeans and nuts May contain traces of nuts, eggs, sesame seeds, soy 4. Amount of specific ingredients or ingre dient categories – – 5. Amount of food product in established units Net weight 150 g Net weight 150 g 6. Minimum expiry date or «take-to» date 10.04.2021 25.05.2021 7. Any special conditions of storage and/or use (if necessary) Store at a temperature not exceeding 25 °С and a relative humidity not exceeding 75 %. After opening the package, use within 1 day Store in a cool dry place. After opening the package, consume within 3–4 weeks 8. Name and location of the food market operator LLC «Eita Natur Organic», Dnipro, Ukraine Wine Bureau LLC, Kyiv, Ukraine 9. Country of origin or place of origin Fleur Alpine, Belgium Wikana Keks und Nahrungsmittel, Germa- ny for Holle baby food GmbH, CH-4125, Reichen, Baselstrasse 11 Switzerland 10. Instructions for use – if the absence of such instructions makes it difficult to properly use the food Belgium – Organic biscuit labeling analysis 41 CHEMICAL ENGINEERING: FOOD PRODUCTION TECHNOLOGY ISSN 2664-9969 So, product labeling complies with Ukrainian legislation. In order to make sure that the applied acronyms and codes of certification bodies are correct, a check was carried out on the site of the Organic Farming Information Sys tem [25] (Fig. 3, 4). It has been established (Fig. 3) that biscuits are pro duced in Belgium in Brussels, as indicated on the package. For Holle biscuits, the information is shown in Fig. Table 1 4. Fig. 3. Checking the Fleur Alpine biscuit information Fig. 4. Checking the acronym for Holle biscuits So, it has been established that organic biscuits meet the requirements of the Law of Ukraine «On information for consumers regarding food products» in terms of label ing and contains the necessary information to designate organic products in accordance with the Law of Ukraine «On organic production». Barcoding is the dominant technology for automatic identification, and the barcode symbol is the most im portant component of this technology, the basis of data for automatic processing [28]. That is why, in the course of the study, the barcode was deciphered with the employer of the site [29] (Fig. 5). Fig. 5. Decoding the barcode of Holle biscuits Fig. 6. Decoding the barcode of Fleur Alpine biscuits So, according to [29], the country of origin of the product is Belgium and Luxembourg, as indicated on the package. Check digits match. The appearance of Fleur Alpine biscuits is shown in Fig. 7. 1 2 3 11. For drinks with an ethyl alcohol content of more than 1.2 % by volume – the actual alcohol content in the drink Method of preparation: the product is ready to eat – 12. Information on the nutritional value of the food – Energy value 1894 kJ/450 kcal. Fat – 14.0 g, including Saturated fatty acids – 8.3 g. Car- bohydrates – 79.5 g, including Sucrose – 14.6 g. Dietary fiber – 3.5 g. Proteins – 8.8 g. Salt – 0.2 g. Thiamin (mg) – 66 Additional labeling requirements for organic products 1. Acronym that identifies the state of origin BE-BIO-01 DE- KO-001 2. The inscription «organic» + + 3. Registration code of the certification body, made the certification of organic production + + Continuation of Table 1 So, product labeling complies with Ukrainian legislation. In order to make sure that the applied acronyms and codes of certification bodies are correct, a check was carried out on the site of the Organic Farming Information Sys tem [25] (Fig. 3, 4). It has been established (Fig. 3) that biscuits are pro duced in Belgium in Brussels, as indicated on the package. For Holle biscuits, the information is shown in Fig. 4. Fig. 3. Checking the Fleur Alpine biscuit information Fig. 4. Checking the acronym for Holle biscuits Fig. 5. Decoding the barcode of Holle biscuits Fig. 6. Table 3 Table 3 The results of the study of products on a scale Threats. It should be noted that the production of or ganic products is more expensive, so every consumer can afford to buy such food. That is why the market for organic products is still limited. Table 3 The results of the study of products on a scale No. Indicator Weight factor Fleur Alpine Holle 1 Shape 1 4.8 5.0 2 surface 1 4.9 5.0 3 Colour 1 4.36 4.1 4 Appearance 1 5 5 5 Fault view 1 4.9 4.9 6 Consistency 0.5 5/2.5 5/2.5 7 Smell 1.5 5/7.5 5/7.5 8 Taste 2 5/10 5/10 The total number of points taking into account the weighting factor 39.96 44 Quality level 0.88 0.97 The results of the study of products on a scale Table 2 Table 2 Organoleptic analysis of organic biscuits Indicator name Fleur Alpine Holle Shape correct, fantasy, oval not clear regular, rectangular, pat- terned, smooth edges Surface smooth interspersed with pieces of chocolate, not burnt, without splashes of crumbs smooth with a clear pat- tern, without inclusions of crumbs Colour beige, uniform light beige, uniform Taste and smell pleasant crunchy taste with hints of chocolate, without foreign smell and taste pleasant taste with the aro- ma of milk and bananas, without foreign smell and taste Fault view baked with uniform po- rosity without voids and traces of non-targeting baked with uniform porosity without voids and traces of non-targeting Table 4 Table 4 Results of the study of physical and chemical indicators Indicator Norm Fleur Alpine Holle Humidity, % 10 ± 2 9.3 8.0 Alkalinity, deg. No more than 2.0 1.6 1.4 Wetness, % Less than 110 120 110 Results of the study of physical and chemical indicators Fig. 8. The appearance of Holle biscuits So, both samples meet the requirements of the stan dard in terms of such physicochemical indicators as humi dity, alkalinity and moisture content. The humidity for Fleur Alpine biscuits was 9.3 % and for Holle biscuits it was 8.0 %. The alkalinity was 1.6 and 1.4 degrees, respec tively, and the wetness was also within the normal range with values of 120 and 110 %. The examination of biscuits for organoleptic indicators was carried out in accordance with DSTU 3781:2014 [19]. The characteristics of the organoleptic characteristics of the biscuits are given in Table 2. 7. SWOT analysis of research results Strengths. Research on organic biscuits has shown that the labeling meets the requirements of current legislation. The barcode is applied correctly, as evidenced by the results of barcode identification according to the source [29]. It was found that both samples meet the requirements of the standard, both in terms of organoleptic and physicochemi cal indicators. Weaknesses. Since organic products are distinguished precisely by the best indicators of safety, the weakness of the study is that these indicators have not been studied and compared with biscuits of traditional technology. Opportunities. Opportunities for further research are in the development of new formulations of organic bis cuits based on the study of the existing range. Market research and expansion of the range of organic products is interesting for many countries of the world, despite the growing popularity of ecological and healthy food. Table 3 shows the results of the study of products on a 45-point scale developed by the authors [26]. Table 3 shows the results of the study of products on a 45-point scale developed by the authors [26]. Table 1 Decoding the barcode of Fleur Alpine biscuits Fig. 4. Checking the acronym for Holle biscuits So, according to [29], the country of origin of the product is Belgium and Luxembourg, as indicated on the package. Check digits match. The appearance of Fleur Alpine biscuits is shown in Fig. 7. So, it has been established that organic biscuits meet the requirements of the Law of Ukraine «On information for consumers regarding food products» in terms of label ing and contains the necessary information to designate organic products in accordance with the Law of Ukraine «On organic production». TECHNOLOGY AUDIT AND PRODUCTION RESERVES — № 1/3(57), 2021 aine matic im data code g. 5). the bers rod but ered Fig. 7. Fleur Alpine Biscuits Barcoding is the dominant technology for automatic identification, and the barcode symbol is the most im portant component of this technology, the basis of data for automatic processing [28]. That is why, in the course of the study, the barcode was deciphered with the employer of the site [29] (Fig. 5). So, according to the code decryption information, the product is made in Switzerland, and the check numbers match. The information provided on the packaged prod ucts indicates that the goods were made in Germany, but ordered by a Swiss company. The code for the Fleur Alpine biscuits was deciphered in a similar way (Fig. 6). Fig. 7. Fleur Alpine Biscuits TECHNOLOGY AUDIT AND PRODUCTION RESERVES — № 1/3(57), 2021 42 ISSN 2664-9969 CHEMICAL ENGINEERING: FOOD PRODUCTION TECHNOLOGY CHEMICAL ENGINEERING: FOOD PRODUCTION TECHNOLOGY The appearance of Holle biscuits is shown in Fig. 8. indicators». The total score for the Fleur Alpine biscuits was 39.96, which corresponds to a quality level of 0.88; and Holle biscuits – 44, with a quality level of 0.97. Fig. 8. The appearance of Holle biscuits q y The results of the study of physical and chemical pa rameters are shown in Table 4. References Available at: https:// ec.europa.eu/agriculture/ofis_public/pdf/EUCBLIST_official_ 2013.pdf?uid = 45EDC919-BA15-14F1-84B7C5EF903233F3 7. Kilnitska, O., Kravchuk, N., Kutsmus, N. (2018). Confectionery market in Ukraine: trends and development prospects. Ekono mika APK, (11), 29–43. doi: http://doi.org/10.32317/2221- 1055.201811029 26. Tkachenko, A. (2015). Formuvannia spozhyvchykh vlastyvostei pechyva tsukrovoho pidvyshchenoi kharchovoi tsinnosti. Lviv, 27. h h h l bl h d h 26. Tkachenko, A. (2015). Formuvannia spozhyvchykh vlastyvostei pechyva tsukrovoho pidvyshchenoi kharchovoi tsinnosti. Lviv, 27. 27. Pechyvo orhanichne. Available at: https://amador.com/pechivo- holle-speltove-organichne-z-8-misyatsiv-150-g-23232/ 27. Pechyvo orhanichne. Available at: https://amador.com/pechivo- holle-speltove-organichne-z-8-misyatsiv-150-g-23232/ 8. Sarkisian, L., Yichyk, Yu. (2016). Osoblyvosti pozshyrennya zovnisnoekonomichnykh zvyazkiv Ukrainy v konteksti hloba lizatsiyi. 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Vidomosti Verkhovnoi Rady, 36, 275. Available at: https://zakon.rada.gov.ua/laws/show/2496-19#Text Available at: https://zakon.rada.gov.ua/laws/show/2496-19#Text 25. Organic Farming Information System (2020). CHEMICAL ENGINEERING: FOOD PRODUCTION TECHNOLOGY ISSN 2664-9969 4. It was revealed that both samples meet the require ments of regulatory documents in terms of such physicochemi cal indicators as humidity, alkalinity and moisture content. The moisture content of the biscuits was determined in an accelerated manner in an oven. Wetness was determined by the ratio of the weight of the items after wetting to the weight of the dry items. Alkalinity was determined by titration of the product filtrate, 9.3 % for Fleur Alpine and 8.0 % for Holle biscuits. The alkalinity was 1.6 and 1.4 degrees, respectively, and the wetness was also within the normal range with values of 120 and 110 %. review of why people purchase organic food. 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Conclusions 1. It has been established that organic biscuits meet the requirements of the Law of Ukraine «On information for consumers regarding food products» in terms of label ing and contain the necessary information to designate organic products in accordance with the Law of Ukraine «On organic production». 2. Using the source [29], the bar coding of both biscuit samples was deciphered and it was established that the check digits of the codes coincide, and the biscuits were produced in the same countries as indicated on the labeling. 3. The results of the study of products on a 45-point scale are shown. The total number of points in the tasting score, taking into accounts the weight factor, for Fleur Alpine biscuits was 39.96, which corresponds to a quality level of 0.88; and Holle biscuits – 44, with a quality level of 0.97. Having processed the results of laboratory research, it can be concluded that the tested samples of organic biscuits meet the requirements of the DSTU 3781:2014 standard «Biscuits. General technical requirements by organoleptic TECHNOLOGY AUDIT AND PRODUCTION RESERVES — № 1/3(57), 2021 43 TECHNOLOGY AUDIT AND PRODUCTION RESERVES — № 1/3(57), 2021 References Customers Purchasing Organic Food – Do They Live Healthier? Results of the German National Nutrition Survey II. European Journal of Nutrition & Food Safety, 5 (1), 59–71. doi: http:// doi.org/10.9734/ejnfs/2015/12734 Khrystyna Kovalchuk, PhD, Associate Professor, Department of Commodity Research and Commodity Expertise, Lviv Institute of Economics and Touris, Lviv, Ukraine, ORCID: http://orcid.org/0000- 0001-6894-9392, e-mail: khristinakovalchuk1985@gmail.com 15. Hughner, R. S., McDonagh, P., Prothero, A., Shultz, C. J., Stanton, J. (2007). Who are organic food consumers? A compilation and TECHNOLOGY AUDIT AND PRODUCTION RESERVES — № 1/3(57), 2021 44
https://openalex.org/W2368319056	https://europepmc.org/articles/pmc5122359?pdf=render	English	null	Identification of polycystic ovary syndrome potential drug targets based on pathobiological similarity in the protein-protein interaction network	Oncotarget	2,016	cc-by	8,929	Hao Huang1,, Yuehan He1,, Wan Li1, Wenqing Wei1, Yiran Li1, Ruiqiang Xie1, Shanshan Guo1, Yahui Wang1, Jing Jiang1, Binbin Chen1, Junjie Lv1, Nana Zhang2, Lina Chen1, Weiming He3 1College of Bioinformatics Science and Technology, Harbin Medical University, Harbin, Heilongjia 3Institute of Opto-electronics, Harbin Institute of Technology, Harbin, Heilongjiang Province, Chi titute of Opto-electronics, Harbin Institute of Technology, Harbin, Heilongjiang Province, China These authors have contributed equally to this work These authors have contributed equally to this work Correspondence to: Lina Chen, email: chenlina@ems.hrbmu.edu.cn Weiming He, email: hewm@hit.edu.cn Keywords: polycystic ovary syndrome, protein-protein interaction network, pathobiological similarity, module, drug target Received: January 04, 2016 Accepted: April 28, 2016 Published: May 13, 2016 Keywords: polycystic ovary syndrome, protein-protein interaction network, pathobiological similarity, module, drug target Received: January 04, 2016 Accepted: April 28, 2016 Published: May 13, 2016 Abstract Polycystic ovary syndrome (PCOS) is one of the most common endocrinological disorders in reproductive aged women. PCOS and Type 2 Diabetes (T2D) are closely linked in multiple levels and possess high pathobiological similarity. Here, we put forward a new computational approach based on the pathobiological similarity to identify PCOS potential drug target modules (PPDT-Modules) and PCOS potential drug targets in the protein-protein interaction network (PPIN). From the systems level and biological background, 1 PPDT-Module and 22 PCOS potential drug targets were identified, 21 of which were verified by literatures to be associated with the pathogenesis of PCOS. 42 drugs targeting to 13 PCOS potential drug targets were investigated experimentally or clinically for PCOS. Evaluated by independent datasets, the whole PPDT-Module and 22 PCOS potential drug targets could not only reveal the drug response, but also distinguish the statuses between normal and disease. Our identified PPDT-Module and PCOS potential drug targets would shed light on the treatment of PCOS. And our approach would provide valuable insights to research on the pathogenesis and drug response of other diseases. www.impactjournals.com/oncotarget/ Oncotarget, Vol. 7, No. 25 Identification of polycystic ovary syndrome potential drug targets based on pathobiological similarity in the protein-protein interaction network Research Paper www.impactjournals.com/oncotarget/ Central importance of genes in PPDT-Modules Degree, closeness, betweenness, and page rank of genes in PPDT-Modules were calculated, and the genes in each module were ranked by these topological metrics, respectively. The G-rank of genes in PPDT-Modules was calculated using the geometric mean rank (G- rank of top 22 genes and all genes in PPDT-Module 2are shown in Table 2 and Supplementary Table 2, respectively). The research on complex diseases from the systems level and biological background might provide new insights into drug target identification, disease gene exploration and pathogenesis elucidation [15–17]. For example, disease genes and drug targets of coronary heart disease (CAD) and T2D could be identified effectively by systems biology approaches [18, 19]. Chan KH et al. identified key drivers that included COL1A1, COL3A1, and ELN in the shared pathways for both CAD and T2D based on the protein-protein interaction network (PPIN) and genome-wide association studies [20]. In addition, the highly central importance proteins in core networks or sub-networks are globally or locally important nodes which could act as functional modules or pathways or key role genes to uncover the pathogenesis and therapeutic ways of disease [21]. For example, Li H et al. presented a novel PPI knowledge-based approach that identified functional modules and hub genes for CAD, revealing several novel pathogenic mechanisms [20]. Wan Li et al. developed a method using combined centrality indices on the systems level to identify essential cancer-related motifs in central roles, which provided a clue for the study of signal transduction in biological pathways [22]. Furthermore, Emad Fadhal et al. have indicated that the genes possessing highly topologically centered in the PPIN may be good therapeutic targets [23]. pp y p y) The PPDT-Modules were the pathobiological similar modules of PCOS and T2D. The higher genes of PPDT-Module were ranked, the more important they might be, and the more kernel roles they would act in the pathogenesis and treatment of PCOS. The genes ESR1, RXRA and NCOA1 were the top three genes not only in the G-rank of genes, but also in four individual topological ranks, which demonstrated the stability of PPDT-Module 2. It was noted that ESR1 (1st) and ESR2 (5th) both ranked highly in the G-rank of genes in PPDT- Module 2. They are the main receptors of estrogen and the metabolism of estrogens was proved to be associated with the pathogenesis of PCOS [24]. Results The genes of PPDT-Module 1 and PCOS disease genes, PPDT-Module 3 and PCOS disease genes are not enriched significantly in the functional categories related to PCOS, respectively (Figure 2A). The genes of PPDT-Module 2 and PCOS disease genes are enriched significantly in functional categories and KEGG pathways associated with the pathogenesis of PCOS (Figure 2A, Introduction that some other features including weight gain, disturbance of hormones and lipid disorders are associated with both PCOS and T2D [6, 7]. In the meantime, PCOS and T2D are closely linked in the epidemiology and etiology as well as in the systems biology [8, 9]. Significant pathobiological similarity between PCOS and T2D was also indicated (−0.2) after determining the pathobiological and clinical relationship between these diseases using a method proposed by Jörg Menche et al. [9]. Polycystic ovary syndrome (PCOS) is one of the most common endocrinological disorders in reproductive aged women, of which the prevalence is 5–10% in the general population [1–3]. The clinical and biochemical characteristics of PCOS are generally grouped into two main categories: (i) reproductive features that mainly include chronic anovulation, and (ii) hyperandrogenic features that mainly include hyperandrogenaemia. Metformin is the only known drug for PCOS in Drugbank. The DrugBank database is a unique bioinformatics and cheminformatics resource that combines detailed drug data with comprehensive drug target information, which was widely recognized and used [10]. As an insulin sensitizer, Metformin can cause reductions in body weight, restore ovulation, increase the rate of pregnancy and reduce the number of pregnancy As important metabolism characteristics, insulin resistance and compensatory hyperinsulinemia are observed in approximately 70% of PCOS women [4], which contribute to the strong association of PCOS with adverse metabolic risk, including dysglycemia, dyslipidaemia and fatty liver, as well as increase the risk for development of type 2 diabetes (T2D) [5]. It is found www.impactjournals.com/oncotarget Oncotarget 37906 complications [11]. In the meantime, metformin is also used for treating T2D, which improves glycemic control by decreasing hepatic glucose production and glucose absorption and increasing insulin-mediated glucose uptake [12]. In addition, Thiazolidinediones (TZDs), including pioglitazone, are peroxisome proliferator- activated receptor (PPAR) agonists that induce adipogenesis and have insulin-sensitizing and antidiabetic properties [13]. Recent studies have demonstrated the beneficial metabolic effects of treatment with pioglitazone in PCOS patients [11, 14]. These indicated that the genes and drugs associated with common metabolism characteristics of PCOS and T2D might be the potential drug targets and drugs of PCOS. PCOS disease genes) and 923 T-Modules (25 T-Modules contained one or more T2D disease genes) were identified. The Jaccard similarity index was calculated for each P-Module and T-Module pairs. 3 candidate PPDT- Modules were identified ultimately, including PCOS disease genes, T2D disease genes and known T2D drug targets simultaneously (Table 1, Supplementary Table 1). Central importance of genes in PPDT-Modules The gene RXRA (2nd) is always binding with PPARG and function together, and the gene PPARG is both the PCOS disease gene and T2D disease gene, is also the known T2D drug target, and ranked 12th in PPDT-Module 2. The gene NCOA1 (3rd) is one of the known T2D drug targets. In addition, the gene PPARA, ranked 11th in PPDT-Module 2, is one of the known T2D targets. So genes which possessed high G-rank in PPDT-Module might play key roles in the pathogenesis, diagnosis and treatment of PCOS. PCOS potential drug targets In this study, based on the pathobiological similarity of PCOS and T2D, a new computational approach (Figure 1) was performed to identify the PCOS potential drug target modules (PPDT-Modules) and PCOS potential drug targets from the systems level and biological background in the PPIN. Our study may reveal novel PCOS therapeutic ways and improve the cure rate of PCOS. From the functional perspective to analyze the association between PPDT-Modules and PCOS, the hyper-geometric test was performed with the genes in PPDT-Modules and the PCOS disease genes in the Gene Ontology (GO) and pathways of Kyoto Encyclopedia of Genes and Genomes (KEGG), and the p value was adjusted with FDR (P < 0.05, Figure 2A). PPDT-Modules www.impactjournals.com/oncotarget Oncotarget 37908 Table 1: Candidate PPDT-Modules Table 1: Candidate PPDT-Modules Candidate PPDT-Module The number of genes in PPDT-Module PCOS disease Gene T2D disease Gene T2D drug Target Candidate PPDT-Module 1 141 TH UCP3 PTPN1, ADRA2C Candidate PPDT-Module 2 85 PPARG HNF4A, PPARG NCOA1, PPARA PPARD, PPARG Candidate PPDT-Module 3 6 INSR INSR, ENPP1 INSR Table 2: G-rank of top 22 genes in PPDT-Module 2 Gene G-rank Rank of Degree Rank of Betweenness Rank of Closeness Rank of Page Rank ESR1 1 1 1 1 1 RXRA 2 2 2 2 2 NCOA1 3 3 3 3 3 NRIP1 4 5 8 4 6 ESR2 5 4 7 12 4 THRB 6 6 10 8 5 RARA 7 8 5 9 7 NR0B2 8 9 12 5 11 NCOA3 9 10 13 6 9 HNF4A 10 11 9 11 12 PPARA 11 12 4 30 10 PPARG 12 7 11 28 8 PPARGC1A 13 13 22 7 13 MED1 14 14 20 10 14 NR2F1 15 28 6 13 24 PNRC2 16 15 19 16 17 PGR 17 19 14 22 15 ESRRA 18 18 21 14 19 ESRRG 19 16 23 39 16 RXRB 20 17 35 27 18 RARG 21 20 26 33 23 VDR 22 21 25 36 21 Table 2: G-rank of top 22 genes in PPDT-Module 2 Gene G-rank Rank of Degree Rank of Betweenness Rank of Closeness Rank of Page Rank ESR1 1 1 1 1 1 RXRA 2 2 2 2 2 NCOA1 3 3 3 3 3 NRIP1 4 5 8 4 6 ESR2 5 4 7 12 4 THRB 6 6 10 8 5 RARA 7 8 5 9 7 NR0B2 8 9 12 5 11 NCOA3 9 10 13 6 9 HNF4A 10 11 9 11 12 PPARA 11 12 4 30 10 PPARG 12 7 11 28 8 PPARGC1A 13 13 22 7 13 MED1 14 14 20 10 14 NR2F1 15 28 6 13 24 PNRC2 16 15 19 16 17 PGR 17 19 14 22 15 ESRRA 18 18 21 14 19 ESRRG 19 16 23 39 16 RXRB 20 17 35 27 18 RARG 21 20 26 33 23 VDR 22 21 25 36 21 Table 2: G-rank of top 22 genes in PPDT-Module 2 “GO:0019216~regulation of lipid metabolic process”, “GO:0004879~ligand-dependent nuclear receptor activity” and “GO:0008289~lipid binding” are associated with lipid metabolism. PPDT-Modules The PPDT-Module identification approach we put forward was performed with 35 PCOS disease genes, 83 T2D disease genes and 62 T2D drug targets in the PPIN. 910 P-Modules (13 P-Modules contained one or more www.impactjournals.com/oncotarget Oncotarget 37907 Supplementary Table 3), such as transcription, steroid hormone receptor signaling pathway (GO biological process, BP), steroid binding, lipid binding and steroid hormone receptor binding (GO molecular function, MF), PPAR signaling pathway, Steroid hormone biosynthesis and “GO:0050681~androgen receptor binding” are also associated with androgen. Androgen receptor signaling is an important mediator in the etiology of PCOS traits [26]. And androgens mediate their steroidal actions via the androgen receptor which are crucial in maintaining female fertility by optimizing follicle growth, health, and ovulation [27]. With PCOS, women typically have high levels of androgens, high levels of androgens affect the development and release of eggs during ovulation [27–29]. “GO:0030520~estrogen receptor signaling pathway” and “GO:0030331~estrogen receptor binding” are associated with estrogen. It is noted that estrogens play important roles in the development and functioning of the male and female reproductive systems [30, 31]. The main mediators of estrogen action are 2 specific high-affinity receptors, the ESR1 and ESR2, are “GO:0030518~steroid hormone receptor signaling pathway”, “GO:0003707~steroid hormone receptor activity”, “GO:0005496~steroid binding” and “GO:0005496~steroid binding” are associated with steroid hormones. Disturbed steroid hormone receptor signaling pathway would lead to the hormonal imbalance which is a main underlying problem with PCOS [25]. The steroid hormones mainly contain the androgens and estrogens and so on. “GO:0030521~androgen receptor signaling pathway” Figure 1: A schematic diagram of PCOS potential drug targets identification and efficiency analysis. Figure 1: A schematic diagram of PCOS potential drug targets identification and efficiency analysis. Figure 1: A schematic diagram of PCOS potential drug targets identification and efficiency analysis. PPDT-Modules Abnormal lipid metabolism is one of the main metabolic characteristics of PCOS patients [39]. The parameters of lipid metabolism were significantly different in women with PCOS from in healthy women, higher triglycerides (TG), and very-low- density lipoprotein cholesterol (VLDL) with lower high- density lipoprotein cholesterol (LDL) in PCOS [40, 41]. Altered expression of genes involved in lipid metabolism could contribute to insulin resistance in non-obese PCOS patients [39]. Based on case-controlled studies, women with PCOS appear to have a high prevalence of the metabolic syndrome including obesity and dyslipidemia, and have a higher risk of diabetes [2]. Insulin resistance necessary for the proper function of the hypothalamic– pituitary–ovarian axis and are expressed in the human ovary, where ESR2 is the predominant receptor and its activation enhances folliculogenesis and ovulation [32, 33]. The expression of ESR2 is lower in follicles derived from women with PCOS compared with healthy women, while ESR1 expression is markedly increased in theca cells of polycystic ovaries, causing alteration in the ESR1/ ESR2 ratio in PCOS and possibly abnormal follicular development [34, 35]. Otherwise, insulin resistance is associated not only with the pathogenesis of T2D, but also with the pathogenesis of PCOS [36, 37]. The role of ESR1 in the development of insulin resistance has been described in human with a null mutation in the ESR1, leading to unresponsiveness to estrogen, who developed impaired glucose tolerance [34, 38]. www.impactjournals.com/oncotarget Oncotarget 37909 features, the average of sensitivity values for normal/ PCOS, PCOS/after pioglitazone treatment were 0.79 and 1, the average of specificity values were 0.68 and 1, the average of AUCs were 0.72 and 1, the average of accuracy values were 0.73 and 1, the average of MCC scores were 0.49 and 1, respectively (Figure 4, Figure 5A, Supplementary Table 5, and Supplementary Table 6). At the same time, the average expression values of PCOS disease genes, T2D disease genes and known T2D drug targets were used as the classification features to classify the same samples of different statuses, respectively (Figure 4, Supplementary Table 5, and Supplementary Table 6). Most of these classification features could classify samples efficiently. i appears to have a pivotal role with multiple studies demonstrating significant associations between altered lipid metabolism and insulin level, which may be induced in part by the insulin-medicated stimulation of lipolysis and altered expression of lipoprotein lipase and hepatic lipase [42, 43]. PPDT-Modules Some studies indicated that disturbance of transcription, disturbance of steroid hormone, lipid abnormalities and so on should lead to promoting the PCOS [44–46]. [ ] Meanwhile, “hsa03320:PPAR signaling pathway” plays an important role in the pathogenesis of PCOS and T2D, and the treatment for T2D. Peroxisome proliferator- activated receptors (PPARs) are nuclear hormone receptors activated by fatty acids and their derivatives [13]. PPAR signaling pathway was shown to associate with the pathogenesis of PCOS, as well as with the pathogenesis of T2D [13]. PPAR has three subtypes (PPARA, PPARD, and PPARG), all of them are known T2D drug targets, are showing different expression patterns. PPARA plays a role in the clearance of circulating or cellular lipids via the regulation of gene expression involved in lipid metabolism in liver and skeletal muscle [47]. PPARD is involved in lipid oxidation and cell proliferation [47, 48]. PPARG promotes adipocyte differentiation to enhance blood glucose uptake [49]. Meanwhile, PPARG is both PCOS and T2D disease gene, the drug targeting to PPARG was used to treat with PCOS patient and had good curative effect [13]. For the original expression profile, expression consistency check was performed via Wilcoxon signed- rank test to test the sample consistency of the same status (13 normal samples, 10 PCOS samples and 10 after pioglitazone treatment samples, FDR P value < 0.05), respectively. 9 of 13 normal samples and 5 of 10 after pioglitazone treatment samples were consistent. These 5 samples may alter to a common status after drug treatment, suggesting similar drug response and good curative effect of drug treatment, so the paired 5 PCOS/after pioglitazone treatment samples were selected and a new SVM was applied to classify the consistent samples of 3 statuses using the above-mentioned classification features (Figure 4). With identified PCOS potential drug targets as classification features, the average of sensitivity values for normal/PCOS, PCOS/after pioglitazone treatment were 0.95 and 1, the average of specificity values for normal/PCOS, PCOS/after pioglitazone treatment were 0.88 and 1, average of AUC values of normal /PCOS and PCOS/after pioglitazone were 0.91 and 1, the average of accuracy values for normal/PCOS, PCOS/after pioglitazone treatment were 0.92 and 1,the average of MCC scores for normal/PCOS, PCOS/after pioglitazone treatment were 0.84 and 1, respectively (Figure 4, Figure 5A, Supplementary Table 5, and Supplementary Table 6). PPDT-Modules The classification performance of consistent samples was much better than that of all samples from the original profile with the same classification features, especially for using the average expression values of identified PCOS potential drug targets and known T2D disease genes as classification features (Figure 4, Figure 5, Supplementary Table 5, and Supplementary Table 6), which demonstrated the effectiveness of identified PCOS potential drug targets. PCOS potential drug targets could effectively reflect drug response, classify the samples between normal and disease and between disease and after drug treatment, as well as represent the treatment effect to PCOS accurately. Interestingly, identified PCOS potential drug targets had a better classification performance than known PCOS disease genes, which demonstrated that these potential drug targets may act as the PCOS disease genes (Figure 4, Figure 5, Supplementary Table 5, and These changes of function and disturbance of pathways were associated not only with PCOS, but also with T2D closely. So, the genes of PPDT-Module 2 enriched significantly in these functions, may act as PCOS and T2D disease genes and drug targets (Figure 2B), such as PPARG. Known T2D drug targets NCOA1 and PPARA, T2D disease genes HNF4A, and the PCOS disease genes AR, CYP11A1 were also enriched in same functional categories and pathways of PPARG. Meanwhile, these disease genes and drug targets in PPDT-Module 2 had a high G-Rank, for instance, NCOA1 ranked 3rd, HNF4A ranked 10th, PPARA ranked 11th, PPARG ranked 12th. Hence, 22 genes of PPDT- Module 2 whose G-Rank was the upper quartile as well as enriched significantly in these PCOS associated functional categories and pathways were identified as the PCOS potential drug targets (Figure 3, Table 2). DISCUSSION PCOS is a disorder of irregular menses, hyperandrogenism and/or polycystic ovary morphology [26]. A large proportion of women with PCOS also exhibit insulin resistance, β cell dysfunction, impaired glucose tolerance and/or T2D [50]. Women with T2D have a higher prevalence of polycystic ovary syndrome compared to the general population [8]. There is also a high rate of T2D in family members of women with PCOS [50]. Some studies have demonstrated that there are common biological background and marker between PCOS and T2D [51]. Based on these, we put forward a new method to identify 3 candidate PPDT-Modules from the systems level in the PPIN based on the pathobiological similarity of PCOS and T2D. Identified PPDT-Modules contained many PCOS and T2D disease genes, and known T2D drug targets, simultaneously. By the functional annotation analysis with the PCOS disease genes, candidate PPDT- Module 2 was identified as the final PPDT-Module. The Dysfunctions of steroid hormone receptor signaling pathway, lipid metabolic process, and fatty acid oxidation are mainly metabolic disturbances of PCOS [44–46]. Identified PPDT-Module 2 had not only a good consistency function with PCOS but also good classification performance for classifying normal/PCOS samples and PCOS/after pioglitazone treatment samples before and after consistent check. These suggested that PPDT-Module 2 responded to drug treatment efficiently, may be used as an integral PCOS potential therapeutic target. 22 PCOS potential drug targets possessed central importance in PPDT-Module 2 as well as had good consistent function with PCOS. It was worth noting that 22 PCOS potential drug targets we identified were all interconnected in PPDT-Module 2, and had good classification performance, as same as one with PPDT-Module2, could represent the whole PPDT-Module 2 well. Figure 2: Main functions annotated by PPDT-Modules. (A) Main functions annotated by 3 candidate PPDT-Modules and PCOS disease genes. The first two columns of gray bars represent no gene of candidate PPDT-Module 1 and candidate PPDT-Module 3 enriched significantly in corresponding functional categories, respectively. The third column of green bars represents the number of genes of candidate PPDT-Module 2 enriched in the functional categories. (B) Main functions annotated by genes in PPDT-Module 2. Main functions annotated by genes in PPDT-Module 2. Each row represents a gene in PPDT-Module 2, and each column represents a functional category: biological process, molecular function and pathway. Arrow represents the ascending trend of G-Rank of genes in PPDT-Module 2. Figure 2: Main functions annotated by PPDT-Modules. Efficiency analysis of PCOS potential drug targets To assess the efficiency of PCOS potential drug targets, SVM was applied to classify samples of different statuses (normal/PCOS, PCOS/after pioglitazone treatment) of GSE8157 with the average expression values of identified PCOS potential drug targets as classification features. 1000 times of five-fold cross-validation were performed (Figure 4). With the average expression values of 22 PCOS potential drug targets as the classification www.impactjournals.com/oncotarget Oncotarget 37910 Supplementary Table 6). Classification using identified PPDT-Module 2 as classification features had good performance for normal /PCOS and for PCOS/after pioglitazone treatment in both original and consistent samples (Figure 4B, Figure 5B, Supplementary Table 5, and Supplementary Table 6). functional categories and pathways that genes of PPDT- Module 2 and PCOS disease genes enriched significantly, are associated not only with pathogenesis and treatment of T2D, but also with PCOS. Considering the central importance and functional annotation of known disease genes and targets, 22 PCOS potential drug targets were identified. These PCOS potential drug targets could describe the drug treatment effect well by an independent profile verified, and the 21 genes (95.45%) had been verified by literature to be associated with the pathogenesis and treatment of PCOS (Supplementary Table 4). DISCUSSION (A) Main functions annotated by 3 candidate PPDT-Modules and PCOS disease genes. The first two columns of gray bars represent no gene of candidate PPDT-Module 1 and candidate PPDT-Module 3 enriched significantly in corresponding functional categories, respectively. The third column of green bars represents the number of genes of candidate PPDT-Module 2 enriched in the functional categories. (B) Main functions annotated by genes in PPDT-Module 2. Main functions annotated by genes in PPDT-Module 2. Each row represents a gene in PPDT-Module 2, and each column represents a functional category: biological process, molecular function and pathway. Arrow represents the ascending trend of G-Rank of genes in PPDT-Module 2. www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 37911 Figure 3: PPDT-module 2 and PCOS potential drug targets. The size of nodes represents their G-Ranks. Different colors represent different functional categories. The green circle represents PCOS disease gene or T2D disease gene or known T2D drug target. Figure 3: PPDT-module 2 and PCOS potential drug targets. The size of nodes represents their G-Ranks. Different colors represent different functional categories. The green circle represents PCOS disease gene or T2D disease gene or known T2D drug target. Oncotarget 37912 ww impactjournals com/oncotarget Figure 4: The sensitivity, specificity, AUC, ACC and MCC score distribution of different classification features with 1000 times five-fold cross-validation. In each figure, the first two boxes represent the distribution of classification of normal/PCOS samples, the next two represent the distribution of classification of PCOS/after pioglitazone treatment samples. Green represents the classification of the samples before consistency check, red represents the classification of the samples after consistency check. Figure 4: The sensitivity specificity AUC ACC and MCC score distribution of different classification features with Figure 4: The sensitivity, specificity, AUC, ACC and MCC score distribution of different classification features with 1000 times five-fold cross-validation. In each figure, the first two boxes represent the distribution of classification of normal/PCOS samples, the next two represent the distribution of classification of PCOS/after pioglitazone treatment samples. Green represents the classification of the samples before consistency check, red represents the classification of the samples after consistency check. www.impactjournals.com/oncotarget Oncotarget 37912 Recent studies have demonstrated that the beneficial metabolic effects of treatment with pioglitazone induce adipogenesis and have insulin-sensitizing and antidiabetic properties in PCOS patients [52, 53]. It is known that different patients’ response to the drug treatment are different, thus the efficacy of treatment is various. The consistent check could identify status-consistent samples to improve the identification accuracy of disease status and effectiveness evaluation accuracy of drug response. The classification performances of samples with consistent expressions were much better than that of all samples with the average expression values of PCOS potential drug targets, PPDT-Module 2, PCOS disease genes, T2D disease genes and known T2D drug targets as classification features to classify 3 statuses samples of the expression profile GSE8157, respectively (Figure 4). www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 37913 Table 3: Drugs targeting PCOS potential drug targets PCOS potential drug targets Drugs ESR1 Mestranolb, Fulvestranta, Danazola, Etonogestrelb, Raloxifeneb, Diethylstilbestrolb, Medroxyprogesterone Acetateb, Genisteina, Desogestrelb, Norgestimateb, Naloxoneb, Levonorgestrela, Ethinyl Estradiolb, Melatonina , Clomifeneb, Progesteroneb, Tamoxifenb, Estradiolb, Estronea, Estriola RXRA Alitretinoina NCOA1 Genisteina ESR2 Raloxifeneb, Diethylstilbestrolb, Genisteina, Tamoxifenb, Estradiolb THRB Levothyroxinea RARA Alitretinoina, Isotretinoinb PPARA Gemfibrozila, Bezafibrateb, Indomethacina PPARG Repaglinidea, Glipizidea, Telmisartana, Indomethacina, Pioglitazoneb, Rosiglitazoneb, Bezafibrateb PGR Mifepristonea, Danazola, Etonogestrela, Megestrol acetateb, Medroxyprogesterone Acetateb, Desogestrelb, Norgestimateb, Levonorgestrela, Drospirenoneb, Spironolactoneb, Norethindroneb, Progesteroneb ESRRG Diethylstilbestrolb RXRB Alitretinoina, Tretinoina RARG Alitretinoina, Tretinoina VDR Alfacalcidola, Calcidiola, Ergocalciferola, Cholecalciferola, Calcitriola a denotes the drug is under experimental investigation; b denotes the drug has been investigated to be used for treating to PCOS in clinical. Table 3: Drugs targeting PCOS potential drug targets Drugs differential pathways instead of differential expressed genes, for analyzing the pathogenesis of PCOS and the way of drug response [52]. It was shown that from the system level and biological background to study the disease statuses and drug response could obtain comprehensive and effective results, especially in the case of no significantly differential expressed genes. ethylene stilbene derivative as an estrogen agonist or antagonist, which is the most commonly used drug for first-line treatment of ovulation induction in women with PCOS [59–61]. Some drugs are targeting several PCOS potential drug targets such as Alitretinoin targeting RXRA, RARA, RXRB and RARG; Tamoxifen targeting ESR1 and ESR2. Retinoid X receptors (RXRs) and retinoic acid receptors (RARs) are nuclear receptors that mediate the biological effects of retinoids by their involvement in retinoic acid-mediated gene activation. These receptors function as transcription factors by binding as homodimers or heterodimers to specific sequences in the promoters of target genes. The proteins encoded by these genes are members of the steroid and thyroid hormone receptor superfamily of transcriptional regulators [62]. Alitretinoin is also known as retinoic acid and derived from maternal vitamin A. Altered retinoic acid synthesis and action could influence the expression of these genes and androgen production in PCOS [63]. Tamoxifen, as one of the selective estrogen receptor modulators (SERM) with tissue-specific activities, is discovered to be a good alternative to clomiphene in women with PCOS and clomiphene-resistant case [64]. These durgs may play key roles in treating PCOS by multiple ways. www.impactjournals.com/oncotarget In the meantime, the classification performances of samples with consistent expressions were also much better than that of all samples of original expression profile with same classification features in another independent expression profile GSE6798 related to PCOS (Supplementary Figure S1). Interestingly, the classification performances of PCOS potential drug targets and PPDT-Module 2 as classification features were better than PCOS disease genes, T2D disease genes and known T2D drug targets in both expression profiles (Figure 5, Supplementary Table 5, Supplementary Table 6 and Supplementary Table 7). The potential drug targets and disease genes were usually identified based on significantly differential expressed genes [54]. It was noted that between normal samples and PCOS samples no significantly differential expressed genes of expression profiles GSE8157 and GSE6798 could be identified, while between PCOS samples and after pioglitazone treatment samples of GSE8157 only few significantly differential expressed genes could be identified, including no known disease genes, known drug targets or our interested genes. So the potential drug targets and disease genes could not be identified from the point of differential expressed genes for these two expression profiles. Nevertheless, identified PPDT-Module 2 and 22 PCOS potential drug targets could distinguish the statuses between normal and disease, evaluate the effectiveness of drug treatment from the systems level and functional point. Just like our research on the PCOS from the biological background, previous researchers used the profile GSE8157 to identify Figure 5: The ROC curves of classification for normal/PCOS with different features. ROC curves of classification for normal/PCOS with (A) PCOS potential drug targets, (B) PPDT-Module 2, (C) PCOS disease genes, (D) T2D disease genes and (E) Known T2D drug targets as classification features, respectively. Green lines represent the ROC curves before consistency check, red lines represent the ROC curves after consistency check. Figure 5: The ROC curves of classification for normal/PCOS with different features. ROC curves of classification for normal/PCOS with (A) PCOS potential drug targets, (B) PPDT-Module 2, (C) PCOS disease genes, (D) T2D disease genes and (E) Known T2D drug targets as classification features, respectively. Green lines represent the ROC curves before consistency check, red lines represent the ROC curves after consistency check. Identification of PPDT-Modules enhance their impacts [20], we shall make efforts in our future work to provide a web-server to displaying findings that can be manipulated by users according to their need. We put forward a new approach to identify PPDT- modules in the PPIN based on the pathobiological similarity of PCOS and T2D. (i) PCOS disease genes and T2D disease genes were set as seeds to filter the no weight protein-protein interaction network and obtained the disease genes related no weight 3-step-neighbor sub-network, respectively. T2D and PCOS related modules (T-Modules and P-Modules) were mined using the Markov Cluster Algorithm (MCL Algorithm) in the 3-step-neighbor sub-network (no requirement of minimum number of seeds each cluster needs to contain), respectively. The MCL algorithm is a fast and scalable unsupervised cluster algorithm for networks, resulting in a collection of densely connected groups of genes [71]. The Markov clustering parameter was set to the default value 1.8. (ii) The pathobiological similar modules were defined as the intersection of P-Module and T-Module pairs, if their Jaccard similarity indexes were larger than their median, and their intersection contained the PCOS disease genes and T2D disease genes simultaneously. The Jaccard similarity index J(P,T) reflected the consistency of each two modules P-Module and T-Module: Our approach was performed based on the existing known diseases genes and drug targets. As the disease genes and drug targets increased, our approach would be more precise. In the meantime, more and more gene expression datasets and novel drugs are available, which will make the result evaluation more objective and precise. The median of the Jaccard similarity indexes was selected as the threshold to identify the pathobiological similar modules in our study, which should be further optimized for the application of other diseases. In addition, the side-effect and toxicology of the drugs identified by identified PCOS potential drug targets were not considered adequately in our study, which will be further improved in our future work. We proposed a new computational approach to identify the PPDT-Module and PCOS potential drug targets from the systems level and biological background based on the PPIN and pathobiological similarity. 21 genes of 22 identified PCOS potential drug targets were verified to be associated with the pathogenesis of PCOS, and 42 drugs targeting 13 PCOS potential drug targets were investigated experimentally or clinically for PCOS. www.impactjournals.com/oncotarget gi y p g In addition, 21 PCOS potential drug targets were verified by literatures to be associated with the pathogenesis and treatment of PCOS, which could reflect the status of disease and drug response. Drugs targeting these potential drug targets may be potential drugs for PCOS. And 42 drugs targeting to 13 PCOS potential drug targets were verified to be investigated experimentally or clinically for treating PCOS (Table 3). As a drug target, PPARG is widely studied and used for T2D, and is also researched to treat PCOS [55, 56]. There are two classes of drugs targeting to PPARG for T2D. One is insulin sensitization agent such as Pioglitazone, Rosiglitazone; the other is correcting the lipid metabolism disorders agent such as Repaglinide, Telmisartan. These drugs targeting to PPARG were highly possible to act as potential drugs for PCOS. Desogestrel, Ethinyl Estradiol, Clomifene and Tamoxifen are targeting to ESR1 (1st). Desogestrel as synthetic progestational hormone component and Ethinyl Estradiol as estrogenic component in oral contraceptives were used to improve clinical hyperandrogenism in PCOS in the first-line medicine [57, 58]. Clomifene is a triphenyl As shown in a series of recent publications [16, 65] in developing new findings and approaches, user-friendly and publicly accessible web-servers will significantly www.impactjournals.com/oncotarget Oncotarget 37914 Identification of PPDT-Modules These PCOS potential drug targets and PPDT-Module could reveal the drug response, distinguish the statuses between normal and disease, and act as PCOS biomarkers. Moreover, further research are expected for the ways of drug response. Our study would shed light on the treatment of PCOS, and provide new insights to research on the pahogenesis and drug response of other diseases that possess high pathobiological similarity. J (P, T) = \|P∩T\| \|P∪T\| (1) J (P, T) = \|P∩T\| \|P∪T\| (1) where P is a P-Module and T is a T-Module. The numerator is the number of common genes in P and T. The denominator is the number of all genes in P and T. (iii) Candidate PPDT-Modules were the pathobiological similar modules that included T2D drug targets, and PPDT-Modules were the candidate modules which possessed consistent function with PCOS. Data source The genes possessing more neighbors, more paths through them and so on, could play the central role in PPDT-Modules. Hence, the G-rank, Ggi, was used to evaluate the central importance of gene gi in these modules, which integrated 4 topological features, degree, betweenness, closeness and page rank, utilizing the geometric mean rank: The Protein-Protein Interaction Network was obtained from the Human Protein Reference Database (http://www.hprd.org/) [66], which contained 37041 protein-protein interaction pairs between 9518 proteins. Our research was based on two gene expression profiles from the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo/) [67] : GSE8157 including 33 samples (13 control samples and 10 PCOS samples before and after pioglitazone treatment, respectively) and GSE6798 including 29 samples (13 control samples and 16 PCOS samples) in GPL570. 35 PCOS disease genes and 83 T2D disease genes were extracted from GAD (http://geneticassociationdb.nih. gov/) [68], DO (http://www.disease-ontology.org/) [69] and OMIM (http://www.ncbi.nlm.nih.gov/omin) [70]; 62 T2D drug targets were extracted from Drug Bank (http:// www.drugbank.ca/) [70]. Ggi = Rankascending (∏j = 1 Rgij) 1 4 4 (2) where Rgij is the rank of gi for the feature j(degree, betweenness, closeness and page rank when j = 1, 2, 3, 4, respectively). where Rgij is the rank of gi for the feature j(degree, betweenness, closeness and page rank when j = 1, 2, 3, 4, respectively). The degree of gi is the number of edges linked to gi. i The closeness of gi, Cgi, is defined as the inverse of the average geodesic distance (or length of the shortest path) to all other genes: Cgi = ( 1 (n − 1) (n − 1) ∑gp ≠ gi dgp gi )−1 (3) www.impactjournals.com/oncotarget Oncotarget 37915 where dgpgi denotes the length of shortest path between gi and gp, n denotes the number of all genes in the module. time, we adopted the five-fold cross-validation as done by many investigators using SVM as the prediction engine [16, 65]. A receiver operating characteristic (ROC) curve was plotted and the area under the curve (AUC), the sensitivity, specificity, accuracy and MCC score were calculated. The five-fold cross-validation was applied 1000 times, and the 1000 ROC curves and AUC scores were obtained to evaluate the robustness of the PCOS potential drug targets. The higher the AUC score was, the better the classification performance was. ACKNOWLEDGMENTS and funding p q The page rank of gi, PRgi, should be highly ranked if other highly ranked genes contained edges to it: This study was supported by the National Natural Science Foundation of China (Grant No. 31301040 and 61272388); the Health Department Funds of Heilongjiang Province China (Grant No. 2012-810); the Science & Technology Research Project of the Heilongjiang Ministry of Education (Grant No. 12541476); and the Master Innovation Funds of Heilongjiang Province (Grant No. YJSCX2015-40HYD). PRgi = d ∑gp ≠ gi PR(gp) L(gp) + 1 − d n (5) (gp) where PR(gp) denotes the rank of gp , L(gp) denotes the number of edges of gp, d ∈ (0, 1) is a fixed parameter. (gp) where PR(gp) denotes the rank of gp , L(gp) denotes the number of edges of gp, d ∈ (0, 1) is a fixed parameter. REFERENCES 1. Allahbadia GN, Merchant R. Polycystic ovary syndrome and impact on health. MEFS. 2011; 16:19–37. 1. Allahbadia GN, Merchant R. Polycystic ovary syndrome and impact on health. 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https://openalex.org/W4387438706	https://zenodo.org/records/8419998/files/ZDIFT%202104.pdf	Russian	null	JO'XORI UNUMDORLIGINI MIKROELEMENTLARDAN FOYDALANISH ORQALI OSHIRISH USULLARI	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	856	` JO'XORI UNUMDORLIGINI MIKROELEMENTLARDAN FOYDALANISH ORQALI OSHIRISH USULLARI Serekeeva Gulzira Qoraqalpog'iston qishloq xo'jaligi va agrotexnologiyalar instituti Agroximiya mutaxassisligi 2-kurs magistranti İkram Nawrizbaev Qoraqalpog'iston qishloq xo'jaligi va agrotexnologiyalar instituti a.x.i.k. docenti Shurenbaeva Qolqanat Qoraqalpog'iston qishloq xo'jaligi va agrotexnologiyalar instituti Agroximiya va tuproqshunoslik mutaxassisligi 3-kurs studenti https://doi.org/10.5281/zenodo.8419998 ` JO'XORI UNUMDORLIGINI MIKROELEMENTLARDAN FOYDALANISH ORQALI OSHIRISH USULLARI Serekeeva Gulzira Qoraqalpog'iston qishloq xo'jaligi va agrotexnologiyalar instituti Agroximiya mutaxassisligi 2-kurs magistranti İkram Nawrizbaev Qoraqalpog'iston qishloq xo'jaligi va agrotexnologiyalar instituti a.x.i.k. docenti Shurenbaeva Qolqanat Qoraqalpog'iston qishloq xo'jaligi va agrotexnologiyalar instituti Agroximiya va tuproqshunoslik mutaxassisligi 3-kurs studenti https://doi.org/10.5281/zenodo.8419998 NUMDORLIGINI MIKROELEMENTLARDAN FOYDALANISH ORQALI OSHIRISH USULLARI Serekeeva Gulzira Annotatsiya. Bu maqolada, jo'xori unumdorligini oshirish uchun mikroelementlardan foydalanishning bir necha usullari haqida ma'lumot berilgan. Bu usullar orasida mikroelementli qo'shimchalarni qo'shish, yer osti suvini mikroelementlar bilan boyitish, mikroelementli o'g'itlarni qo'shish va mikroelementli qo'shimchalarni barglarga sirkulatsiyadan o'tkazish kabi usullar keltirilgan. Bu usullar unumdorlikni oshirish va o'simlikning yaxshi tarzda rivojlanishiga yordam beradi. Kalit so'zlar. Jo'xori unumdorligini mikroelementlardan foydalanish orqali oshirish usullari, mikroelementlar, qo'shimcha, yer osti suvi, o'g'itlar, barglar, sirkulatsiya, unumdorlik, rivojlanish Jo'xori unumdorligi, o'simlikning o'ziga xos va asosiy jismoniy va funksional holatini ifodalovchi bir xususiyatdir. Mikroelementlar esa o'simlikning normal rivojlanishi va o'sishini ta'minlaydigan zarur elementlar hisoblanadi [3, 14-17]. Jo'xori unumdorligi, o'simlikning o'ziga xos va asosiy jismoniy va funksional holatini ifodalovchi bir xususiyatdir. Mikroelementlar esa o'simlikning normal rivojlanishi va o'sishini ta'minlaydigan zarur elementlar hisoblanadi [3, 14-17]. Jo'xori unumdorligini oshirish uchun mikroelementlardan foydalanishning ahamiyati juda katta. Mikroelementlar o'simlikning normal funktsiyasini ta'minlayadi va o'sishini rag'batlantiradi. Bu elementlar o'simlikning fotosintez va respiratsiya jarayonlarida qatnashadi, enzimlar va gormonlar yaratishda muhim rol o'ynayadi. Mikroelementlar esa tuproqda qisqa miqdorda bo'lsa ham, ulardan yetarli miqdorda foydalanish o'simlikning yaxshi rivojlanishi uchun zarurdir. Bu elementlar orqali o'simliklar o'zining genetik potensialini amalga oshirishi mumkin bo'ladi. Misol uchun, azot o'simlikning yashil qismi va o'sishini rag'batlantiradi, fosfor energiya olish va o'sishni ta'minlaydi, kaltsiy o'simlikning qurilishi va rivojlanishiga ta'sir qiladi [4, 20-22]. Bundan tashqari, mikroelementlar o'simliklarning kasalliklarga qarshi immunitetini oshirishda ham muhim rol o'ynaydi. Ulardan yetarli miqdorda foydalanish o'simlikning zararli mikroorganizmlar va xavfli kimyoviy moddalardan himoya qilishga yordam beradi. Shuningdek, mikroelementlardan foydalanish o'simlikning mahsulot sifatini oshirishda ham juda muhimdir. Ulardan yetarli miqdorda foydalanish o'simlikning mevaviy, sabzavotli va o'simlik mahsulotlarini yaxshi sifatda olishga imkon beradi. Shuningdek, mikroelementlardan foydalanish o'simlikning mahsulot sifatini oshirishda ham juda muhimdir. Ulardan yetarli miqdorda foydalanish o'simlikning mevaviy, sabzavotli va o'simlik mahsulotlarini yaxshi sifatda olishga imkon beradi. Mikroelementlardan foydalanish orqali jo'xori unumdorligini oshirishning bir necha usullari mavjud: 1. Yer osti suvini to'g'ri tashkil etish: o'simliklar uchun suv juda muhimdir. Yer osti suvi to'g'ri tuzilishi va unumdorlik darajasi, o'simlikning suvni qabul qilishini va uni o'sishini ta'minlaydi. 15 ` Mikroelementlar, masalan, azot (N), fosfor (P) va kaltsiy (Ca), yer osti suvining to'g'ri tuzilishini ta'minlaydi [1]. 2. Mikroelementli o'g'itlar: o'simliklar uchun zarur mikroelementlarni yetarli miqdorda olib keluvchi o'g'itlar foydalidir. Bu o'g'itlar o'simlikning unumdorligini oshirishda katta ahamiyatga ega bo'ladi. Masalan, temir (Fe), magniy (Mn) va bor (B) kabi mikroelementlar o'simlikning fotosintez jarayonida muhim rol o'ynaydi va unumdorligini oshirishda zarurdir. o simlikning fotosintez jarayonida muhim rol o ynaydi va unumdorligini oshirishda zarurdir. 3. Yer osti unumdorligini oshirish: Yer osti unumdorligini oshirish uchun mikroelementlar qo'shilishi mumkin. Bu usulda o'zgartirilgan yer ostiga zarur mikroelementlar qo'shiladi. Misol uchun, o'simlikning unumdorligini oshirish uchun yod (I) kabi mikroelementlar qo'shilishi mumkin. 4. Sirkulatsiya: o'simliklar uchun zarur mikroelementlarning to'g'ri sirkulatsiyasi juda muhimdir. Bu mikroelementlar o'simlikning o'sish va rivojlanish jarayonlarida foydalaniladi. O'simlikning sirkulyatsiyasini ta'minlash uchun, suv, o'g'itlar va havo to'g'ri tashkil etilishi kerak [3]. 4. Sirkulatsiya: o'simliklar uchun zarur mikroelementlarning to'g'ri sirkulatsiyasi juda muhimdir. Bu mikroelementlar o'simlikning o'sish va rivojlanish jarayonlarida foydalaniladi. O'simlikning sirkulyatsiyasini ta'minlash uchun, suv, o'g'itlar va havo to'g'ri tashkil etilishi kerak [3]. Mikroelementlardan foydalanish orqali jo'xori unumdorligini oshirish usullari, o'simlikning o'sish va rivojlanish jarayonlarini muvaffaqiyatli amalga oshirishi uchun muhimdir. Bu usullar o'simlikning unumdorligini oshirishda katta ahamiyatga ega bo'ladi va o'simlikning yaxshi yig'ilishini va rivojlanishini ta'minlaydi. Xulosa. Jo'xori unumdorligini oshirish uchun mikroelementlardan foydalanishning bir necha usullari mavjud. Yer osti suvini to'g'ri tashkil etish, mikroelementli o'g'itlar, yer osti unumdorligini oshirish va sirkulatsiya bu usullardan ba'zilaridir. Bu usullar o'simlikning normal rivojlanishi va o'sishini ta'minlaydi va o'simlikning yaxshi yig'ilishini va rivojlanishini muvaffaqiyatli amalga oshiradi. References: 1. Золотарёв, С. В., Кобозев, И. В., Кобозева, Т. П., Делаев, У. А., Зузиев, У. Г., & Неустроев, И. И. (2012). Оценка качества семян разных сортов сои северного экотипа с целью их рационального использования. Вестник Алтайского государственного аграрного университета, 87 (1), 8-14. 2. Павлова, А. В. (2011). Влияние макрои микроудобрений на содержание переваримого протеина в горохо-овсяной смеси и её компонентах. Вестник Алтайского государственного аграрного университета, 80 (6), 37-39. 2. Павлова, А. В. (2011). Влияние макрои микроудобрений на содержание переваримого протеина в горохо-овсяной смеси и её компонентах. Вестник Алтайского государственного аграрного университета, 80 (6), 37-39. 3. Спицына, С. Ф., & Павлова, А. В. (2012). Вынос микроэлементов компонентами горохо-овсяной смеси. Вестник Алтайского государственного аграрного университета, 87 (1), 14-17. 3. Спицына, С. Ф., & Павлова, А. В. (2012). Вынос микроэлементов компонентами горохо-овсяной смеси. Вестник Алтайского государственного аграрного университета, 87 (1), 14-17. 4. Спицына, С. Ф., & Павлова, А. В. (2010). Влияние совместного применения макрои микроудобрений на урожайность горохо-овсяной смеси. Вестник Алтайского государственного аграрного университета, 66 (4), 20-22. 4. Спицына, С. Ф., & Павлова, А. В. (2010). Влияние совместного применения макрои микроудобрений на урожайность горохо-овсяной смеси. Вестник Алтайского государственного аграрного университета, 66 (4), 20-22. 16
https://openalex.org/W1915497867	https://quod.lib.umich.edu/cgi/p/pod/dod-idx/ontological-catastrophe-zizek-and-the-paradoxical.pdf?c=ohp;idno=12763629.0001.001;format=pdf	English	null	Ontological Catastrophe: Žižek and the Paradoxical Metaphysics of German Idealism	null	2,014	cc-by	138,121	Ontological Catastrophe: Žižek and the Paradoxical Metaphysics of German Idealism Joseph Carew Ontological Catastrophe: Žižek and the Paradoxical Metaphysics of German Idealism Joseph Carew Ontological Catastrophe: Žižek and the Paradoxical Metaphysics of German Idealism Joseph Carew Ontological Catastrophe: Žižek and the Paradoxical Metaphysics of German Idealism Joseph Carew Ontological Catastrophe New Metaphysics Series Editors: Graham Harman and Bruno Latour The world is due for a resurgence of original speculative metaphysics. The New Metaphys- ics series aims to provide a safe house for such thinking amidst the demoralizing caution and prudence of professional academic philosophy. We do not aim to bridge the analytic- continental divide, since we are equally impatient with nail-filing analytic critique and the continental reverence for dusty textual monuments. We favor instead the spirit of the intel- lectual gambler, and wish to discover and promote authors who meet this description. Like an emergent recording company, what we seek are traces of a new metaphysical ‘sound’ from any nation of the world. The editors are open to translations of neglected metaphysical classics, and will consider secondary works of especial force and daring. But our main inter- est is to stimulate the birth of disturbing masterpieces of twenty-first century philosophy. Joseph Carew Ontological Catastrophe Joseph Carew Ontological Catastrophe www.openhumanitiespress.org OPEN HUMANITIES PRESS First edition published by Open Humanities Press 2014 Freely available online at http://dx.doi.org/10.3998/ohp.12763629.0001.001 Copyright © 2014 Joseph Carew This is an open access book, licensed under a Creative Commons By Attribution Share Alike license. Under this license, authors allow anyone to download, reuse, reprint, modify, distribute, and/or copy this book so long as the authors and source are cited and resulting derivative works are licensed under the same or similar license. No permission is required from the authors or the publisher. Statutory fair use and other rights are in no way affected by the above. Read more about the license at creativecommons.org/licenses/by-sa/3.0 Copyright © 2014 Joseph Carew Design by Katherine Gillieson Cover Illustration by Tammy Lu The cover illustration is copyright Tammy Lu 2014, used under a Creative Commons By Attribution license (CC-BY). ISBN-13 978-1-60785-308-4 Open Humanities Press is an international, scholar-led open access publishing collective whose mission is to make leading works of contemporary critical thought freely available worldwide. Books published under the Open Humanities Press imprint at MPublishing are produced through a unique partnership between OHP’s editorial board and the University of Michigan Library, which provides a library-based managing and production support infrastructure to facilitate scholars to publish leading research in book form. www.publishing.umich.edu www.openhumanitiespress.org OPEN HUMANITIES PRESS www.openhumanitiespress.org OPEN HUMANITIES PRESS Contents Acknowledgements 9 Acknowledgements 9 Introduction 11 To the Memory of Joey Basha Introduction 11 1. The Madness of the Symbolic 19 2. Grasping the Vanishing Mediator Between the Real and the Ideal 37 3. Psychoanalysis and the Enigma of Transcendental Subjectivity 53 4. The Problem of Nature in the Lacanian Subject 67 5. Kant, Todestrieb, and Beyond the Pleasure Principle 85 6. From Transcendental Philosophy to Substance as Subject 108 7. The Logic of Transcendental Materialism 140 8. When the World Opens its Eyes 171 9. The Abyss of Unconscious Decision 193 10. Radicalizing the Subject 223 11. From Radical Idealism to Critical Metaphysics 259 12. The Deadlocks of Ontological Catastrophe 283 1. The Madness of the Symbolic 19 2. Grasping the Vanishing Mediator Between the Real and the Ideal 37 4. The Problem of Nature in the Lacanian Subject 67 12. The Deadlocks of Ontological Catastrophe 283 Bibliography 317 To the Memory of Joey Basha To the Memory of Joey Basha Acknowledgements Firstly, I would like to express my gratitude to two people without whom this project would not have been possible: Dr. Antoinette Stafford and Dr. Sean McGrath. Dr. Stafford is, in many ways, the reason why I am doing philosophy, and it is to her that I owe my knowledge of the history of modern philosophy and German Idealism, without which this book would never have gotten off the ground. Dr. McGrath sparked my interest in psychoanalysis and has been a constant source of inspiration for my reading of Schelling. I must thank him for stressing the notion of the ambiguity of the Real in Lacan and Žižek, which immensely shaped my own engagement with these authors, and for all the encouragement he has given me since. Secondly, I should thank four people who read the manuscript at various stages of its production. Dr. Peter Trnka and the late Dr. James Bradley read a very early version of the manuscript several years ago, and I am much indebted to their extremely kind and useful commentary. While Dr. Bradley gave me insight into further aspects of Žižek's philosophy—some of which has shown up in my other work on Žižek—and encouraged me to continue the project, Dr. Trnka's much appreciated criticism helped me avoid conceptual imprecision at several key points, as well as general difficulties in the presentation of psychoanalytical methodology. Specifically, Dr. Trnka's emphasis on the conflictual relation between materialism and idealism in many ways moulded my own views on the difficult Symbolic-Real relation in Acknowledgements 10 Žižek's transcendental materialism into the form they take here. Dr. Graham Harman's comments and suggestions were of great help in stylistically improving the manuscript's quality, in increasing its overall coherence, and also in avoiding several pitfalls of argumentation. I should also thank him for his always quick responses to various questions I had while preparing the manuscript. Last but certainly not least, I thank Kyla Bruff for her laborious proofreading of the final two drafts. Her careful eye was able to eliminate innumerable ambiguities and awkward turns of phrase, thus making the book much smoother. Thirdly, I would like to express my gratitude to the Social Sciences Research Council of Canada and Europhilosophie for their generous funding, which both directly and indirectly supported the project. Acknowledgements I would also like to thank the Memorial University of Newfoundland's Department of Philosophy and School of Graduate Studies for additional financing in the form of fellowships and awards, which enabled me to begin the first draft. An early version of three strands of argumentation that appear in what follows has been published as “The Grundlogik of German Idealism: The Ambiguity of the Hegel-Schelling Relation in Slavoj Žižek,” International Journal of Žižek Studies, 5, no.1 (2011); “'Why is There Nothing Rather Than Something?' Less Than Nothing's New Metaphysics,” International Journal of Žižek Studies, 8. no.1 (2014); and “Denaturalizing Nature, Dehumanizing Humanity: Lacan, Žižek, and the Metaphysics of Psychoanalysis,” in Natürlichkeit und Künstlichkeit zwischen Tatsache und Ideal, ed. Benedetta Bisol (Bielefeld: transcript, 2014). Introduction A Metaphysical Archaeology of the Psychoanalytico-Cartesian Subject This book is an investigation into Slavoj Žižek's return to German Idealism in the wake of Lacanian psychoanalysis. Its thematic crux is Žižek's attempt to develop, by reading the traditions against one another by means of their mutually compatible notions of Todestrieb, a highly original theory of subjectivity able to explain the subject's simultaneous freedom from and dependence upon its material ground. But it does not stop there: rather than just limiting itself to a recapitulation of Žižek's account of the eruptive, ontologically devastating birth of subjectivity out of nature, it also seeks to systematize the stark metaphysical consequences of this account. The fundamental thesis of this book is that, if the emergence of the Symbolic out of the Real—the passage from nature to culture enacted by the founding gesture of subjectivity—is the advent of a completely self-enclosed, self- sustaining structural system, then not only must its founding gesture withdraw from the scene in the very act of instituting the Symbolic, but further, even to explain this act we must posit the absolute as a fragile not-all wrought by negativity and antagonism. Or, to put it in terms of Žižek's Less Than Nothing (his latest magnum opus, or “big fat Hegel book,” as he says), as a series of less than nothings whose essence constitutes an ontologically incomplete field. By means of a metaphysical archaeology of the psychoanalytico- Cartesian subject, an archaeology that is the necessary supplement to Introduction 12 Freud's own archaeological investigations of the emergence of mind out of the conflict of unconscious drives and their vicissitudes,1 especially in the aftermath of Lacan's structuralist reworkings of it, what we will see is that Žižek's own ontology of the subject goes far beyond the normal constraints of psychoanalytical methodology (which is so concerned with psychogenesis and its various pathologies) and radically challenges our normal conception of self and world, a challenge summarized by the notion of ontological catastrophe, which I extract from it as its key operative moment. In the course of the book this concept takes on a number of different meanings. In a first moment, it refers to Žižek's interpretation of Todestrieb as that which incites the passage from nature to culture, a grotesque excess of life that is unable to control itself according to its own prescribed natural logistics and thus opens up room for the possibility of experience. Introduction Wondering how this void of nothingness could be broken so that creation itself could emerge, Žižek argues that the basic form of ontological catastrophe should be extended A Metaphysical Archaeology of the Psychoanalytico-Cartesian Subject 1 13 from that of the subject as the breakdown of nature in Todestrieb, or the incompletion of nature testified to by the latter's constitutive tension, to the world itself as the necessary disturbance of this void, whereby the classical terrain of metaphysics itself is inverted: “[f]or a true dialectician, the ultimate mystery is not 'Why is there something rather than nothing?' but 'Why is there nothing rather than something?': how is it that, the more we analyze reality, the more we find a void?”2 In broad strokes, this is the terrain we will investigate—a terrain that is not merely difficult because it is nuanced and challenging because it is new, but also primordially uncanny and traumatic, forcing us to encounter aspects of self and world that we not only normally do not acknowledge, or continually disavow, but that we even try to repress. To arrive at and evaluate this notion of ontological catastrophe, my metaphysical archaeology of the Žižekian psychoanalytico-Cartesian subject takes three paths: one that traverses the wider historical context informing Žižek's project, another that internally reconstructs its reactualization of German Idealism through psychoanalysis, and a final one that attempts to extract and problematize the intrinsic originality and daringness of Žižek's metaphysics. The first path consists of chapters 1 through 4. Chapter 1 outlines the ambiguity of the Real in Lacanian psychoanalysis. Displaying a form of radical idealism of a linguistic structuralist variety, it proclaims that the Symbolic operates as a self-enclosed system with no need of any external support. This not only means that human freedom is equivalent to an ontological madness, but also appears to foreclose the very possibility of explaining this passage into madness at its basis. Chapter 2 shows how, although Žižek believes himself able to find resources to overcome this difficulty in German Idealism, he can only do so by psychoanalytically tracing and reconstructing an unconscious history of struggle with the obscure origins of subjectivity he perceives throughout the tradition. Introduction In a second moment, it names the self-positing of subjectivity in nature tearing the latter apart into irreconcilable zones, which, although in a certain sense conditioned by a libidinal-material breakdown of the biological system, is ultimately irreducible to the latter as a pure act. Taken together, these two moments underlying the emergence of subjectivity demand that we delve into the naturephilosophy that this account implies, a nature that shows itself (due to the very extimate presence of Todestrieb and pure difference within its heart of hearts) to be predicated upon painful tension and self-sabotaging tendencies to such a degree that its very being is co- incidental not only with the existence of death, disease, and monstrosities, but also with the always possible unpredictable upsurge of disorder and complete collapse as it risks touching the void. In a third moment, the metaphysical archaeology of the subject is pushed to its ultimate limits. Delving into the question of how being could sustain itself despite its rampant and devastating negativity, what we will see is how the more we move towards the most fundamental level of the universe, the latter proves to be in its depths of depths not a dense, fully subsisting reality that exists by itself by means of a self-explanatory surplus, but a series of indeterminate proto-ontological states only minimally distinguishable from the void of nothingness that serves as its contrast. Introduction Given the psychoanalytical horror that is the basis of subjectivity, chapter 9 explains how Schelling, although the thinker of its abyssal origins, ultimately ends up recoiling just like Hegel from own great insights after coming face to face with its full trauma, which gives further support to the necessity of a psychoanalytical reconstruction of German Idealism. After this concrete exploration of Žižek's methodology, chapter 10 concludes the second path by bringing to the fore his three most significant theoretical contributions: a rich ontology of nature, a new metaphysics of the void developed through quantum mechanics, and a nuanced theory of The second path, which unfolds through chapters 5 to 10, comprises the substantiation of Žižek's claim that there is an identity between the founding insights of German Idealism and psychoanalysis by retroactively rewriting the former's unconscious history. Drawing upon the late Lacan's ruminations concerning the breakdown of nature as the pre-condition of the Symbolic, chapter 5 outlines how Kant finds also himself forced to posit a meta-transcendental ground of the transcendental in organic disorder, even going so far as to anticipate Lacan's mirror stage. Chapter 6 demonstrates how the early Hegel, led by insufficiencies in Kant's and Fichte's transcendentalism and Schelling's Naturphilosophie, attempts to reconcile idealist freedom and realist system by inscribing the subject into the fold of being as an eruptive, world-shattering event, thus radicalizing Kant's insight into the devastating origins of subjectivity. Chapter 7 then illustrates how the mature Hegel psychoanalytically recoils from the ontological catastrophe at the heart of the subject's essence by subsuming it under the self-mediation of the Notion. It is only with the middle-late Schelling, fighting against the perceived threats of Absolute Idealism, that the true kernel of truth unearthed by Kantian idealism is brought to the fore and along with it its stark, even horrifying implications for our understanding of nature, human historicity, and the absolute. Chapter 8 gives flesh to the Schellingian-Žižekian subject as the vanishing mediator between the Real and the Ideal. The symbolic universe of meaning is not a high point of evolutionary achievement, but rather a mistake, the outcome of something having gone horribly wrong in the order of things and to which it is only a defense mechanism. Introduction Insofar as the psychotic non-relation between the Real and the Symbolic is also a rethinking of the cogito, chapter 3 shows why Žižek feels the theoretical obligation to revitalize subjectivity in an intellectual milieu that attacks it from all sides. Chapter 4 tries to understand how the Real could have given rise to the Symbolic. Contra the early and middle Lacan, Žižek argues that the Symbolic cannot be an external parasite that attacks the Real from Introduction 14 nowhere, but must arise from some sort of self-sabotaging tendency always already implicit within it. already implicit within it. The second path, which unfolds through chapters 5 to 10, comprises the substantiation of Žižek's claim that there is an identity between the founding insights of German Idealism and psychoanalysis by retroactively rewriting the former's unconscious history. Drawing upon the late Lacan's ruminations concerning the breakdown of nature as the pre-condition of the Symbolic, chapter 5 outlines how Kant finds also himself forced to posit a meta-transcendental ground of the transcendental in organic disorder, even going so far as to anticipate Lacan's mirror stage. Chapter 6 demonstrates how the early Hegel, led by insufficiencies in Kant's and Fichte's transcendentalism and Schelling's Naturphilosophie, attempts to reconcile idealist freedom and realist system by inscribing the subject into the fold of being as an eruptive, world-shattering event, thus radicalizing Kant's insight into the devastating origins of subjectivity. Chapter 7 then illustrates how the mature Hegel psychoanalytically recoils from the ontological catastrophe at the heart of the subject's essence by subsuming it under the self-mediation of the Notion. It is only with the middle-late Schelling, fighting against the perceived threats of Absolute Idealism, that the true kernel of truth unearthed by Kantian idealism is brought to the fore and along with it its stark, even horrifying implications for our understanding of nature, human historicity, and the absolute. Chapter 8 gives flesh to the Schellingian-Žižekian subject as the vanishing mediator between the Real and the Ideal. The symbolic universe of meaning is not a high point of evolutionary achievement, but rather a mistake, the outcome of something having gone horribly wrong in the order of things and to which it is only a defense mechanism. 1. This, of course, being a constant metaphor throughout Freud’s corpus, span- ning from “The Aetiology of Hysteria” (1896) to “Construction in Analysis” (1937). See, respectively,The Standard Edition of the Complete Psychological Works of Sigmund Freud, translated from the German under the General Editorship of James Strachey, in collaboration with Anna Freud, assisted by Alix Strachey and Alan Tyson (London: Hogarth Press and the Institute of Psycho-analysis, 1953–1974) (hereafter SE), III, p. 192, and XXIII, p. 259. A Metaphysical Archaeology of the Psychoanalytico-Cartesian Subject 15 A Metaphysical Archaeology of the Psychoanalytico-Cartesian Subject 15 unconscious, each of which goes beyond a mere reactualization of German Idealism or psychoanalysis. The third and final path is summarized by the word “paradoxical” in the subtitle of the book—Žižek and the Paradoxical Metaphysics of German Idealism. Chapter 11 highlights that, instead of being opposed to metaphysics, radical idealism not only demands a metaphysics, for thinking in all of its intrinsic paradox and self-referentiality must be seen as existing in the world, but more primordially forces upon us a new domain of metaphysics, which first became explicit in German Idealism. Whereas all metaphysics prior to Kant is dogmatic insofar as it assumes thought's power to reach out and touch the truth of being in virtue of a special capacity (a gesture that is repeated by, amongst others, Badiou's and Meillassoux's elevation of mathematical formalization), what occurs in Schelling and Hegel is an intense reflection upon how the very process by which thought forms an image of being is inscribed into being as an event, whereby even the very philosophical position of theory formation is reflexively thematized both epistemologically and ontologically. What emerges is a metaphysics that can be baptized as critical because it is capable of developing a theory of reality that is maximally realist and idealist and therefore best suited to explicate the metaphysical whole of what is without falling into the downfalls of a theory that is one-sidedly one or the other.3 Chapter 12 explores the paradoxical nature of this endeavour as it articulates itself in the intrinsically original and daring character of Žižek's own variation upon this German Idealist leitmotif and the problems it potentially poses not only for his own philosophy, but perhaps for any radical idealism seeking to break the correlationist circle. Introduction Given the psychoanalytical horror that is the basis of subjectivity, chapter 9 explains how Schelling, although the thinker of its abyssal origins, ultimately ends up recoiling just like Hegel from own great insights after coming face to face with its full trauma, which gives further support to the necessity of a psychoanalytical reconstruction of German Idealism. After this concrete exploration of Žižek's methodology, chapter 10 concludes the second path by bringing to the fore his three most significant theoretical contributions: a rich ontology of nature, a new metaphysics of the void developed through quantum mechanics, and a nuanced theory of 3. Gabriel draws a distinction between critical and dogmatic metaphysics for similar, but different reasons. See Das Absolute und die Welt in Schellings Freiheitsschrift (Bonn: University Press, 2006), p. 8. I take up this distinction at length in chapter 11. 2. Žižek, Less Than Nothing: Hegel and the Shadow of Dialectical Materialism (London: Verso, 2012), p. 925. Chapter 1 The Madness of the Symbolic Transcendental Materialism and the Ambiguity of the Real Chapter 1 The Madness of the Symbolic Transcendental Materialism and the Ambiguity of the Real Re-interpreting Freud through structural linguistics, Lacan radically rethinks the unconscious: no longer a quasi-biological phenomenon centered in drives, it largely becomes associated with the differential system of the Symbolic responsible for the production of meaning. However, since the latter proves to be operationally closed and has no relationship to the world in itself, Lacan himself is forced to proclaim that the founding gesture of subjectivity is a passage through madness. This poses two difficulties that set up the entirety of Žižek's project. First, it points towards a transcendental materialism at the basis of the subject, a self-splitting of being into irreconcilable material and transcendental zones, but one that Lacan fails to systematize. Second, insofar as the Symbolic itself is self-enclosed, it seems methodologically impossible even to explain its own obscure origins, even if such is ultimately required if psychoanalysis is to find an adequate theoretical justification. In this regard, Žižek's primary task is to find a way to explain the madness of the Symbolic without overstepping the constraints of psychoanalysis. Notes 16 Introduction I Death Drive Death Drive The Madness of the Symbolic Žižek's metaphysics originates in his attempt to delve into the material origins preceding the psychoanalytical subject by focusing on this moment If madness is “freedom's most faithful companion” it is precisely because madness in its most primordial sense refers to the specific ontogenetic conditions for the irreducibility of language that makes us distinctly human: that is to say, to the state of affairs by means of which language can solipsistically relate to itself as a self-enclosed differential system of signification “with no an external support.”7 Just as in clinical cases of psychosis or madness, here too the subject has “lost touch” with reality, although reality must be understood in its natural (animalistic) rather than its sociopolitical (human) meaning. It is “the price the Lacanian subject pays for its 'transubstantiation' from being the agent of a direct animal vitality to being a speaking subject whose identity is kept apart from the direct vitality of passions,”8 that which guarantees that the subject is dominated by “non-natural” influences or which is, strictly speaking, at its zero- level abiological (wherein lies its freedom). This has two principal effects. First, because Lacan's self-given task is to formulate the autonomous structures that constitute human subjectivity in opposition to naturalist theories of psychiatry, his philosophy has the formal appearance of a retour to the modern transcendentalism of the cogito. The Lacanian subject is consequently haunted by similar problems as those of the Cartesian subject, both in terms of epistemology (since the relationship to the extra-conscious alterity of the world is problematized, how can we justify the propositions of science?) and the mind-body relation (what exactly is the relationship between symbolic thinking and natural processes?). Second, due to the internal constraints of his project, Lacan left unanswered how reality in itself could incite the generation of these quasi-transcendental structures that make up the universe of human meaning in its psychotic self-enclosure, with the concomitant problem of how we relate to this X that simultaneously precedes our emergence into language and forms its obscure ontologico- foundational basis. Seeing a structural identity with the immediate reactions to Kant, Žižek sees the possibility of confronting the fundamental concepts of psychoanalysis with those of German Idealism. The Madness of the Symbolic If madness is “freedom's most faithful companion” it is precisely because madness in its most primordial sense refers to the specific ontogenetic conditions for the irreducibility of language that makes us distinctly human: that is to say, to the state of affairs by means of which language can solipsistically relate to itself as a self-enclosed differential system of signification “with no an external support.”7 Just as in clinical cases of psychosis or madness, here too the subject has “lost touch” with reality, although reality must be understood in its natural (animalistic) rather than its sociopolitical (human) meaning. It is “the price the Lacanian subject pays for its 'transubstantiation' from being the agent of a direct animal vitality to being a speaking subject whose identity is kept apart from the direct vitality of passions,”8 that which guarantees that the subject is dominated by “non-natural” influences or which is, strictly speaking, at its zero- level abiological (wherein lies its freedom). This has two principal effects. First, because Lacan's self-given task is to formulate the autonomous structures that constitute human subjectivity in opposition to naturalist theories of psychiatry, his philosophy has the formal appearance of a retour to the modern transcendentalism of the cogito. The Lacanian subject is consequently haunted by similar problems as those of the Cartesian subject, both in terms of epistemology (since the relationship to the extra-conscious alterity of the world is problematized, how can we justify the propositions of science?) and the mind-body relation (what exactly is the relationship between symbolic thinking and natural processes?). Second, due to the internal constraints of his project, Lacan left unanswered how reality in itself could incite the generation of these quasi-transcendental structures that make up the universe of human meaning in its psychotic self-enclosure, with the concomitant problem of how we relate to this X that simultaneously precedes our emergence into language and forms its obscure ontologico- foundational basis. Seeing a structural identity with the immediate reactions to Kant, Žižek sees the possibility of confronting the fundamental concepts of psychoanalysis with those of German Idealism. 1.1 A (Transcendental) Materialism of the Psychoanalytical Subject Žižek's return to German Idealism is an investigation of the obscure origins of the Lacanian subject. Žižek is attracted not only to Lacanian Chapter 1 20 psychoanalysis' thematization of the non-coincident “gap” in the Symbolic and its consequences for politics, but also to the conflictual relationship between mind and body that it places at the foundation of psychogenesis. If symbolic structures of language display a radical autonomy from bodily forces and can construct their own world, the essence of human being must be constituted originally by a kind of biological “short circuit” that disrupts man's complete immersion in nature, eternally separating the Innenwelt and Aussenwelt (inner world and outer world), thereby making it so they can never coincide: that is, by a mal-adaptation that “represents the minimum of freedom, of a behaviour uncoupled from the utilitarian-survivalist attitude” insofar as “the organism is no longer fully determined by its environs, that it 'explodes/implodes' into a cycle of autonomous behaviour.”4 If, as conventionally defined in Freudian psychoanalysis, psychosis or madness is taken to be a withdrawal from the objective world into an inner, self- enclosed space (a loss of reality),5 then in Lacanian psychoanalysis psychosis or madness is paradoxically not a mere accidental state seen in certain “sick” individuals, but is the irreducible ontological background of all human existence. More disconcertingly, this is understood by Lacan not only to be the condition of possibility of human experience as such, but also that of freedom, so that the philosophical significance of the two is ultimately identified as dialectically interrelated aspects of the same phenomenon: Thus rather than resulting from a contingent fact—the frailties of his organism—madness is the permanent virtuality of a gap in his essence. And far from being an “insult” to freedom, madness is freedom's most faithful companion, following its every move like a shadow. Not only can man's being not be understood without madness, but it would not be man's being if it did not bear madness within itself as the limit of his freedom.6 For Lacan, the primary question in psychoanalysis is not how various forms of madness arise as a deviation from normal mental health, but how this more originary, irruptive state of nature as that within which freedom and madness magically emerge in a single brushstroke can be regulated so that what we regard as sanity and normality can themselves take hold. 1.1 A (Transcendental) Materialism of the Psychoanalytical Subject The Madness of the Symbolic 2 The Madness of the Symbolic Žižek's metaphysics originates in his attempt to delve into the material origins preceding the psychoanalytical subject by focusing on this moment Žižek's metaphysics originates in his attempt to delve into the material origins preceding the psychoanalytical subject by focusing on this moment of immanent rupture in being—which he links to the Todestrieb—as that which, by opening up a space separating us from nature in the latter, Chapter 1 Chapter 1 22 appears simultaneously to be linked to our freedom, that is, to our madness. Refusing to buy into the claim that all is ultimately reducible to the ebb and flow of matter, he sees his own endeavour as “the necessary step in the rehabilitation of the philosophy of dialectical materialism.”9 Yet this designation is inherently problematic, given what we have just seen: not only does it try to make Žižek's own form of materialism approach that of Marx and Engels without drawing the necessary distinctions between them, it more importantly fails to articulate the essentially paradoxical and innovative manner in which Žižek rearticulates the materialism-idealism debate and therefore risks obscuring his own originality. Consequently, I endorse Adrian Johnston's characterization of Žižek's theory of the subject as a form of transcendental materialism10 for four reasons, but differ in my own understanding in one important way that in turn distinguishes my own project from his. First, it has the benefit of allowing the reader to have a direct intuition of what is truly at stake in Žižek's parallax ontology and its metaphysical implications. Whereas dialectical materialism traditionally views the mind- body relationship as grounded within the dynamic interpenetration of the two as a complex self-unfolding identity within difference, transcendental materialism, by focusing on the ontogenetic conditions of the possibility of the transcendental subject, conventionally understood as in opposition to natural conditions, already suggests the immanent emergence of an irruptive negativity within being, an irreducible transcendence that paradoxically shatters the former's pure immanence from within. Second, a point not mentioned by Johnston, Fichte uses the expression in his 1794 Some Lectures Concerning the Scholar's Vocation to draw attention to the impossibility of explaining the (onto)genesis of the subject: “[i]t is certainly not true that the pure I is a product of the not-I [...]. The Madness of the Symbolic 23 autonomy. Writing in a similar vein, Žižek attempts to show not only how we can, but more primordially why we must develop a metaphysical account of reality that, instead of jeopardizing the (practical) primacy of idealism, would actually found it by inscribing this very dualism of I and not-I into the fold of being as that which makes the absolute divided within itself and whose non-coincidence to self thereby opens up the birthplace for human freedom. Transcendental idealism is—or better, must be said to always already spectrally refer to—transcendental materialism, the difference between them being only that of a parallax shift: the two are negatively linked to one another by an impossible in-between, a disjunctive “and,” the very name of which is for Žižek the subject, so that an idealism must convert itself into a materialism and vice versa if subjectivity is to be fully explained. It is precisely at this conceptual conjuncture of the role of a disjunctive “and” that my own understanding of Žižek's transcendental materialism distinguishes itself from that of Johnston. Although Johnston is right to claim that Žižek's own descriptions of the birth of subjectivity have a propensity to focus on its process as a self-instituting fiat “analogous to the cutting of the Gordian knot” (which in a certain sense obfuscates his project insofar as one of its major questions is how the closed circuit of drives in the Real could result in the transcendence of subjectivity),12 Johnston in my view has a tendency to downplay the intrinsically paradoxical nature of all such inquiry into the obscure origins of the psychoanalytico-Cartesian subject in Žižek's work in two ways. On the one hand, he emphasizes that the subject is rendered possible by a short-circuiting of its libidinal-material ground. But if an emergent breakdown in nature's inner being does give rise to the ontogenetic possibility of the subject, it in no way gives birth to the latter: nature's auto-laceration may be necessary for the self-positing freedom of transcendental subjectivity, but it is not sufficient, for there is no possible transition from nature to subjectivity, a point that—though also raised by Johnston—I believe must be radicalized. The Madness of the Symbolic The assertion that the pure I is a product of the not-I expresses a transcendental materialism which is completely contrary to reason.”11 Fichte's argument is simple: one cannot explain the material conditions of transcendental freedom insofar as that would equate two logically distinct fields irreconcilable with one another—namely, that of unbridled self- determination (the realm of acting) and that of dead contingency (the realm of being), thus causing us to lose sight of the radicality of human The Madness of the Symbolic The Madness of the Symbolic This is why on my reading the reference to Fichte is so important, for if Žižek's metaphysics is an attempt to show how a transcendental materialism can be developed, it nevertheless refuses to give up on the fundamental claim made by Fichte that the upsurge of the pure I in being is executed “by absolute freedom, not through a transition, but by means of a leap.”13 As a result, we can also understand Chapter 1 24 why Žižek displays hesitation concerning Johnston's and Malabou's shared project of merging philosophy and neuroscience—because there is ultimately no emergence of subjectivity possible within his parallax ontology.14 On the other hand, because Johnston attaches less importance than I do to the rupturing free leap into subjectivity, he is simultaneously silent concerning the necessarily mytho-poetic element of Žižek's transcendental materialism, which for me thus becomes central for understanding the nuance of his specific overcoming of radical idealism. This has two important consequences, one methodological and the other metaphysical. Methodologically, if the leap into subjectivity is an ontological passage through madness,15 then there is an upper limit to the power of thought to explain its own emergence in being. What we need is a capacity for fabulative mythologizing, for “the need for the form of mythical narrative arises when one endeavours to break the circle of the symbolic order and to give an account of its genesis ('origins') from the Real and its pre-symbolic antagonism.”16 Metaphysically, if subjectivity is the psychotic night of the world, then any investigation into its underlying ontology simultaneously requires a metaphysical archaeology of madness, that is, a theorizing of what the ultimate structure of reality must be like so that the subject's emergence could occur. In this manner, if Johnston is perhaps more interested in the paradoxical basis of transcendental subjectivity in nature, I am more interested in what occurs methodologically and metaphysically once we inscribe radical idealism as a form of madness into being, in such a way that two similar yet different views of Žižek's system emerge. The Madness of the Symbolic The Madness of the Symbolic 25 materialism, his Philosophies of Nature after Schelling17 offers an alternative account of the materialism-idealism relationship and the immanent emergence of the transcendental subject within nature that challenges not only Žižek's reading of Schelling, but more strongly his entire metaphysics. Zimmerman, a prolific German philosopher little known in the English-speaking world, was in fact the first person to use the concept of “transcendental materialism” in a contemporary context in his 1998 book Die Rekonstruktion von Raum, Zeit und Materie (The Reconstruction of Space, Time and Matter)18 and then fully develop it in his massive 2004 System des transzendentalen Materialismus (System of Transcendental Materialism).19 Zimmerman, like Grant, departs largely from Schelling, but also from Spinoza and Bloch, and offers an understanding of transcendental materialism in dialogue with contemporary science, especially physics, in stark opposition to the one presented here. For both, the thinking subject does not implicate an ontological trembling or pure difference within the field of being, and their respective accounts of the mind-body relationship can in no means be equated with what Žižek would perhaps be tempted to call a return to pre-modern cosmology. Moreover, considering how Žižek's own philosophy, similar in spirit to that of Zimmerman, does not possess mere implications or consequences for how we conduct empirical science, but directly engages with a broad range of disciplines such as quantum mechanics20 and cognitive science,21 by calling Žižek's philosophy a transcendental materialism I further hope to accomplish two things: to emphasize the systematic reach of Žižek's thinking and the exigence that metaphysics must also be an interlocutor with science. materialism, his Philosophies of Nature after Schelling17 offers an alternative account of the materialism-idealism relationship and the immanent emergence of the transcendental subject within nature that challenges not only Žižek's reading of Schelling, but more strongly his entire metaphysics. The Madness of the Symbolic Third, to return to the benefits of the characterization “transcendental materialism,” I endorse it because it draws our attention to Žižek's philosophical relevance outside of the fields of cultural studies, radical politics, and film theory by placing his thinking in direct contact with a series of other contemporary thinkers rethinking metaphysics, whether it be by representatives of the analytic tradition, object-oriented philosophy, new French materialism, or various other forms of the new speculative turn. Two thinkers deserve to be mentioned here by name, since they offer a transcendental materialism radically different from that of Žižek: Iain Hamilton Grant and Rainer E. Zimmerman. Although Grant would have reservations about his thinking being referred to as a “transcendental” The Madness of the Symbolic Zimmerman, a prolific German philosopher little known in the English-speaking world, was in fact the first person to use the concept of “transcendental materialism” in a contemporary context in his 1998 book Die Rekonstruktion von Raum, Zeit und Materie (The Reconstruction of Space, Time and Matter)18 and then fully develop it in his massive 2004 System des transzendentalen Materialismus (System of Transcendental Materialism).19 Zimmerman, like Grant, departs largely from Schelling, but also from Spinoza and Bloch, and offers an understanding of transcendental materialism in dialogue with contemporary science, especially physics, in stark opposition to the one presented here. For both, the thinking subject does not implicate an ontological trembling or pure difference within the field of being, and their respective accounts of the mind-body relationship can in no means be equated with what Žižek would perhaps be tempted to call a return to pre-modern cosmology. Moreover, considering how Žižek's own philosophy, similar in spirit to that of Zimmerman, does not possess mere implications or consequences for how we conduct empirical science, but directly engages with a broad range of disciplines such as quantum mechanics20 and cognitive science,21 by calling Žižek's philosophy a transcendental materialism I further hope to accomplish two things: to emphasize the systematic reach of Žižek's thinking and the exigence that metaphysics must also be an interlocutor with science. Ž Finally, it is worth mentioning that in Less Than Nothing Žižek also endorses Adrian Johnston's coinage of “transcendental materialism” in two passages in the last chapter on quantum mechanics, but with an important qualification that renders explicit for the first time a crucial metaphysical element of his own brand of materialism and its nuanced character. In the first instance, he claims that the key insight gained by contemporary physics is that material reality in itself does not present us with a dense field of fully constituted realities that form the ultimate building blocks of the universe, but rather with irreducibly indeterminate states lacking any substantial being and from which “hard” reality can only emerge if there Chapter 1 26 is a collapse of the wave function.22 In this sense, the micro-universe of quantum particles is strangely “less” than that of the macro-universe that constructs itself from its vicissitudes, in a way that is remarkably similar to how the Kantian subject can only construct a unified, coherent world of appearances from the inconsistent fragments of sensation. The Madness of the Symbolic In a strange logical short circuit, it would appear that not only is there no bottom-up causality at the level of experience (transcendental constitution is more real than what Kant calls “a rhapsody of perception”23), but even the most fundamental level of the universe is metaphysically more chaotic than the ordered macro-level physical world that science classically described. It is as if all reality is transcendentally constitutive, so that the only way to break free of the correlationist circle is to push “this transcendental correlation into the Thing itself:” “[i]t is against this background that one can make out the contours of what can perhaps only be designated by the oxymoron 'transcendental materialism' (proposed by Adrian Johnston).”24 The second instance adds some clarification to the metaphysical implications of this idea by stating that the “the only true consistent 'transcendental materialism' which is possible after the Kantian transcendental idealism” is one that risks the following difficult wager: “[w]hat if we posit that 'Things-in-themselves' emerge against the background of the Void of Nothingness, the way this Void is conceived in quantum physics, as not just a negative void, but the portent of all possible reality?”25 Although Žižek here takes transcendental materialism in a different direction than Johnston by introducing it to make a metaphysical point concerning the absolute rather than a more broadly ontological one concerning the subject, part of my project in what follows will be to show that his metaphysics of the void is not only completely compatible with his older ontology of the subject, but can be seen as its organic elaboration. If Žižek's approach differs significantly from that of Grant or Zi i i b hi i diff h h hi l d hi If Žižek's approach differs significantly from that of Grant or Zimmerman, it is because his game is different, even though this leads him to cover much of the same thematic domains—and hence it is interesting to label all three as “transcendental materialists” for the same reasons as it is to refer to Kant, Fichte, Hegel, and Schelling as “German Idealists” insofar as subsuming such irreconcilably different thinkers under a single category reveals a dynamic, pulsating movement, a battleground of theoretical The Madness of the Symbolic 27 positions around a shared set of problems rather than a shared doctrine, and thus something living. 1.2 The Lacanian Subject and the Irreducible Ambiguity of the Real As the fundamental presupposition of Žižek's philosophy, the Lacanian subject is to be radically distinguished from the philosophical subject of modernity. Although the former exhibits many traits that link it to early transcendental philosophy (it grounds the symbolic structures that constitute phenomenal reality through a free idealization) it is in direct opposition to the self-conscious transparency of the Cartesian cogito, the self-legislation of the Kantian rational agent, or the Hegelian account of free personality. For Lacan, the freedom of the I as witnessed in phenomenological self-experience is an illusion: completely determined by cultural and linguistic influences, the ego is an object and is constantly trying to construct a fantasmatic narcissistic space within which it can (falsely) perceive itself as a centre of self-effectuating action. Although this does not prevent the existence of human freedom for either Lacan or Žižek, it means that freedom itself gets largely displaced from consciousness into the unconscious, in a move formally similar to the middle-late Schelling of the Freiheitsschrift and Weltalter, but with an important twist. The subject is not an energetic, productive will that precedes the constitution of phenomenal reality, but is in one of its most important modalities nothing but an impersonal abyss that, uncannily, renders possible a minimal consistency of self. In this sense, the self is infinitely split at its core: when one looks inside oneself one not only finds an “extimacy,” material coming from elsewhere, where one should find one's innermost core, but if one looks long enough one only finds a void staring back. The Lacanian-Žižekian subject has no intrinsic content because it is pure form: the entire “plenitude” of cultural and psychological experience only emerges as a kind of defence formation against this primordial nothingness of the subject as an attempt to fill in its constitutive “gap” with false positive substantiality. But because this “gap” can never be filled in, it is ultimately repressed due to its traumatic, personality-shattering quality, so that we find traces of its disavowed knowledge in the slips and slides of speech, symbolic inconsistencies and non-coincidences in writing, the images of fantasies and dreams, and other phenomena. This creates two levels to any given (personal, ethical, political, or even philosophical) discourse: its surface movement, grounded in the As the fundamental presupposition of Žižek's philosophy, the Lacanian subject is to be radically distinguished from the philosophical subject of modernity. The Madness of the Symbolic What so strongly distinguishes Žižek's form of transcendental materialism from others is that, finding a fundamental structural identity between Lacanian psychoanalysis and post-Kantian idealism, he tries to illustrate the uncanny identity that exists between the psychoanalytical subject haunted by the Todestrieb as its constitutive basis and the unconscious, disavowed Grundlogik of German Idealism, with the conviction that a psychoanalytical dialogue between the two could open up a radically new possibility for metaphysics. By falling upon premonitions of the psychoanalytical experience in concepts such as Kantian unruliness, the Hegelian “night of the world,” and the Schellingian notion of the Grund, Žižek psychoanalytically reinterprets the late German Idealist attempts to give a metaphysical vision of reality compatible with the ontological emergence of the pure I in order to make us not only rethink what is at stake in the tradition of modern philosophy, a truth repressed and haunting its very history, but more primordially what is revealed with the advent of subjectivity as such: the notion of ontological catastrophe, the auto-disruption of reality into a painful not-all, at the origin of experience, with all the metaphysical implications that entails for our understanding of world in itself. Although Žižek's interpretations are heterodox, he believes that he is justified in singling out and radicalizing these premonitions, which often only appear in textual margins and often officially lack the theoretical primacy that Žižek bestows upon them, by means of the methodological application of various psychoanalytical techniques to the texts of German Idealism, these enabling him to plunge into and reveal the non-coincidences internally plaguing their symbolic space and thus retroactively restructuring their surface appearance in a manner similar to the analyst-analysand relation in therapy. We must traverse the fantasy of tradition if we are to arrive at its truth—this is Žižek's provocative claim and one that we will explore throughout most of this book. Thus, if we are to gain a preliminary sense of the driving forces of Žižek's reactualization of German Idealism and the systematicity of its method in spite of its apparent self-serving selectiveness, we must briefly pass through the Lacanian subject. 28 Chapter 1 28 Chapter 1 28 The Madness of the Symbolic The Madness of the Symbolic 29 narcissistic, self-deceiving orbit of the ego, and its “latent,” underlying truth, which shows itself negatively within the holes and inconsistencies of the former and can only be brought forth après-coup. In therapy, the task of the analyst is to make the analysand encounter and appropriate the second, the Real that afflicts the analysand often to a degree of painful agony, thereby forcing them to realign the symbolic structures underlying their personality so that the latter are more in tune with that which they reject, given that this (unconscious) act of recoil and exclusion has begun to obstruct their life. After all, the repressed always returns. To explain this complex, Lacanian psychoanalysis categorizes experience in terms of the three registers of the Imaginary, the Symbolic, and the Real. All three exist in dialectical simultaneity, so that they all depend upon and interpenetrate one another. Lacan uses a Borromean knot to illustrate this level of mutual co-existence, the point of which is to preclude the possibility of arguing for the primacy of one register over the other, as it is unclear if either can have logical priority insofar as the cutting off or isolating of one destroys the whole. The Imaginary is roughly equivalent to phenomenological experience and perception but is also related to the cogito and its “narcissistic” fantasy of existential self-mastery. It is identified with a necessary moment of misrecognition and irremovable untruth in one's everyday being and knowledge of self, world, and others, for it projects completion where there is lack. The Symbolic constitutes the logical fabric of language and the laws of culture that transcend and are anterior to the concretely existing personal subject. It therefore precedes the imaginary orbit of experience insofar as the individual phenomenological constitution of objects in a strong sense presupposes language. 1.2 The Lacanian Subject and the Irreducible Ambiguity of the Real Although the former exhibits many traits that link it to early transcendental philosophy (it grounds the symbolic structures that constitute phenomenal reality through a free idealization) it is in direct opposition to the self-conscious transparency of the Cartesian cogito, the self-legislation of the Kantian rational agent, or the Hegelian account of free personality. For Lacan, the freedom of the I as witnessed in phenomenological self-experience is an illusion: completely determined by cultural and linguistic influences, the ego is an object and is constantly trying to construct a fantasmatic narcissistic space within which it can (falsely) perceive itself as a centre of self-effectuating action. Although this does not prevent the existence of human freedom for either Lacan or Žižek, it means that freedom itself gets largely displaced from consciousness into the unconscious, in a move formally similar to the middle-late Schelling of the Freiheitsschrift and Weltalter, but with an important twist. The subject is not an energetic, productive will that precedes the constitution of phenomenal reality, but is in one of its most important modalities nothing but an impersonal abyss that, uncannily, renders possible a minimal consistency of self. In this sense, the self is infinitely split at its core: when one looks inside oneself one not only finds an “extimacy,” material coming from elsewhere, where one should find one's innermost core, but if one looks long enough one only finds a void staring back. The Lacanian-Žižekian subject has no intrinsic content because it is pure form: the entire “plenitude” of cultural and psychological experience only emerges as a kind of defence formation against this primordial nothingness of the subject as an attempt to fill in its constitutive “gap” with false positive substantiality. But because this “gap” can never be filled in, it is ultimately repressed due to its traumatic, personality-shattering quality, so that we find traces of its disavowed knowledge in the slips and slides of speech, symbolic inconsistencies and non-coincidences in writing, the images of fantasies and dreams, and other phenomena. This creates two levels to any given (personal, ethical, political, or even philosophical) discourse: its surface movement, grounded in the The Madness of the Symbolic As a self-enclosed structural system capable of reproducing and propagating itself, the Symbolic displays an irreducible autonomy that displaces the role of nature in understanding human psychology and cultural phenomena because it is able to articulate itself in utter isolation from it: the “incessant sliding of the signified under the signifier,”26 the solipsistic dance of language always in step with itself, means that the essential link between signifier and transcendent, extra-linguistic signified has been violently ruptured and that the mere chains of signifiers relating to themselves are capable of producing/ constructing meaning by themselves in an ontological void. In its simplest Chapter 1 30 form, the Real is that which does not fall under either the Imaginary or the Symbolic, whereby its upsurge is associated with experiences of breakdown and inconsistency not only of the transcendental unity at the basis of phenomenological experience, but even of language or culture itself. Lacan and Žižek therefore use a plethora of adjectives to describe it, which attempt to capture this element of irrevocable logical and existential rupture: “traumatic,” “monstrous,” “horrifying,” and “impossible,” to name a few. attempt to capture this element of irrevocable logical and existential rupture: “traumatic,” “monstrous,” “horrifying,” and “impossible,” to name a few. Yet there is an irreducible ambiguity in Lacan's definition of the Real, which serves as the starting point for Žižek's own philosophical endeavour, for it is precisely in trying to resolve this ambiguity that his metaphysical project gets off the ground. The Real elicits two potentially incompatible interpretative possibilities, and we often see Lacan oscillating back and forth between them. In its first guise, the Real is the excluded Other of the Imaginary and Symbolic, which only truly “comes to be” when the subject constitutes itself. In this sense, the Real is not only dependent upon the symbolic matrix of language and the orbit of phenomenological experience but also only shows itself negatively through their immanent obstruction. This Real-as-lack is distinctly Hegelian: it corresponds to concepts such as “tarrying with the negative” and the suffering that consciousness undergoes when it runs up against non-coincidence, paradox, and limitation in social and political action or scientific thought about the world. The Madness of the Symbolic 31 the idealizing process of human meaning makes it impossible to reach. As something that overreaches the idealizing, linguistic activity of the subject, in this modality the Real, corresponding to the Schellingian concept of the indivisible remainder (der nie aufgehende Rest), that pre-experiential darkness that can never be brought into light of consciousness yet upon which all consciousness rests, is the Real-as-excess. But such a free ciphering activity simultaneously creates the condition of the possibility of its own breakdown insofar as it will not always be capable of idealizing the Real in a way that enables its own autonomous, smooth functioning.28 These “kinks” in the Symbolic correspond with the Real-as-lack or Real2, something that cannot be integrated because it presents itself as intrinsically and paradoxically non-relational, as an inassimilable kernel within the self-referential matrix of the symbolic relations within which it emerged. The problem is as follows: Is the Real1 a necessary, illusory construct of the Symbolic designed to give a fantasmatically fabricated sense of “positivity” to a world that exists beyond its grasp (rendering it a mere secondary effect of a solipsistically self-contained structuralist metapsychology)? Or is it, more primordially, the pre-subjective, ontological basis of the Symbolic, to which we have access despite the apparent impossibility of reaching the pure Real through the differential system of language (thus showing the obscure origins of the Real2 in an ontology)? If the second is possible, what does this mean in terms of Lacan's declaration of the logical equivalence and interpenetration of the registers? The Madness of the Symbolic It has absolutely no positive content in itself even though, as an internal limit within a given symbolic space, it may effectuate an overhauling of the latter's structure and possibilities as the subject attempts to overcome its deadlock so that it is potentially productive in its very trauma. In its second guise, we could also understand the Real as the pre-subjective life of pure jouissance from which the human infant exiles itself by becoming a linguistic subject, yet upon which the Imaginary and the Symbolic logically depend, even if they only relate to it negatively through its primordial foreclosure from experience and language as the founding gesture of subjectivity itself. Bruce Fink refers to this as the Real1 because it is the necessary posit of the Symbolic despite the fact that it is inaccessible from within the latter, whose ciphering activity doesn't merely “reconstitute” objective reality by meditating it like a camera obscura. Given that this pre-subjective Real must be said to be without lack (only with language can we speak of absence and presence),27 The Madness of the Symbolic 1.3 From Logico-Dialectical Simultaneity to the Ontogenesis of the Subject Even if all three registers exist in a logico-dialectical simultaneity, within the development of Lacanian psychoanalysis we see a gradual shift in emphasis in the thematization of the registers largely due to internal reasons. Lacan's early work is largely an attempt to come to terms with the mirror stage and its implications for understanding psychogenesis. In the mirror stage, which happens around the age of six months, there is a recognition of an immanent blockage in nature that tears apart the organic unity of the body. The human infant lacks motor coordination—its self-experience is infinitely fragmented and lacking in any internal unity. Lacan's provocative Chapter 1 Chapter 1 32 thesis is that the only way out of this biological short circuit is a vel, a misrecognition of the primordial helplessness of the human organism in the “specular image”29 of its mirror self in which the child finds a mesmerizing and captivating lie of false mastery into which it libidinally invests itself. Already at this early stage we see why Lacan is so critical of the modern conception of subjectivity and rationality. The result of the mirror stage is a reorganization of the fragmented being of the child through a virtual and therefore illusionary schema as the self becomes alienated from its real chaotic being. Yet Lacan comes to see that the imaginary beginnings of psychogenesis are themselves necessarily grounded in the Symbolic: the only reason why the child becomes tantalized by his image is because their parents provoke the response. “Look, it's you!” In this sense, the entire genesis of the self is preceded by a carving out of a space for the child within the symbolic universe of familial relations even before the child was born. After this “linguistic turn,” Lacan turns all of his attention to the nature of the Symbolic and seems, in many respects, largely to leave the Imaginary behind. Inspired by the work of Levi-Strauss, who argued that “[s]tructural linguistics will certainly play the same renovating role with respect to the social sciences that nuclear physics, for example, has played for the physical sciences,”30 Lacan then begins to apply the methodology of Saussure's structuralism to psychoanalysis, accomplishing this feat largely by a retour to Freud. 1.3 From Logico-Dialectical Simultaneity to the Ontogenesis of the Subject What Lacan finds so intriguing is that, despite all of Freud's attempts to ground the unconscious within a natural vitalism of the body or biological movement of instincts, his texts themselves orbit around the analyses of images and language. Lacan's fundamental thesis is that, retroactively, we can see that Freud already had an implicit idea of the importance of linguistics for understanding the unconscious but was unable fully to articulate this fundamental insight because he lacked the appropriate methodology. This in turn creates a fundamental and irremovable non- coincidence within Freud's texts as they oscillate between purely structural analyses of language and obscure vitalistic biologism. Consequently, Lacan argues that if we read Freud against Freud, structural linguistics can give psychoanalysis the scientific rigor that it needs by systematizing the logic of the unconscious, which is the origin of Lacan's famous saying “the The Madness of the Symbolic 33 unconscious is structured like a language.” Linked to this linguistic turn are his critiques of ego-psychology as an attempt to strengthen the ego and post- Freudian attempts to biologize the unconscious. For Lacan, the unconscious is, strictly speaking, an irreducibly linguistic phenomenon: it only emerges after or alongside the advent of language, in the split between the subject of enunciation and the enunciating subject. It has nothing to do with deep- lying personality structures determining how the ego relates to the external world or instinctual energetics. Although this suggests an obvious superseding of the Imaginary by the Symbolic, commentators such as Richard Boothby and Alexander Leupin warn against this, arguing that Lacan is much more complex and subtle than he may initially appear. Lacan never backs away from the claim that all registers mutually depend upon one another in order to have any efficacy at all. Even if the self-generating matrix of language and culture historically precedes and conditions the possibility of any concretely existing person, its differential network of meaning is only possible through an originary phenomenological perception of signifiers.31 The colonization of the body by the transcendentally alienating structures of the Symbolic requires the activity of the Imaginary so that the various phonetic differences that allow signs to be intelligible in contradistinction from one another can be established in the first place. 1.3 From Logico-Dialectical Simultaneity to the Ontogenesis of the Subject Moreover, the late Lacan's topological formalizations of the psyche, as already mentioned, proclaim a strict equivalency between them, so that “the symbolic order's supremacy appears as an aporia, an ethical decision that logic does not support.”32 But how can Žižek then, as a Lacanian philosopher of the Real, justify his own theoretical preference for it over the other registers where Lacan's texts seem to contradict such an approach? Although Žižek's work holds an uncertain relation to Lacanian orthodoxy, it would be wrong to claim that this troublesome problematic is just a direct consequence of Žižek's own reading of Lacanian psychoanalysis. By the very act of embarking on a metaphysics of the Real Žižek does seem to imply that we must find a way to overstep Lacan's attempt to conserve the equivalency of the registers if psychoanalysis is to find a sufficient theoretical grounding, even if doing so means that we risk making the entire psychoanalytical edifice he constructed collapse. But Žižek's own Chapter 1 34 thinking is not as radical a rupture with Lacan as it may appear, for the late Lacan too ruminates about the understanding of nature necessarily implied by his theory and therefore himself gestures towards the possibility of a metaphysics of the Real consistent with it.33 Even as early as the seventeenth seminar, we can find Lacan proclaiming that one of the logical implications of the psychoanalytical experience is that substance is not-all (that is, nature does not present us with a spherical totality—un tout, une sphère).34 Yet if the Real is only lack, and the essential link between signifier and transcendent, extra-linguistic signified has been cut, how can Lacan even legitimately make such statements? How can he philosophically justify such a “direct touching” of the Real given the epistemological solipsism intrinsic to the cybernetic ciphering of the Symbolic? 6. Lacan, “Presentation on Psychic Causality,” in Écrits, trans. Bruce Fink (New York: W. W. Norton, 2006), p. 176/144. 5. See Freud, “The Loss of Reality in Neurosis and Psychosis” (1924), SE, XIX, especially pp. 185–87. 4. Žižek, The Parallax View (Cambridge: MIT Press, 2009), p. 231. 1.3 From Logico-Dialectical Simultaneity to the Ontogenesis of the Subject Žižek’s wager is that one can develop a metaphysics of the Real that not only does not jeopardize the equivalency of the registers, but even explains their emergence, thereby implying that to grasp the essence of psychoanalysis and draw out its philosophical implications we need to do two things: (i) metapsychologically explicate the ontogenesis of the subject in terms of a materialism compatible with the founding insights of a radical idealism or, in other words, explain the relation of the Real1 of the apparent excess of the ontological to the linguistic to the Real2 of symbolic or notional lack, since this is the great question left unanswered by Lacan and upon which his entire theoretical apparatus ultimately depends; (ii) instead of focusing on Lacan's relationship to nineteenth- and twentieth-century psychology, French structuralism, the Prague school of linguistics, or existentialism, we should return to German Idealism, since it is only in a direct dialogue with this tradition that we can find a way out of the impasses of Lacanian psychoanalysis. But to develop such a metaphysics, we must leave psychoanalysis and venture into German Idealism. Notes The Madness of the Symbolic 35 The Madness of the Symbolic 3 The Madness of the Symbolic 35 7. Žižek, Less Than Nothing, p. 77. 8. Ibid., p. 197. 9. Žižek, The Parallax View, p. 4. 10. For Johnston’s justification of “transcendental materialism,” see Žižek’s Ontology: A Transcendental Materialistic Theory of Subjectivity (Evanston: Northwestern University Press, 2000), pp. 273–74. 11. Fichte, Fichte: Early Philosophic Writings, trans. Daniel Breazeale (Ithaca: Cornell University Press, 1988), p. 147. 12. See Johnston, Žižek’s Ontology, pp. 80–92. 12. See Johnston, Žižek’s Ontology, pp. 80–92. 13. Fichte, Science of Knowledge, ed. and trans. Peter Heath and John Lachs (Cambridge: University of Cambridge Press, 1982), p. 262. 14. Žižek, The Parallax View, p. 214. 15. Žižek, The Ticklish Subject: The Absent Centre of Political Ideology (New York: Verso, 2000), pp. 33–41. 16. Žižek, The Indivisible Remainder: On Schelling and Related Matters (New York: Verso, 2007), p. 8. 17. Grant, Philosophies of Nature after Schelling (New York: Continuum, 2006). 18. Zimmerman, Die Rekonstruktion von Raum, Zeit und Materie. Moderne Implikationen Schellingscher Naturphilosophie (Frankfurt am Main: Lang, 1 19. Zimmerman, System des transzendentalen Materialismus (Paderborn: Mentis, 2004). 20. See Žižek, chapter 3 (“Quantum Physics with Lacan”) of The Indivisible Remainder, pp. 189–236; and chapter 14 (“The Ontology of Quantum Physics”) of Less Than Nothing, pp. 905–62. 21. See part 2 (“The Solar Parallax: The Unbearable Lightness of Being No One”) of The Parallax View, pp. 145–250; and interlude 6 (“Cognitivism and the Loop of Self-Positing) of Less Than Nothing, pp. 715–37. 22. Žižek, Less Than Nothing, p. 724. 23. Kant, The Critique of Pure Reason, trans. Paul Guyer and Allen W. Wood (Cambridge: Cambridge University Press, 1998), A 156/B195. 24. Žižek, Less Than Nothing, p. 906. 25. Ibid., p. 935. 25. Ibid., p. 935. 26. Lacan, “The Instance of the Letter in the Unconscious, or Reason since Freud,” in Écrits, p. 502/419. 27. One of many possible quotes: “By definition, the Real is full.” Lacan, Le séminaire, Livre IV: La relation d’objet et les structures freudiennes, 1956–1957, ed. Jacques-Alain Miller (Paris: Seuil, 1994), p. 218. I capitalize “the Real.” 28. See Fink, chapter 3 (“The Creative Function of the Word: The Symbolic and the Real”) of The Lacanian Subject: Between Language and Jouissance (New York: Princeton University Press, 1995), pp. 24–31. 29. Notes Lacan, “The Mirror Stage as Formative of the I Function as Revealed in Psychoanalytical Experience,” in Écrits, p. 94/76. 36 Chapter 1 36 Chapter 1 36 Chapter 1 30. Lévi-Strauss, “Structural Analysis in Linguistics and in Anthropology,” in Structural Anthropology, trans. Claire Jacobson and Brooke Grundfest Schoepf (New York: Basic Books, 1963), p. 33. 31. See Boothby, Freud as Philosopher: Metapsychology after Lacan (New York: Routledge, 2001), pp. 86–94. 32. See Leupin, Lacan Today: Psychoanalysis, Science and Religion (New York: Other Press, 2004), p. 27. 33. See chapters 4 and 5. 34. See Lacan, Le séminaire, Livre XVII: L’envers de la psychoanalyse, 1969–1970, ed. Jacques-Alain Miller (Paris: Seuil, 1991), p. 36. Chapter 2 Grasping the Vanishing Mediator Between the Real and the Ideal Žižek and the Unconscious Truth of German Idealism Chapter 2 Grasping the Vanishing Mediator Between the Real and the Ideal Žižek and the Unconscious Truth of German Idealism Chapter 2 Grasping the Vanishing Mediator Between the Real and the Ideal Žižek and the Unconscious Truth of German Idealism Seeing a structural homology between the contemporary concerns of psychoanalysis and those of late German Idealism's response to Kant, Žižek turns to the latter to find the resources he needs to give an account of the ontogenesis of the psychoanalytical subject. However, to do so he not only has to go against mainstream interpretations of this tradition, but also has to do great damage to the founding texts themselves. Outlining the methodology behind Žižek's reactualization, what we will see is that the only reason Žižek can apply psychoanalytical tools to restructure its symbolic space and thereby develop his own philosophy is that this tradition itself is haunted by a spectral history of an encounter with an underlying trauma: namely, the subject as the vanishing mediator between the Real and the Ideal, which both Hegel and Schelling primordially reveal in their own manner, but ultimately recoil from and disavow. In this regard, German Idealism presents us with an unconscious Grundlogik that we can only now, with the aid of Freud and especially Lacan, reconstruct, thus giving us a profoundly new and controversial view of its internal development and theoretical preoccupations. 38 Chapter 2 38 2.1 The Methodology of a Psychoanalytical Dialogue: Or, Lacan with Hegel and Hegel with Lacan What is amazing about this psychoanalytical dialogue, however, is the heterodox readings of German Idealism and Lacanian psychoanalysis it produces. To many critics, Žižek simply shows no concern for textual faithfulness or the history of ideas in his readings of Kant, Hegel, Schelling, and Lacan. His methodological approach appears, if anything, to function through a deliberate misunderstanding or liberal reconstruction that purposefully overlooks key conceptual distinctions that challenge his own philosophical outlook. Although there is some superficial truth in these critiques—one must admit that Žižek focuses on often marginal selections from texts and raises them to a level of logical priority that they do not have in the original—one of Žižek's rare comments on his own methodology is very helpful for dispelling confusion on how he proceeds: Hegel didn't know what he was doing. You have to interpret him. Let me give you a metaphoric formula. You know the term Deleuze uses for reading philosophers—anal interpretation, buggering them. Deleuze says that, in contrast to other interpreters, he anally penetrates the philosopher, because it's immaculate conception. You produce a monster. I'm trying to do what Deleuze forgot to do—to bugger Hegel, with Lacan [chuckles] so that you get monstrous Hegel, which is, for me, precisely the underlying radical dimension of subjectivity which then, I think, was missed by Heidegger. But again, the basic idea being this mutual reading, this mutual buggering [chuckles] of this focal point, radical negativity and so on, of German Idealism with the very fundamental (Germans have this nice term, grundeswig35) insight of psychoanalysis.36 Even if Žižek describes his own philosophy as an act of textual violence, almost of rape (it is also worth mentioning that the word “bugger” originates the old French bougre, meaning heretic, and acquires its colloquial sense from heresy being associated with deviate, outlawed sexual practices), this quote reveals a hidden methodological presupposition that guides all Grasping the Vanishing Mediator Between the Real and the Ideal Grasping the Vanishing Mediator Between the Real and the Ideal 39 of Žižek's interpretative work. The comparison of his own philosophy to that of Deleuze is of crucial importance and is not to be downplayed. It demonstrates that, even if Žižek is intentionally going against surface textual movements in his reading, he does not understand his own philosophy as in any sense arbitrary, a deliberate misunderstanding of the philosophers with whom he is engaging, or even as exhibiting any disregard for the tradition. Žižek recognizes that he is not doing traditional history of philosophy or any kind of philologico-exegetical interpretation, but is, instead, attempting to do something that is productive of new concepts by revealing their disavowed insights. But this generative activity of concept-creation can bring forth something unexpected, unsettling, even traumatic—we may produce monsters. Žižek is not directly interested in what the texts of the German Idealist tradition “have to say,” that is, their intended meaning, because this level of their discourse—like most discourses that fail to reflect upon the psychoanalytical conditions within which a discourse as such can take place—usually operates largely on the level of the Imaginary and its illusionary fantasy and can thus, perhaps even at most crucial junctures of conceptual argumentation, display a psychoanalytical superficiality. What therefore concerns him are hitherto unrealized textual potentialities, premonitions of which we can see in “marginal” comments or in various structures that often obstruct the general flow of a given philosophical system and consequently can be said to protrude out of its symbolic universe, negatively contorting it from the inside. Yet it is only by means of a thorough familiarity with this system and its surface affirmations that one can arrive at such unearthed possibilities and “reactualize” them. The analyst must, after all, listen to the analysand, even if, especially in its Lacanian mode, it often appears as if they are unconcerned or ignoring your needs and demands (for the goal of therapy is not the adaptation of the ego but rather a confrontation with unconscious, often traumatic truth). Žižek's own methodological approach to the history of philosophy, however, drastically differs from that of Deleuze insofar as it takes as its starting point the fundamental Lacanian claim that we can never say what we mean because there is an irremovable gap between the imaginary moi (“me” as the subject of self-conscious awareness who expresses the intention to say Chapter 2 That is what Lacan means when he says that “interpretation hits the cause”: it hits that around which the analysand is revolving without being able to “put it into words.”38 Keeping this in mind, we can easily see that Žižek's reading of German Idealism, by following its unconscious Grundlogik, is an attempt to psychoanalytically construct what the tradition in fact tries to say but cannot, by revealing what has been repressed in the first-level propositional Grasping the Vanishing Mediator Between the Real and the Ideal 41 affirmations of its texts. In other words, to borrow a Lacanian phrase, it tries to hit their cause. Although one can, of course, take issue with this methodology, one must admit that critics who take issue with Žižek's “selective” reading or “obvious misinterpretations” often just miss the point: they fail to see the underlying systematicity evident in his engagement with the tradition or understand why the conceptual moments he draws upon are so important. In the spirit of Hegel and Schelling, who, in the uncertain aftermath of Kant, sought to articulate a philosophical system that could guarantee the latter's intuition of the irreducible autonomy of the subject,39 Žižek like Lacan before him is convinced that there is something genuinely real and insurmountable in the experience of human freedom. Starting from psychoanalysis' insight into the constitutive disharmony between mind and body as a key to understanding its true essence, Žižek returns to German Idealism to see how the notion is conceptually refracted there. But in this respect Žižek's method is not a textual reconstruction of their arguments through an act of classico-philological retrieval nor is it an attempt to polish them up by using new resources at our disposal. What he notices is something primordially at odds with their surface content, which rather than rendering the texts of German Idealism logically inconsistent is more profoundly indicative of some kind of internal unconscious struggle, a struggle that is philosophically revealing and whose exploration—now made possible by the clinical tools of psychoanalysis—promises to unearth new ways of approaching its fundamental concepts. Chapter 2 Chapter 2 40 something, but somehow says something different and unintended) and the symbolic je (the elusive I as the subject that one deduces from what has in fact been said). Not only is language something that exerts control over us more than we have power over it (as an ego, rather than existing as a speaking linguistic subject, we are in a strong sense spoken), but the surface content of our own words often belies a greater (consciously) disavowed (but unconsciously known) truth, a truth that is not “hidden” in some deep, elusive place, but is so obvious that we often do not see it: “[t]he psychoanalyst is not an explorer of unknown continents or of grand depths, but a linguist: he learns to decode the writing that is already there, under his eyes, open to the look of everyone.”37 In the slips and slides of discourse, in seemingly meaningless hints and gestures, we catch a glimpse of the Real as that which cannot be said directly in discourse but around which it eternally moves, acting as a black hole drawing in everything towards it, or what Lacan refers to as its “cause” or “truth”: Lacan's theory of interpretation is based, to some extent, on a formulation similar to that of the caput mortuum: an analysand speaking in the analytic setting is often unable to say, formulate, to come out with certain things; certain words, expressions, or thoughts are unavailable to him or her at a particular moment and he or she is forced to keep circling around them, beating around the bush, as it were, never enunciating what he or she senses to be at issue. The analysand's discourse traces a contour around that which it hovers about, circles, and skirts. Those words or thoughts may become accessible to the analysand in time, in the course of analysis, but they may also be introduced by the analyst in the form of an interpretation. Chapter 2 Finding premonitions of Todestrieb protruding out of the imaginary-symbolic space of its discourse, Žižek sees a disavowed truth lurking “beneath” the tradition's own attempts to think substance as subject by means of a dialectical interconnecting of mind and body, a disavowed truth that reduces the latter to a mere reaction formation, a defence from the true horror of subjectivity finally brought to the fore by Kant's account of transcendental freedom. After all, if German Idealism's breakthrough is the freedom of the subject, according to Žižek we must also recognize that “our experience of freedom is properly traumatic.”40 Consequently Žižek gives us resources to think how, if we attentively follow the tradition's conceptual movements, we can see traces of an operative logic ephemerally emerging from time to time at crucial moments only to be Chapter 2 42 covered up by preceding steps, so that our task is to reconstruct retroactively this hidden Grundlogik constantly interfering with what the tradition takes itself to be, if we are to come to terms with the disturbing metaphysics of freedom demanded by its founding intuitions. However, the methodology of such a psychoanalytical reconstruction of philosophical trauma around which an entire tradition circulates as its unconscious cause or truth displays an important paradox that deserves to be highlighted, which we can elucidate by means of Žižek's discussion of the legend of Eppur si muove: The legend has it that, in 1633, Galileo Galilei muttered, “Eppur si muove” (“And yet it moves”), after recanting before the Inquisition his theory that the Earth moves around the Sun[. ...] There is no contemporary evidence that he did in fact mutter this phrase, but today the phrase is used to indicate that although someone who possesses true knowledge is forced to renounce it, this does not stop it from being true. But what makes this phrase so interesting is that it can also be used in the exact opposite sense, to assert a “deeper” symbolic truth about something which is not true—like the “Eppur si muove” story itself, which may well be false as a historical fact about Galileo's life, but is true as a designation of Galileo's subjective position [... B]eyond the truth of reality, there is the reality of the fiction.41 Likewise, the alternative history of German Idealism that I develop in the following in a strong sense did not happen as a historical fact. Chapter 2 Little if any evidence points towards it, and often the philological references that I have gathered can be easily opposed by numerous counter-examples. However, this is not the point: although this account of German Idealism's failed encounter with its inner truth is in many ways nothing but a fiction, this does not prevent it from directly expressing the subjective position that each of its representatives holds (even if it falsely capitulates their conscious interpretation of what they are doing) in a manner that other philosophical methodologies cannot, for what concerns us are traces of the Real in imaginary-symbolic constructs and their logic. After all, “when truth is too traumatic to be confronted directly, it can only be accepted in the guise of a fiction.”42 Grasping the Vanishing Mediator Between the Real and the Ideal 43 44 Chapter 2 Than Nothing.45 The effect of this comment is twofold: not only does it strike those immersed within “conventional,” textbook accounts of the history of philosophy as essentially wrong (which, it must be said, are losing credibility as Fichte and Schelling research has finally gained an autonomy of its own), but it also highlights a fundamental difficulty at the core of Žižek's reading of German Idealism and by consequence his own philosophy, insofar as his parallax ontology is perceived as a reactualization or continuation of this movement. This problem is only amplified when one engages with Žižek's major works on German Idealism. In both The Ticklish Subject and The Parallax View, for example, there are praises of Schelling as the greatest philosopher of the pre-symbolic Real, of the nature of the impossible X, the je ne sais quoi, which precedes consciousness as the most central theme in post-Kantian philosophy and whose problematic uncannily reappears in the wake of the Lacanian subject. Žižek even goes as far as to say that Schelling was “the first to formulate this task”46 and the philosopher who “gave the most detailed account of this X in his notion of the Ground of Existence,”47 which is why his philosophy and not that of Hegel is “at the origins of dialectical materialism.”48 Given that Žižek in Less Than Nothing describes “the key question” of philosophy as that of “how thought is possible in a universe of matter,” so that we should focus our efforts on “the very rise of representation or appearing out of the flat stupidity of being” if we are to avoid “the very rise of representation or appearing out of the flat stupidity of being” if we are to avoid “a regression to a 'naive' ontology of spheres or levels,”49 the issue of whether this project is most radically accomplished by Schelling or Hegel is more than a matter of intra-textual consistency or classico-philological accuracy, but touches the very heart of what Žižek takes to be the program of speculative philosophy. On account of this ambiguity, Žižek's own project of describing this process of the auto-disruption of the Real displays an undeniable oscillation between the characterization of this project as a Schellingian or a Hegelian one, even though he claims outright that Schelling is the culmination of this line of thought. 44 Chapter 2 44 Than Nothing.45 The effect of this comment is twofold: not only does it strike those immersed within “conventional,” textbook accounts of the history of philosophy as essentially wrong (which, it must be said, are losing credibility as Fichte and Schelling research has finally gained an autonomy of its own), but it also highlights a fundamental difficulty at the core of Žižek's reading of German Idealism and by consequence his own philosophy, insofar as his parallax ontology is perceived as a reactualization or continuation of this movement. This problem is only amplified when one engages with Žižek's major works on German Idealism. In both The Ticklish Subject and The Parallax View, for example, there are praises of Schelling as the greatest philosopher of the pre-symbolic Real, of the nature of the impossible X, the je ne sais quoi, which precedes consciousness as the most central theme in post-Kantian philosophy and whose problematic uncannily reappears in the wake of the Lacanian subject. Žižek even goes as far as to say that Schelling was “the first to formulate this task”46 and the philosopher who “gave the most detailed account of this X in his notion of the Ground of Existence,”47 which is why his philosophy and not that of Hegel is “at the origins of dialectical materialism.”48 Given that Žižek in Less Than Nothing describes “the key question” of philosophy as that of “how thought is possible in a universe of matter,” so that we should focus our efforts on “the very rise of representation or appearing out of the flat stupidity of being” if we are to avoid “the very rise of representation or appearing out of the flat stupidity of being” if we are to avoid “a regression to a 'naive' ontology of spheres or levels,”49 the issue of whether this project is most radically accomplished by Schelling or Hegel is more than a matter of intra-textual consistency or classico-philological accuracy, but touches the very heart of what Žižek takes to be the program of speculative philosophy. 2.2 A Metaphysics of the Real: An Hegelian or a Schellingian Project? Central to Žižek's metaphysics of the Real as a psychoanalytical reactualization of German Idealism is his stark emphasis on the indispensable significance of Hegel. In various places, Žižek characterizes his own project as strictly Hegelian because, like Hegel, the enigma that occupies him is the possibility of appearance itself: that is, how the realm of phenomenal reality could emerge from the self-actualization of substance. Hegel is said to be the first to understand this question in terms of inscribing the subject into the absolute: a subject that, in the Kantian aftermath, is understood as intrinsically irreducible to the immanent ontological field that brought it forth, and that can freely mediate the latter for itself through its own idealizing activity. In this sense, Hegel's metaphysics could structurally supply the missing principle of a Lacanian metapsychology. Through Hegel, Žižek believes he can show that subjectivity is not illusory by situating idealism into the heart of materialism as an irruptive event, the claim being that premonitions of this can be seen throughout Hegel's philosophy. Apparently arguing by means of resources found in the latter for a self-splitting or of the noumenal within the dark pre-history of subjectivity, Žižek tries to show how the only consistent way to explain why there is experience is to posit an ontological catastrophe at the basis of the subject. Consequently, it would appear that Žižek makes the following his axiomatic first principle thanks to a direct confrontation with Hegel: Freedom is not a raw, brute fact, but an expression of the caustic collapse of material being, a brisure in the heart of Real, which is synonymous with the subject itself; “it designates [...] the primordial Big Bang, the violent self-contrast by means of which the balance and inner peace of the Void of which mystics speak are perturbed, thrown out of joint.”43 But the overtly Hegelian nature of Žižek's project does not go without saying. In one essay, Žižek makes a claim that to many readers must appear out of place insofar as he gives it no qualification: “Hegel's 'overcoming' of Schelling is a case in itself: Schelling's reaction to Hegel's idealist dialectic was so strong and profound that more and more it is counted as the next (and concluding) step in the inner development of German Idealism.”44 What is more, this claim is repeated almost verbatim in Less 44 Chapter 2 Not only does it potentially suggest that Žižek tries to disavow too strong a relationship between a Lacanian-inspired metaphysics of the Real and Schelling, but it also shows that he might downplay the role that Schellingian notions play (or undermine possible roles that others Grasping the Vanishing Mediator Between the Real and the Ideal Grasping the Vanishing Mediator Between the Real and the Ideal 45 could) in such a metaphysics, in terms of either our understanding of ontology (the universal), natural sciences (the particular), or politics (the singular), the trinity that constitutes the conceptual fold of The Parallax View, his first theoretical magnum opus. In other respects, however, it appears that Žižek may have recently changed or distanced himself from his earlier interpretation of Schelling, perhaps precisely for these reasons. Although Žižek, despite his constant insistence that his fundamental question is a “properly Hegelian one: how does appearance itself emerge from the interlay of the Real?,”50 oscillates in his early major theoretical works The Ticklish Subject and The Parallax View in his descriptions of who most adequately answers this question, in his new masterpiece Less Than Nothing this oscillation suddenly stops. While this project is again and again characterized as Hegelian,51 the former glory once bestowed upon Schelling as the great thinker of the “phenomenalization of being”52 is not just downplayed, but apparently completely forgotten, with Žižek even going so far as to say that thinking the emergence of appearance is “what at his most radical Hegel does,”53 thus contradicting himself. However, despite appearances to the contrary, the ambiguity does not thereby disappear. Not only does Žižek claim that “[t]he minimal definition of materialism hinges of the admission of a gap between what Schelling called Existence and the Ground of Existence,”54 but the ambiguity of the Hegel-Schelling relationship in fact gets radicalized in the middle of the second chapter on Hegel, when Žižek describes the latter's dialectic as “the science of the gap between the Old and the New,” and then without explanation jumps into a two-and-a-half page discussion of Schelling's theosophic odyssey of the emergence of “the cosmos (of fully constituted reality, ruled by logos) out of the proto-cosmic pre-ontological chaos.”55 Although to many readers this may appear as just another “typical” Žižekian digression with no inner logic, the argument that I develop in this book attempts to show that it is anything but that. Grasping the Vanishing Mediator Between the Real and the Ideal Insofar as Žižek himself claims that the only way to avoid mystification is not to abandon the project of the Weltalter but rather “to reformulate it so as to avoid the mystification of the theosophic mytho-poetic narrative,”56 it appears that the proximity of his reading of Hegel and Schelling, when coupled with this brief and rare methodological elaboration, points to the core of his heterodox reading of could) in such a metaphysics, in terms of either our understanding of ontology (the universal), natural sciences (the particular), or politics (the singular), the trinity that constitutes the conceptual fold of The Parallax View, his first theoretical magnum opus. In other respects, however, it appears that Žižek may have recently changed or distanced himself from his earlier interpretation of Schelling, perhaps precisely for these reasons. Although Žižek, despite his constant insistence that his fundamental question is a “properly Hegelian one: how does appearance itself emerge from the interlay of the Real?,”50 oscillates in his early major theoretical works The Ticklish Subject and The Parallax View in his descriptions of who most adequately answers this question, in his new masterpiece Less Than Nothing this oscillation suddenly stops. While this project is again and again characterized as Hegelian,51 the former glory once bestowed upon Schelling as the great thinker of the “phenomenalization of being”52 is not just downplayed, but apparently completely forgotten, with Žižek even going so far as to say that thinking the emergence of appearance is “what at his most radical Hegel does,”53 thus contradicting himself. However, despite appearances to the contrary, the ambiguity does not thereby disappear. Not only does Žižek claim that “[t]he minimal definition of materialism hinges of the admission of a gap between what Schelling called Existence and the Ground of Existence,”54 but the ambiguity of the Hegel-Schelling relationship in fact gets radicalized in the middle of the second chapter on Hegel, when Žižek describes the latter's dialectic as “the science of the gap between the Old and the New,” and then without explanation jumps into a two-and-a-half page discussion of Schelling's theosophic odyssey of the emergence of “the cosmos (of fully constituted reality, ruled by logos) out of the proto-cosmic pre-ontological chaos.”55 Although to many readers this may appear as just another “typical” Žižekian digression with no inner logic, the argument that I develop in this book attempts to show that it is anything but that. Grasping the Vanishing Mediator Between the Real and the Ideal Insofar as Žižek himself claims that the only way to avoid mystification is not to abandon the project of the Weltalter but rather “to reformulate it so as to avoid the mystification of the theosophic mytho-poetic narrative,”56 it appears that the proximity of his reading of Hegel and Schelling, when coupled with this brief and rare methodological elaboration, points to the core of his heterodox reading of Chapter 2 Grasping the Vanishing Mediator Between the Real and the Ideal 47 47 process of the dialectic, the third term is already the second, understood as a negativity or internal limit inscribed in the first, insofar as it has merely taken over, usurped, the primary position from which the movement began by asserting itself as such. In terms of substance and subject, this means that “this very reversal is the very definition of subject: ‘subject' is the name for the principle of Selfhood which subordinates to itself the substantial Whole whose particular moment it originally was.”61 Nothing at the level of content changes: it expresses a purely formal change at the level of structure due to the tension generated by the contorting presence of negativity. The dialectical movement that goes (i) immediacy → (ii) negation → (iii) negation of negation is superior because there is no genuine return movement to the first, although it all takes place in a single, logically immanent field. The beginning and end do not overlap because something irreducibly different emerges within the first moment (negativity now being made foundational to identity), namely, to take the example that concerns us here, an “out of joint” spirit that has a degree of notional self-reflexivity in opposition to its ontogenetic ground, the whole within which it gave birth to itself. There is no need to posit a state of “originary health” of which the devouring restlessness of the negative cannot be predicated (absolute indifference as that which stands in the difference between Grund and existence and thus is equally in one as in the other) in order to explain the dialectical movement of reality and its relation to the absolute. As we shall see, Žižek's criticism of Schelling gets more complicated because it does not universally apply to the entirety of Schelling’s work, since for Žižek the latter is not characterized by an organic unity or continuity, but rather by a series of irreconcilable ruptures. He draws a distinction between a Schelling1 of the period of quasi-Spinozism (the philosophy of indifference), a Schelling2 of the radical ontology of freedom as seen in the second draft of the Weltalter and the Freiheitsschrift, and a Schelling3 of the philosophy of mythology and revelation, which he qualifies—very violently—as a return to pre-modern essentialism. Chapter 2 46 being purely Hegelian or purely Schellingian, Žižek’s philosophy thus proves to be a hybrid of the two, an attempt—in some as yet unidentified way—to use Hegel to formalize Schelling's thinking, “saving” it from its theosophic commitments, and to use Schelling to radicalize Hegel, allowing him to draw out implicit but disavowed moves in his own philosophy and thus in German Idealism as a whole. But why does this ambiguity exist in the first place? Immediately after his remarkable and provocative reading of Schelling in the first chapter of The Indivisible Remainder, Žižek goes on to argue for the supremacy of Hegelian dialectics over Schellingian metaphysics. For him, although there are premonitions of a radical breakthrough in works such as the Freiheitsschrift and the Weltalter,57 Schelling remains philosophically inferior to his great rival because Grund and existence ultimately remain distinct from one another only by being founded within absolute indifference, which itself “is not a product of opposites, nor are they contained in it implicite; rather it is a being of its own, separated from all oppositions, on which all oppositions are broken, which is nothing other than their very non-being, and which therefore has no predicate except predicatelessness.”58 Indeed, Schelling claims that “from this neither-nor, or from this indifference, duality [...] immediately breaks forth, and without indifference, i.e., without an unground, there would be no twofoldness of the principles.”59 For Žižek, however, this means that “the [Schellingian] Absolute is primarily the 'absolute indifference' providing the neutral medium for the coexistence of the polar opposites” of the Real and the Ideal60 and, as such, is at odds with the potential latent in his middle-late philosophy, a potential Žižek sees in its articulation of the eruptive logic of Grund and its relationship to existence at the basis of subjectivity. In this regard, Hegel provides a superior logic in which there is no need for a principle of meditation exterior to Grund and existence as two reflective pairs that determine themselves through their internal dynamic, which in turn allows us to internally restructure Schelling's own descriptions of the interrelation of the two. In Hegel the very category of “and” changes—it becomes, in essence, tautological, thereby enabling us to identify Grund and existence instead of rendering them mere opposites or dual principles founded in something external to their own inner movement. In the logical Grasping the Vanishing Mediator Between the Real and the Ideal 47 For Žižek, what distinguishes the middle-late period of Schelling's thinking is, strictly speaking, the articulation of an ontogenetic emergence of self- positing freedom in a manner remarkably similar to the Hegelian dialectical movement from abstract immediacy to notional self-reflexivity, which goes Chapter 2 48 against the surface-level theosophic inclinations of the texts. It protrudes out of them and makes them non-coincidental with themselves in such a way that we can psychoanalytically reactualize their fundamental movement by “tarrying with the negative”—that is, by encountering the Real within them. What this means is that even if the self-operative logic of the Grund contains premonitions of a radical transcendental materialism, Schelling is at the same time the father of “New Age obscurantism.”62 Expressing a reliance upon and indebtedness to Schelling would, in essence, open up a possible connection between Žižek’s own thinking and everything he denies—the non-Freudian unconscious (in its Bergsonian, Jungian, Deleuzian, and other forms), “pre-modern” cosmology, Romantic theories of nature, theosophy and even its pop-culture descendant, New Age spirituality. In this sense, the very act of placing the logic of the Grund at the heart of the psychoanalytic subject appears to risk “destabilizing” the primacy of the Lacanian mode of the unconscious insofar as it opens up the possibility of interpreting it through a different account of one's relation to the pre-subjective life of the Real; covering up the pivotal importance of Schelling would seem to be nothing but a certain ideological act on Žižek's part. But here two remarks must be made. First, although it is perhaps a bit underhanded on Žižek's behalf to downplay the importance of Schelling for the development of his transcendental materialism, it must be added that this obfuscation is consistent with his overall interpretation of Schelling, for he rejects most of the latter's philosophy and needs recourse to Hegel even to reactualize the logic of the Grund that he sees as its breakthrough. But second, and more importantly, such an explanation does not solve the problem of the Hegel- Schelling relation insofar as this emphatic shift is simultaneously ambiguous, given the fact that Žižek does not distinguish which Schelling he is arguing against or justify how he is able to read the second draft of the Weltalter as an ephemeral breakthrough. In this regard, by characterizing his own philosophy again and again as against the surface-level theosophic inclinations of the texts. Grasping the Vanishing Mediator Between the Real and the Ideal 47 It protrudes out of them and makes them non-coincidental with themselves in such a way that we can psychoanalytically reactualize their fundamental movement by “tarrying with the negative”—that is, by encountering the Real within them. What this means is that even if the self-operative logic of the Grund contains premonitions of a radical transcendental materialism, Schelling is at the same time the father of “New Age obscurantism.”62 Expressing a reliance upon and indebtedness to Schelling would, in essence, open up a possible connection between Žižek’s own thinking and everything he denies—the non-Freudian unconscious (in its Bergsonian, Jungian, Deleuzian, and other forms), “pre-modern” cosmology, Romantic theories of nature, theosophy and even its pop-culture descendant, New Age spirituality. In this sense, the very act of placing the logic of the Grund at the heart of the psychoanalytic subject appears to risk “destabilizing” the primacy of the Lacanian mode of the unconscious insofar as it opens up the possibility of interpreting it through a different account of one's relation to the pre-subjective life of the Real; covering up the pivotal importance of Schelling would seem to be nothing but a certain ideological act on Žižek's part. But here two remarks must be made. First, although it is perhaps a bit underhanded on Žižek's behalf to downplay the importance of Schelling for the development of his transcendental materialism, it must be added that this obfuscation is consistent with his overall interpretation of Schelling, for he rejects most of the latter's philosophy and needs recourse to Hegel even to reactualize the logic of the Grund that he sees as its breakthrough. But second, and more importantly, such an explanation does not solve the problem of the Hegel- Schelling relation insofar as this emphatic shift is simultaneously ambiguous, given the fact that Žižek does not distinguish which Schelling he is arguing against or justify how he is able to read the second draft of the Weltalter as an ephemeral breakthrough. In this regard, by characterizing his own philosophy again and again as a Hegelian, but never a Schellingian project Žižek belies his overt reliance on texts such as Schelling's Freiheitsschrift and the Weltalter for his project. Grasping the Vanishing Mediator Between the Real and the Ideal 47 What I propose, therefore, is to read Žižek against Žižek not only to demonstrate the implicit, complex intertwining of Schellingian ontology and Hegelian logic throughout his thinking, wherein I perceive the nuance Grasping the Vanishing Mediator Between the Real and the Ideal 49 of his theoretical philosophy, but also to show how by unravelling this ambiguity we can find new resources to explore the obscure origins of the psychoanalytical subject and the fundamental structures and essence of the world within which it emerges. Yet if embarking upon a reconstruction of the Hegel-Schelling relation within Žižek's work can achieve this, it is only because Žižek sees his own work as an attempt to culminate the Grundlogik of German Idealism itself, which gestures towards the hypothesis that the intra-textual ambiguity of the Hegel-Schelling relation within his thinking is perhaps less expressive of an internal inconsistency in his philosophy than of a fundamental problem inherent in the self-unfolding of that tradition itself in its endeavour to overcome radical idealism; so that, if by a dialectical reversal we turn an epistemological limit into a positive ontological condition, apparent textual confusion into a real feature of the movement itself in its historical development, the ambiguity will prove itself in a second moment to be a side effect of a sustained methodological engagement with the fundamental insights of German Idealism which thus deserves to be investigated in its own right. As we will see, this ambiguity is in fact due to a single traumatic kernel of truth that both Hegel and Schelling, attempting to ground the Kantian subject, bring to the fore in different ways, but ultimately have to repress or force into the unconscious, a fact that therefore requires us to use a mode of inquiry that goes beyond philologico-exegetical interpretation if we are to penetrate it: The notion of Schelling's Grundoperation—the “vanishing mediator” between the two poles (the Real and the Ideal, B and A)—opens up the possibility of establishing a connection with Hegelian dialectics: the founding gesture “repressed” by the formal envelope of the “panlogicist” Hegel is the same as the gesture which is “repressed” by the formal envelope of the “obscurantist” Schelling, yet which simultaneously serves as its unacknowledged ground. [...] Does the gesture of “vanishing mediation” not point, therefore, towards what, following some German interpreters, one could call the Grundoperation des Deutschen Idealismus, the fundamental, elementary operation of German Idealism? 35. Neither this word nor a comparable one appears to actually exist in modern or old German. A simple Google search is enough to be suspicious: it only comes up with this quote. Grasping the Vanishing Mediator Between the Real and the Ideal 51 the fore and deals explicitly with the critical difficulties posed by attempting to give an account of the emergence of the Ideal out of a Real that has been, from within the former, always already lost. The psychoanalytical fusion of Schelling and Hegel implicit in his thinking—a fusion which points towards a certain unconscious struggle inherent to German Idealism itself—either reveals or resolves a potentially fatal ambivalence in the conception of the Real, an ambivalence that risks jeopardizing not only Žižek's project, but also the development of any form of metaphysics from within a radical idealism. According to Lacan, the Symbolic exhibits what may be called a form of ontological solipsism insofar as it is an irreducibly self-referential matrix of signifiers whose relation to an external, extra-ideal world has been utterly cut off. Yet to give a theoretical grounding of our inability to access objective reality we must describe its dark pre-history in the orgasm of forces that is the pre-symbolic Real. Is the Real that which precedes and exceeds consciousness, or a pure lack that only presents itself through the breakdowns of the Symbolic? Can these two modalities be reconciled within a metaphysics? If so, can a hybridization of Hegel and Schelling help us conceive of the emergence of the Symbolic out of the Real from within the Symbolic by overcoming the opposition between materialism and idealism? But before we answer these questions, we should step back and ask: why would we even want to proclaim such a Cartesian dualism of mind and body, nature and spirit, the Real and the Symbolic, especially since such a form of subjectivity has been criticized for so long in the contemporary intellectual scene as no longer sustainable? What are its theoretical advantages? 36. Žižek, “Liberation Hurts: An Interview with Slavoj Žižek (with Eric Dean Rasmussen).” Retrieved Feb. 23, 2010, from http://www.electronicbookreview. com/thread/endconstruction/desublimation. 50 Chapter 2 It is our endeavour to articulate clearly the Grundoperation of German Idealism which necessitates reference to Lacan; that is to say, our premiss is that the “royal road” to this Grundoperation involves reading German Idealism through the prism of Lacanian psychoanalytical theory.63 In this manner, to raise the question of whether Žižek's project is Schellingian or Hegelian is misleading, for what interests Žižek is not Kant, Schelling, or Hegel as particular historical thinkers per se, but a psychoanalytically retrievable unconscious truth self-unfolding throughout their works, a truth inaugurated by the Cartesian cogito but from which the entire tradition of modern philosophy has been repelled because of its horror, but to which we can now have access thanks to Freud's and Lacan's groundbreaking work on the original trauma of the human subject. In short, my wager is that Žižek forces us to consider that what makes German Idealism so singular in the history of philosophy is that it is haunted by an unconscious history that is nothing other than one of the sustained encounters with the ontological catastrophe at the very basis of human subjectivity in human thinking, this concept thus presenting itself as a Lacanian cause or truth around which the surface structures of its great epics circulate as their repressed traumatic Real and whose movement demand being systematically reconstructed retroactively. But the ambiguity of the Hegel-Schelling relationship has another advantage: it not only allows us to construct an original and perhaps controversial understanding of German Idealism and its legacy by articulating its unconscious history, a dynamic spectral history of repression and the return of the repressed, but it also allows us to evaluate the philosophical significance of Žižek's work by situating him directly within its concerns. Žižek is able to rely upon Hegel and Schelling to explicate the origins of subjectivity and their metaphysical implications because he sees them as embarking upon a metaphysics from within a self-grounding transcendental framework, whereby German Idealism and the psychoanalytical tradition in its Lacanian legacy become uncannily close due to their underlying epistemologies and the need to make sense of them ontologically. In this respect, unpacking the ambiguity of the Hegel- Schelling relation is of profound methodological importance, for it brings to Grasping the Vanishing Mediator Between the Real and the Ideal 37. Lacan, “Clefs pour la psychoanalyse (entretien avec Madeleine Chapsal).” Retrieved May 16, 2010, from http://www.ecole-lacanienne.net/docu- ments/1957-05-31.doc. Notes 37. Lacan, “Clefs pour la psychoanalyse (entretien avec Madeleine Chapsal).” Retrieved May 16, 2010, from http://www.ecole-lacanienne.net/docu- ments/1957-05-31.doc. Chapter 3 Žižek's reactualization of German Idealism represents an avid attempt to revitalize Cartesian transcendental subjectivity. In the face of a contemporary discourse that attacks from all sides the very possibility of a non-material I that stands in its own autonomous register, Žižek's philosophy aims to articulate the theoretical currency still implicit within the groundbreaking intuition that heralds modernity. But what warrants this repetition/resurgence of such an apparently “outmoded” idea? What kind of explanatory efficacy could it still exhibit in a theoretical field that believes itself to have largely overcome its shortcomings? By exposing the various theoretical holes revealed in the contemporary intellectual scene by Lacanian psychoanalysis, Žižek rethinks the modern materialism-idealism debate by showing how movements such as those in current phenomenology of the body, postmodernism, and neurobiology are philosophically insufficient and thus force us to reconceptualize transcendentalism radically. insufficient and thus force us to reconceptualize transcendentalism radically. 52 Chapter 2 38. Fink, The Lacanian Subject, p. 28. 38. Fink, The Lacanian Subject, p. 28. 39. Žižek, Less Than Nothing, p. 266. 39. Žižek, Less Than Nothing, p. 266. 40. Ibid., p. 265. 40. Ibid., p. 265. 41. Ibid., pp. 3–4. 41. Ibid., pp. 3–4. 42. Ibid., p. 24. 42. Ibid., p. 24. 43. Žižek, The Ticklish Subject, p. 31. 44. Žižek, “Fichte’s Laughter,” in Mythology, Madness and Laughter: Subjectivity in German Idealism (New York: Continuum, 2009), p. 122. 45. Žižek, Less Than Nothing, p. 137. 45. Žižek, Less Than Nothing, p. 137. 46. Žižek, The Parallax View, p. 166. 46. Žižek, The Parallax View, p. 166. 47. Žižek, The Ticklish Subject, p. 55. 47. Žižek, The Ticklish Subject, p. 55. 48. Žižek, The Indivisible Remainder, p. 11. 48. Žižek, The Indivisible Remainder, p. 11. 49. Žižek, Less Than Nothing, p. 905. 50. Žižek, The Parallax View, p. 106. 50. Žižek, The Parallax View, p. 106. 51. See, for instance, Žižek, Less Than Nothing, pp. 13f. and 642ff. 52. See Žižek, The Indivisible Remainder, p. 14; and Žižek, The Abyss of Freedom, in The Abyss of Freedom/Ages of the World (Cambridge: MIT Press, 2008), p. 15. 53. Žižek, Less Than Nothing, p. 239. 53. Žižek, Less Than Nothing, p. 239. 54. Ibid., p. 912. 54. Ibid., p. 912. 55. Ibid., p. 273. 55. Ibid., p. 273. 56. Ibid. 56. Ibid. 57. Žižek, The Indivisible Remainder, p. 8. 58. Schelling, Philosophical Investigations into the Essence of Human Freedom and Other Related Matters, trans. Priscilla Hayden-Roy, in Philosophy of German Idealism, ed. Ernst Behler (New York: Continuum, 2003) (hereafter Freiheitsschrift), p. 276. 59. Ibid., p. 278. 59. Ibid., p. 278. 60. Žižek, The Indivisible Remainder, p. 105. 60. Žižek, The Indivisible Remainder, p. 105. 61. Ibid., p. 106. 61. Ibid., p. 106. 62. Žižek, The Abyss of Freedom, p. 4. 62. Žižek, The Abyss of Freedom, p. 4. 63. Žižek, The Indivisible Remainder, p. 92. 3.1 Postmodernism and an Uncanny Defence of Transcendentalism Although phenomenologists like Merleau-Ponty and more recently Michel Henry and Jean-Luc Marion argue for the primordial unity of consciousness and the lived body or the self's immanent auto-determination from the unfolding of givenness of the flesh (which indicates the disappearance Chapter 3 54 of a radical subject-object distinction by the interpenetration of both in embodiment), Žižek makes the claim that such descriptions are intrinsically lacking insofar as they fail to take account of the experience of the monstrous and the traumatic irrevocably tied to the essence of human being. One cannot merely replace classical subjectivity with a more organic theory of experience that intertwines consciousness with a phenomenological auto-affection of the flesh. The rift between the spiritual and the material uncovered by the I destabilizes the very possibility of such a primordial union or identity: if we take transcendental reflexivity seriously we see, according to Žižek, a pure self-positing that tears itself away from any kind of immersion within the field of corporeal activity. The very possibility of the psychoanalytical experience as that which reveals a split between the energetic dynamics of the body and subjective, experiential reality presupposes an irreducibly antagonistic interrelation, a discordant dichotomy, between mind and body, which proclaims that the human subject must in some sense “protrude” out of its carnal materiality and be understood on its own terms. The claim is that the modern subject not only paves the way for the psychoanalytical discourse of the unconscious, but also sets up the possibility of a genuine encounter with what it means to be a human subject existing with an intrinsic relation to a bodily substrate that paradoxically presents itself as Other. We cannot just think the self that comes after the subject in a way that does away with the latter, for transcendental subjectivity brings to our attention a primordial truth of what it means to be a free human being that, in many ways, we have yet to come to terms with: that of a structural conflict between mind and body which divides our being into two incommensurate spheres. However, the implications of Žižek's reactualization of German Idealism are not just limited to the phenomenology of the body. Psychoanalysis and the Enigma of Transcendental Subjectivity 55 55 sociopolitical activity. The unceasing play of cultural difference, the non- finite proliferation of identities and discourses, can only be adequately understood through the transcendental framework offered by Cartesian subjectivity because, as that which prevents human activity from being explicable through solely natural or biological grounds, it supplies the formal-universal structure through which such change is rendered possible. The cogito is necessary for deciphering not only human embodiment, but also human historicity insofar as it gives us a clue to what kind of ontological event could have led to the emergence of something like a symbolic universe in contrast to the “cyclical” movement of nature. Here we see another advantage of the cogito that transcends its theoretico-explicative currency, or more strongly, coincides with it: as the transcendental condition of the possibility of historical contingency, the pure I always stands above all fixed, particular sociopolitical constellations and thus presents the always possible basis of ideological critique and political revolution. sociopolitical activity. The unceasing play of cultural difference, the non- finite proliferation of identities and discourses, can only be adequately understood through the transcendental framework offered by Cartesian subjectivity because, as that which prevents human activity from being explicable through solely natural or biological grounds, it supplies the formal-universal structure through which such change is rendered possible. The cogito is necessary for deciphering not only human embodiment, but also human historicity insofar as it gives us a clue to what kind of ontological event could have led to the emergence of something like a symbolic universe in contrast to the “cyclical” movement of nature. Here we see another advantage of the cogito that transcends its theoretico-explicative currency, or more strongly, coincides with it: as the transcendental condition of the possibility of historical contingency, the pure I always stands above all fixed, particular sociopolitical constellations and thus presents the always possible basis of ideological critique and political revolution. But perhaps the greatest threat to subjectivity comes from contemporary cognitive science. In light of decades of groundbreaking research in neurobiology, there is a growing tendency to turn towards scientific models of explanation to explain away the uniqueness of human subjectivity. 3.1 Postmodernism and an Uncanny Defence of Transcendentalism Arguing against postmodern theorists like Derrida and Foucault, who claim that the subject itself is merely an empty, accidental construction that arises out of the flux of historical experience, Žižek contends that by forgetting the ontological schism between mind and body that enables the self to be determined according to linguistic and political forces in the first place, they lose sight of the very formal structure of the I that is required even to speak of the endless temporal variations of selfhood within the contingent upsurge of 56 Chapter 3 Žižek's argument here is twofold. Firstly, founding itself upon classical biology, contemporary cognitive science presupposes that every organism is a self-contained system in harmony with itself seeking homeostasis and self-preservation, which prevents it from coming to terms with the psychoanalytical concept of Todestrieb. Representing a malfunction in biology, whereby a person no more strives for pleasure and satisfaction, for the minimal possible level of distress and affliction, but rather for pain and even self-destruction, psychoanalysis identifies this apparently negative moment of short circuit within the biological machine with one of the defining traits of human subjectivity and thus of culture itself.64 Instead of being a mere haphazard disorder or a contingent feature of a sick mind, Todestrieb comes to represent a necessary feature of the singularity of our being: the condition of the possibility of psychopathological self- destruction is ultimately linked to our very freedom because the two are structurally homologous. Secondly, what Žižek adds to this argument for the supremacy of psychoanalysis over reductionist biology is the following insight: if there were nothing but the self-contained, deterministic system of the neuronal interface of the brain, then why is there (self-)experience at all?65 Why is there not just blind existence, a mere mechanism that auto- develops according to its own laws? Why does the nonconscious trembling of brute matter in its dynamic pulsations need to be aware of itself? Since the category of subjective experience is superfluous, unnecessary, to the materialism displayed by science, the mere fact of experience proclaims that neurobiological activity is not-all—that there is a gap, a series of interstices, which arise within its logical fold as a kind of unpredictable short circuit to which, perhaps, phenomenal reality arises as a response. Naming the place of this rupture the subject itself, Žižek's own work on cognitive science consequently tries to underline the inherent difficulty that the discipline has (for this very reason) to explain the emergence of consciousness, insofar as it points to a limit-situation within which the discourse itself breaks down. Neither the phenomenology of the body, postmodernism, or cognitive science can just dispel the “myth of subjectivity” because all three of them, Neither the phenomenology of the body, postmodernism, or cognitive science can just dispel the “myth of subjectivity” because all three of them, in distinct ways, need it to account for the very subject matter they take as their own. Psychoanalysis and the Enigma of Transcendental Subjectivity 55 Researchers are not just constantly downplaying the role of systematic self-observation and autonomous discourses that deny the supremacy of experimental science; rather, what is increasingly coming into question is the infinite array of material offered by self-consciousness and the meaning of philosophical investigations into its culture and politics as structurally free from biological concerns. Instead of having recourse to first-person experience as it shows itself to us in the irreducibility of its complex dynamics as the site of personality (phenomenology), or the labyrinthine network of the symbolic universe of discourses informing our sense of self and other (postmodernism), they are able to explain the entire range of emotional and social characteristics through the nonconscious, asubjective pulsation of brute matter, the mere non-personal movement of neurochemico-electrical activity wherein the I becomes an epiphenomenal illusion created by a closed biological system of response mechanisms pre- determined by genetic code. 56 Chapter 3 If the phenomenology of the body is to understand the very field of the (self-)appearing of phenomena to consciousness as embodied, then it Psychoanalysis and the Enigma of Transcendental Subjectivity 57 has to come to terms with the breakthrough of transcendental reflexivity as a form of pure self-positing that institutes a subject-object schism rendering the very body that we live in Other to ourselves: “the subject (Self) is [...] immaterial: its One-ness, its self-identity, is not reducible to its material support. I am precisely not my body: the Self can only arise against the background of the death of its substantial being, of what it 'objectively' is.”66 Postmodernism needs to presuppose the pure I as something over and above the contingent field of non-finite cultural difference that it sets up even to talk about the complex network of discourses irreducible to naturalistic influences. Lastly, cognitive science cannot discard the subject if it is to explain the very possibility of how a gap in (material) being could emerge so that there is the basic distance from self that is necessary for the phenomenalization of reality. If we are to understand the true nature of the human being, Žižek's contention is that we must reread transcendental philosophy through the psychoanalytical category of Todestrieb, for both seem to cover the same theoretical set of problems in an uncanny manner; this unholy marriage of German Idealism and psychoanalysis aims to reconfigure the contemporary intellectual scene by offering a comprehensive system that is able to respond to the intrinsic limitations of all three disciplines. However, although Žižek's retrieval of subjectivity thus superficially appears to be an attempt to assert the unshakeable supreme position of the human subject against the various de-centerings that it has undergone in the twentieth century, this is an overt vel. Whereas most defenders of the transcendental ego return to Descartes, Kant, or Hegel to regain and unpack their conviction in the self-grounding ipseity at the core of human activity in face of its dissipation within flux of radical historicism, the all- engulfing force of structuralism, or the brute determination of mechanical nature, what intrigues Žižek are the various ways in which the cogito fails at its own task and the implications of this failure for our understanding of self and world. 56 Chapter 3 Instead of presenting an intuition of a self-positing substantiality that persists behind all representational content of thought while being co-given, co-equal, and ultimately fully coinciding with my own personal self, what we truly find in the thinkers of subjectivity according to Žižek is the affirmation of a negatively charged void that holds together the Chapter 3 58 sedimentations of personality through its sheer self-reflexive nothingness. Whether this be Descartes' inability to establish the positive characteristics of the I that necessarily thinks,67 Kant's failure to penetrate into the kernel of the subject an sich that makes experience possible,68 or Hegel's early account of the night of the world,69 what is obvious to Žižek is that the impervious, vacuous core of subjectivity is simultaneously its condition of possibility and impossibility. Consequently, what interests Žižek are two things: (i) the traces of this abyssal nature of subjectivity that can be found in modern philosophy, which immanently problematizes all attempts to ground experience in the intuition of a self-positing substantial core of existential familiarity and transparency; and (ii) how this grounding insight into transcendental reflexivity and the deadlocks it generates are schematized under the textual surface in different modalities by Kant and his followers, yet in such a way that they display in them a hidden logic implicitly working itself out throughout the entire tradition that can be retroactively constructed and used today in order to combat a range of theoretical problems that plague us. 3.2 Idealism and its Shadow: Materialism in the Cracks Taking the inevitable failure of self-grounding idealism to fully posit itself as a guide, Žižek's reading of German Idealism focuses on what he perceives as an ambiguous relationship in its texts: between such a self-grounding idealism, and the spectral presence of what appears as a form of materialism within its ideal-synthetic fold that must somehow be posited as theoretically more fundamental and primordial après-coup. The agonizing tension within ideality as it tries to constitute the world of experience suggests that, although a non-mediated, purely material reality has been forever lost due to the idealizing activity rendered possible by the subject, we can still, through the very limitations, blockages, and obstructions of this activity, negatively catch a glimpse of it, the very inconsistency of thought guaranteeing that we gain knowledge of reality “outside” our representations. The figure of Fichte is of utmost importance in this context for comprehending the conceptual contours of Žižek's position. Instead of being a contradictory, deficient concept within the internal trajectory of German Idealism that automatically reveals Fichte's inconsequentiality and impotence as a thinker, the concept of Anstoß manifests the truth of the materialism-idealism relation expressed within the tradition, albeit in a manner that he does not explicitly thematize. Taking the inevitable failure of self-grounding idealism to fully posit itself as a guide, Žižek's reading of German Idealism focuses on what he perceives as an ambiguous relationship in its texts: between such a self-grounding idealism, and the spectral presence of what appears as a form of materialism within its ideal-synthetic fold that must somehow be posited as theoretically more fundamental and primordial après-coup. The agonizing tension within ideality as it tries to constitute the world of experience suggests that, although a non-mediated, purely material reality has been forever lost due to the idealizing activity rendered possible by the subject, we can still, through the very limitations, blockages, and obstructions of this activity, negatively catch a glimpse of it, the very inconsistency of thought guaranteeing that we gain knowledge of reality “outside” our representations. The figure of Fichte is of utmost importance in this context for comprehending the conceptual contours of Žižek's position. 3.2 Idealism and its Shadow: Materialism in the Cracks However, to return to German Idealism and its “rampant” transcendentalist inclinations with the intention of retrieving a conception of the subject able to respond to perceived dead ends in contemporary intellectual discourse presents an immediate problem. In order to articulate an account of experience that can combine psychoanalysis and modern subjectivity in a manner capable of combating the phenomenology of the body, postmodern discourse theory, and cognitive science, Žižek must fight against the anti- materialistic tendencies evident in the German Idealist tradition itself. Žižek's fundamental thesis is—perhaps counter-intuitively—that it is the latter's very descriptions of the non-material, purely spiritual I that point the way. The nothingness of the cogito reveals more than a form of ontological dualism, the horrifying split nature of subjectivity that forever haunts psychic life: not only is the cogito unable to find an existential or ontological interiority at the heart of one's own most personal being— being a mere formal void that guarantees the self-sameness of identity through phenomenal time, the I is an empty set—but the very gesture of a Psychoanalysis and the Enigma of Transcendental Subjectivity 59 self-grounding idealism that it sets into play shows itself to be structurally incomplete and riddled with holes. In modernity's irreversible establishment of the primary function of the cogito (as a synthesizing activity that idealizes our contact with the world in such a way that we lose contact with real entities non-mediated by mind) Žižek argues that we immediately come face to face with the internal limitation and contradiction of idealism as such, insofar as absolute mediation is not possible for the subject. Instead of exhibiting a smooth transcendentally constituted fabric of experience freely brought forth by the subject on its Godlike throne, idealism generates in and of itself irremovable and unpredictable moments of blockage and obstruction that infringe upon the subject's totalizing claims for autocratic autonomy and a complete, undisturbed idealization of experience. In other words, every attempt of idealism to affirm itself as “all,” to set the stage for the transcendental constitution of phenomenal reality as an almighty, full-blown “hallucination,” fails: within idealism there is always something uncannily more than ideal, an extimacy that corrodes it from the inside and cannot be explained from within its free idealization of the world. 3.2 Idealism and its Shadow: Materialism in the Cracks Instead of being a contradictory, deficient concept within the internal trajectory of German Idealism that automatically reveals Fichte's inconsequentiality and impotence as a thinker, the concept of Anstoß manifests the truth of the materialism-idealism relation expressed within the tradition, albeit in a manner that he does not explicitly thematize. Chapter 3 60 In Fichtean subjective idealism, the I is the axiomatic starting point of philosophy. Even the not-I, the entire sphere of everything that presents itself within consciousness as “extra-subjective” content, is freely posited and brought forth by the self-grounding activity of the absolute subject, in effect robbing it of any alterity that it would possess in itself as a material, non-mediated being with its own interiority that thrusts itself upon consciousness. In other words, the not-I does not affect the I through a form of causality. Yet because of the double meaning of Anstoß as a hindrance and impetus, as something that obstructs and incites our activity, one cannot just say that the Anstoß is a mere obstacle created by the subject so that it can have the necessary degree of resistance needed in order concretely to assert and actualize its own freedom. Although the not-I depends upon the pure I for its meaning, the former of itself cannot be reduced to the idealizing activity set up by the latter: it is not “like the games the proverbial perverted ascetic saint plays with himself by way of inventing ever new temptations and then, by overcoming them, confirming his strength.” For if this were so, “it would present a case of the hollow playing of the subject with itself, and we would never reach the level of objective reality.”70 There is therefore an element of speculative truth in Fichte that Žižek wants to bring to the fore: even if the circle of idealism is a self-enclosed, free creation rendered possible by the subject, due to the painful curvature of this space we must nevertheless speak of a negative materialism in Fichte's thinking.71 Here, we encounter a paradoxical coincidence of internal-external limitation and inside-outside: “Anstoß thus designates the moment of the 'run-in,' the hazardous knock, the encounter of the Real in the midst of the ideality of the absolute I: there is no subject without Anstoß, without the collision with an element of irreducible facticity and contingency—'the I is supposed to encounter within itself something foreign,”72 some inassimilable body that sticks in its throat like a bone. According to Žižek, this truth is then later radicalized by Hegel's and Schelling's own response to Fichte's thinking insofar as they realize it gives us the necessary resources we need to develop a metaphysics while never leaving the transcendental idealist framework. Chapter 3 Žižek's own theoretical position is therefore a rather precarious one. It contends that it is only from within the deadlock and limitations of a full- In Fichtean subjective idealism, the I is the axiomatic starting point of philosophy. Even the not-I, the entire sphere of everything that presents itself within consciousness as “extra-subjective” content, is freely posited and brought forth by the self-grounding activity of the absolute subject, in effect robbing it of any alterity that it would possess in itself as a material, non-mediated being with its own interiority that thrusts itself upon consciousness. In other words, the not-I does not affect the I through a form of causality. Yet because of the double meaning of Anstoß as a hindrance and impetus, as something that obstructs and incites our activity, one cannot just say that the Anstoß is a mere obstacle created by the subject so that it can have the necessary degree of resistance needed in order concretely to assert and actualize its own freedom. Although the not-I depends upon the pure I for its meaning, the former of itself cannot be reduced to the idealizing activity set up by the latter: it is not “like the games the proverbial perverted ascetic saint plays with himself by way of inventing ever new temptations and then, by overcoming them, confirming his strength.” For if this were so, “it would present a case of the hollow playing of the subject with itself, and we would never reach the level of objective reality.”70 There is therefore an element of speculative truth in Fichte that Žižek wants to bring to the fore: even if the circle of idealism is a self-enclosed, free creation rendered possible by the subject, due to the painful curvature of this space we must nevertheless speak of a negative materialism in Fichte's thinking.71 Here, we encounter a paradoxical coincidence of internal-external limitation and inside-outside: “Anstoß thus designates the moment of the 'run-in,' the hazardous knock, the encounter of the Real in the midst of the ideality of the absolute I: there is no subject without Anstoß, without the collision with an element of irreducible facticity and contingency—'the I is supposed to encounter within itself something foreign,”72 some inassimilable body that sticks in its throat like a bone. Psychoanalysis and the Enigma of Transcendental Subjectivity 61 61 to matter—here we must think of Žižek's philosophy as a turning inside- out of Jacobi's challenge that idealism must become a speculative egoism by following it to its logical conclusion: “[t]he transcendental idealist [...] must have the courage to assert the strongest idealism that has ever been taught, and not even to fear the charge of speculative egoism”73—that we can develop a truly speculative materialism up to the task of explaining the emergence of experience, for it also suggests that only a full-blown idealism is truly capable of disclosing crucial facts about the nature of materiality. However, such a materialism rests upon the apparently paradoxical claim that it is the very auto-collapse of idealism that is epistemologically capable of overcoming itself and describing the world in itself. As Adrian Johnston says, “materialism [...] formulates itself vis-à-vis the deadlocks internal to radical transcendental idealism. On this account, materialism is philosophically tenable solely as the spectral inverse of idealism, accompanying it as the shadow cast by idealism's insurmountable incompleteness.”74 But the stark consequences of this thesis must be brought to the fore: it claims that all forms of radical idealism always already contain traces of materialism in their blind spots, even if they do not recognize this. Yet how are we to grasp this overlapping of the Real and the Ideal, this contradictory mode of inclusion/exclusion, internal/external, presence/ absence, from within a logical space that by its very essence proclaims to exclude it? How can this impossible feat of a materialism within the cracks of idealism be accomplished? Chapter 3 According to Žižek, this truth is then later radicalized by Hegel's and Schelling's own response to Fichte's thinking insofar as they realize it gives us the necessary resources we need to develop a metaphysics while never leaving the transcendental idealist framework. Žižek's own theoretical position is therefore a rather precarious one. It contends that it is only from within the deadlock and limitations of a full Žižek's own theoretical position is therefore a rather precarious one. It contends that it is only from within the deadlock and limitations of a full- blown idealism asserting the ultimate irreducibility of mind and subjectivity Chapter 3 62 more, the very nature of idealizing activity must tell us something about the nature of the world as it is in itself and this in two ways: firstly, we cannot just understand our own inability to grasp the Real as a mere limitation due to the finitude of human cognition, but must see this inability as revelatory of an ontological state of affairs—the subject is, after all, an event in the world, such that our entrapment in the solipsistic circuitry of ideality is always already minimally revelatory of that which we are searching for once we make a parallax shift in perspective. Secondly, the very inconsistency of our notional apparatus points to the fact that there can be no absolute constructionism, which appears to epistemologically justify the possibility of using the internal limitations and obstructions of ideality to overcome ideality's very self-enclosure. The goal of Žižek's materialism is to seek to understand the precise philosophical status of the free ideal constitution of the world and how and why this autonomous register could collapse. In other words, its defining feature is that, although it strives to maintain the ontological significance of the irreconcilable difference between materialism and idealism, it searches for a way to overcome this absolute opposition at the epistemological level so that the conflict between them does not infringe upon knowledge. If the self-grounding gesture of idealism expresses a constitutive opposition to materiality, and its failure is indicative of the phantom-like presence of the latter in the former, then this disharmonious non-relation must reveal a method of explaining it. The underlying question guiding Žižek's ontology is therefore the following: What do the internal obstructions in idealization and the fact of ideality itself tell us about the mind-body relation: that is, our connection to the vital movement of being? Whereas there have been for some time in the contemporary intellectual scene two options for understanding the relationship of consciousness and world—their dynamic interconnectivity and unity in phenomenological accounts of the lived body or the outright rejection of the importance of lived first-person experience as a mere epiphenomenal effect due to the mechanical movement of nature or the structures guiding discourse, both of which comprise a disavowal of the primordial self-reflexive ipseity of the subject—Žižek opts for a third. 3.3 A Metaphysics of the Disjunctive “and” By zoning in on the limitations of idealization, the experiences of internal resistance within its own self-enclosed phenomenal space (experiences that reveal a difficult truth concerning the impotence of self-positing idealist freedom), Žižek tries to construct his own metaphysics. Only able to sustain itself from within the cracks of transcendental synthesis, his parallax ontology functions within the impossible in-between of spectral materialism and full-blown subjective idealism. But Žižek does not just claim that the latter is wholly untenable because its internal tension proclaims that there is necessarily an extimate, spectral presence of the Real within the Ideal haunting it due to its impotence to hallucinate its own world. What is Chapter 3 What is noticeable in all of these approaches is not only an inability to grasp the true insight unveiled by a transcendental freedom, but also the latter's direct Psychoanalysis and the Enigma of Transcendental Subjectivity 63 Psychoanalysis and the Enigma of Transcendental Subjectivity 63 consequence: namely, the experience of idealistic disintegration and failure, the two types of experiences being so crucial in Lacanian psychoanalysis for our understanding of psychopathologies and psychogenesis that for Žižek they demand the rehabilitation of the ontologically schismatic duality opened up by Cartesian subjectivity. These are not merely revelatory of the structure of an individual psyche lost in disarray, but rather reveal a basic metaphysical truth of subjectivity by presenting a world where “the mind and body are, so to speak, negatively related—oppositional discord is, obviously, a form of relation.”75 Pointing towards the subject being more than the matter it inhabits, insofar as the symbolic structures constituting psychic life display a quasi-absolute degree of freedom from purely naturalistic activity, the psychoanalytical experience proclaims that these two zones must resist, must be in perpetual conflict with, one another, so that the structure of psychoanalytical subjectivity is brought close to an archaic form of modern dualism while calling for a radical reconfiguration of the latter's split between mind and body. Accordingly, Žižek's attempt to think materialism and idealism requires a far-reaching remodulation of the logical conjunctive between the two into a form of psychotic non-relation, insofar as transcendentalism implies a kind of negative space isolated unto itself and alienated from external reality, an isolation that is simultaneously the logical structure of normal and pathological subjective reality. But what exactly is this disjunctive “and”? Žižek's answer is unequivocal: the place of non-coincidence between mind and body, the break or rupture between these two zones of independent activity, is nothing but the subject itself, where the subject is transformed from a mere transcendental- epistemological construct grounded through concerns in a theory of knowledge into some kind of self-positing negativity in material being, a bone in substance forever holding apart materialism and idealism. Psychoanalysis and the Enigma of Transcendental Subjectivity 63 The lacuna in contemporary thinking is therefore immense: by failing to take into account the experience of psychopathology whose possibility coincides with that of freedom (as that which institutes an infinite conflict between mind and body) its representatives fail to see the terrifying truth intrinsic to our very relation to the world: something that proclaims the need for a new paradoxical form of materialism developed within idealism to be able t consequence: namely, the experience of idealistic disintegration and failure, the two types of experiences being so crucial in Lacanian psychoanalysis for our understanding of psychopathologies and psychogenesis that for Žižek they demand the rehabilitation of the ontologically schismatic duality opened up by Cartesian subjectivity. These are not merely revelatory of the structure of an individual psyche lost in disarray, but rather reveal a basic metaphysical truth of subjectivity by presenting a world where “the mind and body are, so to speak, negatively related—oppositional discord is, obviously, a form of relation.”75 Pointing towards the subject being more than the matter it inhabits, insofar as the symbolic structures constituting psychic life display a quasi-absolute degree of freedom from purely naturalistic activity, the psychoanalytical experience proclaims that these two zones must resist, must be in perpetual conflict with, one another, so that the structure of psychoanalytical subjectivity is brought close to an archaic form of modern dualism while calling for a radical reconfiguration of the latter's split between mind and body. Accordingly, Žižek's attempt to think materialism and idealism requires a far-reaching remodulation of the logical conjunctive between the two into a form of psychotic non-relation, insofar as transcendentalism implies a kind of negative space isolated unto itself and alienated from external reality, an isolation that is simultaneously the logical structure of normal and pathological subjective reality. But what exactly is this disjunctive “and”? Žižek's answer is unequivocal: the place of non-coincidence between mind and body, the break or rupture between these two zones of independent activity, is nothing but the subject itself, where the subject is transformed from a mere transcendental- epistemological construct grounded through concerns in a theory of knowledge into some kind of self-positing negativity in material being, a bone in substance forever holding apart materialism and idealism. Psychoanalysis and the Enigma of Transcendental Subjectivity 63 The lacuna in contemporary thinking is therefore immense: by failing to take into account the experience of psychopathology whose possibility coincides with that of freedom (as that which institutes an infinite conflict between mind and body) its representatives fail to see the terrifying truth intrinsic to our very relation to the world: something that proclaims the need for a new paradoxical form of materialism developed within idealism to be able to combat phenomenological accounts of embodiment, postmodern theories Psychoanalysis and the Enigma of Transcendental Subjectivity 65 fundamental problematic guiding Žižek is consequently the following: what is the relationship between (i) this abyssal void of the I; (ii) the pre-symbolic, material X; and (iii) the mediating activity of conscious ideality? What are the “meta-transcendental” conditions of possibility for the emergence of the subject that could explain the systematic discord that persists between mind and body? Can the psychoanalytical notion of the Todestrieb as a form of biological malfunction and its avatars in the tradition tell us something about the extra-ideal state of affairs logically preceding experience? But in order to grasp the stakes at hand in this endeavour, we must first understand the problem of nature bequeathed by Lacan and how it sets up the coordinates of his own procedure. Chapter 3 64 of discourse, and cognitive science, a materialism that would be at the same time a metaphysics of the disjunctive “and.” Since it is Lacanian psychoanalysis that most strongly brings forth this disjunctive “and” as an irreducible element of human experience, Žižek's controversial thesis is not merely that Lacan brings to the fore something previously underdeveloped or neglected in other theories of subjectivity. Rather, it is much stronger—he claims that Lacan constitutes a true breakthrough in the history of thought that irreversibly restructures the field within which we can do philosophy, in a gesture that rivals that of Kant. Yet the great downfall of Lacan is that he is only interested in the systematization of the breakdowns and structural inconsistencies that define psychoanalytic experience, the antagonistic interaction of the three registers as the condition of the (im)possibility of phenomenal reality, and largely leaves unanswered the ontological question of the subject. Realizing that the Lacanian subject is lacking any explicit account of its genesis—which leads many commentators to argue that Lacan is a transcendental idealist or linguistic phenomenalist—Žižek is driven back to German Idealism to do away with this theoretical deficiency. But if German Idealism, alongside Lacanian psychoanalysis, is able to help us elucidate the paradoxical “and” negatively linking together materialism and idealism, it is because there must be something homologous in their attempts to think the subject that would allow them to be creatively read through one another. By drawing out various passages that link the basis of subjectivity in late modern philosophy to fundamental concepts of psychoanalysis, Žižek gives flesh to this disjunctive “and” by showing how the very fact of the existence of the field of ideality illustrates the necessity of the existence of the former, a fact from which many recoil. In this respect, if the Symbolic cannot assert itself as all, it is not only because of the ever-present pressure exerted from the inside upon its constitution of phenomenal reality, but also because the very “and” that precedes and sets the stage for the autonomous universe of meaning prevents the latter from being able to posit itself as a self-sustaining, total positivity. Idealization always collapses upon itself, and it is due to its self-foundering that we are led to see that there is a disharmonious relationship between mind and body that requires a new materialism to complement its internal limitations. The 73. Jacobi, “Idealism and Realism,” in Kant’s Early Critics: The Empiricist Critique of the Theoretical Philosophy, ed. Brigitte Sassen (Cambridge: Cambridge University Press, 2000), p. 175. Notes 64. Lacan, “A Theoretical Induction to the Functions of Psychoanalysis in Criminology,” in Écrits, p. 127/104. 65. Žižek, The Parallax View, p. 168. 66. Žižek, Less Than Nothing, p. 905. 67. As Žižek points out, as soon as Descartes makes the breakthrough to the cogito, he is forced to conclude: “[b]ut I do not yet understand sufficiently what I am—I, who now necessarily exists.” What follows is a discussion that prevents the confla- tion of the cogito with any specific experiential content—in short, the recognition of its intrinsic vacuous nature. However, Descartes is unable to hold onto this groundbreaking insight and quickly ends up reifying the subject into a res cogitans. See Meditations on First Philosophy, trans. Donald A. Cross (Indianapolis: Hackett Publishing, 1993), pp. 18ff.; and Žižek, “Cartesian Subject versus Cartesian Theater,” in Cogito and the Unconscious, ed. Slavoj Žižek (Durham: Duke University Press, 1998), pp. 259–61. 68. On Žižek’s reading, not only is any intimate self-relation in Kant precluded in- sofar as the self qua phenomenal is denied any experience of its “true being,” but subjectivity is reduced to a positive void: the ground of self-reflexive consciousness becomes a mysterious “this I or he or it (the thing) that thinks” that strangely guarantees the condition of possibility of experience. See Kant, The Critique of Pure Reason, A346/B404; and Žižek, Tarrying with the Negative: Kant, Hegel, and the Critique of Ideology (Durham: Duke University Press, 2003), pp. 12–18. 69. This concept will be explored in detail in chapter 6. 70. Žižek, The Abyss of Freedom, p. 45. 71. See Johnson, Žižek’s Ontology, pp. 16–20. 72. Žižek, The Abyss of Freedom, p. 45. 74. Johnson, Žižek’s Ontology, p. 19. 75. Ibid., p. 56. This is Johnston’s thesis about the nuance of Lacanian-Žižekian subjectivity. Chapter 4 The Problem of Nature in the Lacanian Subject The Obscure Origins of the Symbolic Chapter 4 The Problem of Nature in the Lacanian Subject The Obscure Origins of the Symbolic In this chapter I will lay out various theoretical problems plaguing Lacan that serve as the starting point for Žižek's own endeavour. Beginning with a precursory analysis of Žižek's identification of the Cartesian and psychoanalytical subjects, I move on to a discussion of the intrinsic limitations of Lacan's psychoanalytical methodology for understanding the ontologico-foundational basis of subjectivity. By focusing on the self- grounding, non-natural function of images and words, especially the transcendental reflexivity underlying both, Lacanian psychoanalysis has a tendency to perceive them as an external parasite that derails the substantial unity of the body. But this tendency renders problematic any account of how images and words could successfully implant themselves, thus leaving the Symbolic itself potentially without theoretical justification. Seeing an uncanny structural homology here between post-Kantian idealism and the legacy bestowed upon us by Lacan, Žižek returns to Hegel and Schelling with the conviction that a combination of psychoanalysis with late German Idealist metaphysics could help us articulate the paradoxical emergence of subjectivity. 68 Chapter 4 68 4.1 An Uncanny Pair: The Cartesian and Psychoanalytical Subjects Lacan's and Žižek's situating of the psychoanalytical discourse within the trajectory of modern transcendental philosophy could appear surprising. Following the works of Freud, it is clear that the father of psychoanalysis understands the psychoanalytical subject as intrinsically anti-Cartesian. Whereas the cogito conjures forth the idea of an untouchable conscious freedom, of complete self-mastery and perfect self-transparency as guaranteed by our capacities of reason, ratiocination, and deliberation, the Freudian unconscious does not merely haphazardly obstruct existential self-familiarity like some kind of accidental feature of human being, but rather puts all conscious activity in the clutches of a mysterious, obscure Other that by definition resists all ideational (re)presentation. The goal of modern philosophy as the articulation of a sound foundation for systematic philosophy through the free mediation/idealization of the world by means of the rational, self-grounding activity of consciousness is by definition foreclosed, for the very process of (re)constituting reality by the I rests upon a structurally non-representational process, a process that constantly risks interfering with the subject's synthetic mediation of self and world. It is no longer the subjective I that “thinks,” but the impersonal, non- subjective Es (the id): the “it” thinks in the same way that “it” rains. So how can “the subject of the unconscious [be] none other than that of the Cartesian cogito”?76 Although this may be true it is evident that, despite the qualitative remodulation that subjectivity undergoes in Freudian psychoanalysis, especially in terms of the nature and scope of conscious personality, there is nevertheless a series of homologous structures at the heart of both the psychoanalytical and modern Cartesian subjects. Most fundamentally, both rely on the splitting of being into two separate zones, the mental and the material, and the inability to reduce one to the other. Even if Freud's later theoretical works exhibit a propensity to reduce psychic processes to the vital force of biological activity, this very attempt self-destructs (as Lacan makes clear) in the face of the brute autonomy of images and words in his analyses over and against their subsumption within the pulsating activity of nerves or the movement of instincts. There always remains a chasm The Problem of Nature in the Lacanian Subject 6 70 Chapter 4 In Žižek's view, the theoretical advantage of Cartesianism and psychoanalysis is their defence of the profound ontological difference between the registers of objective material corporeality and (inter)subjective cultural and psychic experience. Difference is just another word for an irresolvable two-way resistance that constitutes a battleground of conflicting, oppositional systems and thereby sets the very stage for the experiential reality that we find ourselves thrown into. To speak in Freudian parlance, one cannot understand consciousness merely in terms of the id-forces that comprise the bodily substrate of the libidinal-material basis of human existence. Although they may serve as the ontologico-foundational basis for all psychic and ultimately phenomenological life, they are philosophically inadequate to explain the full essence of personality and its pathologies insofar as the latter are constituted through the antagonistic interaction of forces that emerge within the tripartite field of id, ego, and superego. None of these alone would be able to articulate what it is to be a human subject, but must be implicated within an intricate psychological trinitarian logic if we are to hope to come to terms with ourselves and our world of experience. Even though the ego and superego arise out of the pre-subjective activity of the id—the id immanently generates the ego out of itself as a way to cope with its own organic insufficiency and primordial Hilflosigkeit to deal with the demands of its external environment—they are not dependent upon it for their being, their very vitality, each constituting a self-subsisting system enjoying a level of genuine autonomy and irreducibility to the other. This very structure of independence is also hinted at in the breakthrough that is the Cartesian subject, which argues that although we could attempt to understand the ideality of spirit by recourse to the mechanics of nature, this attempt would ultimately collapse upon itself because something essential would be lost. It is not that the mechanical externality that forms the causal determinacy of the body's extensive field as the obscure ground of mind and its sensations is a mere underdetermination of mental activity. 70 Chapter 4 Descartes’ claim is much stronger: it is ultimately incapable of helping us establish the nature of subjectivity, even if some kind of paradoxical non-relation must nevertheless be said to hold between the two; the cogito's founding gesture exhibits an irreducible self-grounding that rejects mechanical externality in all of its modes (or however it may be reworked by In Žižek's view, the theoretical advantage of Cartesianism and psychoanalysis is their defence of the profound ontological difference between the registers of objective material corporeality and (inter)subjective cultural and psychic experience. Difference is just another word for an irresolvable two-way resistance that constitutes a battleground of conflicting, oppositional systems and thereby sets the very stage for the experiential reality that we find ourselves thrown into. To speak in Freudian parlance, one cannot understand consciousness merely in terms of the id-forces that comprise the bodily substrate of the libidinal-material basis of human existence. Although they may serve as the ontologico-foundational basis for all psychic and ultimately phenomenological life, they are philosophically inadequate to explain the full essence of personality and its pathologies insofar as the latter are constituted through the antagonistic interaction of forces that emerge within the tripartite field of id, ego, and superego. None of these alone would be able to articulate what it is to be a human subject, but must be implicated within an intricate psychological trinitarian logic if we are to hope to come to terms with ourselves and our world of experience. Even though the ego and superego arise out of the pre-subjective activity of the id—the id immanently generates the ego out of itself as a way to cope with its own organic insufficiency and primordial Hilflosigkeit to deal with the demands of its external environment—they are not dependent upon it for their being, their very vitality, each constituting a self-subsisting system enjoying a level of genuine autonomy and irreducibility to the other. This very structure of independence is also hinted at in the breakthrough that is the Cartesian subject, which argues that although we could attempt to understand the ideality of spirit by recourse to the mechanics of nature, this attempt would ultimately collapse upon itself because something essential would be lost. It is not that the mechanical externality that forms the causal determinacy of the body's extensive field as the obscure ground of mind and its sensations is a mere underdetermination of mental activity. The Problem of Nature in the Lacanian Subject 69 69 separating human psychological life from the mere flux of corporeal or instinctual energetics, because ideality and materiality are operationally different registers in terms of their inner articulation or logical makeup. For Žižek, it is not a question of the actual doctrine espoused by Descartes with respect to his own doctrine of the cogito and the essence of consciousness it entails, but rather of various theoretical potentialities and possibilities exhibited by the very structure inherent in his concept of subjectivity, which come to the fore in the aftermath of psychoanalysis. Consequently, what Žižek perceives in the Cartesian subject and its various reworkings is the possibility of extracting theoretical resources from it that could be used to help explicate the enigma of human freedom and the essence of personality as revealed in the psychoanalytical experience of the conflictual (non-)relation that holds between mind and body. In Cartesianism and psychoanalysis, we do not come across a mere conceptually regulative difference between the arena of human subjective activity and the raw processes of brute material nature, which would enable both to enjoy a strong degree of epistemic autonomy insofar as both could self-actualize themselves in terms of a standalone language game with the appearance of being intrinsically incommensurate to one another despite being on the ontological level fundamentally one. Žižek is unwilling to reduce this difference to the level of useful “working distinctions” that have the status of an “as if,” for consciousness refuses to be completely naturalized. In a similar vein, in contradistinction to a theory of monism according to which both are merely seen as attributes of a single, overarching whole (however it may be conceived) and whereby both would offer differing expressions of an ultimately identical self-same reality, Žižek wants to bestow ontological heft upon the difference. The claim is that any account of subjectivity that attempts to reduce conscious experience to the register of a pure material or substance caught within self-sustaining laws, whether it be the neuronal Real, vitalistic energetics, or nature as all, not only does not take heed of the radical metaphysical implications of the psychoanalytical experience, but is also by consequence unable to propound a satisfactory account of what it is to be a human and our relation to the world. 70 Chapter 4 Descartes’ claim is much stronger: it is ultimately incapable of helping us establish the nature of subjectivity, even if some kind of paradoxical non-relation must nevertheless be said to hold between the two; the cogito's founding gesture exhibits an irreducible self-grounding that rejects The Problem of Nature in the Lacanian Subject 71 reinterpreting it as nature, vitalistic energetics, the neuronal Real, etc.) in a process that not only makes it distinctively non-natural in terms of its inner structuration, but also thereby guarantees its capacity for transcendental self-determination and its incommensurability with other logical registers. The question is how we can explain this ontological difference opened up by Cartesianism with the aid of psychoanalysis, and vice versa. 4.2 Lacan, antiphusis, and the Parasite of Images and Words In order to expand upon this problematic, two comments can be made that will enable us to uncover the conceptual limitations inherent to the psychoanalytical theory of subjectivity and that allow us in turn to draw out its similarities with the central conceptual problems plaguing post- Kantian German Idealism as bestowed upon it by its Cartesian heritage. First, for psychoanalysis, the language that we use mediates our very experience of the biological constitution of the body in such a way that there is a radical degree of separation between the idealization of the givenness of the flesh and its objective, extra-subjective existence in itself. Freud speaks of people who suffer from paralysis of a certain part of their body, although scientifically speaking the paralysis does not coincide with where the nerve anatomically ends, and when this is pointed out the pain ceases. Here it is the common, everyday belief of where a nerve ends and begins that structures the symptom. Thus, understood as a complex cultural phenomenon and not just grammatical structures of syntax and morphology, language shows itself as an irreducible element of experience and points to a discordant relationship between mind and body. Second, as arising from the facticity of cultural, historical experience, language appears to emerge out of a symbolic interpersonal network of meaning that operates according to its own intrinsic and self-unfolding logical matrix. Even if it has a basis in our physiology (vocal folds) and neurology (language centres in the brain), words themselves appear to exist in an autonomous world of their own that, in many ways, shows itself as a kind of external parasitic attachment to the biological unity of the body. Taken together, these two points assert more than the fact that both registers, the Symbolic and the corpo-Real, exist in disunion: the former even appears to manifest itself as a “psychotic” withdrawal from the latter into some kind 2 Chapter 4 72 of self-enclosed sphere. What is obvious from this is not only the intrinsic Cartesian structure of the psychoanalytical subject—here the deadlock of ontological dualism is amplified, brought to its extreme, by the advocation of a constitutive dissonance—but that the latter's philosophical legitimacy depends upon the precise articulation of the delicate relation between these two mutually incommensurate spheres. The Problem of Nature in the Lacanian Subject 73 73 the Husserlian transcendental ego grounds itself and the fabric of the world of its experience in a moment of absolute freedom that establishes an insurmountable rupture between reflexive self-consciousness and the causal mechanics of nature, within Lacanian psychoanalysis we encounter the same logical move, but merely displaced. In the latter it is not a subject in any traditional sense, but rather the self-generating play of signifiers within the web of the Symbolic that expresses the impossibility of articulating a comprehensive, satisfactory psychological theory of the self only by recourse to the pulsations of neurological activity or biological instincts. There are two principal reasons for this. First, the Symbolic constitutes the possibility of the orbit of the Imaginary within phenomenological experience in such a way that it autonomously mediates all contact with the outside world, thus diminishing the latter's importance; second, as a consequence, it represents the predominance of non-natural (= irreducible to the ebb and flow of the material-objective world) influences in the explanation of the essence of psychic life. It is in this precise manner that the ciphering activity of the Symbolic can be said to obey its own self-grounding transcendental logic and be that which sustains the primordiality of the real-ideal divide: “subjectivity has no relationship to the Real, but rather to a syntax which is engendered by the signifying mark.”77 Accordingly, one can now understand why Lacan defines the object of psychoanalysis in “The Direction of the Treatment and the Principles of its Power” as “antiphusis.”78 With this terminological choice he is drawing our attention to the fact that what analysis deals with is not something natural in any sense of the nature investigated by science or understood by our common sense. Analysis operates at a level that is, by definition, incommensurate with natural laws. The effect of this designation is much greater than it may initially appear, for the human subject is characterized as “non-natural” not only insofar as the structural fabric forming the base of conscious experience enjoys a freedom over and above, or an ontological heft irreducible to, the flux of nature, but also because it transcendentally alienates us from the immediacy of the corpo-Real of our body, the ground of our objective substantial being, as the very condition of our possibility of what we call experience. 4.2 Lacan, antiphusis, and the Parasite of Images and Words Although these zones are infinitely conflictual and never to be harmonized, they must nevertheless be somehow related if the system is to be held together. The Cartesian problem of the mind-body relation is consequently inverted: the issue at hand is not how both can be in unison with one another through some kind of occasionalism, but how the conflictual gap that separates them emerged in the first place so that there can never be a harmony or overlapping of each sphere. Both Freud and Lacan spend the majority of their careers elaborating this field of disparate spheres whose interaction generates and constitutes personality. Rereading Freud through contemporary developments in linguistics and structuralism and even drawing upon cybernetics, Lacan attempts to reconfigure our understanding of the self in what he believes to be a more scientific manner than Freud could by excavating the strict logical structures constituting the condition of the possibility of the psychoanalytical subject: that is, the irreducibly non-natural basis of experience and the mind-body discord it reveals. There is something intrinsically transcendental about the project, even if the end result is apparently quite removed from the Kantian analysis of consciousness in the Critique of Pure Reason, Fichte's Wissenschaftslehre, or later attempts such as Husserl's transcendental idealism. Any project that advocates the autonomy of subjectivity over materiality and a form of self-grounding idealization or symbolization of reality must at some level seem so, for the question of the condition of the possibility of experiential reality does not pose itself in more “realist-inclined” philosophies insofar as experience is here seen as just the effect or another instantiation of a large set of dynamic relations between objects in the world. As a consequence, fighting against Freud's attempt to biologize/naturalize the unconscious by highlighting that one can never escape the ultimate primacy of images and of words in psychoanalysis, Lacan relentlessly maintains what he perceives as the Cartesian essence of subjectivity. Just as the Kantian, the Fichtean, and even to a certain extent The Problem of Nature in the Lacanian Subject The Problem of Nature in the Lacanian Subject 73 Images and words not only precede us historically in terms of the temporal genesis of our identities (we are often named before Chapter 4 74 we are conceived: a space is always already carved up for us in the symbolic universe before we can be said to exist in the Real) but also structure the logico-formal space that makes personality possible in such a way as to prevent us from having a genuine access to the brute reality of our own “intimate” being as it is an sich. After the I is spoken, that is, after self- reflexive subjectivity emerges, an experience of the Real is only possible through the ideational (re)presenting capacity of a symbolic network of signifiers, thus becoming impossible: a mysterious, ever-elusive X. As soon as we enter language, the Real as a time of “fullness,” “immediacy,” and “complete immersion,” if ever there was one, is subsequently lost forever, leaving us trapped within the fragmented, ontologically solipsistic world opened up through the Symbolic-Imaginary matrix. Insofar as the symbolic network simultaneously constitutes the underlying structural support of experience and mediates all our contact with an external, subject-independent reality, Lacanian psychoanalysis appears to be a completely self-grounding transcendental idealism in the spirit of Kant. Whatever the human subject is, it is not natural in the same sense as other objects within phenomenal reality, even those that appear to have some kind of spontaneous connection with that which they find around them. Indeed, according to Lacan even animals have a kind of knowledge in the Real79 that is missing in us due to the Symbolic. Although language is intimately linked with “our” freedom—and “our” madness, we must never forget, the two being structurally identical—nevertheless it thus appears as some kind of alien, foreign presence that cannot be understood in terms of the categories of the world because it has no analogue within it. But then the question arises: where do these transcendental structures come from? How and why do they irredeemably separate the human subject from the vital ebb and flow of nature? The Problem of Nature in the Lacanian Subject 73 As a science of the symbolic network that constitutes the nature of personality and self-conscious experience, if psychoanalysis is unable to articulate the origins of these non-natural structures and the process of their genesis, it itself rests groundless insofar as the object of its inquiry remains unable to explain its own theoretical, ontogenetic basis: that is, how it itself is possible as a discourse. As Fichte, Schelling, and Hegel looked upon Kant's critical philosophy, they themselves were taken by a similar problem concerning the heritage The Problem of Nature in the Lacanian Subject 75 bestowed upon them. They asked themselves how the transcendental structures of subjectivity could be explained in a more comprehensive manner, how and why they emerged in the first place, and why they were as such necessary for the very possibility of experience. They held the brute givenness of the categories of understanding to be philosophically unacceptable and took it upon themselves to explore the Achilles' heel of the entire critical system. Their guiding intuition was that by undertaking an explanation of their genesis they would not only save the Kantian breakthrough, but also delve into the wider implications of the subject as propounded by it. Similarly, there is a brute givenness in Lacan's account of the transcendentality of the symbolic matrix because he never gives an account of its origins. When one reads his early and middle works, one is consequently struck by the impression that images and words are a purely external force that attacks the raw unity of the body and devastates it from the outside, thereby creating a denaturalized human subject out of a preexisting substantial whole. There is a movement from uncontaminated instinctual being within the positive order of the Real—the oneness of the body, the immediacy of an organic nature—to a being that speaks, to words as intruders that radically split the subject, destroying its immersion in objective being. The network of the Symbolic comes from an infinite elsewhere ravaging the unity of substance and precluding the possibility of a biologically closed libidinal economy as an inborn, innate movement of energy defined by materially articulated, instinctual schemata. The Problem of Nature in the Lacanian Subject 73 Whereas in the animal kingdom this schema directs bodily energy towards various objects that it needs within its immediate environment through a kind of immanent causal push, language eternally obstructs the dialectic of need and satisfaction, fullness and lack, by instituting an ontological going-haywire in being. Preventing the flow of the search for homeostatic constancy, the subject is thrown into the endless deferral of desire and structurally loses the possibility of attaining its object. Instead of predetermined, biological goals, we are left with the objet petit a: [T]he standard Lacanian theme in the 1950s and 1960s was h bl i i b h i l i [T]he standard Lacanian theme in the 1950s and 1960s was the unsurmountable opposition between the animal universe of imaginary captivity, of the balanced mirror-relationship between Innenwelt and Aussenwelt, and the human universe 76 Chapter 4 of symbolic negativity, imbalance. Lacan thereby fully participates in the line of thought that begins with Hegel, according to which man is “nature sick unto death,” a being forever marked by traumatic misplacement, thrown “out of joint,” lacking its proper place, in contrast to an animal which always fits into its environment, that is to say, is immediately “grown into” it. Symptomatic here is Lacan's “mechanistic” metaphorics: an almost celebratory characterizing of the symbolic order as an automaton that follows its path, totally impervious to human emotions and needs—language is a parasitic entity that battens on the human animal, throwing his or her life rhythm off balance, derailing it, subordinating it to its own brutally imposed circuit.80 Within this advocacy of a split between the cyclical, balanced world of nature and the derailed being of man, the following theoretical concerns immediately emerge: how does the symbolic-imaginary matrix give rise to itself through a transcendental act of self-positing? How is it able to take control of and infiltrate the body, apparently lacerating its objective unity? If the symbolic order is really an external force, an alien blow, that in some way tears apart or obstructs the body's knowledge in the Real, how is it able to sustain itself in face of the corpo-Real of the biological organism? The Problem of Nature in the Lacanian Subject 73 To put it another way, why is it not “rejected by this economy in a manner analogous to failed organ transplants”?81 The problem of the psychoanalytical constitution of human experience opens up unto the extra- psychological/meta-transcendental conditions or state of affairs that must have preceded the emergence of the subject if we are to give a complete account of the psyche as such. Psychoanalysis must explain its own beginnings in a lost ontological time in a move similar to how the categories of the understanding must not be taken as mere elementary facts liberated from the theoretical exigency of explaining their genesis. Just as the history of the reception of Kantian idealism has shown that it must open up onto a comprehensive metaphysics that is inclusive of it, so too we must try to develop a metaphysics in the wake of psychoanalysis. The Problem of Nature in the Lacanian Subject 77 4.3 German Idealism, Psychoanalysis, and the Quest for the Birthplace of the Transcendental Yet after the 1950s and 1960s, we see within Lacanian psychoanalysis a developing preoccupation with the ontologico-foundational basis of the subject. After the period of Lacan's “The Direction of the Treatment and the Principles of its Power” (1958), which defines the object of psychoanalysis as “antiphusis,” there is a profound shift in the meaning of this concept that is highly significant for the development of a metaphysics capable of explicating and thus justifying the psychoanalytical subject. Whereas the early to middle Lacan focuses on the derailing capacity of images that set the stage for words as that which parasitizes the corpo-Real of the body, which makes the human subject radically non-natural by preventing its immersion in the autopoietic, self-regulating unity of positive organic being by creating an alienating self-distance within it, we encounter in the late Lacan a reconceptualization of the problematic. His new emphasis on the Real proclaims that one must radicalize this notion of self-distance. The object of psychoanalysis and by consequence the human subject are not antiphysical because images and words are an infinite Other to nature, an external alterity that arises ex nihilo only to penetrate into its secret chamber like a vandal and deface its sacred inviolability. The incommensurability lies elsewhere: images and language cannot be the cause of the denaturalization of the human subject; there must be something in nature itself that immanently moves it toward denaturalization. In short, nature itself is antiphusis, self-sabotaging, self-lacerating. Although this represents a fundamental change in the meaning of the category of the non-natural, it only serves as a beginning. Insofar as the psychoanalytical experience is engulfed by images and words, which means that it is predominately ridden by non-biological influences, the only way to philosophically ground the primacy of psychoanalysis is to elaborate an underlying ontology that could explain how the subject could have emerged out a biological field that it presents itself as irreducible to. The theoretical merit of the Lacanian project is dependent upon the articulation of the workings of substantial reality that renders possible the birth of a more-than-material subjectivity out of its material Grund (to borrow an expression from Adrian Johnston), a paradoxical ontologization of the Chapter 4 78 transcendental structures that constitute and found experience by separating themselves from and effacing their ontological foundation. 4.3 German Idealism, Psychoanalysis, and the Quest for the Birthplace of the Transcendental If our generation of The Problem of Nature in the Lacanian Subject 79 experience proves itself to be unable to posit itself as an absolute and unbridled hallucination of objects, then we must encounter some kind of paradoxical limit to our autonomous ciphering and constitution of phenomenal reality from within; the impasses obstructing the self-grounding idealization of the world demonstrate that, although we are forever stuck within ideality, we are not simply prisoners of the completely solipsistic sphere of the self-referential, masturbatory play of thought within thought and that a metaphysics of the Real, an account of the noumenal, appears to be theoretically possible. The inassimilable kernel of the Real within our notional, symbolic code points to the paradoxical negative coinciding of inside with outside, the Real and the Ideal, within thinking: the cracks of ideality cast an abyssal shadow that opens up onto the materiality of being, albeit only as refracted through the impossibilities of the Ideal, in such a way that tarrying with the latter offers a way to develop idealism into a science of the Real. Idealism has been overcome because the materialism-idealism distinction has become intra-discursive, that is, internal to idealism itself. In this sense, we should not understand Žižek's proclamation of Hegel's “transcendentalism” to say that Hegel, like Kant, remains at the level of a pure subjectivism: due to the constitutive movement of notional tension intrinsic to the concept of dialectics, not only is idealism prevented from being subjective, but, more strongly, “[t]he opposition between idealistic and realistic philosophy is therefore without meaning,” which is why idealism can be said to be absolute.83 Embracing the paradox of the critical system, its breakthrough articulation of the irreducible idealizing activity of consciousness, the mediative structures that freely transcendentally constitute the fabric of experience, Hegel's philosophy is a daring attempt to rethink the possibility of a metaphysics in the wake of the Kantian revolution, to carve a space between idealistic ontological solipsism and a speculative materialism within the former. It is only within such a space that the question: “What is the transcendental subject's relation to nature?” or, in Lacanian parlance, “What is the Symbolic's relation to the pre-symbolic Real?” can be asked. 4.3 German Idealism, Psychoanalysis, and the Quest for the Birthplace of the Transcendental It is exactly this problematic that thematically binds together the theoretical concerns of the psychoanalytical and German Idealist traditions, for according to the latter the only way to save the breakthrough of the Kantian critical system is to find a way, from within the self-founding idealism that it advocates, to reach out into the extra-subjective Real, or to speak in its parlance, the field of noumena, and ground it within a system that does not represent a return to previous modes of thinking (naïve dogmatic realism) but rather somehow manages to reach the Real through the Ideal despite the self-enclosed, self- subsistent ideal activity of consciousness. But how? The very trap in which we find ourselves proves to hold the way out: We can now see in what sense Hegel's logic remains “transcendental” in the strict Kantian sense—that is, in what sense its notional network is not merely formal, but constitutive of reality itself, whose categorical structure it describes. Hegel's Logic is the inherent tension in the status of every determinate/limited category: each concept is simultaneously necessary (i.e. indispensable if we are to conceive reality, its underlying ontological structure) and impossible (i.e. self-refuting, inconsistent: the moment we fully and consequently “apply” it to reality, it disintegrates and/or turns into its opposite). This notional tension/contradiction is simultaneously the ultimate spiritus movens of “reality” itself: far from signalling the failure of our thought to grasp reality, the inherent inconsistency of our notional apparatus is the ultimate proof that our thought is not merely a logical game we play but is able to reach reality itself, expressing its inherent structuring principle.82 Looking back at the history of the inner development of modern transcendental philosophy, Žižek sees a structural homology to his own task of searching for the ontogenetic condition of the possibility of the psychoanalytical subject, a question left unanswered by Freud and Lacan, in the tradition's attempt to find a way to construct a metaphysics from within the internal deadlock of transcendental idealism. 4.3 German Idealism, Psychoanalysis, and the Quest for the Birthplace of the Transcendental Žižek's retrieval of the cogito and his reactualization of German Žižek's retrieval of the cogito and his reactualization of German Idealism are therefore irrevocably caught up in his own attempt to develop a metaphysics of the Real able to explicate the obscure grounds of the Chapter 4 80 psychoanalytical subject. Attempting to excavate the ontological edifice implied by the latter, his more overtly theoretical works pass through modern to late modern philosophy by means of an intuition of a basic structural parallelism binding together such seemingly different traditions. Although the starting point is always psychoanalysis—“psychoanalysis is ultimately a tool to reactualize, to render actual for today's time, the legacy of German Idealism”84—it is clear that Žižek does not understand his work as an intrusion or unmethodological tearing apart of the tradition according to extrinsic philosophical principles he is imposing upon it. In fact, for him, the psychoanalytical subject is not merely a direct consequence of German Idealism's grounding insights—there is actually something intrinsically identical in these traditions, so much so that it seems that the only way to fully realize the former is to supplement it with the latter and vice versa, insofar as they are both really dealing with the same central issues that each fail in their own way to adequately thematize. Describing his own project, Žižek says: If you were to ask me at gunpoint, like Hollywood producers who are too stupid to read books and say, “give me the punchline,” and were to demand, “Three sentences. What are you really trying to do?” I would say, Screw ideology. Screw movie analyses. What really interests me is the following insight: if you look at the very core of psychoanalytic theory, of which even Freud was not aware, it's properly read death drive—this idea of beyond the pleasure principle, self- sabotaging, etc.—the only way to read this properly is to read it against the background of the notion of subjectivity as self-relating negativity in German Idealism. 4.3 German Idealism, Psychoanalysis, and the Quest for the Birthplace of the Transcendental That is to say, I just take literally Lacan's indication that the subject of psychoanalysis is the Cartesian cogito—of course, I would add, as reread by Kant, Schelling, and Hegel.85 By zoning in on this basic identity, Žižek's philosophy doesn't lose methodological rigor, as if it made him blind to other, more broad-reaching concerns in the tradition, thereby reducing his thinking to a mere haphazard picking out of various conceptual structures and ideas that are useful for his own project to the neglect of others. Rather, it enables him to show The Problem of Nature in the Lacanian Subject Chapter 4 82 not only questions our conventional reading of this movement, but more fundamentally challenges our very conception of ourselves and the world. not only questions our conventional reading of this movement, but more fundamentally challenges our very conception of ourselves and the world. The Problem of Nature in the Lacanian Subject 81 81 how there is an unconscious, disavowed Grundlogik that repeats and self- unfolds throughout modern transcendental philosophy and finally comes out into the open with the advent of psychoanalysis, which shows itself as the culmination of this entire lineage. Because Žižek perceives some kind of self-developing truth inherent within the tradition that binds it together with psychoanalysis, his reading is pushed on by a sense of fidelity to the movement as such so that his return to German Idealism resembles Fichte's, Hegel's, and Schelling's return to Kant86 just as much as Lacan's return to Freud: what is at stake is never merely textual faithfulness, but a hidden kernel of truth that has been simultaneously opened up and obscured by the tradition itself. The effect here is twofold: while psychoanalysis allows us to reconstruct retroactively the Grundlogik inherent in the German Idealist account of subjectivity by bringing to the fore the relevance of previously underemphasized concepts and textual moves, this reconstruction of its fundamental concepts also allows us to elaborate our understanding of the psychoanalytical subject. It is not merely that psychoanalysis reveals a hidden unconscious logic at work throughout tradition; the backward retroactive glance is simultaneously a forward-looking task that tries to pave the way for the new. Žižek's investigation into the history of philosophy is never a mere activity of philological exegesis, the retrieval of the “primordial” meaning of a text, but a creative generation of new concepts that, paradoxically, must be said to have been already present in the now lost factical past, yet to have existed there in the paradoxical temporality of a futur antérieur (“I will have been”) whose contours are now first visible in the après-coup reconstruction of said past. A repetition is always the establishment of a difference, so that the legitimacy of Žižek's reading rests not so much upon his apparent “lack” of faithfulness to the words or the spirit of the great German philosophers, but how this temporal intersection of philosophico-psychoanalytical interpretation, the space within which Žižek effectuates his reactualization, functions and realizes itself. But what is the true breakthrough of the German Idealist and psychoanalytic traditions and why can only a psychoanalytical dialogue between the two enable us to see the conceptual impetus underlying the former's unconscious Grundlogik and also to come to terms with the ontology of the subject? Žižek's answer Chapter 4 Notes 76. Žižek, Less Than Nothing, p. 632. 77. See Lacan, “Seminar on ‘The Purloined Letter,’” in Écrits, p. 51/38. I capitalize “the Real” for consistency. 78. Lacan, “The Direction of the Treatment and the Principles of Its Power,” in Écrits, p. 615/514. 79. Knowledge in the Real (savoir dans le réel) refers to the fact that “it is as if there is a knowledge of the laws of nature directly inscribed into the Real of natural objects and processes—for instance, a stone ‘knows’ what laws of gravity to obey when it is falling.” Žižek, How to Read Lacan (New York: W. W. Norton, 2007), pp. 74–75. 80. Žižek, The Indivisible Remainder, p. 218. 81. Johnston, Žižek’s Ontology, p. 271. 81. Johnston, Žižek’s Ontology, p. 271. 82. Žižek, The Ticklish Subject, pp. 98–99. 83. Hegel, The Science of Logic, trans. George di Giovanni (Cambridge: Cambridge University Press, 2010), p. 124. 84. Žižek, “Liberation Hurts.” 85. Ibid. 85. Ibid. 86. “The Kantian philosophy needed to have its spirit distinguished from its letter.” Hegel, The Difference between Fichte’s and Schelling’s System of Philosophy, trans. H. S. Harris and Walter Cerf (Albany: SUNY Press, 1977), p. 79. Fichte’s says the same thing: Kant should be “studied, not as the Kantians without exception have studied him (holding on to the literal text [...]), but rather on the basis of what he actually says, raising oneself to what he does not say but which he must assume in order to be able to say what he does.” Fichte, The Science of Knowing: Fichte’s 1804 Lectures on the Wissenschaftslehre, trans. Walter E. Wright (Albany: SUNY Press, 2005), p. 30. II Nature Torn Apart Nature Torn Apart Chapter 5 Kant, Todestrieb, and Beyond the Pleasure Principle The Unruly Basis of Transcendental Freedom Chapter 5 Kant, Todestrieb, and Beyond the Pleasure Principle The Unruly Basis of Transcendental Freedom In this chapter I begin an examination of Žižek's metaphysics by showing how the thematic intersection between German Idealism and psychoanalysis allows him to develop a highly original approach to the transcendental. Beginning with a brief discussion of Adrian Johnston's summary of Lacan's passing remarks on nature in largely unavailable seminars stemming from the late period, I then precede to analyze how these remarks lay the foundation for Žižek's own radical rethinking of the Symbolic-Real relationship. Trying to comprehend the emergence of the Symbolic, Žižek attempts to demonstrate that the very fact of its existence must be revelatory of some ontological process that set the stage for its immanent genesis out of the Real as an extimate Other, even if, in turn, it makes the very Real from which it arose an impossible concept. Perceiving the key for understanding this paradoxical point of discordant (non-) relation between the pre-subjective Real and the transcendental matrix of the Symbolic in the Todestrieb, and following Lacan's claim that “Kant's practical philosophy [is] the starting point of the lineage culminating in Freud's invention of psychoanalysis,”87 Žižek reactualizes the legacy of German Idealism in order to articulate his own parallax ontology through an intuition of a fundamental identity between Kantian transcendentalism and psychoanalysis as established in the former's concept of the primordial unruliness and diabolical evil at the core of subjectivity. 86 Chapter 5 86 5.1 From the Rottenness of Nature... Although Lacan lacks an explicitly developed philosophy of nature as a complement to his structuralist metapsychology, throughout his career we see a growing interest in and appreciation for the underlying ontology implied by his theory of the subject. Adrian Johnston succinctly describes the theoretical situation plaguing the late Lacan in his shift from the primacy of the Symbolic to that of the Real, a problematic of utmost importance for understanding the deficiency in Lacanian psychoanalysis that guides Žižek's own project: A psychoanalytically influenced theory of the subject that fails to furnish a basic delineation of human nature as the precondition for the genesis of subjectivity is groundless, incapable of explaining a foundational dimension of its object of inquiry. In the later seminars of the 1970s, a series of somewhat cryptic remarks testify to Lacan's awareness of the need to redefine nature itself in order to account for why human nature is predisposed to being thoroughly altered by the denaturalizing mediation of socio-symbolic structures. In both the twenty-first and twenty-fourth seminars, Lacan contends that nature is far from being entirely natural. However, this isn't just a slightly reworked reiteration of his earlier remarks from the 1950s about humanity's denaturalized nature. Rather than grounding his assertions here by invoking the externally imposed intrusion of images and signifiers as the ultimate cause of the denaturalization involved in subjectification, Lacan takes the additional step of pointing to something within nature itself that inclines it in the direction of its own effacement.88 What is missing in Lacan, however, is a fully worked out account of the consequences of this shift, a detailed investigation into the paradoxical ground of the subject intrinsic to the very gesture of psychoanalysis. Yet he makes a crucial advance by suggesting that the Real is not to be, despite the fact that we can only posit its existence from within the differential network Kant, Todestrieb, and Beyond the Pleasure Principle 87 Kant, Todestrieb, and Beyond the Pleasure Principle 87 of signifiers, merely taken as that which must be said to logically precede the emergence of the linguistic subject, but also as that which renders the latter in a certain sense possible by virtue of a self-destructive tendency always already within it that opens up the space for its infinite loss to self through the colonizing activity of images and words. We encounter a metaphysical thesis: subjectivity does not come on the scene as a scar inflicted upon an otherwise harmonious nature, as a disturbance of its symphonized order by means of a haphazard intrusion into its sphere of non-natural influences that produce an accidental zone of ontological non-coincidence. The psychoanalytical experience is, rather, revelatory of something much more primordial: namely, that nature itself must be always already antiphusis, self-sabotaging, self- lacerating, and responsible for its own demise in the human being's denaturalized essence. But why? Lacan provides only a few hints. At one point, he identifies “liberty” (liberté) with “the non-existence of the sexual relationship,” which, in light of the above, can be understood as indicating that the freedom enjoyed by the autonomous subject is made possible by the lack of an integrated organic foundation as the grounding basis of the subject's being. Similarly, several years later, Lacan speaks of nature as not all that natural due to being internally plagued by “rottenness” (pourriture), by a decay or defect out of which culture (as antiphusis) bubbles forth (bouillonner). Viewed thus, human nature is naturally destined for denaturalization. Put differently, more-than-material subjectivity immanently arises out of the dysfunctionality of a libidinal-material ground.89 Yet it would perhaps be erroneous to say that this theoretical awareness is limited to the late Lacan's move from the primacy of the Symbolic towards that of the Real. Kant, Todestrieb, and Beyond the Pleasure Principle 87 As early as 1949 in his work on the Imaginary and the mirror stage, Lacan had already said that “these reflections lead me to recognize in the spatial capture manifested by the mirror stage, the effect in man, even prior to this social dialectic, of an organic inadequacy of his natural reality—assuming we can give some meaning to the word 'nature,'” an inadequacy that points to “a certain dehiscence at the very heart of the organism, a primordial Discord betrayed by the signs of malaise and motor Chapter 5 88 uncoordination of the neonatal months”90 which is, in fact, responsible for the alteration we see in our relationship to nature in comparison with other animals insofar as it represents “the shattering of the Innenwelt and Umwelt circle”91 and thus functions as the true “intersection of nature and culture.”92 Here it is noteworthy to mention that the French word stade does not completely map onto the English stage. Although it does correspond in one of its principal meanings to the latter (a distinct stage in a process of evolution: les stades de la vie) it also means stadium (a terrain or area where something takes place) and thus signifies a primordial scene constituting the foundation or arena within/through which an activity unfolds. Consequently, Lacan's thesis is that the mirror stage can never be subsumed in a later phase of development, forcing us to conclude that, even in the early period, it must necessarily refer to some kind of self-effacing force immanent within nature that gives rise to and simultaneously sustains the ontogenetic condition of the possibility of the paradoxical emergence of a more-than-material subject. This force thereby institutes the infinitely denaturalizing process of flirtation with images and symbolic castration, so that organic discord in the motor coordination of the body is not a mere failure of the biological system but also a “positive” support that persists in its very non-naturalness even after the Imaginary and the Symbolic have taken hold as their dark origin. If the quasi-experience of dismemberment is to be taken as originary, as that which incites the libidinal investment of the captivating mirror picture the human infant sees of itself as the beginning of psychogenesis by letting itself be alienated by the Otherness of images and words, then nature here must also be seen—at least in the case of human being—as a festering, half-living corpse. Kant, Todestrieb, and Beyond the Pleasure Principle 87 The shift of emphasis in the late seminars is already contained within the founding texts of Lacanian psychoanalysis, which not only suggests their central thematic unity, despite the stark differences that they may exhibit, but more strongly a historical unfolding that follows an internal development conforming to the model of the Hegelian movement from the in-itself to the for-itself. But we should avoid looking at the various hints and suggestions in the l L h d h h f h i l difi But we should avoid looking at the various hints and suggestions in the late Lacan that gesture towards the character of the material edifice upon which the subject rests as a mere immanent elaboration of the implications of his previously laid out position, given that there are important changes Kant, Todestrieb, and Beyond the Pleasure Principle 89 Kant, Todestrieb, and Beyond the Pleasure Principle 89 of position in the development of his thinking. What is important is not the unity or disunity of Lacan, but rather the radicality and nuance inherent in his thinking of the subject as brought to the fore when we focus on this very specific constellation of problems hovering around the obscure relation between nature and the essence of human being, a constellation that proposes a frightening metaphysical conception of the world, albeit only implicitly. Synonymous with the irrevocable organic inadequacy of its biological prematurity at birth, and functioning as such as the basis for full-fledged subjectivity, the primordial Hilflosigkeit of the human infant already points toward a vision of the world that exceeds the constraints of psychoanalysis as a mere investigation into psychogenesis and its pathologies as to be dealt with in the psychiatric setting. Driven by its own concerns, psychoanalysis—indeed, perhaps like any strong theory of subjectivity— offers a metaphysics, or at least must become a metaphysics, since we can never safely isolate the subject under investigation from the greater scheme of ontology within which it is inscribed as a thing, process, or event. The subject is. What Lacan proclaims about its modality of “being” is that subjectivity can no longer be perceived as unnatural in the sense of an external-parasitical invasion into the vital movement of nature through the alienating effects of flirtatious images and castrating words, which somehow spoil or disfigure its pure unity by disrupting the smooth functioning of its immanent laws. The necessary theoretical posit of an originary rottenness of nature contends that there was never a realm of innocence, a pre-symbolic whole whose peaceful in-itself precluded division, a self-pervasive oneness whose unbroken energetic flow was then interrupted through the advent of language, which would be said not only to forever ideally fragment and lacerate it through artificial categories, but more disconcertingly to upset the very positivity of its movement, the cyclical repetition of things in the Real of nature, by short-circuiting the body's self-determining laws striving after homeostatic balance by giving rise to desire. No: it is not that it is only here that we see a snag, a breakdown, in the natural flow of things. Lacan's claim is much stronger: nature, in some sense, was never completely natural (it is in this spirit that we should interpret Lacan's hesitation in “The Mirror 90 Chapter 5 For Žižek, it is precisely this intuition of a necessary moment of negativity, which simultaneously rots nature from the inside out and gestures towards its constitutive weakness, that allows us to come to terms with the Symbolic-Real relation in Lacanian psychoanalysis. It alludes to the necessity of a metaphysics of the Real to explicate what the subject truly is and sketches its contours. Yet due to his conceptual reworking of Lacan, Žižek is led to part ways with and challenge many conventional ways of understanding this relation. Bruce Fink, for example, says: So too, Lacan's Real is without zones, subdivision, localized highs and lows, or gaps and plenitudes: the Real is a sort of unrent, undifferentiated, fabric, woven in such a way as to be full everywhere, there being no space between the threads that are its “stuff.” It is a sort of smooth, seamless surface or space which applies as much to a child's body as to the whole universe. The division of the Real into separate zones, distinct features, and contrasting structures is a result of the symbolic order, which, in a manner of speaking, cuts into the smooth facade of the Real, creating divisions, gaps, and distinguishable entities and laying the Real to rest, that is, drawing or sucking it into the symbols used to describe it, and thereby annihilating it.94 For Žižek, if we are to understand how language emerges out of/within the Real, this pre-symbolic, pre-logical Real sans fissure does not go far enough and must be argued against for two reasons, even if traces of it can be found in Lacan. First, it is a necessary posit created by the Symbolic at the moment of its free self-instituting, just as the transcendental subject posits the notion of a pure noumenon as a consequence of its (re)constitution of phenomenal reality. In this sense, the idea of the extra-subjective Real as an undifferentiated “mass” exhibiting no absence and negativity, just like the noumenon, risks being a mere fantasy of some kind of positive state of ontological completion outside of symbolization and idealization, which psychoanalytical experience (the mind-body discord) disproves. The Real prior to language may not possess linguistic and conceptual determination into a system of strict symbolic differences, but it cannot be said to be a substantial reality fully existing unto itself in such a way that language Kant, Todestrieb, and Beyond the Pleasure Principle 91 Kant, Todestrieb, and Beyond the Pleasure Principle 91 “pierces” its smoothness by “cutting into it” it like a flesh wound, which such presentations of the problematic appear to imply. This means that the above reading (represented by Lacanians such as Fink) is not false, but must be qualified. The Real sans fissure and the noumenon represent a compensation for the impossibility of an intimate experience of the Real within the Symbolic by claiming that, outside the reach of this synthetic (re)constitution of reality, it can still be said to persist in a state lacking contradiction and antagonism. It safeguards us from the realization that the Real itself is morcelé: it does not merely get itself into traps, producing monsters that disrupt the flow of knowledge in the Real by making the latter howl under ontological pain (chaotic states such as black holes, wherein the laws of physics seem to break down, or states in which animals, misreading meteorological conditions, perceive warm days in winter as the beginning of spring and act accordingly, “not only rendering themselves vulnerable to later onslaughts of cold, but also perturbing the entire rhythm of natural reproduction”95) but is always already riddled with internal differences, in such a manner that symbolic categories, due to a certain kind of “family resemblance,” cannot be said to be some kind of lacerating agent that first cuts up the stuff of the world into a system of divisions. Speaking of quantum mechanics (which Žižek is interested in precisely because it gives us resources that prevent us from having recourse to “a 'naive' ontology of spheres or levels”96 and challenges our understanding of nature and culture/ the Real and the Symbolic), he says: According to [our] “spontaneous ideology,” nature stands for the primacy of actuality over potentiality, its domain is the domain of the pure positivity of being where there are no lacks (gaps) in the strict symbolic sense; if, however, we take the ontological consequences of quantum physics seriously, then we have to suppose that the symbolic order pre-exists in a “wild” form, albeit in what Schelling would have called a lower potency.97 Žižek outlines four precise ways in which the symbolic order unexpectedly “pre-exists” in the Real as according to quantum mechanics, which deserve to be paraphrased in full in this context: 92 Chapter 5 92 Chapter 5 1. Possibility as such possesses actuality, that is, has effects. Just like parental authority imposes itself all the time despite the fact that it is normally only virtually and not actually present, understanding a particle's trajectory at the quantum level presupposes that we already know its possible trajectories within its wave function, which have a “being” of their own. What is more, the actualization of one of these latter does not do away with the rest: similar to the case of parental authority, as various phenomena of guilty conscience arising from an act or thought that no parental authority (or their stand-in) could ever find out or demonstrate, “what might have happened continues to echo in what actually happens as its virtual background.”98 2. Both possess knowledge in the Real. As the now (in)famous double- slit experiment testifies, if we observe a particle to see through which slit it will pass, it will always behave as a particle, but if we do not observe it, it will always behave as a wave; it is as if the particle knows when it is and when it is not been watched by scientists. We display similar behaviour in the Symbolic—often, for instance, when others project certain roles on us, we act appropriately, being aware of the projection and assuming it. 3. Each exhibits the phenomenon of registration. In the symbolic universe of meaning, an event only truly occurs when the surrounding “external” environment takes note of or registers it, that is, if it can leave a trace. In order to explain the phenomenon of the collapse of the wave function, physicists must also resort to such metaphors: even at the quantum level, an event only “fully actualizes itself only through its symbolic registration, its inscription into a symbolic network, which is external to it.”99 For this reason, particles can pop into and out of existence, just as long as the universe does not notice—like it is possible to cheat a banking system if one does not violate normal functioning. 4. Both exhibit an irreducible openness. In the Symbolic, there is always a delay between an event and its symbolic registration. The rise of a new master signifier that rewrites the entire logical field 4. Both exhibit an irreducible openness. In the Symbolic, there is always a delay between an event and its symbolic registration. Kant, Todestrieb, and Beyond the Pleasure Principle 93 within which it occurs is not a substantial, fully constituted and self-unfolding process that was determined from the get-go, like a plant growing from a seed: it was not until the precise moment when it fully actualizes itself (when it inscribes itself into its surroundings as a master signifier) that it comes to be that which it retroactively always already was, thus rewriting its own entire past. Similarly, in the double-slit experiment, when a particle is observed, it “will not only (now) behave as a particle, its past will also retroactively become (‘will have been’) that of a particle,”100 so that beforehand it could be said to have only existed as a form of proto-reality. within which it occurs is not a substantial, fully constituted and self-unfolding process that was determined from the get-go, like a plant growing from a seed: it was not until the precise moment when it fully actualizes itself (when it inscribes itself into its surroundings as a master signifier) that it comes to be that which it retroactively always already was, thus rewriting its own entire past. Similarly, in the double-slit experiment, when a particle is observed, it “will not only (now) behave as a particle, its past will also retroactively become (‘will have been’) that of a particle,”100 so that beforehand it could be said to have only existed as a form of proto-reality. Secondly, if we try to understand the pre-logical Real as in itself an undifferentiated “mass” or ontologically complete, we just cannot comprehend the possibility of the emergence of the Symbolic in the first place. If something like the human subject is to emerge, then nature must be self-divided, wrought with tension-ridden zones of inner laceration, for otherwise we cannot account for its ontogenesis in any adequate manner, insofar as the subject, intrinsically exhibiting an originary ontologico-natural discord, could not be inscribed into the world. 92 Chapter 5 The rise of a new master signifier that rewrites the entire logical field Kant, Todestrieb, and Beyond the Pleasure Principle 93 Kant, Todestrieb, and Beyond the Pleasure Principle 93 The subject and its linguistic capacities must be seen as expressive of the underlying ontological status of the Real, even if they represent within Lacanian metapsychology the loss and impossibility of such a non-mediated, pre-symbolic reality: the Symbolic is not merely some kind of extraneous, self-unfolding construct (a self-generating matrix of “meaning” that can assert itself in complete freedom from the Real as such) for the mere fact that we are entrapped within it must be revelatory of the essence of objective reality at some level since it must have given birth to it. The meaning of this is twofold: firstly, descriptions of the Symbolic-Real relation such as Fink's risk obfuscating what is for Žižek of essential importance for understanding what is at stake because they describe language as “encrusted upon the living,”101 thereby reducing language to an external reflection upon substance and rendering the task of explicating its obscure origins impossible; secondly, the apparently self-grounding idealism of Lacanian psychoanalysis—the autonomous, self-positing ciphering of the Real by the Symbolic—already points to a way of explaining its emergence, and thus of breaking free from Chapter 5 94 its correlationalist prison, by zoning in on that very feature that seems to prohibit such an inquiry: that is, its ontological solipsism now understood as an ontological event. It is necessary to explain how the Real can open itself up and give rise to a force seemingly Other to itself, to explain how images and words can “colonize” being from within, but this process forces us to include a moment of non-coincidence and antagonism with the Real. Although it may be without meaning to say that in the Real there is lack—lack only being brought forth in the reign of the Symbolic—it would nevertheless be erroneous to deduce from this that there is no negativity or difference within it. Yet how are we to understand this element of negativity in the all- pervasive fabric of the pre-symbolic Real, this self-sabotaging moment in the very heart of being? 5.2 …to a Denaturalized Monstrosity We can now begin to see why the psychoanalytical experience of the infinite dichotomy between the structures underlying personality (subjectivation, culture) and the vital flow of energy sustaining us in objective being (nature, the corpo-Real of the body) is so pivotal for Žižek. The two registers of the Symbolic and the Real may function without any degree of reciprocal interaction or mutual interconnectivity, but the simple declaration of this unstable Cartesian bipolarity actually belies a third element that subsists throughout the discord and, in fact, paradoxically ties them together in their very antagonistic dialectical (non-)relationship. Here, following another hint given by Lacan— While psychoanalysis cannot, since its experience is limited to the individual, claim to grasp the totality of any sociological object or even the whole set of forces currently operating in our society, the fact remains that it discovered in analytical experience relational tendencies that seem to play a basic role in all societies, as if the discontent in civilization [das Unbehagen in der Kultur] went so far as to lay bare the very meeting point of nature and culture.102 Kant, Todestrieb, and Beyond the Pleasure Principle 95 Self-positing autonomy, as both freedom from the laws of a closed libidinal-material economy and the relatively closed structural determinism of the symbolic law of culture, rests upon the subject as a self-relating point of infinite negativity, a positively Chapter 5 96 charged, excessive void, but it can only beget itself if there is first of all a short circuit, a breakdown, in the dynamic flow of energy constitutive of nature's rhythms as that which carves up room for its more-than-materiality. Full-fledged subjectivity is rendered possible by a devastating ontological violence and is consequently nothing but a denaturalized monstrosity (Todestrieb) logically existing above and beyond the flux of being (due to its non-coincidence with the latter) while never able to leave its immanent plane within which it is primordially inscribed as out of joint: representing the failure, the collapse, of a self-enclosed biological system based upon the homeostatic self-preservation of the organism, it is an extimate inassimilable body in nature that exhibits the double feature of inclusion/exclusion, internal/external, presence/absence so characteristic of the Real in Lacan, which simultaneously demonstrates why the pre-symbolic Real must be said to be morcelé. In other words, set up as nature turned against itself, the first dull stirrings of the human subject refer to some kind of trauma that eventually incites the growth of individual ego life and culture in such a way that the latter exhibit the structural form of a reaction formation against this dysfunctionality in being and thus can never prevent themselves from being a negative, symptomatic expression of their basis in an ontological crisis, no matter how they may try to occult this fact. The psychoanalytical experience by definition presupposes an emergent schism in the fabric of the world between substance and subject, matter and mind, the Real and the Symbolic. But how can psychoanalysis—explicitly a theory of psychogenesis and its pathologies—explain where this denaturalization comes from, since such an investigation by principal must be external to its theoretical field and methodology? charged, excessive void, but it can only beget itself if there is first of all a short circuit, a breakdown, in the dynamic flow of energy constitutive of nature's rhythms as that which carves up room for its more-than-materiality. Kant, Todestrieb, and Beyond the Pleasure Principle 95 Furthermore, is not the object of psychoanalysis precisely this gap between first and second nature—the insecure position of a human subject who, after losing his footing in the first nature, can never feel fully at case in the second: what Freud called das Unbehagen in der Kultur, the different way the subject's passage from first to second nature can go wrong (psychosis, neurosis ...)? There is thus a core that resists the subject's full reconciliation with his second nature: the Freudian name for this kernel is drive, the Hegelian name for it is “abstract negativity” (or, in more poetic terms of the young Hegel, the “night of the world”).103 The conceptual contours of this passage are much more complex than they may originally appear. Žižek is saying that central to any psychoanalytical theory of psychogenesis and psychopathology is the claim that the subject is out of joint with both the biological needs of the corpo-Real of its body (the anorexic eats nothing, the romantic is willing to die for love) and the symbolically constituted “second” nature that is created to compensate for the primordial Hilflosigkeit of human organic insufficiency. Understood in the context of seeking the obscure origins of the Symbolic in the Real, the subject, as the very gap between first nature (Real) and second nature (Symbolic), cannot be said to fit into either: it is a paradoxically self-standing space of non-relation that protrudes out of and obstructs both. Strictly speaking, the subject is neither Real nor Symbolic—it is a pure logical non-coincidence that possesses no place in either, so that the question of its upsurge within being must also go beyond a mere exploration of the breakdown of the libidinal-material fold of its biological nature as that which sets the stage for its emergence by opening up a liberating space within nature's hold. What is crucial and groundbreaking here is the outlining of the ontological edifice that grounds human subjectivity qua cogito: we see the articulation of the “site” or “juncture” from which transcendental freedom and spontaneity emerge and take told. Kant, Todestrieb, and Beyond the Pleasure Principle 97 We just have to look at how the latter's key representatives have recourse to various concepts (unruliness and diabolic evil in Kant, the night of the world in Hegel, or the Entscheidung in Schelling) that indicate a necessary disruption, breach, or violence at the very basis of the founding attributes they bestow upon the subject (self- legislative reason, the irreducibility of spirit, or freedom as the capacity for good and evil). It is exactly this paradoxical connection between real discord and ideal freedom throughout both traditions that enables Žižek to develop a new metaphysics by working in the intersections of both traditions, a metaphysics whose first conceptual contours we already see in Kant. Kant, Todestrieb, and Beyond the Pleasure Principle 97 as having two goals.105 First, because the Lacanian subject is lacking any account of its ultimate origins, Žižek turns to German Idealism to develop a transcendental materialism insofar as there exists a structural parallelism in the underlying problematic plaguing both post-Kantian idealism and contemporary psychoanalysis. Second, and more strongly, Žižek's claim is that this parallelism is more than a mere shared set of theoretical concerns hovering around the grounding of the subject. If we read Kant, Schelling, and Hegel through Freud and Lacan, we actually see that there is a fundamental identity between the psychoanalytical subject haunted by the Todestrieb as constitutive of its very existence and the unconscious Grundlogik of German Idealism. We just have to look at how the latter's key representatives have recourse to various concepts (unruliness and diabolic evil in Kant, the night of the world in Hegel, or the Entscheidung in Schelling) that indicate a necessary disruption, breach, or violence at the very basis of the founding attributes they bestow upon the subject (self- legislative reason, the irreducibility of spirit, or freedom as the capacity for good and evil). It is exactly this paradoxical connection between real discord and ideal freedom throughout both traditions that enables Žižek to develop a new metaphysics by working in the intersections of both traditions, a metaphysics whose first conceptual contours we already see in Kant. as having two goals.105 First, because the Lacanian subject is lacking any account of its ultimate origins, Žižek turns to German Idealism to develop a transcendental materialism insofar as there exists a structural parallelism in the underlying problematic plaguing both post-Kantian idealism and contemporary psychoanalysis. Second, and more strongly, Žižek's claim is that this parallelism is more than a mere shared set of theoretical concerns hovering around the grounding of the subject. If we read Kant, Schelling, and Hegel through Freud and Lacan, we actually see that there is a fundamental identity between the psychoanalytical subject haunted by the Todestrieb as constitutive of its very existence and the unconscious Grundlogik of German Idealism. Kant, Todestrieb, and Beyond the Pleasure Principle 95 Full-fledged subjectivity is rendered possible by a devastating ontological violence and is consequently nothing but a denaturalized monstrosity (Todestrieb) logically existing above and beyond the flux of being (due to its non-coincidence with the latter) while never able to leave its immanent plane within which it is primordially inscribed as out of joint: representing the failure, the collapse, of a self-enclosed biological system based upon the homeostatic self-preservation of the organism, it is an extimate inassimilable body in nature that exhibits the double feature of inclusion/exclusion, internal/external, presence/absence so characteristic of the Real in Lacan, which simultaneously demonstrates why the pre-symbolic Real must be said to be morcelé. In other words, set up as nature turned against itself, the first dull stirrings of the human subject refer to some kind of trauma that eventually incites the growth of individual ego life and culture in such a way that the latter exhibit the structural form of a reaction formation against this dysfunctionality in being and thus can never prevent themselves from being a negative, symptomatic expression of their basis in an ontological crisis, no matter how they may try to occult this fact. The psychoanalytical experience by definition presupposes an emergent schism in the fabric of the world between substance and subject, matter and mind, the Real and the Symbolic. But how can psychoanalysis—explicitly a theory of psychogenesis and its pathologies—explain where this denaturalization comes from, since such an investigation by principal must be external to its theoretical field and methodology? Using Lacan's gesture towards an originary rottenness plaguing nature as a theoretical starting point, Žižek seeks to expand this structuralist metapsychology in order to secure the means of articulating the ontogenetic possibility-conditions of the Symbolic in the Real. Seeing psychoanalysis as conceptually unable to fulfill this task, he expands its horizon by recourse to modern philosophy, seeing therein a certain homology that enables him to draw upon its resources.104 Following Lacan's claim that “Kant's practical philosophy [is] the starting point of the lineage culminating in Freud's invention of psychoanalysis,” Žižek's project could be described Kant, Todestrieb, and Beyond the Pleasure Principle 97 Chapter 5 98 nothing to do with the unconscious in the Lacanian sense; on top of that, it displays great hope in the modern Enlightenment project of establishing the self-transparency and powers of reason—albeit through reason's own self-critique—as a means of historical progress and concretizing man's perfectibility. However, even if this might be the image of Kant's practical philosophy that always comes to mind, the matter at hand is far more complicated. Kant's practical philosophy is of central importance because it is an expression and systematization of the experience of freedom in its irreducible essence, freedom understood as the self-legislative spontaneity at the core of human subjectivity, a faculty that separates us from the rest of mechanical nature insofar as we generate and obey our own laws. One must also remember that, for Kant, the Critique of Pure Reason is an attempt to make room for faith by limiting knowledge and reason, a point that directs the entirety of the critical enterprise from beginning to end by penetrating into the originary self-positing of human liberty at all costs, something that the late German Idealists were retroactively convinced revealed a decisive deficiency present in all the great thinkers in the history of philosophy. It is not the Copernican revolution—indeed, Schelling's106 and Hegel's107 projects are founded upon an attempt to escape its consequences—which forces us to rethink the very possibility of philosophy, but rather Kant's account of freedom.108 This is also true of Žižek: “Kantian practical reason provides a glimpse into the abyss of freedom beyond (or beneath) the constraints of traditional metaphysical ontology.”109 It is all a matter of how one understands the Kantian breakthrough. Even if it is true that it is a response to specific epistemological and scientific concerns that emerge out of modern philosophy (the [im]possibility of universal and necessary knowledge, the nature of the correlation of our ideas to reality in the genesis of concepts, etc.) it is clear that Kant's revolution cannot merely be reduced to his innovative way of rethinking the question of the subject-object relation, for the radical reflexivity of the cogito that imposes upon us the task of reconceptualizing what it is for an object to be present in the field of experience is also at the basis of what it means to be a practical subject. 5.3 Kant, Unruliness, and the Cry of the Newborn Lacan's claim that the beginning of psychoanalysis is in Kant's practical writings appears, at first, counter-intuitive. Especially given Lacan's structuralist bent, one would perhaps expect the clearest elaboration of the subject to be found in Kant's philosophical treatises on the mediating structures of consciousness. What do we see by delving into his practical philosophy except an attempt to found the ethical in the self-legislative spontaneity of human reason through articulating the self-imposing impetus of the categorical imperative and a listing of the a priori duties that automatically follow from its law? How could such a cold, machine-like way of determining the legitimacy of existential action be the immediate origin of psychoanalysis? Although we do encounter traces here of the irrevocably split nature of subjectivity in the tension between reason and sensible inclination, Kant's practical philosophy appears to have absolutely Chapter 5 Kant, Todestrieb, and Beyond the Pleasure Principle 99 Kant, Todestrieb, and Beyond the Pleasure Principle 99 99 Kant appears to be making the self-legislative freedom we witness in concrete, existential situations the very basis of theoretical philosophy— instead of a passive thinking subject that receives external reality as a kind of inert receptacle, a mere spectator, we have a reflecting subject that spontaneously and freely generates the very fabric of its own experience into a continuous, unitary whole through an activity of synthetic integration in a way similar to how it gives itself its own laws. The theoretical subject that is unearthed in transcendental apperception is ultimately identical with the practical subject of self-legislative freedom—or in other words, one cannot speak of one without the other, because they form a dialectical whole: if one wants to plunge into the labyrinthine depths of subjectivity, one should not read the Critique of Pure Reason in isolation from later works such as Lectures on Pedagogy and Religion within the Limits of Reason Alone. In a similar vein, Žižek locates the true Kantian breakthrough in Kant’s practical thinking on subjectivity instead of his epistemologico- transcendental destruction of metaphysics insofar as it is in the former that we most directly see the abyss of freedom at the obscure origin of subjectivity. But what is so primordial in the former that could bestow upon Kant's pedagogical writings such a privilege for understanding the radicality of critical philosophy, while also enabling us to shed light on the basis of the Cartesian-psychoanalytical subject? Finding numerous textual traces of the death-drive understood as a self-sabotaging tendency in nature as logically prior to subjectivity in both Kant, Fichte, Schelling, and Hegel, what interests Žižek in Kant's pedagogical writings is how they set the stage for what he claims to be the unconscious Grundlogik of German Idealism: that is, its founding intuition of the passage from nature to culture centred around a disturbing moment of irreducible negativity inscribed within the palpitating heart of being, which suggests that what first appears as a mere homology in conceptual structure between psychoanalysis and German Idealism is in fact a strict identity. Chapter 5 The movement goes both ways: if we read the Critique of Pure Reason through the later ethical, pedagogical, and religious writings, Kant, Todestrieb, and Beyond the Pleasure Principle 99 What they bring to the fore is a thematization of the subject as some kind of disjunctive “and”: The key point is thus that the passage from “nature” to “culture” is not direct, that one cannot account for it within a continuous evolutionary narrative: something has to intervene between the two, a kind of “vanishing mediator,” which 100 Chapter 5 is neither nature nor culture—this In-between is silently presupposed in all evolutionary narratives. We are not idealists: this In-between is not the spark of logos magically conferred on Homo sapiens, enabling them to form his supplementary virtual symbolic surroundings, but precisely something that, although it is also no longer nature, is not yet logos, and has to be “repressed” by logos—the Freudian name for this In-between, of course, is the death drive. Kant, Todestrieb, and Beyond the Pleasure Principle 99 Speaking of this In-between, it is interesting to note how philosophical narratives of the “birth of man” are always compelled to presuppose such a moment of human (pre)history when (what will become) man is no longer a mere animal and simultaneously not a “being of language,” bound by symbolic Law; a moment of thoroughly “perverted,” “denaturalized,” “derailed” nature which is not yet culture.110 According to Žižek, within Kant this “in-between” finds its expression in the necessity to discipline the excessive “unruliness” (Wildheit) of human nature, the “wild, unconstrained propensity to insist stubbornly on one's own will, cost what it may.”111 What is to be emphasized here is the drastic nature of this claim: if we do not tame this primordial rawness (Rohigkeit) that presents itself as the zero-level of human spontaneity, not only do we fail to become full-fledged, fully adjusted subjects in the sociopolitical field of the world, but our freedom even threatens to devour itself in its frenzy in such a way that we become failed subjects: we are not born as humans, but rather become human—or, as Kant says, “[m]an only becomes man by education,”112 which leads him to contend that “with education is involved the great secret of the perfection of human nature”113 insofar as it is only through the principles offered by this act of disciplining, a means of schematizing our originary unruliness, that a “second nature”114 can emerge as a response to the grounding ontological dilemma of human being, “nature ha[ving] placed no instinct in [man] for that purpose.”115 Even if the dysfunction of nature, this ontological abortion that is a monstrous, uncontainable excess of life, can in a second dialectical moment serve as a “positive” foundation or support, it can also collapse upon itself like a dying the dysfunction of nature, this ontological abortion that is a monstrous, uncontainable excess of life, can in a second dialectical moment serve as a “positive” foundation or support, it can also collapse upon itself like a dying star. Yet this “unruliness” cannot be equated with the brute reality of animal “positive” foundation or support, it can also collapse upon itself like a dying star. Kant, Todestrieb, and Beyond the Pleasure Principle 99 In this context, we only need to cite a passage from the 02 Chapter 5 Anthropology from a Pragmatic Point of View to show how explicitly Lacanian Kant's point is: The cry of a newborn child is not the sound of distress but rather indignation and furious anger; not because something hurts him, but because something annoys him: presumably because he wants to move above and his inability to do so feels like a fetter through which his freedom is taken from him.—What could nature's intention be here in letting the child come into the world with loud cries which, in the crude state of nature, are extremely dangerous for himself and his mother? For a wolf or even a pig would thereby be lured to eat the child, if the mother is absent or exhausted from childbirth. However, no animal except the human being (as he is now) will loudly announce his existence at the moment of birth [...]. One must therefore assume that in the first epoch of nature with respect to this class of animals (namely, in the time of crudity), this crying of the child at birth did not yet exist; and then only later in a second epoch set in, when both parents had already reached the level of culture necessary for domestic life; without our knowing how, or through what contributing causes, nature brought about such a development.117 Not only does Kant relate the dark unruliness that sets the stage for full- fledged human freedom to some kind of ontologically disjointed state of natural being, but he more radically links the cry of the newborn to the infinite dis-coordination of the corpo-Real of the human neonate, its feeling of utter dismemberment so central to the mirror stage in Lacan, and even suggests that this direct expression of painful negativity immanent in the fold of material being is fundamental to the passage from nature to culture. These passages could be further drawn out by supplementing them with a number of possible citations from Religion within the Limits of Reason Alone, the first book in which Kant attempts to deal with the insurmountable propensity to evil that lies at the core of human subjectivity and ethical action. Kant, Todestrieb, and Beyond the Pleasure Principle 99 Yet this “unruliness” cannot be equated with the brute reality of animal Kant, Todestrieb, and Beyond the Pleasure Principle 101 existence—even if it exists within nature, it is strictly speaking something non-natural: existence—even if it exists within nature, it is strictly speaking something non-natural: The love of freedom is naturally so strong in man, that when once he has grown accustomed to freedom, he will sacrifice everything for its sake [...]. Owing to his natural love of freedom, it is necessary that man should have his natural roughness smoothed down; with animals, their instinct renders this unnecessary.116 For Žižek, this extract shows that the enigma of the emergence of subjectivity in German Idealism cannot be reduced to a mere dichotomy between nature and culture, as if in order to conform to the symbolic law of our own making we must first tame the blind, egotistical pleasure-seeking principles of our animal nature. The self-creative, logically autonomous milieu of culture is only possible through a prior, infinitely uncontainable freedom that acts as the “vanishing mediator” between brute animal reality and structured human intersubjective existence. The passage to culture does not consist in a sublimation of animalistic needs, but rather in a disciplining or symbolic re-articulation of a monstrous and logically irreducible unruliness that marks the essence of the human being, a disciplining that, when it succeeds (the possibility of neurosis always lurks in the air), simultaneously functions as that which once and for all separates us from nature by causing this denaturalized Grund (our ontogenetic “origins”) to withdraw from the scene. It is worth pointing out that Žižek strangely overlooks an important passage on the first page of the transcript we have of Kant's Lectures on Pedagogy, the very text that he makes use of at such a crucial point in his argument, which actually further supports his own Lacanian-inspired and ontologically oriented rereading of the vision of practical freedom it offers. In a perhaps unexpected move, Kant defines the human neonate as non- natural, claiming that, if an animal came to the world crying as a human does, it would merely attract attention to itself as potential prey, thus establishing that there is something off, primordially non-advantageous from a biological point of view, about the obscure ontogenetic beginnings of human subjectivity. Kant, Todestrieb, and Beyond the Pleasure Principle 99 It argues two major points: (i) “the ground of this evil cannot be placed, as is so commonly done, in man's sensuous nature” and (ii) “neither can the ground of this evil be placed in a corruption of the morally Kant, Todestrieb, and Beyond the Pleasure Principle 103 legislative reason.”118 As such, this diabolic evil can neither be linked to the corpo-Real of the body and its pleasure-seeking tendencies or to the self's being within the law of a criminal or coercive symbolic order, but must itself be a foundational, constitutive part of the subject insofar as, being an infinitely self-asserting activity irreducible to and incommensurate with either zone, it articulates this same paradoxical structure of the in-between. Protruding out of nature and culture and failing to be understood except by its own self-positing, self-determining logic, the diabolic evil at the basis of freedom can only appear as an uncontrollable urge threatening to devour everything, even itself, in its self-destructive forward thrust. But here we see one crucial difference: whereas before we were at the strict level of ontogenesis (the “origins” of transcendental subjectivity) we now encounter full-fledged speaking subjects acting in a world (the ambiguity of freedom). Linking this consuming fire at the core of subjectivity to the perverse truth hidden in the Cartesian gap between mind and body, nature and culture, we already see in Kant the outlines of a radical materialist ontology, for it is as if in the movement from the former to the latter we see unruliness positing or owning itself in its own attempt at self-domestication, the structural consequences of which have profound metaphysical consequences for our understanding of reality as a substantial whole or totality. Since the ontogenetic origins of the subject are linked to a denaturalized unruliness and a full-fledged subject only comes on the scene when the latter posits itself as such in this endeavour of schematizing itself, which in turn institutes a pure difference in being by forever sustaining the gap it attempts to fill in, subjectivity exhibits an insurmountable propensity to evil because its very founding gesture is structurally evil. It is a radical, egotistical “No!” that reverses the order of the world according to its own self-assertion and cannot be undone without undoing itself. 104 Chapter 5 What is so difficult to come to terms with in the theoretical positing of this state of unruliness, and the ambiguity at the core of the self-positing of the subject, is its proclamation that the good itself is only possible through the gentrification or taming of evil. In order for good to be truly good, it too must present itself as a non-deducible act that breaks free from any order within which it could be contained by refusing all inscription within a field of heterogeneous, external forces that could impose itself upon the absolute originarity of its uncompromising self-assertion. The Yes of union depends upon the No of separation, but the process of converting the latter into the former can never be complete, since this would reduce freedom to a mere moment of cultural law as a kind of quasi-natural immersion within a pre-given logistics, thereby robbing human spontaneity of its untouchable autonomy in face of everything else that may be said to have influence upon it. Posited as that which transcendentally precedes and even conditions the possibility of its (logico-symbolic) articulation, at the very heart of the good there paradoxically lies evil as its extimate Other and menacing ground, threatening at any moment to erupt and disturb its smooth surface. Evil and good are not infinitely different and opposed, but merely two logical modalities of freedom, a freedom that in and of itself knows no law except its own uncontainable upsurge. If subjectivity is evil and the subject is an What is so difficult to come to terms with in the theoretical positing of this state of unruliness, and the ambiguity at the core of the self-positing of the subject, is its proclamation that the good itself is only possible through the gentrification or taming of evil. In order for good to be truly good, it too must present itself as a non-deducible act that breaks free from any order within which it could be contained by refusing all inscription within a field of heterogeneous, external forces that could impose itself upon the absolute originarity of its uncompromising self-assertion. Kant, Todestrieb, and Beyond the Pleasure Principle 99 What is more, not only did Kant identify the intrinsic break from the order of substantial being that sets the stage for the cogito's very autonomy and sketch this movement, he also already saw the paradox at the heart of subjectivity. That is to say, he implicitly understood that we can only grasp the latter as a pure act non- deducible from the obscure libidinal-material grounding that renders it possible (after all, for Kant evil is intrinsically enigmatic insofar as once as it 104 Chapter 5 Kant's breakthrough is the following: if human subjectivity is to be truly self-legislative, at its zero-level there can be no formal distinction between a good and evil free act insofar as both are self-chosen in a non-disciplinable frenzy that has no definitive (real or symbolic) status and knows no influence exterior to its own self-asserting, self-positing activity. In itself, freedom is indifferent to both: there is no intrinsic difference between a will that wills evil and a will that wills good in terms of the pure act itself insofar as both are merely following their own self-given causality, an unconstrained self- legislation that tears apart the very fabric of nature in its self-imposition.119 A will that wills evil is not merely giving itself over to the animalistic impulses of the body, nor is it expressing its ignorance or even corruption of the symbolic fold of cultural laws; it is merely forcefully upholding its own diabolical evil for the sake of it, basking in its own self-grounding tyranny, even when it has complete knowledge of its nature and repercussions. Kant's breakthrough is the following: if human subjectivity is to be truly self-legislative, at its zero-level there can be no formal distinction between a good and evil free act insofar as both are self-chosen in a non-disciplinable frenzy that has no definitive (real or symbolic) status and knows no influence exterior to its own self-asserting, self-positing activity. In itself, freedom is indifferent to both: there is no intrinsic difference between a will that wills evil and a will that wills good in terms of the pure act itself insofar as both are merely following their own self-given causality, an unconstrained self- legislation that tears apart the very fabric of nature in its self-imposition.119 A will that wills evil is not merely giving itself over to the animalistic impulses of the body, nor is it expressing its ignorance or even corruption of the symbolic fold of cultural laws; it is merely forcefully upholding its own diabolical evil for the sake of it, basking in its own self-grounding tyranny, even when it has complete knowledge of its nature and repercussions. Kant, Todestrieb, and Beyond the Pleasure Principle 105 event in the world, then the world must not be understood as not-all, for the latter is not incapable of subsuming the former within its smooth touch. It is precisely for this reason that Žižek, along with Lacan, believes that Kant's practical philosophy is the beginning of the lineage that culminates in psychoanalysis, the latter being understood as the second great revolution in philosophy that inflicts upon us the task of radically reconfiguring how we view ourselves and our relation to the world. Not only can we already therein see its traits principaux being thematized in an implicit way, but paradoxically one could even say that it in any uncanny manner develops them in different directions from Freud or Lacan so that bringing psychoanalysis and the German Idealist tradition together promises to produce something new. Just as Kant asserts an ultimate identity between the theoretical and practical ego, Žižek argues for the interpenetration of modern transcendental philosophy and psychoanalysis through the Kantian notion of the original “unruliness” of human nature. Todestrieb becomes a synonym for the transcendental “I,” the cogito, by giving expression to the pre-subjective conditions of the possibility of freedom as some kind of non-masterable excess in nature that must be “tamed” if full-fledged subjectivity is to come on the scene. Exposing an activity uncontainable within positive being, it alludes to an ontologically self-violent “wildness” that serves as the ontogenetically constitutive basis of the subject's self- positing, in such a way that makes the late Lacan's passing remarks over the ontology of the psychoanalytical subject (“the rottenness of nature”) come strikingly close to those of Kant (“[w]hat could nature's intention be here [...] ?”). In Kant, however, just as in Lacan, the actual status of the subject remains ambivalent and theoretically undetermined, even if there are various suggestions littered throughout their texts that programmatically outline how to proceed if one were to develop a materialism of transcendental freedom, despite the insurmountable problem that such gestures surpass the very constraints imposed upon the epistemology that they develop. The exact same set of theoretical questions is brought to the fore by both thinkers but remains unsolved: why does transcendental freedom develop? What is its exact relation to the “unruliness,” synonymous with the excess of life presented by the Todestrieb, at the core of our being that appears to logically precede it? 104 Chapter 5 The Yes of union depends upon the No of separation, but the process of converting the latter into the former can never be complete, since this would reduce freedom to a mere moment of cultural law as a kind of quasi-natural immersion within a pre-given logistics, thereby robbing human spontaneity of its untouchable autonomy in face of everything else that may be said to have influence upon it. Posited as that which transcendentally precedes and even conditions the possibility of its (logico-symbolic) articulation, at the very heart of the good there paradoxically lies evil as its extimate Other and menacing ground, threatening at any moment to erupt and disturb its smooth surface. Evil and good are not infinitely different and opposed, but merely two logical modalities of freedom, a freedom that in and of itself knows no law except its own uncontainable upsurge. If subjectivity is evil and the subject is an Kant, Todestrieb, and Beyond the Pleasure Principle 105 Kant, Todestrieb, and Beyond the Pleasure Principle 105 Insofar as transcendental spontaneity is just as related The exact same set of theoretical questions is brought to the fore by both thinkers but remains unsolved: why does transcendental freedom develop? What is its exact relation to the “unruliness,” synonymous with the excess of life presented by the Todestrieb, at the core of our being that appears to logically precede it? Insofar as transcendental spontaneity is just as related Chapter 5 106 to the synthetic powers of the imagination as it is to self-legislative reason in Kant, what role does the former play in this picture (or, in a Lacanian parlance, how can the Symbolic emerge as that which [re]constitutes the fold of experience)? It is only in Kant's successors that such concerns will be addressed in a more explicit manner, and it is through a psychoanalytical reactualization of their thinking that the transcendental materialist ontology at the heart of Kant's breakthrough can finally come to light. Kant, Todestrieb, and Beyond the Pleasure Principle 107 Kant, Todestrieb, and Beyond the Pleasure Principle 107 105. Žižek, The Ticklish Subject, p. 48. 106. The founding gesture of Schelling’s entire project begins with an attempt to surmount the Copernican revolution. Only the modalities of his response differ: whether it be through an attempt to give a genesis of the categories from the I as the first principle, a daring naturephilosophy, or eventually by a theosophic mytho-poetics of creation, one thing is clear—we must go “beyond simple rep- resentation.” Einleitung in die Philosophie, ed. Walter E. Ehrhardt (Stuttgard-Bad Connstatt: Frommann-Holzboog, 1989). 106. The founding gesture of Schelling’s entire project begins with an attempt to surmount the Copernican revolution. Only the modalities of his response differ: whether it be through an attempt to give a genesis of the categories from the I as the first principle, a daring naturephilosophy, or eventually by a theosophic mytho-poetics of creation, one thing is clear—we must go “beyond simple rep- resentation.” Einleitung in die Philosophie, ed. Walter E. Ehrhardt (Stuttgard-Bad Connstatt: Frommann-Holzboog, 1989). 107. Although people are wont to speak about the tight relationship between Kant and Hegel, one must point out that in the Lesser Logic Hegel badmouths Kant by by throwing upon him the ultimate insult one could give to a systematic philosopher (except, of course, that their absolute is a dead dog)—namely, that he was just too lazy to think through what he says and therefore completely misses the point: “[i]n deal- ing with this highest Idea [teleological causality], however, the laziness of thought, as we may call it, finds in the ‘ought’ an all too easy way out.” The Encyclopedia Logic (with the Zusätze), trans. T. F. Geraets, W. A. Suchting, and H. S. Harris (Indianapolis: Hackett Publishing, 1991), p. 101 (§55A). 108. See chapter 6. 109. Žižek, The Ticklish Subject, p. 48. 110. Ibid., p. 36. 110. Ibid., p. 36. 111. Ibid. 112. Taken from Kant, Kant on Education, trans. Annette Churton (Boston: D. C. Heath, 1900), p. 6; for a more easily findable edition, see Lectures on Pedagogy, trans. Robert B. Louden, Anthropology, History, and Education, ed. Günter Zöller and Robert B. Louden (Cambridge: Cambridge University Press, 2007), p. 439. 113. Ibid., pp. 7 and 439. 113. Ibid., pp. 7 and 439. 114. Ibid., pp. 9 and 440. 115. Ibid., pp. 13 and 442. 115. Ibid., pp. 13 and 442. 116. Ibid., pp. Notes 87. Žižek, The Ticklish Subject, p. 48. 88. Johnston, Žižek’s Ontology, p. 272. Here Johnston is referring to Le Séminaire de Jacques Lacan, Livre XXI: Les non-dupes errent, 1973–1974 (unpublished type- script), pp. 2, 21, and 74; and Le Séminaire de Jacques Lacan, Livre XXIII: Le sinthome, 1975–1976, ed. Jacques-Alain Miller (Paris: Éditions du Seuil, 2005), pp. 5, 17, and 77 (untranslated). 89. Ibid., p. 273. Following Johnston’s bibliography, the first quote comes from Le Séminaire de Jacques Lacan, Livre XVIII: D’un discours qui ne serait pas du semblant, 1971 (unpublished typescript), pp. 2, 17, and 71, while the second seminar he makes reference to is (as in the previous footnote) Le Séminaire de Jacques Lacan, Livre XXIV: L’insu que sait de l’une bévue s’aile à mourre, 1976–1977, ed. Jacques- Alain Miller, in Ornicar? 12-18, pp. 5, 17, and 77. 90. Lacan, “The Mirror Stage as Formative of the I Function,” Écrits, p. 96/77. 91. Ibid., p. 97/78. 91. Ibid., p. 97/78. 92. Ibid., p 100/80. 93. Ibid., p. 96/77. 94. Fink, The Lacanian Subject, p. 24. I capitalize “the Real” for consistency. 95. Žižek, Living in the End Times (New York: Verso, 2011), pp. 350–51. 96. Žižek, Less Than Nothing, p. 905. 96. Žižek, Less Than Nothing, p. 905. 97. Ibid., p. 921. 97. Ibid., p. 921. 98. Ibid., p. 920. 99. Ibid. 99. Ibid. 100. Ibid., p. 921. 100. Ibid., p. 921. 101. Fink, The Lacanian Subject, p. 12. 102. Lacan, “A Theoretical Induction to the Functions of Psychoanalysis in Criminology,” Écrits, p. 127/104. 103. Žižek, The Ticklish Subject, pp. 81–82. 103. Žižek, The Ticklish Subject, pp. 81–82. 104. Žižek, “Liberation Hurts.” 104. Žižek, “Liberation Hurts.” Chapter 6 This chapter comprises a Žižekian-inspired interpretation of the philosophical movement from Kant to Hegel by focusing on Kant's thematization of freedom and how it radically reconfigures the possibility of metaphysical inquiry. In the aftermath of critical philosophy, what is clear is that any philosophy unable to think system and the irreducibility of the human subject is to be rejected. By following certain premonitions within Kant's pedagogical writings that appear to link transcendental spontaneity to the psychoanalytical concept of Todestrieb, Žižek gives us resources to read Hegelian Absolute Idealism against standard interpretations by claiming that Hegel's attempt to think substance as subject implies the ontogenetic emergence of freedom through a self-sundering of being, the immanent advent of a devastating ontological non-coincidence, which forces upon us the necessity of a new kind of metaphysics: a metaphysics of the not-all. This enables us not only to rethink the Kant-Hegel relation in a provocative manner, but also to explain how Žižek is able to draw upon post-Kantian idealism to lay the foundation for the logic of his own transcendental materialism. Kant, Todestrieb, and Beyond the Pleasure Principle 107 4–5 and 438; quoted by Žižek, The Ticklish Subject, p. 36. 117. Anthropology from a Pragmatic Point of View, trans. Robert B. Louden, Anthropology, History, and Education, p. 423. First set of italics is my own. 118. Kant, Religion within the Limits of Reason Alone, trans. T. M. Greene and H. H. Hudson (New York: Harper & Row, 1960), p. 30. 119. Žižek & Daly, Conversations with Žižek (Cambridge: Polity, 2004), p. 124. From Transcendental Philosophy to Substance as Subject 109 From Transcendental Philosophy to Substance as Subject 10 109 penetrating account of the essence of human freedom.120 Although much of what he says concerning freedom is already laid out in Descartes' thinking on the cogito, it was Kant who gave it a full, profound expression. For Žižek, this means that it is here that the principal intuitions that heralded forth modernity—the ontologically shattering schism between the thinking mind and extended substance and subjectivity's irreducible reflexivity as that which institutes this very split—are radicalized and find an overpowering degree of theoretical articulation. After Kant, there is just no going back, for this would be to give up on what it means to be an infinitely self-standing, autonomous subject, to turn one's back on one's own freedom, whose apparently indemonstrable existence has been proven once and for all.121 Any system that regresses into a “primitive,” “pre-critical” way of philosophizing is in effect merely recoiling from the difficulty that is the burden of freedom, “our experience of freedom [being] properly traumatic, even for Kant himself.”122 Herein lies the fundamental undecidability intrinsic to the Kantian breakthrough: not only is the freedom of human subjectivity liberating, but it is also (and perhaps more originally) monstrous, insofar as we are infinitely given over to it and therefore responsible for it, yet can only comprehend it according to the frenzy that is its own self-positing essence. In the wake of the Kantian system, there is only “the uncanny abyss of freedom without any guarantee in the Order of Being”:123 in Kantian ethics, the true tension is not between the subject's idea that he is acting only for the sake of duty, and the hidden fact that there was actually some pathological motivation at work (vulgar psychoanalysis); the true tension is exactly the opposite one: the free act in its abyss is unbearable, traumatic, so that when we accomplish an act out of freedom, in order to be able to bear it, we experience it as conditioned by some pathological motivation. Here I am tempted to bring in the key Kantian concept of schematization: a free act cannot be schematized, integrated into our experience; so, in order to schematize it, we have to “pathologize it.”124 Immediately following the birth of transcendental idealism, however, there is an overall ambiguity as to how to proceed. 6.1 Fichte and the Frailty of Freedom Within the trajectory of modern philosophy, the inheritors of the legacy of the critical system all agree that it is with Kant that we see the first truly From Transcendental Philosophy to Substance as Subject 109 Although there is some general consensus concerning the various ways that the critical system is by itself 110 Chapter 6 Chapter 6 110 incomplete, internal discord quickly arises. Leaving aside Reinhold's and Maimon's own responses to its perceived insufficiencies, Fichte, Schelling, and Hegel, despite all agreeing that (i) the categories of the understanding are dogmatically asserted (they lack a genetic deduction grounding their necessity and universality) and ultimately too static (there is no clear articulation of their systematic interconnectivity) and (ii) concepts such as the thing-in-itself in their Kantian mode are internally contradictory or at least theoretically unnecessary, each offer different strategies to think through the deadlock of Kant's legacy to retain its grounding insights. The early Fichte of the Jena period—the only Fichte who had a significant impact on the internal development of German Idealism— proceeds by removing the extra-subjective alterity of the thing-in-itself, often referred to as the residual trace of a materialism in Kant, by reducing its status to a mere generated effect of subject's purely autonomous activity that knows no outside. Taking as his starting point the immanent field of absolute actuation presented by the subject's radical freedom, whose self- manifestedness is revealed in the unavoidable transcendental reduction of any given fact of consciousness to its activity and most primordially demonstrated by intellectual intuition, and which is best exemplified in the infinite thetic judgement I am,125 subjectivity, as uncontainable freedom, cannot be trumped. Groundless, nothing can get beyond it: it is the ultimate, self-explanatory condition of experience. Yet, although a wondrous fountainhead of activity, the theoretical and practical unconditional beginning that is the I is paradoxically lacking. Not only is its essence (an essence that is its very act of existing) indistinguishable from nothingness insofar as it knows no bounds; more disconcertingly, it is also immensely fragile and immediately threatened by a not-I that risks destroying its very omnipotence from within its own sphere. In the face of the “not-I,” which is transcendentally simultaneous with the “I,” freedom cannot be simply restricted to the undetermined nothingness of the I am, but it must upsurge in an attempt through our very actions and finitude to overcome that which opposes it. From Transcendental Philosophy to Substance as Subject 111 From Transcendental Philosophy to Substance as Subject 111 as the pure act of freedom tries to posit itself as such, it runs up against the impossibility of asserting itself as an autarchic all. In this conflict-ridden battle, the not-I proves to be more than a pure alterity wreaking havoc on the subject's freedom. Instead, it becomes a determined other, an Anstoß (an obstacle and impetus). Through encountering this Anstoß there can be a continual overcoming of the menacing not-I and a never-ending perfecting of the I’s own savage freedom by attaining an ever greater degree of concrete autonomy. In this sense, consciousness emerges from the shock of the not-I on the immanent field of actuation that is the unconscious I in its pure freedom and the latter's defiant cry and refusal to submit when confronted with the possibility of its own demise. The victory of this freedom is never ontologically guaranteed, but can only ever be won again and again in the onslaught of time, whose basic structure is described by the total theoretico- practical syntheses of divisibility opened by the third principle, thus making all principles simultaneous in experience. In this picture there is nothing outside of the pure immanence of the I as freedom and the dynamics of its subjectification as necessitated by the opposition it encounters to its raw, unconditional power. Due to this internal opposition, the I is divided between the absolute I (which is less an egological pole than a faceless, even inhuman activity that only warrants the title of I because it is always spoken of in relation to persons amongst whom it incarnates itself, thus making it in its very essence cryptic126) and the finite (which in turn is divided into passive and active aspects wherein the absolute totality of reality as expressed by the I is never completely annihilated). There is no need for a hard, impenetrable remainder left over from the transcendental constitution of reality: that which threatens to destroy the subject if not gentrified through the syntheses of divisibility, the not-I, is a mere negative magnitude, even if its contorting effects upon the subject can never be predicated and could potentially upsurge as traumatic events. We do not need to go beyond this logical role of an internal pressure proclaiming the possible implosion of the I as freedom to explain experience. From Transcendental Philosophy to Substance as Subject 109 In order to truly sustain its theorectio-practical firstness, to assert its own primordiality against the not-I that “desires” its annihilation, the subject must bring forth the freedom that is its own capacity for absolute actuation in the natural and sociopolitical field of the world, since as soon From Transcendental Philosophy to Substance as Subject 111 Thus, to say that the thing-in-itself becomes a theoretical posit of the subject is to say that its irreducibility to conscious, finite subjectivity and thus its apparently extra-subjective status have been deduced from the self-manifestedness offered by subjectivity as a first principle: its function is 112 Chapter 6 112 Chapter 6 to underline the paradoxical simultaneity of radical spontaneity and fragility in the free constitution of phenomenal reality as it continually comes upon an alien presence within its very intimacy: namely, the irresolvable contradiction that exists in subjectivity between the groundlessness of consciousness as a radically self-grounding idealist freedom and the necessary realist contingency that continually jeopardizes it. For Fichte, the removal of an extra-subjective reality is a necessity imposed upon us not just by Kant's radical articulation of freedom, but more broadly by idealism as such. From within the latter, there can be no coherent assertion of a self-subsisting thing-in-itself which, existing outside of the closed idealist circle of phenomenality in an infinite elsewhere, causes our representations. This would be to transgress the epistemological constraints imposed upon us by the very confines of the idealization of the world; any assertion of the thing-in-itself would constitute a return to the worst kind of dogmatism. The Anstoß is therefore not merely the true limit of idealism, but contains the necessary resources to synthesize idealism and realism into a greater unity:127 instead of proclaiming that all our idealizations are first caused by a foreign intruder pushing in upon subjectivity, it says that if there are immanent obstructions from within the mediating activity of the Ideal, if phenomenal experience is plagued by internal inconsistencies, we can legitimately say that these knots negatively point to some extra-subjective, inassimilable body in subjectivity itself forcing it partially to negate itself and transfer its power to that which is extimately Other in order to save itself from complete collapse.128 Giving the immanent intruder reality is a way to appropriate it: in short, to idealize it. In this way, one of the major tasks of Fichte’s 1794 Wissenschaftslehre is to draw our attention to the radicality of this conditional: if we take idealism seriously then we can without difficulty account for realism, for the realist character of our everyday experience can be entirely explained by the very movement of the Ideal itself. From Transcendental Philosophy to Substance as Subject 111 Idealism does not infringe upon the freedom of the objective world, since it is clear that the latter can in some way make itself known through the very obstructions of our idealizations of it. The epistemic priority of the Ideal is not to be equated with the unknowability of the world, since its autonomous operational self-closure is precisely that which enables us to have a world at all. From Transcendental Philosophy to Substance as Subject 113 From Transcendental Philosophy to Substance as Subject 113 From within this originary psychosis at the heart of subjectivity, there is no escape “except” accepting it and taking the path it opens up to its end by the self-conscious creation of fabulous, imaginative identities (criticism) that can be used Subjectification is nothing more than a spinning in the positively charged void of freedom. In the latter's aftermath, we become irrevocably lost in a series of dream-like images, a rhapsody of sociopolitical phantasmagoria, which give a transcendental structure to the fabric of our experience and thus to our ethical striving, thus even making our own self just one image amongst others, but without having any basis in the world at large. If we take Freud's definition of psychosis as a loss of the causal impact of the world upon the self—a “loosening of the relationship to reality”130—due to a radical withdrawal into primary narcissism, whereby object cathexes are libidinally cut off and the primary process slowly takes over psychic reality, we could venture the claim that the founding gesture of the Fichtean subject is a form of psychosis (the I “posits itself absolutely, and is thereby complete in itself and closed to any impression from without”131), justified in the name of autonomy. Here, just as in the illusionary, image-filled world of the psychotic, the “objective” world is reduced to a mere haphazard obstruction to a self-unfolding and self-sustaining tale creative of its own experiential reality (which for Fichte, “absolutely creates itself [...] in a genesis out of nothing”132), a mere haphazard obstruction that is to be overcome and integrated within the tale if the latter is to sustain its very consistency. What is more, in the same manner that the psychotic must continually refuse new perceptions so as to “autocratically” construct an external and internal world that pleases the id's wishful impulses,133 the Fichtean subject, struggling to subsist as a pure I due to the contemporaneous emergence of the not-I and its violence with its self-positing, strives to rid itself of all influence of the not-I in the syntheses of divisibility and thereby actualize the sphere of absolute self-positing at the empirical level. In this respect, its ideal is a full-blown psychosis, an “alloplastic”134 creation of its own experience, which, though technically impossible (without the influence of the not-I, the I would lack determination), is a structural tendency in all experience, the paradoxical basis of practical freedom. From Transcendental Philosophy to Substance as Subject 113 For Fichte, however, the most decisive consequence to draw from the removal of the thing-in-itself is not a rethinking of the realism-idealism debate. He never takes an interest in the resources that this new way of philosophizing would offer for developing a new form of spectral materialism. The reason is simple: taking the unbridled freedom of the I as his starting point, Fichte agrees with Kant's prohibition on searching for the ontological origins of the subject because, even if it seemingly becomes methodologically possible in Fichte, he argues that it is a futile project, the Ideal being self-explanatory without recourse to the Real. That the subject has no need for the thing-in-itself signifies that the subject is characterized by a constitutive groundlessness: transcendental spontaneity demonstrates that it is totally engulfed within a self-unfolding practical world of its own making, so that what is of primordial importance is never an extra-subjective reality, but the unconditioned freedom of our concrete activity. Fichte does not need to embark upon a metaphysical archaeology of the subject, instead focusing all of his attention on the dynamic inherent in the process of subjectification itself, which inflicts upon us a frightening realization ridden with stark ontological and political implications. Not only are all our actions—and thereby the identities and the collectives that we construct through them—irreducible in themselves insofar as they have no foothold in objective reality, but they become, as it were, mere parts of the free play of an infinite imagination so engrossed by its own self-composing stories that it almost lacks the power to know its self-narrating fiction as a fiction: There is no being. I myself do not know at all and don't exist. There are images: they are all that exists and they know about themselves in the manner of images—images which drift by without there being anything by which they drift; images which hang together through images; images which do not represent anything, without meaning and purpose. I myself am one of these images. No, I am not even that, but only a distorted image of these images. All reality is transformed into a fabulous dream, without there being any life the dream is about, without there being a mind which dreams; a dream which hangs together in a dream of itself. From Transcendental Philosophy to Substance as Subject 113 Intuition is the dream; thought (the source of all being and all reality which I 114 Chapter 6 imagine, of my being, my power, my purposes), thought is the dream of this dream.129 Subjectification is nothing more than a spinning in the positively charged void of freedom. In the latter's aftermath, we become irrevocably lost in a series of dream-like images, a rhapsody of sociopolitical phantasmagoria, which give a transcendental structure to the fabric of our experience and thus to our ethical striving, thus even making our own self just one image amongst others, but without having any basis in the world at large. If we take Freud's definition of psychosis as a loss of the causal impact of the world upon the self—a “loosening of the relationship to reality”130—due to a radical withdrawal into primary narcissism, whereby object cathexes are libidinally cut off and the primary process slowly takes over psychic reality, we could venture the claim that the founding gesture of the Fichtean subject is a form of psychosis (the I “posits itself absolutely, and is thereby complete in itself and closed to any impression from without”131), justified in the name of autonomy. Here, just as in the illusionary, image-filled world of the psychotic, the “objective” world is reduced to a mere haphazard obstruction to a self-unfolding and self-sustaining tale creative of its own experiential reality (which for Fichte, “absolutely creates itself [...] in a genesis out of nothing”132), a mere haphazard obstruction that is to be overcome and integrated within the tale if the latter is to sustain its very consistency. What is more, in the same manner that the psychotic must continually refuse new perceptions so as to “autocratically” construct an external and internal world that pleases the id's wishful impulses,133 the Fichtean subject, struggling to subsist as a pure I due to the contemporaneous emergence of the not-I and its violence with its self-positing, strives to rid itself of all influence of the not-I in the syntheses of divisibility and thereby actualize the sphere of absolute self-positing at the empirical level. In this respect, its ideal is a full-blown psychosis, an “alloplastic”134 creation of its own experience, which, though technically impossible (without the influence of the not-I, the I would lack determination), is a structural tendency in all experience, the paradoxical basis of practical freedom. From Transcendental Philosophy to Substance as Subject 113 From within this originary psychosis at the heart of subjectivity, there is no escape “except” accepting it and taking the path it opens up to its end by the self-conscious creation of fabulous, imaginative identities (criticism) that can be used From Transcendental Philosophy to Substance as Subject 115 From Transcendental Philosophy to Substance as Subject 115 If the human being is an animal, then he is an utterly incomplete animal, and for that very reason he is not an animal.137 What is striking about this quote is not only that Fichte, like Kant before him, draws attention to the utter helplessness of the neonate as an indication 16 Chapter 6 116 of some kind of ontological indeterminacy within nature as that which sets the stage for subjectivity and freedom, an indeterminacy that is non- natural (“nature withdraws her hand”), but also that he is forced to do so from within the confines of a self-grounding idealism. To account for the very transcendental unity of experience, there is a point during the deduction of its laws that something meta-transcendental must be posited whose very existence appears to be responsible for the subject's groundlessness. That there is no possible explication of the leap from not-I to I appears now shaken, for a certain relation between the two has been established despite the fact that such a relation would seem to jeopardize the I's self- standingness. What is at stake is not that Fichte creates an unacceptable subjective idealism within which knowledge of the world is lost and even precluded through being constructed, but that his philosophy ultimately lacks the resources to tie together an ontology of nature with the spontaneity of the I, though such an impossible link has been immanently deduced. Although Fichte too outlines a transcendental materialist ontology of the subject, he, like Kant before him, cannot answer the question “How does appearance itself emerge from the Real?,” even if he stumbles upon the solution. If Fichte and Kant are right in these intuitions in the same way that Lacan could be said to be right in his late musings on the status of nature in light of the psychoanalytical subject, the task facing Hegel, Schelling, and Žižek is thus remarkably similar: how can being and thinking, the Real and the Symbolic, be reconciled to one another, for surely the latter must exist in the former? From Transcendental Philosophy to Substance as Subject 115 to give the subject the resources it needs to actualize itself through the formation of an absolutely free identity liberated from all external causality (dogmatism). What this means is that “the human being is originally nothing:”135 it is free to strive, and should strive, to absolutely create itself according to its own transcendental self-positing/primary narcissism. For both Schelling and Hegel, however, this complete removal of the problematic of the grounding of the subject is not satisfying on two accounts. First, although Fichte's notion of the Anstoß in the 1794 Wissenschaftslehre does allow us to develop something like a spectral materialism, Fichte does not use his own real-idealism/ideal-realism to investigate the obscure origins of the subject, rejecting any transcendental materialist account of the emergence of the I out of the not-I as contrary to reason.136 Transcendentalism must remain purely immanent—we need not investigate its ontological origins, since these will never suffice. But this does not necessarily mean we cannot embark on such an inquiry (a “pre-history” of the Ideal). Second, Fichte himself, despite his remarks concerning its impossibility, like Kant cannot refrain from commenting on the paradoxical ground of the subject's freedom, as if to say that the matter at hand cannot be limited to the transcendental structures at the heart of the self-unfolding conceptual-imaginative fabric underlying subjectification, but must also include the birthplace of the I as causa sui: Every animal, a few hours after its birth, moves and seeks nourishment at the breast of its mother [...]. To be sure, the human being has a plant-like instinct, but he has no animal instinct at all in the meaning given here. He needs the freely given assistance of other human beings, and without it would die shortly after birth. When the human offspring has left its mother's body, nature withdraws her hand from it and cuts it loose, so to speak [...]. For it is precisely nature's abandonment of him that proves that the human being, as such, neither is nor should be nature's pupil. From Transcendental Philosophy to Substance as Subject 117 of its one-sided countermove.141 But what, then, exactly drives Hegel to part ways from Schelling and develop his own way of balancing idealism (radically self-positing freedom) with a philosophy of nature (a system of the world)? And why is this juncture so important for understanding Žižek's own metaphysics of the disjunctive “and”? To answer this question, we must first make a brief detour through the development of modern philosophy. From Transcendental Philosophy to Substance as Subject 115 For the early Schelling, what is necessary is a theoretical project that supplements the ontologically solipsistic Fichtean subject with an account of its emergence out of an unconsciously creative nature, which would implicate the interpenetration and ultimate identity of the Real and the Ideal, so that such a problem is shown to be ultimately moot.138 Interestingly, though he is initially satisfied with Schelling's response to the deadlock of Fichtean idealism,139 Hegel later breaks from what Schelling himself refers to as a “real” or “objective idealism,”140 claiming that by attempting to solve the internal contradictions of Fichte's idealism, Schelling unknowingly becomes its inverted opposite, a mere reactionary form spurred on by the inconsistencies of the former that ultimately fails because From Transcendental Philosophy to Substance as Subject 117 118 Chapter 6 are Hegel and Schelling attempting to demonstrate how it is still possible to do metaphysics within the very breakthrough of critical philosophy without denying any of what they consider to be its necessary/essential presuppositions, but also, and most importantly, why it is necessary to do so: are Hegel and Schelling attempting to demonstrate how it is still possible to do metaphysics within the very breakthrough of critical philosophy without denying any of what they consider to be its necessary/essential presuppositions, but also, and most importantly, why it is necessary to do so: Here, however, a gap between Kant and his followers occurs: for Kant, freedom is an “irrational” fact of reason, it is simply and inexplicably given, something like an umbilical cord inexplicably rooting our experience in the unknown noumenal reality, not the First Principle out of which one can develop a systematic notion of reality, while the Idealists from Fichte onwards cross this limit and endeavour to provide a systematic account of freedom itself. [... F]or the Idealists [this is] just an indication that Kant was not yet ready to pursue his project to the end, to draw all the consequences from his breakthrough. For the Idealists, Kant got stuck half-way.144 Although this may mean a vigorous rethinking of transcendental spontaneity and imagination, noumena and the status of nature, ultimately neither Hegel nor Schelling wants to give up on Kant's descriptions of freedom in order to substitute transcendental idealism with just another classico-metaphysical system. Yet for this endeavour to be successful, they realize that they need to fight against the Critique of Pure Reason's own prohibition of pure ontological inquiry; they deny one of the major claims of critical philosophy in the hope of saving it from itself. This becomes clear when we realize that for both Hegel and Schelling, Spinoza is the very emblem of a philosopher. His rationalist system is admirable not only for its depth and beauty, but for its self-consistency, clarity, and comprehensiveness. Case of Spinoza A common critique of Schelling and Hegel as they attempt to think through the problems bequeathed by Kantian idealism is that they ignore the basic breakthrough of the Critique of Pure Reason: that is, the recognition of the insurmountable finitude of human reason and its inability to grasp the absolute structure of reality. In face of its debilitation of a priori enquiry by pure reason, they return to a metaphysical thinking that has already been debunked. According to the canonical reading, while Schelling talks of the interrelated poles of subject and object in his attempt to balance transcendental idealism with a naturephilosophy, and then of their ultimate identity in the absolute as indifference by developing an account of absolute reason, Hegel supposedly attempts to show that the logical structures that the thinking subject uses to constitute the world transcendentally are actually one with its ontological, that is, its extra-conscious structure. What Hegel does is dialectically “fix up” Schelling by revealing that reality in all its facets (mind and matter, nature and subject) is merely an expression of the rational self-actualization of the absolute as a logical self-unfolding totality, which does away with the Schellingian indifference as an indeterminate void lacking genuine philosophical content, “the night in which, as the saying goes, all cows are black.”142 Žižek, however, refuses this interpretation, calling it “the standard cliché according to which German Idealism pleads the 'pan-logicist' reduction of all reality to the product of the self-mediation of the Notion.”143 Žižek gives us material that allows us to shed new light on the movement from Kant to Hegel by showing that this conventional reading of German Idealism levels off the daring character of the post-Kantian gesture by making it look like just another classical metaphysical system. Not only 118 Chapter 6 118 Chapter 6 In terms of an expression of metaphysics, each looks up to him and sees something fundamentally askew in Kant's preclusion of its possibility—or, as Hegel says “[i]t is therefore worthy of note that thought must begin by placing itself at the standpoint of Spinozism; to be a follower of Spinoza is the essential commencement of all Philosophy.”145 Not only did Kant not show trust in the capacities of human reason, more problematically his critical system was lacking any foundation insofar as it left the origins of the transcendental subject, the very object of its thematic, a mystery and even went so far as to put a ban on their From Transcendental Philosophy to Substance as Subject 119 From Transcendental Philosophy to Substance as Subject 119 investigation. They reject this for two reasons: first, it seems arbitrary, and, second, it threatens to destabilize the very Kantian edifice. Although both Schelling and Hegel believe that metaphysical knowledge of an objective reality is still possible, they are at the same time unwilling to abandon idealism insofar as they are not willing to back down from its articulation of freedom, which leads them to see both questions as intimately intertwined and refuse to separate them as Kant does in order to make room for faith and human autonomy. Schelling, for instance, bemoans the fact that “idealism,” whose founding intuition he identifies in the Freiheitsschrift with the principle of freedom, “is not a work of reason,” and that consequently “the supposedly sad honor of being a system of reason remains only for pantheism and Spinozism.”146 Even if Kant's systematization of freedom is of irreducible importance for the late German Idealists, it must be stressed that of itself it remains theoretically negative in a strong sense: largely remaining at the level of formality, it never really manages to get off the ground and provide a thoroughly developed basis for itself.147 We must provide the missing link between system (Spinozism, absolute determinism) and idealism (Kantianism, the true philosophy of freedom) by showing that freedom itself is a metaphysical possibility. But what does it mean for system if freedom is inscribed within it? Amongst other things, Spinozistic metaphysics represents an avid attempt to rethink the ontological splitting of mind and matter by reconceiving the very notion of substance so that the two categories no longer represent a schismatic split but are subsumable under a single, unified substrate of which they are merely different attributes, all the while preserving as much as possible the basic intuitions of Cartesianism. Mind and matter, the brute material Real of the universe and the self-reflexive powers of ideal spirit, are merely differing “perspectives” on the same, unchanging substance,148 a kind of epistemic parallax shift between two of the countless logical modalities of an all-pervasive “weave” that encompasses all things within its vital ebb and flow. From Transcendental Philosophy to Substance as Subject 119 Although they succeed in manifesting the infinite power of substance through different refraction mediums irreducible to each other, they ultimately must be said to interpenetrate one another insofar as they articulate the same content: there is no possible rupturing chasm between them, but only an untouchable oneness.149 But Chapter 6 120 this oneness is not some kind of undifferentiated, static whole, an abyssal metaphysical void that devours all difference within its cruel awesomeness, like Chronos who eats his own offspring, but rather a pure power or force that is capable of expressing itself in an infinite variety of ways,150 and indeed is only one insofar as it does so. If “nothing exists except substance and its modes,”151 it is because the two constitute one another in an immanent pulsating field teeming, overflowing, with immanent life. Substance is a raw, pulsating activity, an unconstrained upsurge of a dynamic, quivering freedom that is at the very core of the flux that is perceived reality and circulates through its most minute and insignificant features: “God's power whereby he and all things are and act, is his very essence.”152 Substance exists absolutely as a harmonious play of forces even in its most seeming conflict, tension, and struggle. It is a wondrous ballet of cosmic energy whose dance is something peaceful and inspiring, sometimes macabre and dreadful, for us mortals who without choice are engaged in its spectacle. Although this may appear to preclude human liberty, it actually proclaims that human beings are in a certain sense free insofar as they directly participate in the self-actualizing movement of substance (God, nature) as causa sui. In Spinozism, however, there is a crucial modification of the underlying logic of modern subjectivity already seen in Descartes: the intuition of irreducible subjective freedom is said to arise out of a misrecognition of our fundamentally determined character as egos. By locating freedom within the kernel of my being, my will, I am merely misperceiving its notion, for real spontaneity lies in the impersonal self- writing symphony of the universe, the self-creative flow of life and difference (“we are in God's power as clay in the hands of the potter”153), in which I also play a constitutive part insofar as its energy expresses itself through me, animates me, constitutes me, so that any radical distinction disappears. The cause is fully in its effect, for it itself is immanent. From Transcendental Philosophy to Substance as Subject 121 From Transcendental Philosophy to Substance as Subject 121 is an ethics: the whole endeavour of Spinozism is meant to show how, by coming to this realization step by step through clear and precise reasoning, one can liberate oneself from the bondage of one's passive emotions and see how one directly takes part in the freedom of the self-unfolding cosmos, overflowing with uncontainable energy. Showing the structure of the world forces us to act differently—logical proofs are equivalent to opening the mind's eye to the dynamics of substance as totality.154 For the late German Idealists working in the aftermath of Kant, however, this vision reduces the apparent autonomous essence of subjectivity to a mere epiphenomenon, a false appearance, of the vital flux of a more primordial life force that runs through and simultaneously is the universe, thus leaving nothing untouched and no room for a transcendence within its omnipresent pull. This “direct participation” can only be paradoxically interpreted as a passive participation, a forced enactment, which befalls us. We see this most unsettlingly in Spinoza's stone: Furthermore, conceive, if you please, that while continuing in motion the stone thinks, and knows that it is endeavouring, as far as in it lies, to continue in motion. Now this stone, since it is conscious only of its endeavour and is not at all indifferent, will think it is completely free, and that it continues in motion for no other reason than that it so wishes. This, then, is that human freedom which all men boast of possessing, and which consists solely in this, that men are conscious of their desire and unaware of the causes by which they are determined. In the same way that a baby thinks that it freely desires milk [...].155 For the late German Idealists, if this is freedom, it is a grotesque joke: instead of being liberating, it is claustrophobic, for our infinite strivings are reduced to a mere puppet show for an impersonal God sive nature whose power drowns any hope we may have for true self-standing independence in its might. Whatever freedom is here, it is distant from us: “[i]n the mind there is no absolute, or free, will. From Transcendental Philosophy to Substance as Subject 119 Spinoza's account of human freedom, instead of being a pure cancellation of the significance of concrete human ethical striving by its submission to the total system of the world in its self-imposing oneness, is an attempt to show its greater truth, meaning, and role by its inclusion in the intimate life of God or nature: in short, its function within the autonomous and infinitely inventive self- actualization of substance. It is precisely for this reason that metaphysics 122 Chapter 6 What repels him is that freedom is “not my freedom at all but rather that of an alien force outside me:”157 “[t]o stand there cold and dead and merely to look at the change of events an inert mirror or fleeting forms—that is an unbearable existence and I disdain and deplore it. I want to love, I want to lose myself in taking an interest, I want to be glad or sad. [...] Only in love is there life; without it there is death and annihilation.”158 At this juncture, a Spinozist may argue that the German Idealist misgivings of Spinoza's naturalistic pantheism are false. Is not the Ethics itself a profound celebration of life rather than death, love rather than indifference, a text that itself can be read as a quasi-psychotherapeutic intervention that aims, with the aid of its geometrical proofs, to liberate one from the passivity of the passions and in so doing find a new manner of radically asserting the (limited, though existent) freedom of one's own conatus? As Spinoza says, the Ethics “concerns the method, or way, leading to freedom.”159 By demanding that we build a new organ of sight,160 it asks us to undergo a profound change in our relationship to self and world, to become a new species, by giving up ill-founded politico-theological ideals and conceptions of humanity (mere fictions),161 which we can only accomplish by giving into and working with the relational dynamics at the heart of what it means for to be singular essence insurmountably inter- bound with the infinity of other essences. Substance as a causal network of complete interpenetration wherein each being attains its life force reveals the immanent “potentials” of existence: that is, how it can achieve more power, more strength, more force in this or that existent. However, even if in this respect Spinoza does allow for the mind to have some power over the body and thus a certain degree of spontaneous activity (for surely the Ethics is emancipatory for the subject only because it is a work of ideas), according to the German Idealists this does not come close to articulating the irreducibility of freedom attested by Kant.162 Spinoza fails to see the true kernel of human liberty, a failure that not only makes the Spinozistic system insufficient in terms of the lived essence of freedom, but also thereby takes away life and richness from its ontology. From Transcendental Philosophy to Substance as Subject 121 The mind is determined to this or that volition by a cause, which is likewise determined by another cause, and this again by another, and so ad infinitum.”156 It is without a doubt Fichte who most succinctly expresses the German Idealist disdain at this picture. 122 Chapter 6 Without inscribing the difference announced by subjectivity into substance, we miss something essential about the life of the absolute, a critique raised by Schelling in his Freiheitsschrift: From Transcendental Philosophy to Substance as Subject 123 From Transcendental Philosophy to Substance as Subject 123 The error of his system lies by no means in the positing of things in God, but rather in that there are things in the abstract concept of the world's being, instead of infinite substance itself, which in fact is also a thing for him. Thus his arguments against freedom are entirely deterministic, and in nowise pantheistic. He treats the will, too, as a thing, and then naturally proves that it must be determined in its every action by another thing, which, in turn, is determined by yet another thing, etc., into infinity. Hence the lifelessness of his system: the mindlessness of its form, the impoverishment of its concepts and expressions, the unyielding acerbity of its definitions [...]. One could view Spinozism in its rigidity as Pygmalion's statue: it needed to be given a soul by the warm breath of love.163 Hegel makes a similar point in his Science of Logic: Of course, substance is the absolute unity of thought and being or extension; it therefore contains thought itself, but only in its unity with extension, that is to say, not as separating itself from extension and hence, in general, not as determining and informing, nor as a movement of return that begins from itself. For this reason, on the one hand substance lacks the principle of personality—a defect that has especially aroused indignation against Spinoza’s system.164 Humans are not mere passive players in the self-unfolding drama of the universe, but constitutive writers of it, dominating it from the inside out, to such an extent that they even present a challenge to the autonomy of substance. In the face of man, the very fabric of substance appears lacerated, for it encounters a transcendent Other within its heart of hearts that makes it non-coincident to itself, infringes upon its oneness and thereby renders it not-all. According to Žižek, what both Hegel and the middle-late Schelling find unsatisfactory about Spinoza is that he is unable to articulate the ontogenetic condition of the possibility of the emergence of a freely existing transcendent(al) subjectivity out of the purely immanent plane of being and its implications for understanding the metaphysical nature of reality. The problem is that freedom—in its very specific articulation in German Idealism—is Chapter 6 124 not compatible with substance qua devouring totality. From Transcendental Philosophy to Substance as Subject 125 of the Imaginary that often tells itself lies to cover up its dirty spots) and various symbolically non-integrated elements visibly existing in its fold (the ephemeral flickering of a traumatic Real interrupting its normal discourse pointing to its repressed truth), whose integration would demand a radical reconfiguration of its own self-perception (in the spirit of Wo Es war, soll ich werden). Žižek seeks to understand what the role played by this disavowed knowledge could teach us about this crucial turning point in the history of philosophy, the nature of subjectivity, and, ultimately, the very ontological structure of the world we live in. From Transcendental Philosophy to Substance as Subject 123 How, then, are we to think substance and subject/system and freedom if we are to retain the spontaneity first brought to light, albeit for the most part formally, by Kant? For Žižek, the answer is clear: “[t]he passage from the Spinozan One qua the neutral medium/container of its modes [to] the One's inherent gap is the very passage from Substance to Subject.”165 But the recognition of this gap has to be intrinsically traumatic, terrifying—it demonstrates a radical shift in our understanding of the world as some sort of harmonious cosmos that holds itself together in its infinite rational majesty to a world that, lacking totalizing order, must be predicated upon disruption and upheaval. If such an intuition did arise in the history of German Idealism, we would expect to see a series of psychoanalytical defence mechanisms against a conscious acknowledgment of its truth, which in turn obstruct its texts. It is a direct confrontation with this Real of the tradition that will enable Žižek to bring forth the true metaphysical horror of subjectivity that he thinks Descartes had already glimpsed and that has been haunting philosophy like a spectre ever since. It is this specific spin on the dialectical “union” of system and freedom that is of utmost importance for understanding Žižek's psychoanalytical reactualization of German Idealism. It has two functions. First, it demonstrates the heterodox character of Žižek’s appropriation of the tradition insofar as he proclaims that its real truth has always been this disjunctive, parallax relationship between the two terms.166 It clearly shows us how and why Žižek challenges our normal preconceptions of German Idealism's internal historico-theoretical concerns and puts us in a position to evaluate Žižek's own readings more clearly. Second, it demonstrates that Žižek's own specific take on its unconscious Grundlogik is grounded in an extremely coherent reading of the stakes at play in post-Kantian philosophy, even if these have been drastically reformatted along the way according to a perceived and unrealized textual possibility. This makes reading Žižek a strange experience because there is an irreconcilable tension between Žižek's account of German Idealism and what its representatives on the surface appear to accomplish with their systems. Yet through this productive clash, Žižek is trying to create a space of “therapeutic” interplay between German Idealism's surface content (what it takes itself to be in the narcissistic orbit From Transcendental Philosophy to Substance as Subject 125 6.3 The Suffocating Deficiency of a Naturphilosophie The immediate problem facing the late German Idealists is that the Kantian affirmation of transcendental freedom must be grounded in an ontological edifice that can rival Spinozism, for otherwise a Spinozist could argue that freedom is merely the misrecognition of the absolutely free self-unfolding oneness of substance, of man's subsumption within the positive order of being driven by a divinely energized and productive nature as its immanent cause. While both the early Schelling and Hegel offer their own solutions, Hegel remains unsatisfied with the results of his onetime colleague and friend, both in terms of the former's naturephilosophical response to the early Fichte and his quasi-Spinozistic attempts to ground transcendental subjectivity and creative nature in a point of absolute indifference that is neither subjectivity nor nature, but possesses both equally within it. Both projects seem to miss the mark, but why this would lead to a break between Hegel and Schelling is not clear. As hinted by Žižek's vision of Hegel and the middle-late Schelling, Hegel must have implicitly recognized here that Schelling fails to grasp the true radicality of Kantian freedom and its implications by adhering too much to a unitary view of the absolute, the seamless oneness of all that is. Consequently, Hegel tries to save the breakthrough of the critical system by thinking substance as subject, by thinking how the positive order of being ex-ists (existere in the sense of stepping or standing out) in the mode of subjectivity, instead of merely tying two apparently different yet complementary areas together into a precarious, “dead” harmony in indifference, wherein all qualitative difference between Chapter 6 126 subjectivity and objectivity becomes secondary, unimportant, and ultimately lost. As Hegel himself says in his Philosophy of Nature: The cause of [Naturphilosophie's] aberration lay in the fundamental error of first defining the Absolute as the absolute indifference of subjective and objective being, and then supposing that all determination is a merely quantitative difference. The truth is rather, that the soul of absolute form, which is the concept and living reality, is solely qualitative self- sublating differentiation, the dialectic of absolute antithesis. One may think, in so far as one is not aware of this genuinely infinite negativity, that one is unable to hold fast to the absolute identity of life, without converting the moment of difference into a simply external moment of reflection. 6.3 The Suffocating Deficiency of a Naturphilosophie This is of course the case with Spinoza, whose attributes and modes occur in an external understanding; life must then completely lack the leaping point of selfhood, the principle of autonomous movement, of internal self-diremption.167 This citation clearly shows the task Hegel believes must be accomplished: a full actualization of the primordial insight underlying the cogito by instituting the I and the schism it evokes into the very immanent activity of the absolute. For if we follow Fichte's intuition that the subject emerges in being “by absolute spontaneity alone,” that is, “not through a transition, but by means of a leap,”168 in the wake of the subject the life of substance must be said to undergo a process of internal self-diremption. Accordingly, Hegel's project is an attempt to ontologize the Kantian framework by exploring conditions of the possibility of the emergence of the subject out of a ground that remains Other to it (its “pre-history”), insofar as its self-positing must be identical to a liberating self-caused separation from substance that leaves the latter bleeding in its ontological fold. Hegel's goal is to balance Spinoza and Kant by creating a metaphysical system that renders possible rather than precludes freedom in such a way that his “transcendentalism” reaches far beyond the conditions of theoretical knowing or practical action and directly opens up to a metaphysics of the ontological rupture that presents itself as the extra-subjective condition of human autonomy. From Transcendental Philosophy to Substance as Subject 127 From Transcendental Philosophy to Substance as Subject 127 The issue is to explain how a true freely existing subject can arise from within the internal mechanics of substance. Žižek's heterodox and challenging claim is that the only way to do this is by taking the split announced by Cartesian subjectivity and pushing it to its limits by inscribing the non-coincidence of mind with matter into the very heart of being. If human freedom is irreducibly self-reflexive and self-legislative it cannot be understood in terms of the basal energetic pulsating of the absolute. Reading the Hegelian response to Schelling remodulated through psychoanalysis, Žižek suggests that what provokes the movement from transcendental philosophy to Hegelian substance as subject is how Spinozism and the Kantian articulation of freedom reciprocally expose each other's intrinsic limitations, which simultaneously force us to rethink our understanding of ourselves and our relation to the world. While the latter lacks a metaphysics, the former misses the irrevocable (ontological) disturbance of/in nature at the basis of the cogito, which signals that human spontaneity cannot be contained in the positive order of being, something also missed in Schelling's own early Naturphilosophie. For Žižek, the true breakthrough of Kantian idealism, made explicit for the first time in Hegel and in the middle-late Schelling and then most acutely in psychoanalysis, is the proclamation of transcendental freedom as linked to Todestrieb, an excess of being that breaks from all externally given laws, dirempts being from the inside out, and thereby produces a tension-stricken not-all burstin at the seams from inner turmoil. Because of the value Žižek accords to the psychoanalytical experience of the discord between mind and body, he arrives here at a conditional: If freedom exists, substance cannot be all. Substance's auto-disruption is the first-level condition of the possibility of the subject. Although this is perhaps a tenuous claim to make within the context of post-Kantian idealism (it exhibits an abundance of other ways of understanding the substance-subject relation), we should be wary of dismissing Žižek for purely “historico-contextual” reasons. He himself is more interested in another possibility of understanding German Idealism he sees hinted at behind the scenes of its texts. The fact that his reading is not a mere line-by-line commentary is no reason to proclaim that it is outright wrong. As a Lacanian, Žižek does not share the presuppositions that would 128 Chapter 6 28 Chapter 6 128 methodologies and constraints of truth for evaluating his interpretation would, in fact, merely do to Žižek what his critics accuse him of doing. In this respect, their critiques are a performative contradiction. What intrigues Žižek in Hegel's articulation of the subject as self- relating negativity is its connection with the Kantian pedagogical concept of “unruliness” and “diabolic evil.” Insofar as self-relating negativity indicates that human subjectivity is non-natural, it shows that, if we follow the internal course of German Idealism, many of Hegel's concepts such as the “night of the world” or “the activity of dissolution [which] is the power and work of Understanding”169 appear to be nothing other than an elaboration of the subject's origins within an ontological disruption from the closed circuitry of nature's homeostatic laws as already alluded to but not fully developed by Kant. Žižek proclaims that we normally overlook something crucial in Hegel, for what Žižek's Hegel adds to the Kantian notion of the transcendental constitution of experience and rational self-determination is a gesture towards their dark commencement, a glimpse into how the spectral pandemonium of the pre-logical Real we see in “unruliness” precedes and makes possible the autonomy of the cogito and its originary “diabolic evil,” thus drawing a more explicit link between notions whose interdependence Kant merely suggested: The human being is this night, this empty nothing, that contains everything in its simplicity—an unending wealth of many representations, images, of which none belongs to him—or which are not present. This night, the interior of nature, that exists here—pure self—in phantasmagorical representations, is night all around it, in which here shoots a bloody head—there another white ghastly apparition, suddenly here before it, and just so disappears. One catches sight of this night when one looks human beings in the eye—into a night that becomes awful.170 Prior to the self-legislative laws of practical reason and the synthesis of transcendental imagination constituting the unity of phenomenal reality, we must posit some kind of ontological going-haywire that represents a savage tearing apart of the flow of vital being (“here shoots a bloody head—there another white ghastly apparition”) as that which opens up their logical From Transcendental Philosophy to Substance as Subject 129 As a result, the unruliness of the human organism is nothing other than another logical modality of transcendental imagination, its ontological zero-level as a disruption in/of the circuitry of 130 Chapter 6 Chapter 6 130 nature's laws that demands its recombination, a recombination that can only be done in a non-natural (virtual) register; and reading this insight in light of Kant's pedagogical writings on the necessity of disciplining unruliness for the emergence of culture, we can thus further say that the epigenesis of the categories173 as that which bestows upon experience its form cannot be limited to the logico-scientific structure that the latter assumes, but must also extend to the sociopolitical code underlying culture itself. The out- of-control freedom of the subject is intimately linked with the power of imagination (Einbildungskraft) precisely because it is only by means of the latter that the subject is capable of producing images (Bilder or schemata) by which it can give structure to this ghastly state of chaos in the Real, a quasi-phenomenal field lacking subject-object articulation since, although the subject itself has emerged in a primordial sense, as of yet there are no conceptual structures and no symbolic network necessary to mould reality into an integrated, smooth fabric (a process of transcendental Bildung, a schematization of the night of the world). The paradox lies in the following: we can only explain the order of experiential reality in its multifarious modes by presupposing an originary, impenetrable pandemonium that we can only glimpse, but never know (it being always already “overcome” as soon as experience has come on the scene), a pandemonium that logically precedes and ontologically renders possible the consistency of full-fledged subjectivity and psychic life. The I itself as an irreducible core of transcendental reflexivity can only emerge out of this chaos, this macabre seizure of forms that represents ontological mayhem/madness at its finest, which in turn signals that, at its zero-level, the subject is nature in the mode of auto-denaturalization. Hegel's horrifying ontologization of transcendental imagination in the night of the world, however, goes a step further. The night of the world does not merely indicate a radical breakdown in the flux of materiality in the Real; it is also the beginning of an infinite withdrawal of being into itself that cuts all ties with the outside world through obeying its own self-given law. From Transcendental Philosophy to Substance as Subject 129 From Transcendental Philosophy to Substance as Subject 129 possibility. If practical reason and transcendental imagination go hand in hand, it is because both are a response to subjectivity itself, a radicalization of this denaturalizing tendency, to a nature whose fold has been disrupted and thus demands re-articulation (“a night that becomes awful”): The pre-synthetic Real, its pure, not-yet-fashioned “multitude” not yet synthesized by a minimum of transcendental imagination, is, stricto sensu, impossible: a level that must be retroactively presupposed, but can never actually be encountered. Our (Hegelian) point, however, is that this mythical/impossible starting point, the presupposition of imagination, is already the product, the result of, the imagination's disruptive activity. In short, the mythic, inaccessible zero-level of pure multitude not yet affected/ fashioned by imagination is nothing but pure imagination itself, imagination at its most violent, as the activity of disrupting the continuity of the inertia of the pre-symbolic “natural” Real. This pre-synthetic “multitude” is what Hegel describes as the “night of the world,” as the “unruliness” of the subject's abyssal freedom which violently explodes reality into a dispersed floating of membra disjecta.171 This chaotic aggregate of ghastly forms and shapes making up the quasi- phenomenological self-experience of the night of the world is the pure expression of the unruliness/biological short circuitry of the human organism, the German Idealist variation upon Lacan's mirror stage.172 As in the latter, this moment is not to be taken in isolation, but to be supplemented with what it ontogenetically makes possible, namely, the ideal-symbolical realm of ordered experience. What we see here is quasi- phenomenological because, in actuality, there is no fully developed I that stands in relationship to an alterity over and against which it can stand (rather than there being well-defined Gegen-stände, there is nothing but a fragmentary field lacking coherence). This I can only emerge après coup after a free act of synthesis of this initial state of chaotic dispersion, that is, its ideal-symbolic re-articulation. From Transcendental Philosophy to Substance as Subject 131 Just as Kant demonstrates in Religion within the Bounds of Reason Alone, the basis of subjectivity for Hegel is inherently ambiguous, insupportably undecidable: not only does it necessarily bring our own understanding of freedom dangerously close to that of evil, where the difference between them threatens to dissipate, but more disconcertingly it points to the ancestral past of the subject as entangled in some kind of unfathomable ontological crime.175 Whereas early post-Kantian philosophy, namely that of Reinhold and the early Fichte, sought to unify the critical project through elementary philosophy and the absolute self-positing of the I, respectively, Hegel came across something disquieting in his own endeavour to escape its impasses: theoretical and practical reason coincide in a chasm in being that testifies to an unspeakable extra-subjective violence. What both Kant's pedagogic writings and Hegel's night of the world reveal is that in order for idealization/ symbolization to occur as a purely autonomous activity, there must be something in nature that leaves it lacerated, wounded, bleeding, this being the only way that a zone freed from its hegemony could arise. In other words, even if we take the breakthrough of Kantian idealism to be the priority of the symbolic-conceptual categories whereby the subject engenders experience rather than its account of freedom (a move that also assures that culture is always of our own making because not subject to natural determination) even on this reading, this is the dark horizon within which ideality and, by implication, the sociopolitical as a logically autonomous milieu can operate: the death of nature in us. In this respect, if the major insight of German Idealism and psychoanalysis is that “the passage from 'nature' to 'culture' is not direct,”176 then one of its major implications is that the Symbolic is not only a “kingdom within a kingdom” (imperium in imperio), but more radically one that has won its way to ontological sovereignty through a destructive, murdering force, thus establishing the avid anti-Spinozism of both traditions.177 The pure I inaugurated by transcendental philosophy exposes a bone The pure I inaugurated by transcendental philosophy exposes a bone in the throat of substance, a snag in natural causality, proclaiming that the absolute as nature or the physical universe cannot be all. From Transcendental Philosophy to Substance as Subject 129 Consequently, if this ontogenetic mayhem/madness is that which renders possible the absolute spontaneity of the I, then it shares an uncanny structural identity with Hegel's definition of evil in the Jenaer Realphilosophie as an “internal reality, absolute certainty of itself, the pure night of being-for-itself.”174 From Transcendental Philosophy to Substance as Subject 131 132 Chapter 6 constitutes the zero-level of human freedom represents an ontological catastrophe, a catastrophe that is synonymous with the subject itself: “it designates [...] the primordial Big Bang, the violent self-contrast by means of which the balance and inner peace of the Void of which mystics speak are perturbed, thrown out of joint.”178 In this sense, the Žižekian appropriation of the Hegelian attempt to think substance as subject readdresses what really differentiates Hegel from Schelling the Naturphilosoph and sets the stage for his own philosophical career. Although the early Hegel does support Schelling's project to develop a rich teleological account of nature (as various sketches in the Jena period demonstrate), he must be said to have come to some kind of recognition of the inherent limitations of Schelling's endeavour. It is this vital hemorrhaging of nature that, preventing the ideality of the subject from being subsumed within the self-actualizing of the absolute, pushes Hegel away from Schelling: before the transcendental (re)constitution of reality by the I can occur, an internal ontological short circuit, a metaphysical breakdown of substance, must be posited, which not only renders impossible a complete immersion of subjectivity within a natural evolutionary narrative, but declares the ultimate discontinuity between nature and spirit instead of their peaceful identity and mutual interpretation in nature as “the unconscious poetry of spirit.”179 From Transcendental Philosophy to Substance as Subject 131 What must be taken from this is one of the key principles of Žižek's ontology: freedom is not a raw, brute fact, but depends upon the caustic collapse of the vital fold of being, a brisure in the heart of the Real; the chaotic aggregate of ghastly forms that From Transcendental Philosophy to Substance as Subject 133 subject, therefore, has no positive ontological substrate, so that in Hegel the German Idealist attempt to supply a foundation for freedom strangely backfires on itself. But in its very failure, this attempt stumbles upon a great truth: the zero-level of human freedom is a blockage, an obstruction in the mechanics of nature, for the ontological status of the transcendental expresses its dependence upon a self-destructive negativity in substance itself. The Žižekian thesis is that without the articulation of this paradoxical site of excessive negativity (Todestrieb) as emerging immanently within being, all metaphysical accounts of human subjectivity and freedom risk their reductionist-monistic cancelation—otherwise we cannot explain the leap constitutive of the latter without risking their subsumption within an evolutionary transition. If we are to defend the fact of freedom, we must assume it and go all the way, which leads us to a single coherent conclusion: “either subjectivity is an illusion, or reality itself (not only epistemologically) is not-All.”181 subject, therefore, has no positive ontological substrate, so that in Hegel the German Idealist attempt to supply a foundation for freedom strangely backfires on itself. But in its very failure, this attempt stumbles upon a great truth: the zero-level of human freedom is a blockage, an obstruction in the mechanics of nature, for the ontological status of the transcendental expresses its dependence upon a self-destructive negativity in substance itself. The Žižekian thesis is that without the articulation of this paradoxical site of excessive negativity (Todestrieb) as emerging immanently within being, all metaphysical accounts of human subjectivity and freedom risk their reductionist-monistic cancelation—otherwise we cannot explain the leap constitutive of the latter without risking their subsumption within an evolutionary transition. If we are to defend the fact of freedom, we must assume it and go all the way, which leads us to a single coherent conclusion: “either subjectivity is an illusion, or reality itself (not only epistemologically) is not-All.”181 Here we see again how Žižek's reactualization fights against textbook accounts of German Idealism. If Hegel is to be a philosopher of freedom, his metaphysical articulation of the absolute must pass through the Kantian critical system, which means it cannot just be a simple retour to Spinozistic metaphysics; we must witness some kind of brisure within the absolute that disrupts the blind, immanent movement of natural laws and thereby enables the possibility of a freely existing subject. 6.4 The Night of the World/A Monism Bursting at Its Seams Hegel makes a crucial step towards elaborating the paradoxical ground of the subject by demonstrating that it is this going-haywire, this dysfunctioning of substance that makes subjectivity incommensurable with material being and renders possible freedom in the truly “idealist” sense of the word. It is Hegel's account of the advent of the I in nature that is the first to truly explicate the eruption of the subject as an extimacy that cannot afterwards be resubsumed within the oneness of the self-integrating unity of the absolute. There is no smooth union, no ultimate self-penetrating identity within the fabric of all-pervasive being, for we encounter an infinite breakdown, an internal tension, that causes irrevocable havoc in the life of the absolute: “Subject designates the 'imperfection' of Substance, the inherent gap, self-deferral, distance-from-itself, which forever prevents Substance from fully realizing itself, from becoming 'fully itself.'”180 The From Transcendental Philosophy to Substance as Subject 133 From Transcendental Philosophy to Substance as Subject 133 Although this is a rather controversial claim to make in the context of German Idealism, Žižek believes it allows us to reconceptualize its attempt to think substance as subject in such a way that it does not fall into conventional platitudes that smother its theoretical potential: platitudes such as comprehending the absolute as a cosmic subject-like agency that remains constant through all determinate change, preceding and (sub/con)suming the freedom of concrete existence by autonomously actualizing itself from a safe distance from the flux and contingency of reality, thereby guaranteeing the movement of history.182 The subject only emerges from an accidental blockage, an irrevocable moment of self-division, which means that substance and subject are in themselves dirempt and caught in finitude: “Substance is Subject” means that the split which separates “Substance is Subject” means that the split which separates Subject from Substance, from the inaccessible In-itself beyond Subject from Substance, from the inaccessible In-itself beyond 134 Chapter 6 134 Chapter 6 phenomenal reality, is inherent to the Substance itself. [...] The point is not that Substance (the ultimate foundation of all entities, the Absolute) is not a pre-subjective Ground but a subject, an agent of self-differentiation, which posits its otherness and then reappropriates it, and so on: “Subject” stands for the non-substantial agency of phenomenalization, appearance, “illusion,” split, finitude, Understanding, and so on, and to conceive Substance as Subject means precisely that split, phenomenalization, and so forth, are inherent to the life of the Absolute itself.183 Not only does the transcendental reconstitution of reality depend upon a prior upsurge of pure ontological violence, but the very phenomenalization of experience is co-incidental with this rupturing, the not-all, of substance. In other words, subject is substance in its mode of self-alienation: the transcendental reconstitution of reality is nothing but an immanent attempt by the absolute as nature to overcome its split by filling in the gap opened up in the core of its vital being, whereby ego development and concrete experience are reduced to a reaction formation, an attempt to suture a wound. Substance is not subject (as a proclamation of identity): substance becomes subject (in a moment of trauma). The very fact that there is experience demonstrates this for Žižek, for there is no intrinsic reason for experience within the “smooth” self-articulation of substance. From Transcendental Philosophy to Substance as Subject 135 critique of Propositions 12 and 13 of Part I of the Ethics, which argue for the absolute indivisibility and untouchable oneness of substance, but in such a way that we do not fall into the logical conundrum outlined in Propositions 2 through 6, whose goal is to show the impossibility of two substances having nothing in common being able to have an effect upon one another or to produce one another, all the while paradoxically without violating Propositions 14 and 15, which argue that all things must be within one substance. What idealism reveals to us is that freedom expresses something operatively new that has emerged within being and is irreducible to its ebb and flow, yet that does not exist outside of it, a transcendence in immanence that indicates some kind of split within substance itself infringing upon the logical hegemony of its infinite power by means of its internal breakdown. The subject “is not a new name for the One which grounds all, but the name for the inner impossibility or self-blockage of the One.”185 In man, negativity, having attained notional self-reflexivity, shows itself to be foundational to identity rather than a mere subcategory of positivity of the absolute used in the determination of finite things, so that it possesses its own monstrous logic that colonizes the immanent field within which it has emerged from a place that can only appear, from within that field, as an infinite elsewhere. However, what do we see here except the beginnings of a systematic ontologization of Lacanian metapsychology, centred in the mirror stage as a primordial organic disharmony indicative of a disjunctive “and” inscribed within being? Doesn't Žižek imply here that the only possible way to answer Miller's famous question: “Lacan, what's your ontology?” is by a passage through German Idealism, because of the distinctly “psychoanalytical” themes omnipresent in the latter once we psychoanalytically reconstruct its Grundlogik? Yet one other thing should be clear at this juncture. Although Žižek's Yet one other thing should be clear at this juncture. Although Žižek's Hegel glimpses into the ontologico-foundational basis of human spontaneity preceding the transcendental constitution of experiential reality, he is unable to account for two things. First, the immanent generation of this negativity within the material flux of substance that sets the stage for subjectivity by enacting the first lacerations upon substance. How does the vital flow of being itself rupture? From Transcendental Philosophy to Substance as Subject 135 critique of Propositions 12 and 13 of Part I of the Ethics, which argue for the absolute indivisibility and untouchable oneness of substance, but in such a way that we do not fall into the logical conundrum outlined in Propositions 2 through 6, whose goal is to show the impossibility of two substances having nothing in common being able to have an effect upon one another or to produce one another, all the while paradoxically without violating Propositions 14 and 15, which argue that all things must be within one substance. What idealism reveals to us is that freedom expresses something operatively new that has emerged within being and is irreducible to its ebb and flow, yet that does not exist outside of it, a transcendence in immanence that indicates some kind of split within substance itself infringing upon the logical hegemony of its infinite power by means of its internal breakdown. The subject “is not a new name for the One which grounds all, but the name for the inner impossibility or self-blockage of the One.”185 In man, negativity, having attained notional self-reflexivity, shows itself to be foundational to identity rather than a mere subcategory of positivity of the absolute used in the determination of finite things, so that it possesses its own monstrous logic that colonizes the immanent field within which it has emerged from a place that can only appear, from within that field, as an infinite elsewhere. However, what do we see here except the beginnings of a systematic ontologization of Lacanian metapsychology, centred in the mirror stage as a primordial organic disharmony indicative of a disjunctive “and” inscribed within being? Doesn't Žižek imply here that the only possible way to answer Miller's famous question: “Lacan, what's your ontology?” is by a passage through German Idealism, because of the distinctly “psychoanalytical” themes omnipresent in the latter once we psychoanalytically reconstruct its Grundlogik? From Transcendental Philosophy to Substance as Subject 133 Not only does the transcendental reconstitution of reality depend upon a prior upsurge of pure ontological violence, but the very phenomenalization of experience is co-incidental with this rupturing, the not-all, of substance. In other words, subject is substance in its mode of self-alienation: the transcendental reconstitution of reality is nothing but an immanent attempt by the absolute as nature to overcome its split by filling in the gap opened up in the core of its vital being, whereby ego development and concrete experience are reduced to a reaction formation, an attempt to suture a wound. Substance is not subject (as a proclamation of identity): substance becomes subject (in a moment of trauma). The very fact that there is experience demonstrates this for Žižek, for there is no intrinsic reason for experience within the “smooth” self-articulation of substance. But here we should make another addition. When we say that the subject is a transcendent Other created from within the immanence of being, we must be careful. It is not that substance creates or produces another substance, in which case the enigma concerning how one could explain the genesis of one out of the other would remain unsolved. Rather, the subject is nothing but the void of substance, the minimal difference of the absolute to itself,184 which creates a metaphysical vacuum that is able to have devastating effects in the fabric of the Real of nature from which it emerged as soon as it develops the capacity to paradoxically relate to itself (movement from “unruliness” to “diabolic evil,” from the tearing apart of the flow of vital being to its radicalization in “the night of the world”). In this sense we should understand late German Idealism, and by consequence Žižek's own metaphysics, as attempts to think system and freedom as a From Transcendental Philosophy to Substance as Subject 135 136 Chapter 6 the Real, thus making it barred, nothing but a series of membra disjecta? Second, the absolute spontaneity of the very founding gesture of subjectivity that depends on nothing but itself for its own self-positing, a self-positing that presents itself as a fiat that of itself institutes a pure difference in being: the problem for us is how we are to conceive of the founding gesture of subjectivity, the “passive violence,” the negative act of (not yet imagination, but) abstraction, self-withdrawal into the “night of the world.” This “abstraction” is the abyss concealed by the ontological synthesis: by the transcendental imagination constitutive of reality—as such, it is the point of the mysterious emergence of transcendental “spontaneity.”186 To answer these questions, we will need to delve into Žižek's oft-neglected work on Schelling, for it is Schelling who delved most profoundly into the obscure origins of subjectivity. From Transcendental Philosophy to Substance as Subject 135 How does this extimate core germinate within the Real and incite a violent explosion that forever precludes the ontological life of 136 Chapter 6 120. In the Freiheitsschrift, for instance, Schelling says that it is idealism that “we have to thank for the first perfect concept of freedom” and then situates the true Kantian breakthrough in its revolutionary articulation. See pp. 231ff. Hegel also praises Kant’s theory of freedom. See Encyclopedia Logic, p. 101 (§54Z). The same applies to Žižek: “No wonder Kant is the philosopher of freedom: with him, the deadlock of freedom emerges.” Žižek, The Parallax View, p. 9. But all this was stressed by Kant himself, who declares that freedom “constitutes the keystone of the whole architecture of the system of pure reason and even of speculative reason.” Critique of Practical Reason, in Practical Philosophy, trans. and ed. Mary J. Gregor (Cambridge: Cambridge University Press, 1999) p. 139. Notes 120. In the Freiheitsschrift, for instance, Schelling says that it is idealism that “we have to thank for the first perfect concept of freedom” and then situates the true Kantian breakthrough in its revolutionary articulation. See pp. 231ff. Hegel also praises Kant’s theory of freedom. See Encyclopedia Logic, p. 101 (§54Z). The same applies to Žižek: “No wonder Kant is the philosopher of freedom: with him, the deadlock of freedom emerges.” Žižek, The Parallax View, p. 9. But all this was stressed by Kant himself, who declares that freedom “constitutes the keystone of the whole architecture of the system of pure reason and even of speculative reason.” Critique of Practical Reason, in Practical Philosophy, trans. and ed. Mary J. Gregor (Cambridge: Cambridge University Press, 1999) p. 139. 121. In this sense, both Hegel and Schelling are following Fichte: “I live in a new world since I read the Critique of Practical Reason; it destroyed theses that I thought were irrefutable, proved things I thought indemonstrable, like the concept of abso- lute freedom.” Johann Gottlieb Fichte, Gesamtausgabe der Bayerischen Akademie der Wissenschaften, Division III: Briefe, vol. I, ed. R. Lauth, Hans Jacob, and H. Gliwitzky (Frommann: Stuttgart-Bad Cannstatt, 1964–), no. 63, p. 168. 122. Žižek, Less Than Nothing, p. 265. 123. Žižek, The Parallax View, p. 93. 124. Ibid., p. 92. 125. Fichte, Science of Knowledge, p. 114. 126. Maldiney, Penser l’homme et la folie (Paris: Gillon, 2007), p. 114. 127. Fichte, Science of Knowledge, p. 247. 127. Fichte, Science of Knowledge, p. 247. 128. Ibid., p. 152. 128. Ibid., p. 152. 129. Fichte, The Vocation of Man, trans. Peter Preuss (Indianapolis: Hackett Publishing, 1987), p. 64. 130. Freud, “The Loss of Reality in Neurosis and Psychosis,” SE, XIX, p. 183. 131. Fichte, The Science of Knowledge, p. 243. 131. Fichte, The Science of Knowledge, p. 243. 132. Fichte, Wissenschaftslehre 1805, ed. H. Gliwitzky (Hamburg: Felix Meiner, 1984), pp. 127–28. 133. Freud, “Neurosis and Psychosis,” SE, XIX, p. 151. 134. Freud, “The Loss of Reality in Neurosis and Psychosis,” SE, XIX, p. 185. 135. Fichte, Foundations of Natural Right, ed. Frederick Neuhouser, trans. Michael Baur (Cambridge: Cambridge University Press, 2000), p. 74. 136. Fichte, Fichte: Early Philosophic Writings, p. 147. 137. Fichte, Foundations of Natural Right, p. 76. Also compare p. 74. 138. See Schelling, System of Transcendental Idealism, trans. Peter Heath (Charlottesville: University Press of Virginia, 2001), p. 232. 139. Notes See Hegel, The Difference between Fichte’s and Schelling’s System of Philosophy, pp. 143 and 168. 140. Schelling, Darstellung meines Systems der Philosophie, Schellings sämmtliche Werke, ed. K. F. A. Schelling, division I, vol. 4 (Stuttgart and Augsburg: J. G. Cotta’scher Verlag, 1856–1861), p. 109. 141. In his “mature period,” Hegel declares Schelling as merely Fichte’s successor, ironically going against the spirit of his early work on the insurmountable differ- ence between them. Cf. Lectures on the History of Philosophy: Medieval and Modern Philosophy, trans. E. S. Haldane and Frances H. Simson (London: University of Nebraska Press, 1995), p. 529. 142. Hegel, The Phenomenology of Spirit, trans. A. V. Miller (London: Oxford University Press, 1977), p. 9. 143. Žižek, The Ticklish Subject, p. 55. 144. Žižek, Less Than Nothing, p. 266. 145. Hegel, Lectures on the History of Philosophy: Medieval and Modern Philosophy, p. 257. 146. Schelling, Freiheitsschrift, p. 231. 147. Ibid., p. 232. 147. Ibid., p. 232. 148. Spinoza, Ethics and Selected Letters, ed. Seymour Feldman and trans. Samuel Shirley (Indianapolis: Hackett Publishing, 1982), II p7 (p. 66). 149. Ibid., I p13c (p. 39). 149. Ibid., I p13c (p. 39). 150. Ibid., I p16 (p. 43). 150. Ibid., I p16 (p. 43). 151. Ibid., I p15 (p. 40) and I ax1 (p. 31). 151. Ibid., I p15 (p. 40) and I ax1 (p. 31). 152. Ibid., I p34 (p. 56). 152. Ibid., I p34 (p. 56). 152. Ibid., I p34 (p. 56). 152. Ibid., I p34 (p. 56). 153. Ibid., Letter 78 to Henry Oldenburg (p. 254). 154. Ibid., V p23s (p. 216). 154. Ibid., V p23s (p. 216). 138 Chapter 6 155. Ibid., Letter 58 to G. H. Schuller (p. 250). Compare with I p24 (p. 49), I p26 and 27 (p. 50), and I p33 (p. 54). 156. Ibid., II p48 (p. 95). 156. Ibid., II p48 (p. 95). 157. Fichte, The Vocation of Man, pp. 21–22. 157. Fichte, The Vocation of Man, pp. 21–22. 158. Ibid., p. 24. 158. Ibid., p. 24. 159. Spinoza, The Ethics, p. V pref (p. 203). 160. Ibid., V p23s (p. 216). 160. Ibid., V p23s (p. 216). 161. Ibid., I app (pp. 57–62). 161. Ibid., I app (pp. 57–62). 162. Schelling, Freiheitsschrift, p. 227. 162. Schelling, Freiheitsschrift, p. 227. 163. Ibid., pp. 230ff. 163. Ibid., pp. 230ff. 164. Hegel, The Science of Logic, p. 472. 165. Žižek, The Parallax View, p. From Transcendental Philosophy to Substance as Subject 139 186. Žižek, The Ticklish Subject, p. 61. 184. Žižek, Less Than Nothing, p. 905. 184. Žižek, Less Than Nothing, p. 905. Notes 42. 166. See Žižek, The Abyss of Freedom, pp. 11–14. 167. Hegel, Hegel’s Philosophy of Nature, vol. 3, pp. 142–43 (§359A). Translation modified. 168. Fichte, Science of Knowledge, p. 262. 169. See Hegel, Phenomenology of Spirit, pp. 18–19. 170. See Hegel, Frühe politische Systeme (Frankfurt am Main: Ullstein, 1974), p. 204; quoted in Donald Philip Verene, Hegel’s Recollection: A Study of Images in the Phenomenology of Spirit (Albany: SUNY Press, 1985), pp. 7–8, whom Žižek takes it from. For a discussion, see The Abyss of Freedom, pp. 4–14; The Ticklish Subject, pp. 26–48; and The Parallax View, pp. 43–45. 171. Žižek, The Ticklish Subject, p. 33. 172. Kant knew this very well. See Kant, Anthropology from a Pragmatic Point of View, p. 423. 173. Kant, The Critique of Pure Reason, B167. 173. Kant, The Critique of Pure Reason, B167. 174. Hegel, Frühe politische Systeme, p. 262. 174. Hegel, Frühe politische Systeme, p. 262. 175. Žižek and Daly, Conversations with Žižek, p. 126. 176. Žižek, The Ticklish Subject, p. 36. 176. Žižek, The Ticklish Subject, p. 36. 177. Compare Spinoza, The Ethics, III pre (p. 103). 177. Compare Spinoza, The Ethics, III pre (p. 103). 178. Žižek, The Ticklish Subject, p. 31. 178. Žižek, The Ticklish Subject, p. 31. 179. Schelling, System of Transcendental Idealism, p. 12. 179. Schelling, System of Transcendental Idealism, p. 12. 180. Žižek, The Abyss of Freedom, p. 7. 180. Žižek, The Abyss of Freedom, p. 7. 181. Žižek, The Parallax View, p. 168; Less Than Zero, p. 725; and Žižek & Woodard, “Interview,” The Speculative Turn: Continental Materialism and Realism, ed. Bryant, Levi, Nick Srnicek, and Graham Harman (Melbourne: re.press, 2011), p. 407. My italics. 182. See Žižek, Less Than Nothing, pp. 234–35 and 286–92. 183. Žižek, The Ticklish Subject, pp. 88–89. 183. Žižek, The Ticklish Subject, pp. 88–89. 184. Žižek, Less Than Nothing, p. 905. 185. Ibid., p. 380. 186. Žižek, The Ticklish Subject, p. 61. Chapter 7 The Logic of Transcendental Materialism Schelling and the Spectral Other Side of German Idealism Žižek needs to go beyond Hegel to articulate a crucial dialectical moment of transcendental materialism: the emergence of an ontological violence within being that prohibits the indivisibility of the absolute as an infinitely powerful, self-sustaining whole and creates room for irreducible freedom and ideality. To demonstrate this, I will outline the inherent limitations in Hegel's attempt to think substance as subject by focusing on Žižek's criticisms of the mature Hegelian Encyclopedia, thereby showing the theoretical hole simultaneously opened up and concealed by Hegel. Spurred on by the perceived threat of Absolute Idealism to human freedom, what we will see is how Schelling's investigation of the abyssal origins of subjectivity presents us with a passionate attempt to rethink the grounding of the subject and its role in being so that the former is never reducible to the latter. In this regard not only can Schelling's “materialist” response to Hegel be seen as the culminating, concluding step in what Žižek takes to be the unconscious Grundlogik of German Idealism, but it puts us face to face with the spectral Other Side of that tradition which it previously recoiled from to varying degrees: the insurmountable tension between the Real and the Ideal, which imposes a new conception of a never-to-be-reconciled quadruple dialectics with stark consequences for our understanding of nature, human historicity, and the absolute. The Logic of Transcendental Materialism 1 142 Chapter 7 upon Schelling remains largely behind the scenes potentially demonstrates some kind of error, inconsistency, or sleight of hand. Not only does Žižek fail to give any systematic argumentation for the superiority of Schelling over Hegel in terms of the obscure origins of the I out of its pre-subjective Grund, he also levels off the differences between the two insofar as he appears, as will become clear, to read them reciprocally through one other. Here I am thinking specifically of his endeavour in The Parallax View to show that, “far from posing an irreducible obstacle to dialectics, the notion of the parallax gap provides the key which enables us to discern its subversive core. To theorize this parallax gap properly is the necessary first step in the rehabilitation of the philosophy of dialectical materialism.”188 But this quote is suspicious: remarking that Schelling was “the first to formulate the post- idealist motifs of finitude, contingency and temporality,”189 and is even thus “at the origins of dialectical materialism,”190 it is clear that Žižek associates Schelling with the beginning of this tradition rather than Hegel; Schelling occupies an “immediate” place, acting “as a kind of 'vanishing mediator' between the Idealism of the Absolute and the post-Hegelian universe of finitude-temporality-contingency, [such] that his thought—for a brief moment, as it were in a flash—renders visible something that was invisible and withdrew into invisibility thereafter.”191 It is Schelling, not Hegel, who supplies us with the premonitions of a new radical way of philosophizing, a new dialectics. Moreover, the very idea of an insurmountable internal limit (a gap) as constitutive of (onto)‍logical movement—the necessity of positing the non-coincidence and tension of its moments to one another in order for it to function—has a more manifest affinity with Schelling's middle-late philosophy, which, developed as a response to the absoluteness of the Hegelian self-mediating Notion, bases itself on the idea of the indivisible remainder, der nie aufgehende Rest, as an irremovable snag in every system that guarantees its very vitality. 7.1 The Hegelian Recoil, The Schellingian Breakthrough In order to explicate how substance auto-disrupts into a chaotic series of membra disjecta, the ontological zero-level preceding the synthetic modality of transcendental imagination, we have to move outside Žižek's reading of Hegel and confront his works on Schelling. The night of the world is merely a profile portrait of the disarray and pandemonium that pave the way for the transcendental constitution of reality into a (relatively) unified fabric of experience. It does not of itself explain the originary moment of withdrawal from organic immersion in the positive order rendered possible by Todestrieb as the obscure birthplace of an irreducible more-than-material subjectivity; comprehending this event requires us to first plunge headfirst into the immanent pulsation of the vital ebb and flow of being itself if we are to see how it sets the stage for the latter—a project that Žižek explicitly says is most acutely developed in Schelling: Kant was the first to detect this crack in the ontological edifice of reality: if (what we experience as) “objective reality” is not simply given “out there,” waiting to be perceived by the subject, but an artificial composite constituted through the subject's active participation—that is, through the act of transcendental synthesis—then the question crops up sooner or later: what is the status of the uncanny X that precedes the transcendentally constituted reality? F. W. J. Schelling gave the most detailed account of this X in his notion of the Ground of Existence—of that which “in God Himself is not yet God”: the “divine madness,” the obscure pre-ontological domain of “drives,” the pre-logical Real that forever remains the elusive Ground of Reason that can never be grasped “as such,” merely glimpsed in the very gesture of its withdrawal.187 This, however, creates an internal problem for Žižek's work insofar as he describes his own project time and time again as Hegelian and never as Schellingian. If it is Schelling who is the philosopher who most fully describes the material ontogenetic conditions for the emergence of the subject rather than Hegel and thus more predominantly influences Žižek's metapsychology and ontology, then the fact that his own overt reliance 142 Chapter 7 142 Chapter 7 142 Chapter 7 Žižek in many ways appears to be interpreting Hegel retroactively through Schelling, which would explain why the irreducibility of the parallax between moments in contradistinction to their organic interpenetration in the notional structure of any given phenomenon is the “perverse” truth of Hegelian dialectics.192 What is more, in a key passage in Less Than Nothing that describes dialectics as “the science of the gap between the Old and the New,” Žižek abruptly and without The Logic of Transcendental Materialism 143 The Logic of Transcendental Materialism 143 explication jumps into a discussion of the middle-late Schelling, going so far as to say that to avoid mystification properly we should not abandon his project from this period of his thinking, but rather “reformulate it so as to avoid the mystification of the theosophic mytho-poetic narrative,”193 in such a way that it appears that the proximity of his reading of Hegel and Schelling when coupled with this brief and rare methodological explanation points to the core of his heterodox reading of German Idealism and thus his philosophy. This suggests that the core of Žižek's philosophy is a hybridism of Schellingianism and Hegelianism, so that exploring this intersection puts us face to face with the theoretico-ontological stakes underlying his entire project. Yet the following question imposes itself: at what point is Žižek's own metaphysical archaeology of the subject Schellingian or Hegelian? The very posing of this question is relatively misleading within the context of Žižek's reactualization of German Idealism, considering that what interests him is not Kant, Schelling, or Hegel as particular historical thinkers whose respective philosophies often display insurmountable differences to one another, not to mention incompatible concerns (to such a degree that one could even question whether German Idealism constitutes a coherent tradition with a single logical nucleus). What he finds alluring is a psychoanalytical truth self-unfolding throughout their works, a truth inaugurated by the Cartesian cogito and ultimately culminating in Freud and Lacan, but that they are unable to articulate fully due to its traumatic nature, “our experience of freedom” being, after all, “properly traumatic.”194 However, even if what intrigues Žižek is the disavowed Grundlogik implicitly driving the movement as an unconscious formation—something that appears in a flash only to withdraw again into the abyss from which it came—we can nevertheless demonstrate a certain dominating influence of Schelling by showing how the latter fills in a theoretical void opened up by Hegel and thereby radicalizes the founding insight of German Idealism. According to a Žižekian narrative of the untold history of German Idealism, the essence of that impossible X that eternally precedes the birth of consciousness remains underdeveloped in the Hegelian attempt to think substance as subject. The Logic of Transcendental Materialism 143 Although the Hegel of the night of the world hints at the disturbing metaphysical paradoxes that arise out of the ontologization of 144 Chapter 7 Chapter 7 144 transcendental imagination and its concomitant concept of freedom, in The Ticklish Subject Žižek expresses outright dissatisfaction with Hegel's most systematic undertaking to inscribe the subject within a dialectics of nature as propounded in the various versions of The Encyclopedia of the Philosophical Sciences. Since this book is written after The Indivisible Remainder, it would appear that Žižek's critical engagement with Hegel's mature system and its account of the passage to culture is based on the presuppositions that guide his own transcendental materialism as worked out in this vehement work on Schelling published only three years earlier. In other words, his admitted disapproval is an implicit proof of the prioritization given to Schellingian ontology for the theorization of the parallax as a metaphysics of the disjunctive “and.” Pointing to an ambiguity persisting within the movement from self- contained Notion to nature and then to spirit in his mature system, Žižek suggests that Hegel was unable to bring into conceptual fullness the earth- shattering realization that he was on the verge of.195 What is left aside is, strictly speaking, the night of the world that Hegel's earlier Realphilosophie had uncovered. In the Encyclopedia, it is uncertain how this radical negativity, this moment of irremediable ontological breakdown haunted by sanguinary spirits, truly fits in. Instead of the precarious, never-to-be- complete “reconciliation” between nature and finite spirit brought forth by the subject, culture itself becomes a closed circuit, a complete return of the Idea to itself out of its infinite self-outsidedness in nature, a move that, by rendering culture a self-sufficient, self-contained all, completely does away with the unruliness that is the zero-level of freedom as revealed in psychoanalytical experience and potentially jeopardizes the irreducibility of the practico-concrete in Kant and made explicit for the first time in Fichte. The Logic of Transcendental Materialism 14 145 the snag in the machine of the Symbolic, loses all currency: we would paradoxically lose the very condition of free spiritual activity, for a complete sublation of nature into culture would herald the destruction of culture as a process of building an artificial, second nature where one is missing. Losing the obstacle causes us, in turn, to lose the goal; dialectics needs its own inner impediment to get off the ground. Corresponding to the reality/Real distinction in Lacanian psychoanalysis, we may thus venture that there are two forms of Hegelian dialectics: either we have the perfect dialectical triad of the mature system (Logic → Nature → Spirit) or a non-closed tetrad that signals the self-collapse of dialectical logic itself as seen in the Realphilosophie (Logic → Nature → finite Spirit → objective/naturalized Spirit).197 The triad is, strictly speaking, not merely inconsistent with Hegel's—and ultimately Kant's—true earlier insight, but self-defeating: it robs dialectics of its own energy, energy that can only be mobilized due to the structural impossibility of completing the task it sets out to complete. At the most basic level, culture can never utterly sublate the excessive kernel of human being and simply make it a moment of the self- meditation of the Notion as it seeks to actualize itself: there must always be a minimal, insurmountable distance between the unruliness of human nature, the withdrawal into the nocturnal Innenwelt of the world that is the founding gesture of subjectivity, and the symbolic, cultural network that attempts to form and discipline this non-natural violence into a new order after subjectivity has posited itself as such. The two can never overlap within an all-pervasive totality, insofar as for Žižek this overlapping would not merely level out the singularity that marks the subject (namely, the fact that it cannot be fully explained by either material or cultural determinations) but also in the same breath radically preclude the condition of the possibility of human freedom and the exploration of its larger metaphysical implications. The difference between traditional accounts of Absolute Idealism and the quadruple dialectic of the Realphilosophie thus enables us to demonstrate the logic Žižek wants to defend both in terms of his own transcendental materialism and the unconscious Grundlogik of modern philosophy that he psychoanalytically constructs. The Logic of Transcendental Materialism 143 The issue is that the Idea is nothing other than this very act of its own returning to itself, nothing but the attempt at reconciliation, so that not only is this very movement generative of that to which the movement returns, but more drastically the self-alienation of the Idea is a condition of its returning to itself.196 If the ontological zone wherein the fabric of the world is torn apart (“here shoots a bloody head—there another ghostly apparition”) disappears, the claim is stronger than simply the subject as the irreconcilable in-between of nature and culture, the bone in the throat of substance and The Logic of Transcendental Materialism 14 Whereas the former articulates itself according to a series of upward-moving spirals wherein each new turn completely encompasses the previous one in an act of subsumption (so 146 Chapter 7 46 Chapter 7 The Logic of Transcendental Materialism 147 self-development of the absolute by instituting a new age of the world that can never be reconciled with that which came before, in a moment of ontological triumph. As a consequence, if we inscribe culture into the fabric of the universe according to the second model of dialectics, we are forced to conclude that the absolute is open, precarious, and necessarily incomplete, for the symbolic universe is not only constitutively out of joint with nature, but as the always doomed attempt to reconcile itself with the latter, is constantly forced to reinvent itself. The process of subjectification (culture) emerges out of the ontological chasm opened up by the pure I and holds a position of infinite difference with respect to nature insofar as it operates within a zone of logical non- coincidence that has been carved out from within the laws of the latter. Instead of a self-enclosed spiral or circle of circles, we see an immanent “break” that prevents the next dialectical phase of self-appropriation from occurring and by means of which another level of autonomous activity irreducible to the first can take hold. The image is of two cones—one ontologically positive, the other immersed in a virtual zone of nonbeing— linked together by a black hole that is the pure I, the night of the world, whereby nature and culture self-actualize in isolation to one another, but are nevertheless negatively tied together by the abyssal void of subjectivity—that which “protrudes” out of both as an impossible in-between non-explicable in either. The subject stands for the bone in the throat of substance that prevents it from being a devouring all following its own immanent laws (nature's non-coincidence to self) just as much as it stands for that snag in the cultural machine (the non-all of the symbolic Other) that can never be filled in or completely overcome, and that thus constitutes the impetus for all subjectification as a series of reaction formations and the infinite proliferation of the forms it can take on due to its necessary failure of covering up, schematizing, the primordial trauma. In this regard, transcendental materialism presents a radically different view of dialectics as Absolute Idealism, going so far as to claim that what the latter misses is that it is, at best, a mere compensation in fantasy for the traumatic truth of the former. 146 Chapter 7 With culture, we see that nature had immanently produced an eruptive, shattering transcendence (the subject) that bursts The Logic of Transcendental Materialism 147 146 Chapter 7 that we encounter a completely self-enclosed, organic oneness that slowly articulates itself in increasing complexity) the very self-unfolding operation of the later precludes the possibility of such a self-totalizing activity. While Absolute Idealism itself does move forward on the basis of fundamental non-coincidence or immanent contradiction (there is conflict internal to the system), it is always productive of new, evermore comprehensive unity, but in such a manner that its innate teleological push towards greater order and self-coherence is able to project an upper limit. Since it knows no radical inner impediment, the ever-expanding series of upward spirals predicts a point in history when the Idea would attain perfection by returning to itself and in turn overcome its prior self-alienation, that is to say, when nature and culture would become reconciled in a moment of ontological jubilation. Here, understood as the self-development of the structure of the world, the absolute is seen as an immanent processual movement from self-externality to absolute self-mediation: by making itself into the Idea, nature (as a realm of pure contingency) would have succeeded by coming to a complete grasp of itself in the freedom of thought, whereby the end of the movement would see itself in the beginning, thus closing the circle of circles. Within transcendental materialism, however, the passage from nature to culture does not reveal a struggle of notional transmutation as culture endeavours to rid itself of its basis in nature in the onslaught of history with the promise of completion, but rather reveals a standstill in the heart of being that cannot be brought into a higher moment of truth of free spirit that would bring the circle of circles to an end: the ebb and flow of substance ontogenetically incites the birth of a freely existing subject only through a self-sabotaging, self-destructive movement that defies perfect reconciliation, because this unruliness inheres in all culturally achieved unity and disrupts it from within. Conflict, though here too internal to the system, articulates at this juncture of the passage from nature to culture an irrevocable place of rupture, devastation, or laceration in the absolute, which points to a dialectical residue that can never become a vehicle of internal growth of the structure of the world, yet that simultaneously sustains culture as the very attempt to overcome it. 148 Chapter 7 with the Freiheitsshcrift or the Weltalter, this demonstrates the manifest Schellingian character of Žižek's criticism of Hegel, while at the same time locating the germ of the former's logic of the Grund within Hegel's early Realphilosophie: But dependence does not annul autonomy or even freedom. It does not determine essence, but merely says that the dependent, whatever it might be, can only be as a consequent of that upon which it is dependent; it does not say what it is, and what it is not. Each organic individual, as something which has become, has its being only through another, and to this existent it is dependent in terms of becoming, but not at all in terms of being. It is not incongruous, says Leibniz, that he who is God is at the same time begotten, or vice versa; as it is no more a contradiction to say that he who is the son of a man is himself a man.198 The Logic of Transcendental Materialism 147 Here we have a rich account of the emergence of two zones of activity wherein, although the second is dependent upon the first that constitutes its genetic ground, it remains entirely free. To anyone familiar 148 Chapter 7 7.2 The Weltalter and the Systematization of Freedom One of the most interesting aspects of Žižek's reactualization of German Idealism is its claim that the middle-late Schelling's “departure” from the throes of reason and “descent” into theosophical obscurantism does not demonstrate a break from modern rationality as inaugurated by Descartes' search for a self-evident Archimedean starting point for all philosophy (famously developed further in Kant's transcendental conditions of the possibility of knowledge, and ultimately epitomized by Hegel's self- mediating Notion).199 On the contrary, Žižek's reactualization states that Schelling actually makes explicit for the first time the perverse, unconscious truth that remains hidden throughout the entire tradition, but only appears ephemerally through the distortion of its imaginary-symbolic universe: his attempt to present a system that would be able to combat the perceived threat posed by Hegel's horrifying “pan-logicism” presents a radicalization, a completion, of modernity's fundamental insight into the paradoxical origins of subjectivity, an insight that Hegel himself was unable to follow through in his own endeavour to solve the impasse bequeathed by Kant's critical philosophy. In this sense, Žižek implies that it is philosophical orthodoxy The Logic of Transcendental Materialism 149 The Logic of Transcendental Materialism 149 Although, for instance, in the Naturphilosophie Schelling is also interested in the dark side of nature, the project forecloses the possibility of Todestrieb as an 150 Chapter 7 Chapter 7 150 emergent and infinitely disruptive force in being. In this sense, Žižek's rejection of the early Schellingian attempts to balance transcendental idealism with materialism follows the same line of argument as his denial of the theoretical weight of contemporary evolutionary models of self- reflexive consciousness,202 for both share the same fault: the ultimate identification of mind and matter instead of the articulation of their ultimate irreconcilability to one another. From the standpoint of the immanent laws of the pre-symbolic Real (as avowed by Spinozistic monism or reductionistic materialism), Žižek's claim is that the Ideal cannot be explained either on the basis of a teleological or purely naturalistic emergence. The Ideal explodes from within the vital throes of positive being,203 rather than just being one specific (albeit complex) mode of physical organization, for it names an alienating distance to self, a non-coincident split that literally short-circuits the world. Rather than inhering in matter as its implicit structure, mind can only emerge within the void of this ontological scar, thus making it impossible to reconcile with matter. Written in the aftermath of the birth of the Hegelian system with the publication of the Phenomenology of Spirit in 1807, which contains a famous explicit criticism of the Schellingian philosophy of absolute indifference as an attempt to balance the two poles of realism and idealism,204 the Freiheitsschrift (1809) and the Weltalter (1810–1815) radically restructure the problematic that had occupied Schelling's philosophical career. The Logic of Transcendental Materialism 149 that got it wrong: it is not Schelling who is the misfit, but rather Hegel, for it is he who turns away from the abyss brought forth by the idealist account of freedom after gazing too deeply into its traumatic core. After the Realphilosophie, something prevents Hegel, holds him back—there is a recoil, a hesitation. What is so compelling for Žižek about works such as the Weltalter is not their anti-Hegelian character, but their ability to penetrate into the breakthrough heralded by modern philosophy and to bring it to a new, higher level. It is Schelling who gives the complete articulation of its underlying but disavowed core insofar as it is he who most fully outlines the principles of a quadruple dialectical logic, whereas Hegel, going against his own initial tendencies, falls back into a triad at a crucial moment and loses sight of the “deontologized being” of the subject. What thus characterizes the passionate fury of the middle-late period is its embrace of the paradoxes surrounding subjectivity. This is what makes Schelling that which is in Hegel more than Hegel himself, the extimate core deeply entrenched within the body and soul of Hegel's philosophy that he could not own, as if Schelling were the real spectre haunting and destabilizing his mature system. Consequently Schelling, more than anyone else, is the culmination of German Idealism: it is he who most passionately tarries with its Real, for, in their “very failure, [the Weltalter drafts] are arguably the acme of German Idealism and, simultaneously, a breakthrough into an unknown domain whose contours became discernible only in the aftermath of German Idealism”;200 “Hegel's 'overcoming' of Schelling is a case in itself: Schelling's reaction to Hegel's idealist dialectic was so strong and profound that more and more it is counted as the next (and concluding) step in the inner development of German Idealism.”201 In the context of the Žižekian reactualization of German Idealism, the fundamental assertion to be made is that to understand the movement towards the middle-late period in Schelling we must at some level say that Schelling himself came to realize the deficiencies of his previous philosophical endeavours, perhaps either through a rethinking of the Kantian critical system or by being spurred on by Hegel. The Logic of Transcendental Materialism 149 But what complicates the issue at hand is the fundamental ambiguity of the Hegel-Schelling relationship in Žižek's own thinking, which is brought to an extreme at this crucial juncture: while one could say that much of the young Schelling's work is an attempt to rethink the subject's relation to the noumenal thing-in-itself that haunts its representations or to explicate the androgynous complementarity of the ideal-real poles, the middle- late Schelling's problematic, on Žižek's reading, is the one he erroneously attributes to Hegel: “the true problem is not how to reach the Real when we are confined to the interplay of the (inconsistent) multitude of appearances, but, more radically, the properly Hegelian one: how does appearance itself emerge from the interplay of the Real?”205 Although Žižek does oscillate between calling this problematic Hegelian and Schellingian, what should be clear is that it is more accurately Schellingian, insofar as it is the latter— The Logic of Transcendental Materialism 151 The Logic of Transcendental Materialism 151 according to Žižek's own words—who most fully develops the quadruple logic of the passage from the pre-logical Real into the Symbolic as the unconscious Grundlogik of German Idealism, whereas Hegel recoiled at the most crucial moment. Interpreting the Weltalter through this theoretical framework, Žižek is then able to interpret its ontology as an attempt to articulate a transcendental materialism capable of grounding the psychoanalytico- Cartesian subject by thematizating the vanishing mediator between the Real and the Symbolic. He can do this, perhaps surprisingly, because first and foremost the Weltalter manuscripts present themselves as a theosophic exploration of creation. Perceiving Hegelian logic as a purely conceptual artifice that suffocates the freedom under the self-articulating necessity of the Notion, Schelling puts his philosophical prowess to use to give his own account of the emergence of temporality and finitude that could rival the dialectics of his great adversary. His basic thesis is that, although Hegelian logic can express notional necessity (what something ideally is) it ultimately fails to grasp the fact of any being, the thatness of its existence, especially if that being has its primordial basis in the brute, raw reality of freedom, something that forever eludes the self-mediation of conceptuality.206 For Schelling, however, this failure of pan-logistic dialectics in the face of a freely deciding being (the emergence of the subject) does not amount to a mere admittance of the intrinsic limitations of knowledge and human reason. It must be distinguished from a merely negative constraint upon philosophizing because this dialectical dead end we come across in explicating freedom does not arise due to the limited synthesizing activity of the subject and the finite conditions of the possibility of knowledge, but rather through an (onto)logically disruptive and yet productive metaphysical activity: to say that the fact of a specific being that possesses freedom as its essential predicate cannot be conceptualized according to an a priori dialectics is to point to the uncontainable act constituting its very self-positing, which is therefore capable of continually heralding forth the new and tearing apart any given causal matrix in which it finds itself,207 thereby making itself only graspable après-coup in the wake of its own self-instituting revelation in the world. The Logic of Transcendental Materialism 151 The inability to conceptualize the advent of eruptive subjectivity in substance through 152 Chapter 7 pure reason transcends mere epistemological constraints and reaches out unto the ontological: it is not merely that we must be agnostic concerning the existence of a totalizing principle that holds being together, but more disconcertingly, freedom proclaims that there is not any. The subject is an unpredictable event in being that rewrites what we consider to be possible. Thus, in trying to systematize freedom in a way to escape the perceived threat of Hegelian Absolute Idealism, Schelling reaches a contradiction, a contradiction that paradoxically becomes the very vitality of his system itself, insofar as it declares that the totality of being must be understood in terms of a constitutive yet conflict-ridden relation with an immanent Other: “[w]ere the first nature in harmony with itself, it would remain so. It would be constantly One and would never become Two. It would be an eternal rigidity without progress.”208 Both the Freiheitsschrift and the Weltalter give expression to the necessary snag in the dialectical machine, the primordial, unruly excess of the Real over the Ideal that prevents any system of thinking from being self-enclosed unto itself and in the same breath guarantees the dynamic character of the latter by making it inclusive of freedom as an irrepressible, self-rupturing event at its very core. In order to situate ourselves more firmly within the dialectical nuance of the Weltalter and show how, in relation to Žižek's ontology, Schelling holds a position of theoretical primacy over Hegel, we can use the problem of evil as an entry point, since it is perhaps in their respective theories thereof that they most strongly distance themselves from one other. Whereas for the mature Hegel evil becomes a mere sublated moment in the self-developmen of the good, a necessary phase for its establishment, for Schelling evil remains at its very core irrational and illogical. By definition it cannot be sublated as a moment within a higher dialectical standpoint because it is, at its primordial basis, the effect of an irreducible act of will. There is something spontaneous about evil that forever eludes conceptualization, something insurmountable about the wildness of a soul that insists on that which it wants and will sacrifice whatever it can to achieve it. The Logic of Transcendental Materialism 153 articulation of freedom as the capacity for good and evil: freedom in itself must rest intrinsically incomprehensible, that which cannot be dialectically mediated, which means that its pure self-positing can only resemble madness insofar as it precedes and makes possible any articulation of a table of values that could be used to comprehend it. It of itself knows no order, no rationality—if it did, it would be explicable in terms of the principle of sufficient reason and at risk of being thrown into a subordinate position within a greater self-articulating whole of which it is a mere functional part. There is therefore something always essentially impenetrable in every good and evil act done out of freedom, something always irreducible and violent in each act of self-positing: without this intuition, we lose the breakthrough of the critical system as revealed most poignantly in Kant's pedagogical writings and succumb to another form of determinism (dogmatism) that cancels out the primordial meaning of autonomy within a logic of overarching and self-unfolding reason. Insofar as the act itself is concerned, both the modalities of good and evil as expressions of freedom are formally identical; they involve an act logically distinct at its zero-level from any set of values—or in other words, an act done without any guarantee and without any external determination or influence. More radically, this testifies that evil is itself at the core of every good act, that evil is actually more primordial than the good. In order for an act to be truly good and authentically free at the same time, it must “pass” through evil, discipline it, and use it as the tamed Grund for its own expansive power—any Yes (adherence to rationally determined ethical principles) must first be a No (an egoistic self-assertion) if it is to be utterly self-determined and not just a blind following of laws: “the day lies concealed in the night, albeit overwhelmed by the night; likewise the night in the day, albeit kept down by the day, although it can establish itself as soon as the repressive potency disappears. The Logic of Transcendental Materialism 151 Evil has something crazed and frantic about it: it is the capacity to say “No!” with the full knowledge of the implications of one's action. As soon as evil is understood, it fails to be evil; it becomes, rather, misguided good in the Platonic sense that no one does wrong willingly. Hence Schelling's The Logic of Transcendental Materialism Chapter 7 154 co-related concept of the original “unnatural” unruliness of the human organism, so that these original insights become an intrinsic part of his own logic of the Grund as the indivisible remainder, the “incomprehensible basis of reality,” which is missing in mature Hegelian dialectics. As a full- fledged ontologization of Kant's declaration of the radical spontaneity at the basis of human practical activity,211 Schelling's philosophical impulse initiated by the Freiheitsschrift is an attempt to develop a system wherein freedom is irreducible to notional necessity, for “[o]nly he who has tasted of freedom can sense the desire to make everything its analogue, to spread it throughout the whole universe.”212 What intrigues him is the fact that there is an insurmountable enigmatic blind spot at the core of every action, every decision, a blind spot that not only presents the truth, mystery, and potential horror of human freedom, but more primordially reveals a deep hole that has been carved out in the flesh of being, making it tremble from within, for the unpredictable has emerged in its core. It is this strong conviction in freedom that leads Schelling into the abyssal labyrinths of self-exploration that constitute the conceptual fabric of the Weltalter, in the same way the intuition of freedom made Kant limit knowledge in order to make room for faith and embark down the path where he would eventually articulate the necessity of diabolic evil and unruliness in his pedagogical writings after years of original investigation into the essence of self-legislative practical reason. For Žižek, it is not an accident that Schelling's own project in the Weltalter ends up radicalizing Hegel's descriptions of the night of the world or Kant's account of unruliness, which in turn proves that his response to Hegel is the concluding step of German Idealism: all are driven by an attempt to give a philosophically adequate bedrock to freedom,213 with Schelling merely following its intuition right to the metaphysical conclusions it forces upon us in a way other representatives in the tradition were unable to do. What distinguishes Schelling is that he, propelled by an immense energy to battle against what he perceived as the threat posed by Hegelian dialectics on human freedom, had the strength to go further than the others in the symbolization of the unconscious Grundlogik inherent in the tradition. The Logic of Transcendental Materialism Hence, good lies concealed in evil, albeit made unrecognizable by evil; likewise evil in good, albeit mastered by the good and brought to inactivity.”209 In this respect, a priori dialectics must fail: if all birth is a birth from darkness to light, there is always something in the emergence of rational order that remains impervious to the latter.210 From this it becomes clear that the Schellingian concept of freedom From this it becomes clear that the Schellingian concept of freedom is an explicit rethinking of the Kantian notion of diabolic evil and its 154 Chapter 7 Chapter 7 The Logic of Transcendental Materialism than of humanity, it is because it is a work that delves into the ontological implications of the freedom revealed by idealism. Chapter 7 If hi b k k i ll d Phil hi l I i i i H co-related concept of the original “unnatural” unruliness of the human organism, so that these original insights become an intrinsic part of his own logic of the Grund as the indivisible remainder, the “incomprehensible basis of reality,” which is missing in mature Hegelian dialectics. As a full- fledged ontologization of Kant's declaration of the radical spontaneity at the basis of human practical activity,211 Schelling's philosophical impulse initiated by the Freiheitsschrift is an attempt to develop a system wherein freedom is irreducible to notional necessity, for “[o]nly he who has tasted of freedom can sense the desire to make everything its analogue, to spread it throughout the whole universe.”212 What intrigues him is the fact that there 155 The Logic of Transcendental Materialism 7.3 The Problem of the Beginning Itself: Schelling's Uncanny Response to Idealism Just as in the Freiheitsschrift, and using the operative logic that it had already programmatically developed as a guide, Schelling in the Weltalter embarks upon a specific form of introspective analysis with the aim of developing a theosophy, the founding intuition of which is that the same process underlying the birth of human subjectivity is fundamentally structurally identical to God's creation of the world (as exhibited in the alchemical principle “so above, so below”).214 In another vein, the idea is that psychological experience is in some sense directly revelatory of the absolute drama of divine being in all its vicissitudes, even if it must pass through the meditating filters of self-reflexive consciousness: the experience of the relationship of dependence and autonomy that holds between one's pre- subjective, material Grund and free personality is primordially disclosive of an ontological event that is a symbol of God's relation to the finite created world. In this way, the theosophic odyssey of the birth of God out of that which in God is not God himself, is irrevocably intertwined with a parallel investigation into the ontogenesis of subjectivity out of a nature that presents itself as Other to and irreconcilable with its free self-standingness. Since the methodological starting point is similar to the psychoanalytical experience of disharmony between mind and body as the obscure basis of freedom (which hints at the vanishing mediator between them), Žižek is led to discard the entire theosophic scope of the work as ultimately accidental to its “true” philosophical core, so that Schelling's narration of the painful process of the self-begetting of God and the decision of divine creation presents itself as a mere “metapsychological work in the strict Freudian sense of the term.”215 Whether or not Žižek himself is justified in completely removing the theosophic scope from Schelling's argument, one must at the very least admit that Žižek's wager follows the spirit of Schelling's middle-late philosophy to the letter, for Schelling himself declares in the Freiheitsschrift to “have established the first clear concept of personality.”216 156 Chapter 7 What makes Žižek's appropriation of Schelling at times so provocative and compelling is his profound ability to penetrate into the fine details of the conceptual structures that make up the operative logic of the Freiheitsschrift and the Weltalter in a way no one else has. What interests him is how Schelling advances the descriptions in the tradition of the status of the elusive X, which simultaneously haunts transcendentally constituted reality, precedes it, and appears to set the stage for its condition of possibility. These three conceptual aspects of this je ne sais quoi map directly unto the three modalities of the Real: (i) the Real as a “kink” in the Symbolic, which pressurizes phenomenal reality; (ii) the Real as pre-symbolic “immediacy” that is lost through the advent of language; and (iii) the barred Real (R⁄ ) now understood as an auto-disruptive substance (N ≠ N) whose self-laceration creates the necessary room within which the transcendental constitution of reality through the Symbolic-Imaginary matrices underlying self-experience can take place, thus drawing attention to the interconnection of each aspect. Prior to these middle-late works, as we have seen, our relation to this mysterious X had already been partially “schematized” by a list of concepts (from Kantian transcendental freedom, diabolic evil, and unruliness to the Hegelian accounts of the night of the world and substance as subject). However, for Žižek, it is only with Schelling's own additions that we move away from the paradoxes of the ideal representation of the extra-subjective world or from a mere haphazard glimpse into the self-effacing ontological catastrophe that precedes the very possibility of free idealization. With him, we completely plunge into the auto-disruptive logic of the pre-symbolic Real at the basis of subjectivity so that the unconscious Grundlogik plaguing the German Idealist tradition, which had already from time to time appeared only to fall back into the darkness from which it came, finally comes clearly into light, becoming now minimally subjectified, as it were. 156 Chapter 7 The major difficulty, however, is how to articulate a philosophical system that can synthesize the various aspects of the Real together into a stable whole insofar as the very ontological space whose exploration would enable it retreats the very moment that conscious experience begins and is only visible in its very gesture of self-withdrawal.217 It is no accident that the problem that haunts the entirety of the middle-late Schelling of the Freiheitsschrift and the Weltalter is, as Žižek emphasizes: What makes Žižek's appropriation of Schelling at times so provocative and compelling is his profound ability to penetrate into the fine details of the conceptual structures that make up the operative logic of the Freiheitsschrift and the Weltalter in a way no one else has. What interests him is how Schelling advances the descriptions in the tradition of the status of the elusive X, which simultaneously haunts transcendentally constituted reality, precedes it, and appears to set the stage for its condition of possibility. The Logic of Transcendental Materialism 157 the problem of the Beginning itself, the crucial problem of German Idealism—suffice it to recall Hegel's detailed elaboration of this problem and all its implications in his Science of Logic. Schelling's “materialist” contribution is best epitomized by his fundamental thesis according to which, to put it bluntly, the true Beginning is not at the beginning: there is something that precedes the Beginning itself—a rotary motion whose vicious cycle is broken, in a gesture analogous to the cutting of the Gordian knot, by the Beginning proper, that is, the primordial act of decision. The beginning of all beginnings, the beginning kat' exohen—“the mother of all beginnings” as one would say today—is, of course, the “in the beginning was the Word” from the Gospel according to St John: prior to it, there was nothing, that is, the void of divine eternity. According to Schelling, however, “eternity” is not a nondescript mass—a lot of things take place in it. Prior to the Word there is the chaotic-psychotic universe of blind drives, their rotary motion, their undifferentiated pulsating; and the Beginning occurs when the Word is pronounced which “represses,” rejects into the eternal Past, this self-enclosed circuit of drives. 156 Chapter 7 In short, at the Beginning proper stands a resolution, an act of decision which, by differentiating between past and present, resolves the preceding unbearable tension of the rotary motion of drives: the true Beginning is the passage from the “closed” rotary motion to “open” progress, from drive to desire—or, in Lacanian terms, from the Real to the Symbolic.218 the problem of the Beginning itself, the crucial problem of German Idealism—suffice it to recall Hegel's detailed elaboration of this problem and all its implications in his Science of Logic. Schelling's “materialist” contribution is best epitomized by his fundamental thesis according to which, to put it bluntly, the true Beginning is not at the beginning: there is something that precedes the Beginning itself—a rotary motion whose vicious cycle is broken, in a gesture analogous to the cutting of the Gordian knot, by the Beginning proper, that is, the primordial act of decision. The beginning of all beginnings, the beginning kat' exohen—“the mother of all beginnings” as one would say today—is, of course, the “in the beginning was the Word” from the Gospel according to St John: prior to it, there was nothing, that is, the void of divine eternity. According to Schelling, however, “eternity” is not a nondescript mass—a lot of things take place in it. Prior to the Word there is the chaotic-psychotic universe of blind drives, their rotary motion, their undifferentiated pulsating; and the Beginning occurs when the Word is pronounced which “represses,” rejects into the eternal Past, this self-enclosed circuit of drives. In short, at the Beginning proper stands a resolution, an act of decision which, by differentiating between past and present, resolves the preceding unbearable tension of the rotary motion of drives: the true Beginning is the passage from the “closed” rotary motion to “open” progress, from drive to desire—or, in Lacanian terms, from the Real to the Symbolic.218 What Žižek refers to as “Schelling-in-itself: the 'Orgasm of Forces'”219 is the remarkable capacity Schelling's philosophy possesses of being able to descend into the immanent driving forces governing the extra- subjective, material Real, the elusive, obscure phase of darkness that precedes and sets the stage for the birth of the light that is the openness of self-reflexive consciousness. 156 Chapter 7 But what fascinates Žižek is the depth of his materialist response to Hegel, which still remains immersed in the fabric of transcendental idealism, a response that is “at the origins 158 Chapter 7 158 Chapter 7 of dialectical materialism.”220 This is why Žižek describes Schelling as a vanishing mediator between classical philosophy and the contemporary discourse of finitude: Schelling stands in a position of irreconcilable contradiction between the two, a tension that Žižek takes upon himself to further develop insofar as Schelling, according to him, is unable to endure his own breakthrough and recoils.221 If, onto the ground/existence distinction propounded in the Freiheitsschrift and systematically laid out in the Weltalter, we superimpose the real(ity)/ideal(ity) distinction operative within modern philosophy from Descartes onward, we perceive a nuance in the ontologization/grounding of Cartesian subjectivity: this split announced between mind and matter, which makes them non-reconcilable to one another, occurs “within” or “on the side of” the material Real through an ontologico-metaphysical deadlock, a schismatic rupture. The standard debate between idealism (ideality precedes and structurally makes possible the positive order of physical being and is thus the insurmountable metaphysical zero-level as in Platonism and textbook Hegelianism, or constitutes completely self-grounding and self-justifying transcendental conditions or normative values that make discourse possible as in Kantianism or much linguistic philosophy) and materialism (there is nothing but the ebb and flow of brute matter, the rest being reducible to an epiphenomenal production of nature's self-enclosed laws, as in the Greek atomists, conventional cognitive science, and logical positivism) is thus stood on its head in Schelling: idealism posits an ideal Event which cannot be accounted for in terms of its material (pre)conditions, while the materialist wager is that we can get “behind” the event and explore how Event explodes out of the gap in/of the order of Being. 156 Chapter 7 The first to formulate this task was Schelling, who, in his Weltalter fragments, outlined the dark territory of the “prehistory of Logos,” of what had to occur in pre-ontological proto- reality so that the openness of Logos and temporality could take place.222 This quote expresses Schelling as one of the most crucial figures (if not the most crucial figure) in the history of dialectical materialism for Žižek, and thus establishes once and for all the pivotal role Schelling's ontology plays in his own philosophical development, despite his own characterizations The Logic of Transcendental Materialism 159 of his project as Hegelian. So how, then, do the vicissitudes of pure, raw materiality open up unto the irreducible event of the Ideal and the transcendentally constituted reality of phenomenological (self-)experience? 7.4 Grund and Existence: The Pulsating Heart of Nature and the Upward Spiral of Human Temporality The reason for this is not that nature, instead of being characterized by a smooth teleological development, is plagued by unpredictable catastrophism, irruptive disharmonies of widespread murder and extinction that cover up their own traces, so that the history of the world cannot be subsumable under a single, all-pervasive trajectory that, preceding from simple to more complex organizations in the unimaginable passage of aeons and aeons, would crown man as the summit of an unconscious yearning for the Word, that is, the structured logos of the symbolic world (contra the surface structure of Schelling's thinking225). This would effectively prevent nature from being motivated by an unknowing search for the light of self- consciousness from within the darkness of its raw, productive potency,226 but the reason lies elsewhere. There are, of course, differing stages, periods, and epochs in nature—and ones that have been lost forever, never to be recovered—due to varying levels of dynamic evolution and growth within its immanent activity, even if there is always unforeseeable violence risking to wreak havoc. Yet when we look around, we cannot find the subject within the system of nature. It just does not fit, a fact that has stark metaphysical implications: the progressive, transformative time of nature presents itself as radically Other to the distinctiveness of human (spiritual) temporality because it does not display the same intensity of uncontrollable dialectical self-sublation. In nature, beginning and end for the most part coincide: in the darkness of the soil, the seed gives birth to the plant, which, after reaching the life-giving light of the radiant sun, finally dies, leaving behind its fruit and thereby returns to itself, only to burst forth again in an eternal recurrence of the same. Change can only transpire in the span of incomprehensible ages: unmeasurably dilatory, dialectical movement is here “enchained” within the unbreakable spurious infinity of endless circularity and does not display the same frantic upward-moving spiral of human temporality, the never repetitious onslaught of history, where beginning and end exist in a productive non-coincidence that is the very vital force of its unimaginably fast paced metamorphosis and unending creation of nuance.227 A new era of culture displaces and supersedes the previous one, going in an unfathomable number of conflicting directions at once, so that self-transmutation that leads to man, so that he is ultimately included within it. 7.4 Grund and Existence: The Pulsating Heart of Nature and the Upward Spiral of Human Temporality Schellingian nature is more than a mere symbol or paradoxical representation of the eternal Past that precedes consciousness: it is that elusive, impossible X, that je ne sais quoi in the modality of the pre-symbolic Real prior to conceptual-linguistic mediation. Yet when we move from the world of human meaning and into the circuitry of nature's vital ebb and flow, we see that “[e]verything that surrounds us points back to a past of incredibly grandeur. The oldest formations of the earth bear such a foreign aspect that we are hardly in a position to form a concept of their time or origin or of the forces that were then at work,”223 in such a way that the task of philosophy becomes to reconstruct this ancestral trajectory of the immemorial into a system of times: We find the greatest part of [its formations] collapsed in ruins, witnesses to a savage devastation. More tranquil eras followed, but they were interrupted by storms as well, and lie buried with their creations beneath those of a new era. In a series from time immemorial, each era has always obscured its predecessor, so that it hardly betrays any sign of an origin; an abundance of strata—the work of thousands of years— must be stripped away to come at last to the foundation, to the ground.224 It is at this very juncture in the Weltalter that Žižek invites us to risk a daring thesis. If we draw our attention to its operative logic, we see that Schelling's system of times does something more radical than displace the primordiality of the human subject by illustrating its subsumption within the tenebrous pulsating heart of nature's productive potency, whose own self-unfolding takes place in the abyssal dregs of an immemorial time that threatens to engulf they who look into it. The crucial observation to be made here is that nature does not have a history that evolves by means of an activity of internal 160 Chapter 7 160 Chapter 7 Chapter 7 160 self-transmutation that leads to man, so that he is ultimately included within it. The Logic of Transcendental Materialism 16 The Logic of Transcendental Materialism 161 always out of place in the inaccessible residue that is the past. The end, the result, is a qualitative break rewiring the plethora of human thoughts, expressions, and emotions, and even the past itself,228 thus setting up a new beginning to be surmounted in its own right. As Žižek succinctly puts it, “[h]ere we encounter the key feature of the Symbolic: the fundamental 'openness' it introduces into a closed order of reality,”229 in such a manner that saying nature yearns for humanity in its depths as the solution to its enigma230 merely covers up the fact that humanity is an irreconcilable break from it. But if the subject is a break from the system of times that constitutes nature and its catastrophism by bringing forth a new age of self-unfolding activity, this does not mean that we are liberated from all dismay by being brought into the luminous sphere of holy spirit. Instead, bound by the erratic and excessive life of freedom that overflows itself, we face our own non- natural catastrophes. We also encounter traces of this paradoxical upward spiralling and uncontrollable linear time in Kant and Hegel, which establish its integral place within founding intuitions of the tradition. Kant's account of unruliness as the ontogenetic starting point of transcendental freedom not only establishes the non-natural basis of human sociopolitical activity—not only is the human being “the only creature that must be educated,” but due to his unruliness, man “has no instinct” and is therefore separated from the vital throes of nature231—but, what is more, it demands to be disciplined if it is not to devour the subject in its frenzy. Due to this exigence of discipline, there is an intrinsic link between what Kant calls moral education and the historical destiny of man that institutes a new form of temporality driven forward through a productive non-coincidence at the core of what it is to be man. Although presenting itself as an excessive energy whose domestication/ schematization will enable us to attain terrestrial perfection, the very ground of our progress is in actuality its own inevitable obstacle: the meta- transcendental condition of subjectivity, while it opens up a distinctly human sphere within which progress is possible, at the same time tarnishes it with the inevitability of eventual collapse, misfire, failure. 7.4 Grund and Existence: The Pulsating Heart of Nature and the Upward Spiral of Human Temporality The reason for this is not that nature, instead of being characterized by a smooth teleological development, is plagued by unpredictable catastrophism, irruptive disharmonies of widespread murder and extinction that cover up their own traces, so that the history of the world cannot be subsumable under a single, all-pervasive trajectory that, preceding from simple to more complex organizations in the unimaginable passage of aeons and aeons, would crown man as the summit of an unconscious yearning for the Word, that is, the structured logos of the symbolic world (contra the surface structure of Schelling's thinking225). This would effectively prevent nature from being motivated by an unknowing search for the light of self- consciousness from within the darkness of its raw, productive potency,226 but the reason lies elsewhere. There are, of course, differing stages, periods, and epochs in nature—and ones that have been lost forever, never to be recovered—due to varying levels of dynamic evolution and growth within its immanent activity, even if there is always unforeseeable violence risking to wreak havoc. Yet when we look around, we cannot find the subject within the system of nature. It just does not fit, a fact that has stark metaphysical implications: the progressive, transformative time of nature presents itself as radically Other to the distinctiveness of human (spiritual) temporality because it does not display the same intensity of uncontrollable dialectical self-sublation. In nature, beginning and end for the most part coincide: in the darkness of the soil, the seed gives birth to the plant, which, after reaching the life-giving light of the radiant sun, finally dies, leaving behind its fruit and thereby returns to itself, only to burst forth again in an eternal recurrence of the same. Change can only transpire in the span of incomprehensible ages: unmeasurably dilatory, dialectical movement is here “enchained” within the unbreakable spurious infinity of endless circularity and does not display the same frantic upward-moving spiral of human temporality, the never repetitious onslaught of history, where beginning and end exist in a productive non-coincidence that is the very vital force of its unimaginably fast paced metamorphosis and unending creation of nuance.227 A new era of culture displaces and supersedes the previous one, going in an unfathomable number of conflicting directions at once, so that no return to the beginning is even possible: the beginning, the origin, is 162 Chapter 7 162 Chapter 7 being, so that man is forever spurred on in the course of history to reinvent himself because of the insurmountable ontological violence preceding all acts of subjectification: “[t]he human being must therefore be educated to the good; but he who is to educate him is on the other hand a human being who still lies in the crudity of nature and who is now supposed to bring about what he himself needs. Hence the continuous deviation from his destiny with the always repeated returns to it.”232 But the consequence of this is that man is nothing more than this perpetual deviation from his destiny, that “man only becomes man”233 by continually (re)creating his own identity by means of education through the construction of a second nature within the forward onslaught of history, its fragile movement of contraction and expansion, history itself thus circulating around an ideal sociopolitical point that it posits by its very activity as necessary to it but which it can never reach, for beginning and end can never coincide as in the realm of nature. In other words, the impossibility of reaching our destiny is that which constitutes its very possibility, that which gives us a destiny in the first place. Due to this impossibility, we have historical time. This idea of a sharp distinction between the natural and the spiritual in terms of time is also taken up by the mature Hegel, even if, on Žižek's reading, he misses the radicality of the vanishing mediator—the night of the world—that enacts the passage from the former to the latter. There are two sides to the story. First, Hegel's starting point in the philosophy of nature is nature as “the Idea in the form of otherness.”234 This means that nature cannot exhibit the characteristics Hegel associates with the Idea, such as development qua self-unfolding activity, whose image we see in the growth of plants from seeds, wherein we encounter a purposive causality guiding all change and movement. “The abstract universality of nature's self- outsidedness”235 demonstrates that nature lacks any inner structuration that would enable it to realize itself freely according to a pre- or self-given telos. In this sense, there is no teleological activity intrinsic to nature: its zero-level is a deterministic mechanics ruled by the contingency of its relations that are always external to themselves. The Logic of Transcendental Materialism 16 But this is not a mere proclamation of defeatism, resignation, and forfeit: the very impossibility of our task is the impetus for action, that which provokes an infinite plurality of new ways of “purifying” the insupportable surplus of our 62 Chapter 7 The Logic of Transcendental Materialism 16 a meaningless giving birth to and destruction of its own offspring.236 Second, at the level of organic life, although nature has sublated this prior staleness and intrinsic lack of purposiveness within its ebb and flow, it nevertheless exhibits a kind of claustrophobic immanence wherein the freedom distinctive of spiritual temporality is foreclosed by the eternal repetition of sameness within nature's cycle of life and death, fullness and lack, for there is a suffocating coincidence between the birth of one individual and the death of the other. Even though there is teleological self-unfolding explicit within organic living being—there is a universal genus that concretizes itself by constituting itself within the series of particulars that it generates and that generates it in turn, a complex unity that sustains itself within difference throughout the dispersion of time—there is no real difference and thus no history possible, even if we are allowed to speak of gradual sedimentations of change over the course of living being's activity. To put it crudely (borrowing one of Hegel's favourite animal examples) in nature we see nothing but one damn parrot after another,237 which ultimately makes nature uninteresting for him: nature remains immensely poor with regard to its notional reflexivity, for in contrast with historical existence, it is not capable of the faster-than-light transformations constitutive of the latter's essence. The Logic of Transcendental Materialism 16 a meaningless giving birth to and destruction of its own offspring.236 Second, at the level of organic life, although nature has sublated this prior staleness and intrinsic lack of purposiveness within its ebb and flow, it nevertheless exhibits a kind of claustrophobic immanence wherein the freedom distinctive of spiritual temporality is foreclosed by the eternal repetition of sameness within nature's cycle of life and death, fullness and lack, for there is a suffocating coincidence between the birth of one individual and the death of the other. Even though there is teleological self-unfolding explicit within organic living being—there is a universal genus that concretizes itself by constituting itself within the series of particulars that it generates and that generates it in turn, a complex unity that sustains itself within difference throughout the dispersion of time—there is no real difference and thus no history possible, even if we are allowed to speak of gradual sedimentations of change over the course of living being's activity. To put it crudely (borrowing one of Hegel's favourite animal examples) in nature we see nothing but one damn parrot after another,237 which ultimately makes nature uninteresting for him: nature remains immensely poor with regard to its notional reflexivity, for in contrast with historical existence, it is not capable of the faster-than-light transformations constitutive of the latter's essence. Hegel refers to this deficiency as the “impotence of nature,”238 insofar as it is incapable of the power of self-relating negativity and therefore displays a spurious infinity.239 Consequently, we must assert the following “distinction between the spiritual and the natural worlds: that, whilst the latter continues simply to return into itself, there is certainly a progression taking place in the former as well.”240 As such, it is thus only with the rise of human spirit that we see contradiction posited as such, and with it, the possibility for real metamorphosis: “[s]pirit is posited as contradiction existing for itself, for there is an objective contradiction between the Idea in its infinite freedom and in the form of singularity, which occurs in nature only as an implicit contradiction, or as a contradiction which has being for us in that otherness appears in the Idea as a stable form.”241 In German Idealism, a human subject is not merely born and then, by dying, proliferating his progeny in an endless repetition of the same. 162 Chapter 7 Exclusively determined by the conditions that engulf them, the bodies emerging here do not display any capacity for self- reproduction, but merely stumble against one another due to empirical laws, whereby nature presents itself as a dead husk and time is nothing other than In this sense, there is no teleological activity intrinsic to nature: its zero-level is a deterministic mechanics ruled by the contingency of its relations that are always external to themselves. Exclusively determined by the conditions that engulf them, the bodies emerging here do not display any capacity for self- reproduction, but merely stumble against one another due to empirical laws, whereby nature presents itself as a dead husk and time is nothing other than The Logic of Transcendental Materialism 16 The Logic of Transcendental Materialism 16 Strictly speaking, nature is nonconscious.243 In it, we only encounter a pulsation of matter, an annular rotary movement of contraction and expansion that follows its own automatic rhythm—what we see knows no pure upsurge of the irrevocable forward march of time, no dynamic linear temporality, as first witnessed in the human symbolic universe, even if its constitutive openness already appears in a “wild” form at lower levels of being. Nature eternally repeats itself in an infinite, relatively self-enclosed cycle of life and death, day and night, fullness and lack, wherein change sediments excruciatingly slowly over inscrutable eons and eons through a sluggishly self-developing, self-growing activity. Outside of it, there is nothing—everything is caught within an agonizing deadlock insofar as there is no room for completely free movement, for there is nothing but a symphonized flow of energy within the indivisibility of nature that is at the same time, from our perspective, a “blind” oscillation because our singularity is there lost. When one looks into nature as that impossible X, that je ne sais quoi, which sustains our life as subjects, one is almost forced to collapse: in face of the all-encompassing immanent laws of substance, one is pushed into an infinitely claustrophobic space. For Žižek we get a sense of this all-devouring, all-consuming force when we look inside the body and specifically the skull—“the realization that, when we look behind the face into the skull, we find nothing; 'there’s no one at home' there, just piles of grey matter—it is difﬁcult to tarry with this gap between meaning and the pure Real.”244 This raw flow of biochemical and electrical energy is so “terrifying” for two reasons. First, it is faceless, personless—it has absolutely difference representing an irreconcilable rupture with the autopoiesis of nature, a self-legislative spontaneity that defines itself in direct opposition to its self-organizing totality, even if it must rely upon it as a dark, inaccessible ground. Human history begins with a cutting off of immemorial natural history, a tearing itself away from the natural cycle of life and death. Although our biology falters and brings us to our end, we die as men, not as mere creatures immersed in the world of substantial being. The Logic of Transcendental Materialism 16 Hegel refers to this deficiency as the “impotence of nature,”238 insofar as it is incapable of the power of self-relating negativity and therefore displays a spurious infinity.239 Consequently, we must assert the following “distinction between the spiritual and the natural worlds: that, whilst the latter continues simply to return into itself, there is certainly a progression taking place in the former as well.”240 As such, it is thus only with the rise of human spirit that we see contradiction posited as such, and with it, the possibility for real metamorphosis: “[s]pirit is posited as contradiction existing for itself, for there is an objective contradiction between the Idea in its infinite freedom and in the form of singularity, which occurs in nature only as an implicit contradiction, or as a contradiction which has being for us in that otherness appears in the Idea as a stable form.”241 In German Idealism, a human subject is not merely born and then, b d i lif ti hi i dl titi f th In German Idealism, a human subject is not merely born and then, by dying, proliferating his progeny in an endless repetition of the same. During man's slow march towards his oblivion, a frenzy of naturally uncontainable and unfathomable activity articulating itself emerges, a Chapter 7 164 difference representing an irreconcilable rupture with the autopoiesis of nature, a self-legislative spontaneity that defines itself in direct opposition to its self-organizing totality, even if it must rely upon it as a dark, inaccessible ground. Human history begins with a cutting off of immemorial natural history, a tearing itself away from the natural cycle of life and death. Although our biology falters and brings us to our end, we die as men, not as mere creatures immersed in the world of substantial being. Schelling testifies to this insight, and advances it further than other representatives of the tradition (despite the fact he simultaneously recoils from, represses, the radicality of his own thematization of it242) insofar as, when we examine the genealogy of natural history in the Weltalter, we realize that nature is not the unconscious proper. The Logic of Transcendental Materialism 16 Schelling testifies to this insight, and advances it further than other representatives of the tradition (despite the fact he simultaneously recoils from, represses, the radicality of his own thematization of it242) insofar as, when we examine the genealogy of natural history in the Weltalter, we realize that nature is not the unconscious proper. Strictly speaking, nature is nonconscious.243 In it, we only encounter a pulsation of matter, an annular rotary movement of contraction and expansion that follows its own automatic rhythm—what we see knows no pure upsurge of the irrevocable forward march of time, no dynamic linear temporality, as first witnessed in the human symbolic universe, even if its constitutive openness already appears in a “wild” form at lower levels of being. Nature eternally repeats itself in an infinite, relatively self-enclosed cycle of life and death, day and night, fullness and lack, wherein change sediments excruciatingly slowly over inscrutable eons and eons through a sluggishly self-developing, self-growing activity. Outside of it, there is nothing—everything is caught within an agonizing deadlock insofar as there is no room for completely free movement, for there is nothing but a symphonized flow of energy within the indivisibility of nature that is at the same time, from our perspective, a “blind” oscillation because our singularity is there lost. When one looks into nature as that impossible X, that je ne sais quoi, which sustains our life as subjects, one is almost forced to collapse: in face of the all-encompassing immanent laws of substance, one is pushed into an infinitely claustrophobic space. For Žižek we get a sense of this all-devouring, all-consuming force when we look inside the body and specifically the skull—“the realization that, when we look behind the face into the skull, we find nothing; 'there’s no one at home' there, just piles of grey matter—it is difﬁcult to tarry with this gap between meaning and the pure Real.”244 This raw flow of biochemical and electrical energy is so “terrifying” for two reasons First it is faceless personless—it has absolutely The Logic of Transcendental Materialism The Logic of Transcendental Materialism 165 nothing to do with either the orbit of phenomenal experience or the human universe of meaning. There is no indication of any genuine human quality: we are only confronted with anonymous, dull palpitations, which resemble the industrial buzzing of automatic machinery, a machinery that may amaze us with its complexity and dynamism (the plasticity of the neuronal network) but that nevertheless exists as a matrix of closed circuitry locked within its own self- enclosed, self-sustaining movement, a movement that is not only greater than us but also thereby appears to “threaten” our very existence as free subjects at every step. Second, the passage from the pure, senseless Real of nature in its mechanism to the absolute spontaneity of the I—the rupturing advent of a dialectical leap—is stricto sensu inexplicable, for given our inability to locate the full-fledged human subject in nature, there is always a moment of arbitrariness and fiat. In the contemporary scientific scene, however, these menacing dimensions of the writhing, pulsating material of the Grund and its irreconcilable tension with free existence are constantly being brought into a new power, because neuroscience puts the very gap itself in question: the neuronal Grund, as a seething, all-devouring force, comes closer and closer to annihilating the distance from nature necessary for the autonomous transcendental constitution of reality, insofar as the fact of experience here risks being reduced to a mere epiphenomenon of a complex biological interface that uses the I as a system of representation to mediate itself to the world. As Žižek reiterates time and time again, “there are two options here: either subjectivity is an illusion, or reality itself (not only epistemologically) is not-Al.”245 That being said, Žižek's reactualization of Schelling's revamped Cartesian positing of the difference between nature (cyclical time, body, ground) and human being (dialectical temporality, mind, existence) allows him to rethink the significance of contemporary neuroscience. The divide between our world of experience and the mechanisms of the natural world does not proclaim the irrelevance of the latter for our understanding of human subjectivity in face of the pure power of scientific explanatory models, their efficacy and statistical guarantee, as perhaps various representatives of phenomenological psychiatry or even psychoanalysis would advocate. The Logic of Transcendental Materialism On the contrary, according to Žižek, these models adequately describe the Real of our lives with a rigorous vigor and precision 166 Chapter 7 Chapter 7 166 Chapter 7 166 C 166 never before imaginable by penetrating into the true ontologico-foundational basis of experience. Žižek criticizes attempts to respond to the threat announced by neuroscience that merely assert the irreducible character of the subject, seeing instead the only feasible way to find a solution being to “develop one approach to its extreme, radically abstracting from the other—to develop the logic of brain science, for instance, at its purest.”246 The question is how approach to its extreme, radically abstracting from the other—to develop the logic of brain science, for instance, at its purest.”246 The question is how a parallax gap could emerge from within the self-regulated biochemical and electrical activity inside the skull, how “the 'mental' itself explodes within the neuronal through a kind of 'ontological explosion.'”247 The question and problematic here is distinctly Schellingian: what is the nature of the copula in judgement?248 Grund is existence in exactly the same way that the neuronal is the mental: the copula here does not primordially distinguish a relation of identity or pure equivalence, so that the latter is entirely subsumable under the former (ground = existence; the neuronal = the mental). It represents an activity that, through the logical self-withdrawal of its pervasiveness and primordiality, results in the production of irreducible difference (ground generates existence; the neuronal immanently gives rise to the mental), wherein each exists as opposite and therefore autonomous to one another, although the unpredictably new—a pure difference—that emerges nevertheless retains an internal thread of logical dependence upon that which gave birth to it at an originary level of theoretical investigation: that is, one of ontogenesis, whereby a productive or creative (schöpferisch) identity emerges between the irreducibly different terms.249 But one must be careful. Grund and existence are just as much “contemporaneous” logical relations as stages of historical development. The Logic of Transcendental Materialism Although the emergent split institutes two autonomous zones of activity—or, to speak in the parlance of the Weltalter, although the divide in being created by the irreducible spontaneity of the unconscious decision or de-scission (Entscheidung as Ent-Scheidung) sets the stage for the irreconcilability of the Present and the Past as epochs or ages in nature that forever alter it—they both exist simultaneously after the act of separation, despite the fact that the Grund also represents the dark “pre-history” of existence. It is in this way that the body as an independent entity existing in infinite contradistinction to mind can still follow its own laws, even if mind ultimately proves itself to be superior to its ontogenetic origins by existing in its own free register only in the a parallax gap could emerge from within the self-regulated biochemical and electrical activity inside the skull, how “the 'mental' itself explodes within the neuronal through a kind of 'ontological explosion.'”247 The question The Logic of Transcendental Materialism 1 aftermath of its hegemonizing self-positing or usurping of the primacy of body—or, to put it differently, natural cyclical time can still exist alongside, albeit in tension with, the out-of-joint dialectical temporality of spirit in the same creature. The neuronal interface can subsist in two paradoxical times, in the non-coincident two-way pull of the parallax as a multistable figure, the eternal “Past” of nonconscious material pulsation and the eternal “Present” of self-consciousness, both being “held” together in the positively charged void that is the subject as the impossible in-between (the vanishing mediator) generated within/by the negativity of being, so that the gap that sustains the subjective consistency of the universe of meaning can be maintained without denying the autonomy and power of cognitivism to describe the pure Real of biochemical and electrical activity that is the brain. The two zones are not to be confounded with one another, even though in a certain sense there is only the brute matter of the neuronal interface. The Logic of Transcendental Materialism Here we must recognize an implicit wordplay in Schelling: the copula in judgement (Urteil) is not merely an act of mind, a mental synthesis bringing a subject and predicate into relation with one another, but the expression of an act of primordial ontological division (Ur-Teil as ursprüngliche Teilung) exhibited by the thing in question with itself.250 The principle of identity should be able to explain eruptive breaches in the fold of being instead of being doomed to subsume everything under the dead univocity of a claustrophobic immanence: “[t]his principle does not give expression to an unity which, revolving in a circle of sameness, would be unprogressive, and thus insensitive or unalive. The unity of this law is immediately creative.”251 The copula in judgement is, in this sense, one of Schelling's many expressions for a metaphysics of the disjunctive “and.” But what does this moment of the breaking of existence out of Grund, of the explosion of the Ideal out of the Real, look like? This leads us into a detailed analysis of the role of ontological catastrophe in the emergence of experience in being. expressions for a metaphysics of the disjunctive “and.” But what does this moment of the breaking of existence out of Grund, of the explosion of the Ideal out of the Real, look like? This leads us into a detailed analysis of the role of ontological catastrophe in the emergence of experience in being. 189. Žižek, The Indivisible Remainder. Taken from the book description. The Logic of Transcendental Materialism Here we must recognize an implicit wordplay in Schelling: the copula in judgement (Urteil) is not merely an act of mind, a mental synthesis bringing a subject and predicate into relation with one another, but the expression of an act of primordial ontological division (Ur-Teil as ursprüngliche Teilung) exhibited by the thing in question with itself.250 The principle of identity should be able to explain eruptive breaches in the fold of being instead of being doomed to subsume everything under the dead univocity of a claustrophobic immanence: “[t]his principle does not give expression to an unity which, revolving in a circle of sameness, would be unprogressive, and thus insensitive or unalive. The unity of this law is immediately creative.”251 The copula in judgement is, in this sense, one of Schelling's many expressions for a metaphysics of the disjunctive “and.” But what does this moment of the breaking of existence out of Grund, of the explosion of the Ideal out of the Real, look like? This leads us into a detailed analysis of the role of ontological catastrophe in the emergence of experience in being. aftermath of its hegemonizing self-positing or usurping of the primacy of body—or, to put it differently, natural cyclical time can still exist alongside, albeit in tension with, the out-of-joint dialectical temporality of spirit in the same creature. The neuronal interface can subsist in two paradoxical times, in the non-coincident two-way pull of the parallax as a multistable figure, the eternal “Past” of nonconscious material pulsation and the eternal “Present” of self-consciousness, both being “held” together in the positively charged void that is the subject as the impossible in-between (the vanishing mediator) generated within/by the negativity of being, so that the gap that sustains the subjective consistency of the universe of meaning can be maintained without denying the autonomy and power of cognitivism to describe the pure Real of biochemical and electrical activity that is the brain. The two zones are not to be confounded with one another, even though in a certain sense there is only the brute matter of the neuronal interface. 188. Žižek, The Parallax View, p. 4. 187. Ibid., p. 55. 187. Ibid., p. 55. 188. Žižek, The Parallax View, p. 4. 189. Žižek, The Indivisible Remainder. Taken from the book description. 188. Žižek, The Parallax View, p. 4. 213. Žižek, Less Than Nothing, p. 266. 168 Chapter 7 190. Ibid., p. 11. 191. Ibid., p. 8. 192. The other important figure here is, of course, Lacan. Since Lacanian psycho- analysis declares that the Symbolic is never all, it is only natural that dialectical logic for Žižek would necessarily include an irremovable moment of irreconcilabil- ity. After all, for Lacan “when one gives rise to two, there is never a return. They don’t revert to making one again, even if it is a new one. The Aufhebung is one of philosophy’s pretty little dreams.” The Seminar. Book XX. Encore, On Feminine Sexuality, The Limits of Love and Knowledge, 1972–3, ed. Jacques-Alain Miller, trans. Bruce Fink (New York: Norton, 1998), p. 86. Yet insofar as Žižek’s project rests on the claim that psychoanalysis is the culmination of a lineage that begins in German Idealism, to assert that he merely reads Hegel through Lacan does not suffice, especially given the similarities between his own dialectics and his reading of Schelling. 193. Žižek, Less Than Nothing, p. 265. 194. Ibid. 194. Ibid. 195. See The Ticklish Subject, pp. 79–86. 196. Žižek, Less Than Nothing, pp. 234–35. 197. Žižek, The Ticklish Subject, p. 82. 198. Schelling, Freiheitsschrift, p. 227. 199. Žižek, Less Than Nothing, p. 922. 200. Žižek, The Abyss of Freedom, pp. 3–4. 201. Žižek, “Fichte’s Laughter,” p. 122. 202. See, for instance, The Parallax View, pp. 197–99. 03. Žižek, The Parallax View, p. 197 and 210. 203. Žižek, The Parallax View, p. 197 and 210. 203. Žižek, The Parallax View, p. 197 and 210. 204. Hegel, The Phenomenology of Spirit, p. 9. 204. Hegel, The Phenomenology of Spirit, p. 9. 205. Žižek, The Parallax View, p. 106 (repeated in Less Than Nothing, pp. 13f. and 642ff.). Contrast this with what he says of Schelling at ibid., p. 166;The Ticklish Subject, p. 55; The Indivisible Remainder, p. 14; and The Abyss of Freedom, p. 15. 206. This stays the same in the late philosophy. See The Grounding of Positive Philosophy, trans. Bruce Matthews (Albany: SUNY Press, 2007), p. 207. 207. Žižek, Less than Nothing, p. 265. 208. Schelling, The Ages of the World: Third Version (c. 1815), trans. Jason M. Wirth (Albany: SUNY Press, 2000) (hereafter Weltalter III), p. 219. 209. Ibid., p. 217. 210. Schelling, Freiheitsschrift, p. 239. 211. Ibid., p. 232. 212. Ibid. 213. Žižek, Less Than Nothing, p. 266. The Logic of Transcendental Materialism 169 214. Schelling, Ages of the World, in The Abyss of Freedom/Ages of the World, trans. Judith Norman (Ann Arbor: University of Michigan Press, 2008) (hereafter Weltalter II), p. 121. 214. Schelling, Ages of the World, in The Abyss of Freedom/Ages of the World, trans. Judith Norman (Ann Arbor: University of Michigan Press, 2008) (hereafter Weltalter II), p. 121. 215. Žižek, The Indivisible Remainder, p. 9. 215. Žižek, The Indivisible Remainder, p. 9. 216. Schelling, Freiheitsschrift, p. 281. 216. Schelling, Freiheitsschrift, p. 281. 217. Schelling, Weltalter II, p. 6. 218. The Indivisible Remainder, p. 13. 218. The Indivisible Remainder, p. 13. 219. Ibid. 219. Ibid. 220. Ibid., p. 11. 220. Ibid., p. 11. 221. Ibid., pp. 35–39. I take up this point in chapter 9. 222. Žižek, The Parallax View, p. 166. 222. Žižek, The Parallax View, p. 166. 223. Schelling, Weltalter II, p. 121. 223. Schelling, Weltalter II, p. 121. 224. Ibid. 224. Ibid. 225. Schelling, Freiheitsschrift, pp. 241–42. 225. Schelling, Freiheitsschrift, pp. 241–42. 226. Ibid., p. 239. 226. Ibid., p. 239. 227. Žižek, Less Than Nothing, p. 233. 227. Žižek, Less Than Nothing, p. 233. 228. See Žižek, The Parallax View, pp. 201–4. 228. See Žižek, The Parallax View, pp. 201–4. 228. See Žižek, The Parallax View, pp. 201–4. 229. Žižek, Less Than Nothing, p. 558. 229. Žižek, Less Than Nothing, p. 558. 230. Schelling, Freiheitsschrift, p. 239. 231. Kant, Kant on Education, p. 1; Lectures on Pedagogy, p. 437. For my discussion, see chapter 5. 232. Kant, Anthropology from a Pragmatic Point of View, p. 420. 233. Kant, Kant on Education, p. 6; Lectures on Pedagogy, p. 439. 234. Hegel, Hegel’s Philosophy of Nature, vol. 1, p. 205 (§247). 235. Ibid., p. 223 (§254). Translation modified. 235. Ibid., p. 223 (§254). Translation modified. 236. Ibid., p. 257 (§257A). 236. Ibid., p. 257 (§257A). 237. This example comes from my first Hegel teacher and philosophy mentor, Toni Stafford. 238. Hegel, The Science of Logic, p. 536. 238. Hegel, The Science of Logic, p. 536. 239. Hegel, The Encyclopedia Logic, p. 293 (§221Z). 239. Hegel, The Encyclopedia Logic, p. 293 (§221Z). 240. Ibid., pp. 302–3 (§234Z). 240. Ibid., pp. 302–3 (§234Z). 241. Hegel, Hegel’s Philosophy of Nature, vol. 1, p. 206 (§247Z). 242. Žižek, Indivisible Remainder, p. 92. 242. Žižek, Indivisible Remainder, p. 92. 242. Žižek, Indivisible Remainder, p. 92. 243. The Logic of Transcendental Materialism 169 The full argument for this will be spilled out in chapter 10. 244. Žižek, The Parallax View, p. 7. 244. Žižek, The Parallax View, p. 7. 245. Ibid., p. 168 (again in Less Than Zero, p. 725; and Žižek & Woodard, “Interview,” The Speculative Turn: Continental Materialism and Realism, p. 407). 250. Hegel draws upon the same wordplay in his Logic. Cf. The Encyclopedia Logic, p. 244 (§166A); and The Science of Logic, p. 552. 170 Chapter 7 246. Ibid., p. 175. 247. Ibid., p. 210. 248. Schelling, Freiheitsschrift, pp. 223–25. 249. Ibid., p. 227. 249. Ibid., p. 227. 250. Hegel draws upon the same wordplay in his Logic. Cf. The Encyclopedia Logic, p. 244 (§166A); and The Science of Logic, p. 552. 251. Schelling, Freiheitsschrift, p. 227. 251. Schelling, Freiheitsschrift, p. 227. Chapter 8 When the World Opens its Eyes The Traumatic Fissure of Ontological Catastrophe Chapter 8 When the World Opens its Eyes The Traumatic Fissure of Ontological Catastrophe Žižek's quadruple dialectics sets forth a conception of the absolute that is incomplete, insofar as it has split itself through a moment of primordial division into two irreconcilable zones of activity: body and mind, nature and spirit, the Real and the Symbolic. However, to explain this moment of self-sundering, Žižek must reappropriate various elements from Schellingian ontology to articulate the pre-conditions of subjectivity in the throes of being, something that the mature Hegel, recoiling from his Jenaer night of the world, fails to do. Žižek's provocative claim is that if we attentively read Schelling's account of the eruption of a freely existing subjectivity out of a nature that becomes infinitely Other, we encounter two startling insights. First, the emergence of desire in being as the ontogenetic condition of the possibility of subjectivity and phenomenalization displays a structural parallel to Schelling's own theory of disease and evil. Second, we must follow the implications of this to the end: rather than exhibiting a great triumph at the end of the odyssey of being, self-consciousness is merely the possible aftereffect of a cancerous negativity in being, an ontological catastrophe, which points to an irreversible fracturing of the very essence of the world. We will also explore the consequences of this for evolutionary theory and contemporary philosophy of mind. 172 Chapter 8 8.1 Desire, the Disease-Stricken Body of Being Following Schelling's descriptions of the eternal Past forever anterior to consciousness and language, a concern immediately arises: when we look at the elusive X of nature in the immemorial epochs of cosmological and geological time or the evolutionary strata of biological auto-development, we encounter an all-encompassing/all-consuming whole that precludes the absolute freedom of the subject. Insofar as this self-totalizing causality immanent in nature represents a relatively closed circle, how is this deterministic “deadlock” surpassed so that autonomy is possible? How exactly can the Grund/neuronal interface act in the self-effacing yet world- giving mode of existence/the mental? Although Žižek's own descriptions of the passage in The Indivisible Remainder and The Abyss of Freedom focus on the founding gesture of subjectivity as a self-instituting fiat, this is not enough. It is only one side of the story. The question is how the undifferentiated circuit of drives that constitute the pre-logical Real could paradoxically ground—give rise to or help incite—the irreducible self- positing act of decision. As Adrian Johnston argues, although this self- positing is ultimately an arbitrary, groundless act “analogous to the cutting of the Gordian knot”—which, as Žižek himself says, “can be described (narrated) only post festum, after it has already taken place, since we are dealing not with a necessary act but with a free act which could also not have happened”252—Schelling himself searches for a way to inscribe the very condition of the possibility of the act itself within the material palpitations of nature, in works that Žižek for the most part does not discuss.253 In this sense, Žižek's own account is not satisfactory because it has a tendency to present the drives as an irrevocably closed and blind system without explaining how of themselves they could short-circuit, a theoretical point that would be advantageous to his overall project. As Johnston points out, within the Schellingian ontogenetic narrative, the self-positing of the subject is first possiblized by the emergence of desire (Begierde) within being. Desire marks the first juncture of some kind of primordial blockage in the heart of blind necessity that upsets the automatic, unbridled oscillation of drives by shattering their pure immanence. This has two effects. 8.1 Desire, the Disease-Stricken Body of Being First, in psychoanalytical terms, it means that instead of a relative homeostasis as the inner telos guiding the entirety of an organism's When the World Opens its Eyes 173 biological life, we see for the first time a relative short-circuiting within the pleasure principle, an inability to find satisfaction through the mere repetition of the same constitutive of the movement of instincts. Second, in place of a smooth, determined relation to the environment wholly programmed by instincts (the coincidence of Innenwelt and Aussenwelt through a predetermined set of biological schemata that hardwire the organism into its “exterior” surroundings) we get a degree of liberation from the various sense data of perception that normally mechanically determine an organism's actions as it enters into a state of denaturalization that is contemporaneous with an act of withdrawing from its immersion in being, and thereby the first stirrings of a free creation of a world of experience. Desire in its Schellingian mode is thus an intermediary stage between nature and the violent unruliness that is the dark birthplace of the transcendental I. But what must be noted here is how desire, as the beginning of the idealization of reality, is essentially identical to the conventional definitions of psychosis as withdrawal from objective reality into self, but here at the ontological level instead of that of sociopolitically structured reality. Consequently, it is Schelling and not Hegel who most succinctly describes the ontological passage through madness insofar as it is the former and not the latter who describes how the night of the world could disrupt the world into a series of membra disjecta. In this respect, when Žižek in The Ticklish Subject and Less Than Nothing proclaims that it is Hegel who is the most radical philosopher of the abyss of madness at the core of subjectivity and the minimal paranoia at the basis of order itself,254 he appears to be completely unaware of how strongly his reading of Schelling influences his own reactualization of German Idealism. 8.1 Desire, the Disease-Stricken Body of Being On his own account, not only is the Schellingian concept of the erratic oscillation of drives that precedes fully constituted reality the expression of the abyss of madness at the core of subjectivity, which Schelling himself supplements with the emergence of desire/ontological madness in being, but at the end of the first chapter of The Indivisible Remainder Žižek discusses Schelling's great insight into the necessarily paranoiac structure of universality as such, a point made explicit for the first time by Lacan, thus establishing a link between the two.255 In this sense, in both Žižekian and Schellingian ontology we can speak of something like a spectrum of subjectivity or ideality inherent within In this sense, in both Žižekian and Schellingian ontology we can speak of something like a spectrum of subjectivity or ideality inherent within Chapter 8 174 nature. Following this claim literally, we must say that we come across traces of desire within other organisms to varying degrees: there is a kind of quantitative accumulation of desire (an evolutionary genesis) that may lead to a complete qualitative break with nature (the splitting act of Ent- Scheidung) but in such a way that the possibility of the latter is not logically contained within the former as a kind of hidden, self-unfolding kernel— rather, the former can only incite it, so that only after the fact can we establish a “relation” between the two. There is no guarantee for freedom in the realm of mere being. Yet, what desire shows us is that the pure Real is not completely all-consuming: it is teeming with crevices within its positive fold, interstices within its being, which present a restless negativity tearing it apart. However, these sites of negativity are to be distinguished from human subjectivity insofar as despite expressing a minimal level of liberation from nature's cycles, they are unable to completely liberate themselves from nature's biological hardwiring and thus seek a new form of non- natural (virtual) organization. In this respect, they would only resemble the primordial unruliness prior to the advent of the Symbolic insofar as they would not exhibit a violent process of utter denaturalization, although we must nevertheless speak of a tension-ridden trembling of the organic system as it begins to quiver under its own weight. 8.1 Desire, the Disease-Stricken Body of Being It is for this reason that Žižek can adopt Heidegger's claim in The Fundamental Concepts of Metaphysics that animals feel “the ‘poorness’ of their relating to the world” in such a way that we see “an infinite pain pervading the whole of living nature.”256 As Žižek points out, this shares common ground with Schelling's notion of “the veil of despondency that spreads itself over nature.”257 But we must understand this pain or despondency in nature in two separate but interrelated ways. First, one could say that in desire as the beginnings of the idealization, that is, of the world's psychotic replication of itself within itself, animals have a kind of implicit yet nonconscious “knowledge” that they are unfree because they exist in a mode of unfathomable tension between the Real (instinctual-deterministic schemata) and the Ideal (desire as a blockage), but without this distinction being posited as such, as if here the Ideal has begun to inhere/persist within the Real, disrupting it from the inside, without yet being autonomous. The Real rules supreme—and not only has full-fledged ideality not yet emerged When the World Opens its Eyes 1 175 in being, but what we encounter here may only be referred to as a form of ontological proto-ideality once ideality in its freedom has come on the scene, that is, retroactively. By consequence, the slightest tinge of desire within the biological system that is supposed to determine the animal's reaction with its environment in advance through a pre-set logistics gives it, as it were, a taste of its own possible, but unreachable freedom, the animal being liberated yet confined in its movement. It is this simultaneous nonconscious “dreamlike” premonition of subjective freedom and its ultimate ontological foreclosure that constitutes nature's despondency or melancholy (Schwermut). But it must be noted that the “despondency” or “melancholy” of nature is not a moment of mere poetical rhetoric or uncalled-for anthropomorphism. As Henri Maldiney points out, Heidegger's descriptions of living beings as plagued by a constitutive Benommenheit (captivation, dazedness) coincide with that “mode of being of the type melancolicus” elaborated by Tellenbach.258 If nature is overcome by a veil of despondency, it is because non-human life is wrought by a structure that is, for us, distinctively pathological: it is as if it is held back, stuck in its tracks, because it is incapable of a truly effective willing. 176 Chapter 8 The Žižekian night of the world emerges as the nonconscious drives of nature for the first time liberate themselves from their blind rotation in being through an immanently generated pandemonium within the corpo-Real of the body. Properly speaking, desire is an impasse within the ontological life of substance—“[s]ince there is consequently an unremitting urge to be and since it cannot be, it comes to a standstill in desire, as an unremitting striving, an eternally insatiable obsession with Being”259—that prevents substance from encompassing all, for the organism now obeys its own non-natural logic. It stands for the irremovable and impenetrable kernel of the Real qua logical paradox/internal limit that disturbs the annular circulation of drives through its own self-assertive violence. Here, the analogue with the body again proves to be useful to perceive the radicality of Žižek's reactualization of Schelling. Although the biological unity of the corpo-Real can astound us with its organic dynamism and systematic efficacy, the very awe-inspiring force of this self-organizing totality can cast a shadow over its dark underbelly, whose traumatic fact is often attested by nature's production of monstrosities, degenerative diseases, the mindless proliferation of cancerous tumors, or even the emergence of various forms of mental illness and psychosis caused by pure organic dysfunction, a fact that demonstrates how, from within the closed totality of a determinist system, a part can assert itself from within and hegemonize the organic whole, restructuring it according to its own “unruly” whim and thereby perturbing its harmonious, symphonized flow. Even if everything is logically pre-determined (the ebb and flow of matter can only follow certain paths carved out by genetics, the neuronal interface of the brain, and various different autopoietic systems), the laws that normally regulate the body can by themselves immanently generate a (bio)logical short circuit, thereby opening up a negative space within the body's corpo-Real that can assert itself and wreak havoc over its self-governing unity through a glitch in its programming. That which guarantees the placid functioning of everyday life and the health of body can suddenly turn into a ghastly apparition of true terror. 176 Chapter 8 Like an illness or disease within Schellingian ontology, desire does not stand for a positive ontological unity, but for an internal scrambling of the biological system that does not follow its supposed path within the whole and instead stubbornly asserts its own self at all costs—even its own The Žižekian night of the world emerges as the nonconscious drives of nature for the first time liberate themselves from their blind rotation in being through an immanently generated pandemonium within the corpo-Real of the body. Properly speaking, desire is an impasse within the ontological life of substance—“[s]ince there is consequently an unremitting urge to be and since it cannot be, it comes to a standstill in desire, as an unremitting striving, an eternally insatiable obsession with Being”259—that prevents substance from encompassing all, for the organism now obeys its own non-natural logic. It stands for the irremovable and impenetrable kernel of the Real qua logical paradox/internal limit that disturbs the annular circulation of drives through its own self-assertive violence. Here, the analogue with the body again proves to be useful to perceive the radicality of Žižek's reactualization of Schelling. Although the biological unity of the corpo-Real can astound us with its organic dynamism and systematic efficacy, the very awe-inspiring force of this self-organizing totality can cast a shadow over its dark underbelly, whose traumatic fact is often attested by nature's production of monstrosities, degenerative diseases, the mindless proliferation of cancerous tumors, or even the emergence of various forms of mental illness and psychosis caused by pure organic dysfunction, a fact that demonstrates how, from within the closed totality of a determinist system, a part can assert itself from within and hegemonize the organic whole, restructuring it according to its own “unruly” whim and thereby perturbing its harmonious, symphonized flow. Even if everything is logically pre-determined (the ebb and flow of matter can only follow certain paths carved out by genetics, the neuronal interface of the brain, and various different autopoietic systems), the laws that normally regulate the body can by themselves immanently generate a (bio)logical short circuit, thereby opening up a negative space within the body's corpo-Real that can assert itself and wreak havoc over its self-governing unity through a glitch in its programming. That which guarantees the placid functioning of everyday life and the health of body can suddenly turn into a ghastly apparition of true terror. 8.1 Desire, the Disease-Stricken Body of Being Second, with emergence of desire, the self-sustaining circuitry of nature becomes disturbed by the interruptive presence of an extimacy, an inassimilable Real, within its very ebb and flow, causing its relatively balanced rotation to fall into a painful deadlock, an erratic oscillation of conflictual tendencies that become more and more uncontrollable as desire increases. If the animal gets its first taste of freedom in desire, then nature in the same moment gets the first taste of the madness that awaits it if this freedom were to fully actualize itself (the night of the world). Nature's pain is the unsteady, unpredictable palpitation of its heart of hearts threatening to explode in one excessive outburst as Todestrieb begins to awaken itself, but because it has yet to self-accumulate to a great intensity, and nothing has arisen to tame this propensity, there is a bleak darkness of antagonism. What is at issue here is the state of nature that precedes and sets the stage for humanity as a response; the emphasis is not on us, but rather on ontogenetic conditions, so that the apparent anthropomorphization of nature is coincident with the dehumanization of 176 Chapter 8 Like an illness or disease within Schellingian ontology, desire does not stand for a positive ontological unity, but for an internal scrambling of the biological system that does not follow its supposed path within the When the World Opens its Eyes 177 dismal downfall by obstructing its own life source. A false unity, it simultaneously represents an ontological perversion and a metaphysical distortion: freedom is a devouring black hole, a mere disturbance, in the vital throes of being. In this context, Žižek talks of Jacques-Alain Miller's remarks on an unsettling rat experiment mentioned in one of Lacan's unpublished seminars, where it is only through a kind of neurological mutilation that a rat can be made to behave like a human. Formally, the specific character that distinguishes human freedom and separates it from the rest of the world is identical to rampant malfunction, a violent ontological disfiguring.260 As the force of desire is raised to a higher and higher degree of ideality, matter enters into a self-lacerating rage (sich selbst zereißende Wut) like a cancer-ridden, disease-stricken body howling under its own out-of-control energy.261 Desire is a self-destructive mania that tears apart the smooth fabric of the world. This is why Žižek finds Schelling's “Wagnerian” vision of God so terrifying. It depicts a being that, by means of the painful, crippling amplification of desire into Todestrieb, becomes completely denaturalized and can thus posit itself as distinct from the anonymous, faceless pulsation of substance by the carving up of a self: “[t]he horror of the rotary motion resides in the fact that it is no longer impersonal: God already exists as One, as the Subject who suffers and endures the antagonism of drives,” “a state of an endless 'pleasure in pain,' agonizing and struggling with Himself, affected by an unbearable anxiety.”262 The primordial unruliness of human nature and its coequal term diabolical evil are therefore synonymous with this grotesque excess of life that we witness in the breakdown of the corpo- Real in times of illness, with what occurs when the self-enclosed logic of nature auto-disrupts through pure dysfunction. 178 Chapter 8 178 Chapter 8 Žižek. What I am currently engaged with is the paradoxical idea that, from a strict evolutionary standpoint, consciousness is a kind of mistake—a malfunction of evolution—and that out of this mistake a miracle emerged. That is to say, consciousness developed as an unintended by-product that acquired a kind of second-degree survivalist function. Basically, consciousness is not something which enables us to function better. On the contrary, I am more and more convinced that consciousness originates with something going terribly wrong—even at the most personal level. For example, when do we become aware of something, fully aware? Precisely at the point where something no longer functions properly or not in the expected way. Daly. Consciousness comes about as a result of some Real encounter? Žižek. Yes, consciousness is originally linked to this moment when “something is wrong,” or, to put it in Lacanian terms, an experience of the Real, of an impossible limit. Original awareness is impelled by a certain experience of failure and mortality—a kind of snag in the biological weave. And all the metaphysical dimensions concerning humanity, philosophical self-reflection, progress and so on emerge ultimately because of this basic traumatic fissure.264 176 Chapter 8 In this regard, the freedom of the subject—that anarchic state that precedes the birth of the Symbolic as a form of retroactive damage control—is not a positive characteristic or attribute: it is the failure of the auto-actualization of the essence of nature, its inability to contain itself within its own preset logistics, which causes an ontological catastrophe.263 As Žižek makes clear in an interview, when viewed from the standpoint of the natural world we can only even understand the peculiarity of (self-) consciousness and human intelligence by positing something going horribly wrong in its internal development: 178 Chapter 8 8.2 Malfunction, Mal-adaptation, Breakdown: Žižek and the Sciences We should pause for a moment and consider this Žižekian notion of a “snag in the biological weave” in order to draw out its full meaning and implications for our understanding of the sciences, which will simultaneously allow us to show that such an ontogenetic account of the emergence of Todestrieb is not enough to explain the emergence of subjectivity. Taken at a purely formal level, both standard accounts of evolution and Žižek's theory of the ontogenesis of the subject appear to share a fundamental presupposition. Functioning through random mishaps When the World Opens its Eyes 179 When the World Opens its Eyes 179 179 in the self-replication of genetic code producing new, unforeseeable ontological differences in living being, neo-Darwinian evolution is rendered possible by a moment of malfunction, error, the always possible upsurge of inconsistency in the “rhythmic” flow of nature. Irreversible mutations form the basal and primordial stuff from which life gains its mercurial character. Consequently, for modern-day biology, nature too is not-all insofar as there is no eternal, overarching unity in the vital energetics of substance, no all-inclusive weave with a preset structure, plan, or predictable movement: there are constant slips and slides in positive reality, glitches and functional disturbances. Evolution is nothing other than nature encountering its own real limit, which proves to be the actual driving force of material creation and change: its weakness does not prove to be a mere limitation, a deficiency, but is often even its strength, allowing its forms to endure by adaption. However, what we could call the biological movement of the negative is only able to ground this transformative activity insofar as it sets up a new “dialectic” between organism and environment. As malfunction slowly and contingently gives rise to new characteristics by the sedimentation of small changes over the sluggish march of evolutionary time, an automatic rubric of natural selection occurs: those who are, by pure chance, more “fit” for their particular niche survive and pass on the biological glitch that, paradoxically, shows itself retroactively to have been a source of force and power, making those that were not struck by accident with the given mishap of self-replication in question disadvantageous, leading them to die out. Though here the possibility of monstrosities coincides with the possibility for new natural forms, a new emergent logistic is able to subsume both within a greater system of the struggle for life and death, even if this struggle is, in and of itself, non-teleological. For Žižek, however, this naturalistic account of biological “negativity” within being at the level of the self-reproduction of organisms does not go far enough. Although he agrees with its fundamental presuppositions— nature as not-all, as driven forward by an internal yet productive inconsistency—he believes that the mechanism of neo-Darwinian evolution is unable to explain the emergence of subjectivity because it falls back into the trap of an organic theory of nature. When the World Opens its Eyes 179 Even if it does not posit an indwelling, necessary tendency for progression, a forward-moving 180 Chapter 8 Chapter 8 180 advancement towards more sophisticated life-forms, the “dialectic” between organism and environment is all-inclusive: the law of self-preservation and homeostasis becomes so primordial that even a mishap in reproduction becomes ultimately subsumed under a species' attempt to assert itself and stay alive, so that the moment of the negative retroactively posits itself as being always already a teleologically guided, rational development. In other words, we can always give reasons as to why this or that evolutionary feature benefited the organism as a being immersed in the natural cycle of life and death. Even if within the naturalistic viewpoint nature/substance is in a certain respect not-all—there is nothing guaranteeing its completion— it does not go far enough, for nothing transcends its grasp. There may be conflict and negativity within the system, but this in no way poses a problem to the inner algorithm of its biological “dialectics.” The problem with this theoretical position is that it fails to see how the subject, haunted by Todestrieb, demonstrates that there is no single overarching principle of explanation, that there is a “place” in nature that is, strictly speaking, non- natural and defines itself against the rhythm of life and death, fullness and lack: the negativity that opens up the space for self-consciousness cannot be, even retroactively, subsumed within an evolutionary narrative of survival. Scientific methodology cannot explain the human, for the zero-level (= unruliness) of experience not only provides no functional advantage in nature, but even disturbs the dialectic of organism and environment because it cuts the link between Innenwelt and Aussenwelt. As Žižek says, “we should bear in mind the basic anti-Darwinian lesson of psychoanalysis repeatedly emphasized by Lacan: man’s radical and fundamental dis-adaptation, mal- adaptation, to his environs,”265 a point not only argued for by Lacan, but also by Kant and Fichte at the beginning of the German Idealist tradition.266 Ž advancement towards more sophisticated life-forms, the “dialectic” between organism and environment is all-inclusive: the law of self-preservation and homeostasis becomes so primordial that even a mishap in reproduction becomes ultimately subsumed under a species' attempt to assert itself and stay alive, so that the moment of the negative retroactively posits itself as being always already a teleologically guided, rational development. When the World Opens its Eyes 179 In other words, we can always give reasons as to why this or that evolutionary feature benefited the organism as a being immersed in the natural cycle of life and death. Even if within the naturalistic viewpoint nature/substance is in a certain respect not-all—there is nothing guaranteeing its completion— it does not go far enough, for nothing transcends its grasp. There may be conflict and negativity within the system, but this in no way poses a problem to the inner algorithm of its biological “dialectics.” The problem with this theoretical position is that it fails to see how the subject, haunted by Todestrieb, demonstrates that there is no single overarching principle of explanation, that there is a “place” in nature that is, strictly speaking, non- natural and defines itself against the rhythm of life and death, fullness and lack: the negativity that opens up the space for self-consciousness cannot be, even retroactively, subsumed within an evolutionary narrative of survival. Scientific methodology cannot explain the human, for the zero-level (= unruliness) of experience not only provides no functional advantage in nature, but even disturbs the dialectic of organism and environment because it cuts the link between Innenwelt and Aussenwelt. As Žižek says, “we should bear in mind the basic anti-Darwinian lesson of psychoanalysis repeatedly emphasized by Lacan: man’s radical and fundamental dis-adaptation, mal- adaptation, to his environs,”265 a point not only argued for by Lacan, but also by Kant and Fichte at the beginning of the German Idealist tradition.266 In his own work on cognitive science, Žižek attempts to show for this very reason how a variety of cognitive scientists and neurophilosophers falter at the enigma of consciousness and thereby create a plurality of approaches at odds with one another. He divides the latter into four groups, which cover the logical possibilities of explaining consciousness in terms of the standard natural model (sadly, the details of each on the Žižekian reading must be left aside here): When the World Opens its Eyes 181 I. eliminative/reductive materialism: the proclamation that there is no such thing as consciousness and qualitative experience is a “naturalized” illusion (Patricia and Paul Churchland); II. the antimaterialist position: the necessity of positing a new fundamental force of nature yet to be discovered (David Chalmers); II. the antimaterialist position: the necessity of positing a new fundamental force of nature yet to be discovered (David Chalmers); III. 182 Chapter 8 182 Chapter 8 182 Chapter 8 its singular character as ideal self-reflexivity—both an attempt to show the theoretical prowess of materialism) or by tackling it head-on ([ii] declaring the irreducibility of mind or [iii] making a possible materialist explanation disappear—both an attempt to demonstrate the necessity of idealism), what each option shows, positively or negatively, is that the self-reflexivity constitutive of consciousness is a disruption of natural laws that demands a new metaphysical vision of the world. For these reasons, Žižek does not locate the moment of malfunction constitutive of the subject at the level of genetic mutation and, in a second step, he refuses to equate it completely with a breakdown of the libidinal- material ground of nature. It goes beyond a scientifically measurable “unit of change” (even if is ultimately incited by a certain series of genetic mutations) insofar as it breaks with all totalizing, homogenizing principles or laws. As a closed loop of infinite self-relating, consciousness can only be made possible by a prior psychotic withdrawal of the world into its nocturnal, ontologically solipsistic self, the primordial rupture dividing Innenwelt and Aussenwelt. It is this founding gesture that sets an internal limit to scientific models because it obstructs the “dialectic” of organism and environment: the self-organizing, instinctual schemata offered by our genetic code go haywire in the life of the organism, so that we are no longer immersed in the world through a preset logistical program, but open up a space within which we are able to freely relate to it as an Other. The point is not to search for how the raw immediacy of the brute Real of neurons and their ancestral history in immemorial time dissipates the primordiality of self-experience, but how the irreducible reflexivity that sustains consciousness itself emerges from within the brute, faceless abyss of asubjective brain matter in such a way that the former remains incommensurate with latter, even if the latter is its obscure birthplace. Relying upon John Taylor's theory of consciousness, Žižek locates this primordial act of ontological division at a very specific point: through the relation of past working memory to present input, present experience spontaneously acquires the ability to relate to itself through a detour through the (its) past, the condition of which is “bubbles” of neuronal activity in local cortical regions establishing complex feedback systems. When the World Opens its Eyes 179 the inherent inexplicability of consciousness: given the “cognitive closure” limiting knowledge, we must assert that the birthplace of conscious awareness is unknowable, even if it did arise out of materiality (Colin McGinn, Steve Pinker); III. the inherent inexplicability of consciousness: given the “cognitive closure” limiting knowledge, we must assert that the birthplace of conscious awareness is unknowable, even if it did arise out of materiality (Colin McGinn, Steve Pinker); IV. an “as-if” non-reductive materialism: consciousness exists, and we can use teleological language to describe it, but it has emerged entirely due to natural laws and can ultimately be completely subsumed within them (Daniel Dennett).267 IV. an “as-if” non-reductive materialism: consciousness exists, and we can use teleological language to describe it, but it has emerged entirely due to natural laws and can ultimately be completely subsumed within them (Daniel Dennett).267 What remains common to each of these approaches, according to Žižek, is the failure of a purely scientific account of consciousness. In typical psychoanalytical style, it is the places where these discourses reach an impasse, where their symbolic space is internally obstructed, that are the most revealing; paradoxically, this occurs whenever they face the essentials of the very object of their investigation. Whether it be (i) the outright dismissal of self-consciousness reflexivity as a pseudo-problem, (ii) the thesis that the only way to inscribe the singularity of consciousness within nature is to posit a new force comparable to gravity or electromagnetism, (iii) entirely giving up any possible material explanation of consciousness, or (iv) the forced attempt to understand conscious reflexivity as a purely natural phenomenon, the closed loop of infinite self-relating that is the condition of the possibility of self- consciousness is itself the impasse of each discourse, for consciousness cannot be naturalized. This leads Žižek to say: “I am therefore tempted to apply here the dialectical reversal of epistemological obstacle into positive ontological condition: what if the 'enigma of consciousness,' its inexplicable character, contains its own solution? What if all we have to do is to transpose the gap which makes consciousness (as the object of our study) 'inexplicable' into consciousness itself?”268 Either by trying to bypass the problem by fiat ([i] completely eliminating the object, “consciousness,” or [iv] stripping it of 8.3 Terror, Perplexity, and the Awakening of the World All at once and as if in a cosmic flash, through an accident of overlapping in the neuronal network, material being encounters its own internal limit amplified to the maximum and the world opens its eyes: with the rise of subjectivity it appears that an unsolvable glitch in the smooth functioning of its own inward-dwelling logic posits itself as such, enabling the world for the first time to find the distance to Situating his own philosophical project within the heritage of German Idealism, Žižek's psychoanalytical reactualization of the Schellingian eruptive logic of the Grund is an attempt to show how the subject is not external to the absolute. In other words, the gaze of the subject in Žižekian ontology must be seen as the material universe finally “gaining” the power to look upon itself through an internal reflection: “the whole domain of the representation of the world (call it mind, spirit, language, consciousness, or whatever medium you prefer) needs to be understood as an event within and of the world itself. Thought is not at all opposed to being, it is rather being's replication within itself.”271 As the dense, closed circuitry of nature dissolves under the impact of the Todestrieb, it gives way to the possibility of the “miracle”272 of human thinking and its free idealization of the world as immanent in the latter. However, this moment of parallax shift from mere being to thought must be taken for what it is. Within the ancestral genealogy of forgotten, phenomenologically inaccessible time, the system of nature slowly and contingently grows in complexity, eventually reaching its autopoietic apotheosis in the structure of the brain, which, because of its some one hundred billion neurons possessing a total of at least one hundred trillion synaptic connections, thereby displaying one hundred times more synapses than the estimated number of stars in our galaxy, is unable to hold itself together according to its own immanent, self-regulating laws. Unintended loops and gaps in its processes become visible as they writhe under the infinite pressure of their own labyrinthine intricacy, nature failing to find its own way in its own production. 182 Chapter 8 It is this direct self-instituting short-circuiting that allows for free, infinitely self-standing thought, the ease, energy and speed of mental operations, in When the World Opens its Eyes 183 contradistinction to the complex mediative channels of neuronal activity that produced the organism as a biological system, for it allows present experience to liberate itself radically from a mere attachment to input.269 In a flash, the organism stops being determined through external conditions, but creates a zone within which it can relate to itself as a self so that bit by bit this self-relation takes charge of instinctual schemata. For Žižek, this “short circuit” or “(bio)logical glitch” between various faculties within the brain—a kind of naturally inexplicable overlapping that creates the possibility of psychotic self-relation, which, just like Schelling's account of the self-begetting of God, “can be described (narrated) only post festum, after it has already taken place, since we are dealing not with a necessary act but with a free act which could also not have happened”270—as a meta- transcendental condition of consciousness is related only in a derivative or secondary sense to the “malfunction” so fundamental to the process of neo-Darwinian evolution.This is true for two reasons. First, it makes itself superior to the natural basis that paves the way for it and thereby elevates itself above it. Second, it is not predictable from the mere level of genetic mutation. In this sense, not only is it a pure self-positing that depends on nothing but itself (even if it is incited by a genetic mutation, it remains irreducible to and logically distinct from it: it is a mere neuronal mishap, a disruptive emergence in the interstices of the logical network sustained by the biochemical and electrical interface of brain matter), but this capacity for self-assertion also allows it to liberate itself from its immersion/ imprisonment in the natural world (the generated closed loop of self-relation breaks the interpenetration of Innenwelt and Aussenwelt in such a way that it cannot be retroactively subsumed within a survivalist narrative of newfound functionality, even if it does manage to acquire a second-degree survivalist function). Genetic mutation, the “dialectics” of organism and environment, and with it the breakdown of the libidinal-material ground of the organism, may be necessary for explaining the origins of subjectivity, but they are not sufficient. For full-fledged subjectivity to emerge, natural breakdown must paradoxically double itself 184 Chapter 8 8.3 Terror, Perplexity, and the Awakening of the World Situating his own philosophical project within the heritage of German Idealism, Žižek's psychoanalytical reactualization of the Schellingian eruptive logic of the Grund is an attempt to show how the subject is not external to the absolute. In other words, the gaze of the subject in Žižekian ontology must be seen as the material universe finally “gaining” the power to look upon itself through an internal reflection: “the whole domain of the representation of the world (call it mind, spirit, language, consciousness, or whatever medium you prefer) needs to be understood as an event within and of the world itself. Thought is not at all opposed to being, it is rather being's replication within itself.”271 As the dense, closed circuitry of nature dissolves under the impact of the Todestrieb, it gives way to the possibility of the “miracle”272 of human thinking and its free idealization of the world as immanent in the latter. However, this moment of parallax shift from mere being to thought must be taken for what it is. Within the ancestral genealogy of forgotten, phenomenologically inaccessible time, the system of nature slowly and contingently grows in complexity, eventually reaching its autopoietic apotheosis in the structure of the brain, which, because of its some one hundred billion neurons possessing a total of at least one hundred trillion synaptic connections, thereby displaying one hundred times more synapses than the estimated number of stars in our galaxy, is unable to hold itself together according to its own immanent, self-regulating laws. Unintended loops and gaps in its processes become visible as they writhe under the infinite pressure of their own labyrinthine intricacy, nature failing to find its own way in its own production. Substance falls into deadlock: with the emergence of the complex neuronal interface of man, it can no longer successfully posit itself as a fragile all, and it breaks into a series of membra disjecta, which opens up the space within which the self-enclosed loop of experience asserts itself out of nowhere, further intruding upon and obstructing its activity from within. 8.3 Terror, Perplexity, and the Awakening of the World Substance falls into deadlock: with the emergence of the complex neuronal interface of man, it can no longer successfully posit itself as a fragile all, and it breaks into a series of membra disjecta, which opens up the space within which the self-enclosed loop of experience asserts itself out of nowhere, further intruding upon and obstructing its activity from within. All at once and as if in a cosmic flash, through an accident of overlapping in the neuronal network, material being encounters its own internal limit amplified to the maximum and the world opens its eyes: with the rise of subjectivity it appears that an unsolvable glitch in the smooth functioning of its own inward-dwelling logic posits itself as such, enabling the world for the first time to find the distance to When the World Opens its Eyes 18 185 self necessary for its own self-phenemonalization by virtue of a shock, a blow, a violence, for which it itself is responsible. The claustrophobic immanence that drowns everything in the blind void of non-experience is finally shattered—yet when the world finally opens its eyes for the very first time in the mode of subjectivity it does not rejoice, it does not celebrate, nor does it feel a passive, grateful joy from the beauty surrounding it and bask in the bliss aroused by the wonder and amazement of the brute fact of its existence. It whimpers under its own weight, hearing its own inarticulate cry as it experiences itself in a moment of unbearable agony and catastrophic self-diremption, “a mixture of terror and perplexity” that Žižek compares to the atrocity of sexual abuse and the horrific pictures of children dying from radiation exposure in Chernobyl: Although one of today's main candidates for the figure of Evil is child sexual abuse, there is nevertheless something in the image of a hurt, vulnerable child which makes it unbearably touching: the figure of a child, between two and five years old, deeply wounded but retaining a defiant attitude, his face and poise remaining stubborn, although he is barely able to prevent an outburst of tears—is this not one of the figures of the Absolute? 8.3 Terror, Perplexity, and the Awakening of the World One thinks here about the photos of children dying from exposure to radiation after the Chernobyl accident, or—also from Ukraine—one of the photos on a child-porn website showing a really young child, no more than four years old, confronting a big ejaculating penis, face covered with fresh sperm. Although the shot probably plays on the link between the penis ejaculating sperm and the mother’s breast full of milk, the expression on the child's face is clearly a mixture of terror and perplexity: the child cannot make out what is going on.273 What the world first sees is not its own awe-striking unity and oneness, the spiritual fullness of a self-seizing, self-actualizing centre that holds everything together in an all-encompassing totality. All it discerns is the tumultuous uproar of erratic pulsation, an insurmountable, non-masterable chaos resulting from the degradation or collapse of its own positive, autopoietic activity. The self-awareness of the world, its self-experience 186 Chapter 8 186 Chapter 8 But it must be insisted that this can only be accomplished at the level of the virtual: “[t]he third moment which 'resolves' the contradiction is by definition 'prothetic' (virtual, artificial, symbolic, not substantially natural).”278 When the world “perceives itself” as lost to self and infinitely When the World Opens its Eyes 187 dirempt (N ≠ N as the originary [onto]logical violence preceding and possibilizing transcendental imagination) in the mode of subjectivity, it recoils into culture as a defence mechanism to try to sublate this gap in its being, to fill in this hole in its depths and thereby posits itself as such as a full-fledged subjectivity.279 In short, the passage from darkness to light only occurs at the level of the Symbolic: in the Real, nothing changes, unruliness (our break from nature) is left untouched. It is this aspect of the intrinsic madness of culture, language, and phenomenal reality, its psychotic lack of contact with the world, that Žižek claims we forget, that we must necessarily forget, if the transcendental misrecognition of reality necessary to subjectification as a reaction formation is to be a successful “compensation.” All our discourses, all our “truths,” are nothing but the deluded ravings of the asylum unaware of their true origin within the founding gesture of subjectivity as a recoil spurred on by the brutal trauma of violently awakening up into a dismembering hemorrhaging of being, the ultimate ontological catastrophe. All the beauty of the world merely belies its true, unbearable horror: “[i]f we take into consideration the many terrible things in nature and the spiritual world and the great many other things that a benevolent hand seems to cover up from us, then we could not doubt that [the ego] sits enthroned over a world of terrors.”280 In this respect, “the true point of 'madness' [...] is not the pure excess of the ‘night of the world,' but the madness of the passage to the Symbolic itself, of imposing a symbolic order onto the chaos of the Real. 186 Chapter 8 6 Chapter 8 186 in the first person as made possible by the existence of subjectivity and thus all experience as such, is necessarily preceded by this irreducible and irreversible auto-disruption that must be seen as catastrophic. The ontogenetic basis of subjectivity is the trauma of primordial loss, where nature is forever alienated from itself through complete breakdown (N ≠ N), so that we must say that the subject is paradoxically that which has survived its own death, for “the past traumatic loss of substance [...] is constitutive of the very dimension of subjectivity”274—or as Schelling himself puts it, “[p]ain is something universal and necessary in all life, the unavoidable transition point to freedom [...]. It is the path to glory.”275 But this is no mere rhetoric on behalf of Žižek: as his Schellingian-inspired argument makes clear, this psychosis-inducing auto-disruption of being is a necessary theoretical posit if free experience is to be possible instead of a blind experiential void: We cannot pass directly from nature to culture. Something goes terribly wrong in nature: nature produces an unnatural monstrosity and I claim that it is in order to cope with, to domesticate, this monstrosity that we symbolize. Taking Freud's fort/da as a model: something is primordially broken (the absence of the mother and so on) and symbolization functions as a way of living with that kind of trauma.276 In short, the ontological necessity of “madness” resides in the fact that it is not possible to pass directly from the “animal soul” immersed in its natural life-world to “normal” subjectivity dwelling in its symbolic universe—the vanishing mediator between the two is the “mad” gesture of radical withdrawal from reality that opens up the space for its symbolic reconstitution.277 The implication of this is that the Symbolic (the reconstitution of reality, the world's self-replication within itself) is nothing but an attempt to tame, to gentrify this constitutive mayhem/madness in the immanent fold of being. 186 Chapter 8 If madness is constitutive, then every system of meaning is minimally paranoid, 'mad.'”281 Paradoxically, the world can only become known to itself—being can only replicate itself within thought—if its medium of self-disclosure operates “with no external support of its truth,”282 without ever touching the Real.283 Accordingly, Schellingian desire must be said to be the beginning of being's withdrawal into its nocturnal, irreal self and that which opens up the space necessary for ego development as symptom formation (a defence). Following various hints and gestures in the middle-late Schelling, Žižek takes premonitions of the psychoanalytical experience of mind-body discord and brings to the fore their underlying eruptive logic. This leads him to the idea of Grund and existence as a dialectically irreconcilable pair emerging through the caustic collapse of being as it gains notional self-reflexivity, 188 Chapter 8 188 Chapter 8 The subject When the World Opens its Eyes 1 189 is therefore infinitely non-coincidental and contradictory: having no place in either mind or body, it can only show itself as the caustic breakdown of substance (N ≠ N) or in the fleeting, ephemeral distortions of symbolic space between signifiers ($), but these two traces fail to grasp its essence in its purity, for the subject is more than the material collapse of being and the limits of discourse. But what is more, not only is the subject outside the Symbolic because it is minimally non-coincident with it, but insofar as it is at its very origin and must withdraw in order for the Symbolic to take hold, we need a new form of language even to discuss its genesis out of pre-logical antagonism, since not even the inconsistencies of our notional apparatus could thus aid us to explain its pure upsurge. The subject is “a non-provable presupposition, something whose existence cannot be demonstrated but only inferred through the failure of its direct demonstration.”289 As a result, the metaphysical archaeology of the subject I have been executing contains a necessary moment of speculative fabulation to fill in the unavoidable gaps of the narrative of how thought arises within the flat plane of being. Here, however, we encounter a major methodological difficulty. Here, however, we encounter a major methodological difficulty. Drawing upon Schellingian ontology to embark upon such a mytho-poetic articulation of how substance could act in the self-effacing, self-sundering mode of subjectivity presents an immediate problem to those who are familiar with the texts upon which Žižek relies, for the Freiheitsschrift and the Weltalter display the structure of a quaternity, with a principle exterior to the dynamic of Grund and existence that ties them together as androgynous opposites in a point of absolute indifference, an Ungrund, whose conceptual contours appear to have starkly different consequences for human self-consciousness and culture, namely, that humans participate in the theo-cosmogonic drama of God's search for self-manifestedness in creation, or to put it differently, in the emergence of “the cosmos (of fully constituted reality, ruled by logos) out of the proto-cosmic pre-ontological chaos.”290 According to this picture, ontological catastrophe (the subject) is less a cataclysmic breakdown than part of the inherent negativity in the intimate life of God that makes His personality truly alive. 188 Chapter 8 188 this being the fundamental logical moment for understanding how his own parallax ontology can bring together a materialism and a self-grounding idealism. It is not the mediating filters of language that are primordially responsible for our lack of access to extra-subjective reality due to some internal limitation—rather, it is reality itself that renders impossible its own access to itself. The Symbolic is always already revelatory of the nature of the world, albeit in an intrinsically negative manner: the prison house of language, our inability to “transcend” it and grasp reality as it is in itself,284 necessarily refers to the material processes that gave birth to the alienating linguistic-conceptual structures that permanently rob us of the immediacy of being. Psychosis is not the feature of a single isolated subject lost in disarray—it is the rite of passage of becoming a subject, a point explicitly brought to the fore by Lacan285 and that justifies rereading Schelling through Lacanian psychoanalysis. It is no accident that both Schelling's Stuttgarter Privatvorlesungen and the third draft of the Weltalter contain at a crucial point a meditation on the relationship between subjectivity and madness and the latter's irreducibility (in the former when discussing the soul's higher faculties, in the latter at the very end of the book of the Past): “[u]nderstanding, if it is to be an actual, living and active understanding, is therefore properly nothing other than a coordinated madness”;286 “[f]or in what does the intellect prove itself than in the coping with and governance and regulation of madness? [...] Without continuous solicitation of it, there would be no consciousness.”287 The difficult paradox of Žižek’s metaphysical archaeology of the subject is that it is only through this ontological catastrophe that the true “miracle”288 of human thinking can emerge, so that there is a speculative identity between the highest (that which sustains and creates the autonomous sphere of spirit in its dialectical creativity) and the lowest (the irrevocable alienation of being to itself, its schismatic auto-disruption that produces a non-natural excess). As that which thereby negatively binds together materialism and idealism, the impossible in-between that is the subject is neither Real nor Symbolic insofar as it dialectically coincides with both modalities simultaneously, yet is also outside of them, thus rendering its status as a vanishing mediator undecidable. In both, it is internally contained as external, present as absent, included as excluded. 188 Chapter 8 Only through the preclusion of a theosophic philosophy of nature as the illusion that there is “a secret, invisible, all-powerful agent who effectively 'pulls the strings' behind the visible, public Power... [an] obscene, invisible power structure Chapter 8 190 acts the part of the 'Other of the Other' in the Lacanian sense, the part of the meta-guarantee of the consistency of the big Other (the symbolic order that regulates social life),”291 can Žižek ground his reading of the ontogenesis of the subject by identifying the act of decision—the pure self-positing of the I that separates Grund from existence for the first time—with the self-assertive violence of evil and disease. Because there is no God who needs a being like Himself in order to recognize Himself as Himself through a mirror that is His Other, the irreducible freedom of the human subject can only be seen as an ontologically narcissistic disturbance of the relatively ordered whole of nature: there can be no internal teleology guided by divine understanding and will whereby man participates in the divine drama of being's search for self-disclosure. Nature is not the primordial, first revelation of God,292 but the obscure basis of subjectivity, that against which it defines itself by a self-caused break. Rejecting the theosophic Ungrund central to Schelling's middle-late texts, Žižek sees traces of a disavowed analytic of finitude within them that, wrought with self-destructive, self-sabotaging tendencies, can paradoxically efface itself in the production of an extimate kernel of negativity, which then, in another moment, hegemonically takes over. In this sense, Žižek's own ontology—just like his work on Hegel—must be understood as an attempt to psychoanalytically construct the unconscious truth of Schelling's thinking by demonstrating how “his thought—for a brief moment, as it were in a flash—renders visible something that was invisible beforehand and withdrew into invisibility thereafter,” and how this tension constitutes the central difficulty of his theosophic epic.293 What, then, motivates and philosophically founds Žižek's violent “reactualization” of the Schellingian subject? To come to terms with this, we will have to explore the motivates and philosophically founds Žižek's violent “reactualization” of the Schellingian subject? To come to terms with this, we will have to explore the psychoanalytical conflict omnipresent in the Weltalter project. 252. Žižek, The Indivisible Remainder, pp. 22–23. 253. See Johnston, Žižek’s Ontology, pp. 80–92. 254. Žižek, The Ticklish Subject, p. 78; and Less Than Nothing, p. 331. 255. Žižek, The Indivisible Remainder, p. 76. 255. Žižek, The Indivisible Remainder, p. 76. 254. Žižek, The Ticklish Subject, p. 78; and Less Than Nothing, p. 331. When the World Opens its Eyes 191 256. Žižek, The Fragile Absolute: Or, Why Is the Christian Legacy is Worth Fighting For? (London: Verso, 2008), pp. 86–89. 257. Schelling, Freiheitsschrift, p. 271. 257. Schelling, Freiheitsschrift, p. 271. 258. Maldiney, Penser l’homme et la folie, p. 270. 259. Schelling, Weltalter III, p. 232. 259. Schelling, Weltalter III, p. 232. 260. See Žižek, The Indivisible Remainder, pp. 219–20. 260. See Žižek, The Indivisible Remainder, pp. 219–20. 261. Schelling, Weltalter III, p. 322. 261. Schelling, Weltalter III, p. 322. 262. Žižek, The Indivisible Remainder, p. 24. 262. Žižek, The Indivisible Remainder, p. 24. 263. Or, as Johnston succinctly puts it, “[t]he surplus of autonomy is made possible by the deficit of heteronomy. Freedom emerges from the dysfunctioning of determin- ism.” Žižek’s Ontology, p. 114. 264. Žižek and Daly, Conversations with Žižek, p. 59. 265. Žižek, The Parallax View, p. 231. 265. Žižek, The Parallax View, p. 231. 266. See chapters 5 and 6. 267. Žižek, The Parallax View, pp. 177–78. 267. Žižek, The Parallax View, pp. 177–78. 268. Ibid., p. 241. 268. Ibid., p. 241. 269. See ibid., pp. 210–14, for a rather interesting, detailed discussion. 270. Žižek, The Indivisible Remainder, pp. 22–23. 271. Gabriel and Žižek, “Introduction: A Plea for a Return to Post-Kantian Idealism,” in Mythology, Madness and Laughter, p. 3. 272. Žižek and Daly, Conversations with Žižek, p. 59. 273. Žižek, The Parallax View, p. 73. Strangely enough, this passage is omitted in the French translation. Cf. La Parallaxe (Paris: Fayard, 2008). 274. Žižek, Living in the End Times, p. 312. 274. Žižek, Living in the End Times, p. 312. 275. Schelling, Weltalter III, p. 335. 275. Schelling, Weltalter III, p. 335. 276. Žižek and Daly, Conversations with Žižek, pp. 64–65. 276. Žižek and Daly, Conversations with Žižek, pp. 64–65. 277. Žižek, The Ticklish Subject, p. 35. 277. Žižek, The Ticklish Subject, p. 35. 278. Žižek, Less Than Nothing, p. 314. 279. Žižek, Living in the End Times, p. 398. 280. Schelling, Weltalter III, p. 291. 280. Schelling, Weltalter III, p. 291. 281. Žižek, Less Than Nothing, p. 331. 282. Ibid., p. 77. 282. Ibid., p. 77. 283. Ibid., p. 959. 283. Ibid., p. 959. 284. Žižek, The Ticklish Subject, p. 33. 284. Žižek, The Ticklish Subject, p. 33. 285. Lacan, “Presentation on Psychic Causality,” in Écrits, p. 176/144. 286. Schelling, Stuttgart Seminars, in Idealism and the Endgame of Theory, trans. 286. Schelling, Stuttgart Seminars, in Idealism and the Endgame of Theory, trans. Thomas Pfau (Albany: SUNY Press, 1994), p. 233. When the World Opens its Eyes 191 Thomas Pfau (Albany: SUNY Press, 1994), p. 233. 192 Chapter 8 287. Schelling, Weltalter III, pp. 338–39. 288. Žižek and Daly, Conversations with Žižek, p. 59. 289. Žižek, Less Than Nothing, p. 730. 289. Žižek, Less Than Nothing, p. 730. 290. Ibid., pp. 273ff. 290. Ibid., pp. 273ff. 291. Žižek, “The Big Other Doesn’t Exist,” Journal of European Psychoanalysis, Spring- Fall 1997. Retrieved Jan. 7, 2013, from www.lacan.com/zizekother.htm. 292. Schelling, Freiheitsschrift, p. 284. 292. Schelling, Freiheitsschrift, p. 284. 293. Žižek, The Indivisible Remainder, p. 8. 9.1 Into the Void: The Frenzy of God's Self-Diremption The ambiguity of the Hegel-Schelling relationship within Žižek stems from his critique of Schellingian metaphysics. Even if Žižek's own transcendental materialism is founded upon a notion of emergent ontological catastrophe at the origin of subjectivity, a notion he largely derives from the Freiheitsschrift and the Weltalter, Žižek is quite adamant in distancing himself from these texts, even if in the same breath he praises Schelling's profound ability to penetrate into the pre-symbolic material of the Real.294 Indeed, immediately after his remarkable and provocative reading of Schelling in The Indivisible Remainder, Žižek argues for the supremacy of Hegelian dialectics. Despite the ephemeral moments of genuine breakthrough that emerge “as it were in a flash,”295 Žižek insists that Schelling remains philosophically inferior to his great rival: he ultimately fails to conceive the radicality implicit in the self-positing act of separating Grund from existence and tries to cover up the non-coincidence in being it indicates by making the two distinct from one another only by being founded within absolute indifference, which itself, as Schelling says, “is not a product of opposites, nor are they contained in it implicite; rather it is a being of its own, separated from all oppositions, on which all oppositions are broken, which is nothing other than their very non-being, and which therefore has no predicate except predicatelessness.”296 The eruptive logic of the Grund, intrinsic to the notion of ontological catastrophe, is thereby completely lost: “from this neither-nor, or from this indifference, duality [...] immediately breaks forth, and without indifference, i.e., without an unground, there would be no twofoldness of the principles,”297 in such a way that “the [Schellingian] Absolute is primarily the 'absolute indifference' providing the neutral medium for the coexistence of the polar opposites” of the Real and the Ideal.298 As such, it provides a way out of the abyss of freedom as a kind of metaphysical violence: the act of unconscious decision, the vanishing mediator that mediates between the real and ideal poles, “'repressed' by the formal envelope of the ‘obscurantist’ Schelling,” becomes relegated to a mere secondary position in a theosophic drama that subsumes it.299 The ambiguity of the Hegel-Schelling relationship within Žižek stems from his critique of Schellingian metaphysics. Chapter 9 The Abyss of Unconscious Decision Schelling's Weltalter and Psychoanalytical Horror of Substance as Subject Chapter 9 The Abyss of Unconscious Decision Schelling's Weltalter and Psychoanalytical Horror of Substance as Subject Since Schelling's own account of the Grund has theosophic tendencies in contradiction with the Lacanian subject, and by consequence with the disavowed yet formative Grundlogik of German Idealism, Žižek is only able to reactualize Schelling by “formalizing” its content. By elaborating Žižek's psychoanalytical methodology, I will show how Žižek is able to legitimate such a violent overhauling of Schelling insofar as he sees an inability to assimilate an encounter with the Real within key conceptual moments, a maddening struggle that simultaneously causes the “failure” of his middle-late philosophy and testifies to his prowess as the greatest thinker of subjectivity, thus presenting us with a nuanced and controversial reading of Schelling's philosophical development and his relation to German Idealism as a whole. What is more, reconstructing this “therapeutic space” will enable us to not only come to a greater understanding of the systematic rigour of Žižek's approach in opposition to what his critics want us to believe, but also bring us face to face with the psychoanalytical horror of the founding insight of modernity—the ontologically catastrophic nature of the subject as the vanishing mediator between the Real and the Symbolic—and the mechanisms by which even the greatest thinker of subjectivity can be seduced by fantasies as he tarries with its essence. 194 Chapter 9 The Abyss of Unconscious Decision 195 night of the world, nevertheless Hegel develops a superior logic because within it there is no need to leave the internal dynamic of Grund and existence and posit some transcendent principle. All can be done at the level of (a self-destructive, negativity-wrought) immanence. What Hegel fails to bring to full conceptual expression is how in dialectics the very category of “and” changes, along with the full range of implications this presents for any metaphysical system of the world: through the paradoxical identification of Grund and existence “and” becomes, in essence, tautological,300 which not only prevents Grund and existence from being mere opposites existing alongside one another through their foundation in something external to their own movement, but also excludes the possibility of any self-totalizing activity. In the logical process of the dialectic, the third term is the second, understood as a negativity or internal limit inscribed in the first, but only insofar as it has successfully taken over, usurped, the originary position from which the movement began by asserting itself as such—that is, the passage from the second to the third is that of an emergent extimacy within the first moment, which renders it non-coincident to itself, and thereby not-all, by owning itself and taking over its originary position to which it was once held at bay through an uprising. In terms of substance and subject, this means that “this very reversal is the very definition of subject: ‘subject’ is the name for the principle of Selfhood that subordinates to itself the substantial Whole whose particular moment it originally was.”301 Nothing at the level of content changes: it expresses a purely formal self-relationality giving birth to itself from within the radical non-coincidence of the absolute with itself, a self-begetting that resets the latter's logical hardwiring in an unpredictable way from within an unforeseeable self-posited zone of operation. The dialectical movement from (i) immediacy → (ii) negation → (iii) negation of negation is superior to Schellingian metaphysical narrative because there is no genuine return movement to the first, for everything takes place within a self-effacing, yet productive immanent field: the beginning and end do not overlap because something irreducibly different emerges within the first moment (negativity being now made foundational to identity): namely, an “out of joint” spirit that has a degree of notional self-reflexivity. 9.1 Into the Void: The Frenzy of God's Self-Diremption Even if Žižek's own transcendental materialism is founded upon a notion of emergent ontological catastrophe at the origin of subjectivity, a notion he largely derives from the Freiheitsschrift and the Weltalter, Žižek is quite adamant in distancing himself from these texts, even if in the same breath he praises Schelling's profound ability to penetrate into the pre-symbolic material of the Real.294 Indeed, immediately after his remarkable and provocative reading of Schelling in The Indivisible Remainder, Žižek argues for the supremacy of Hegelian dialectics. Despite the ephemeral moments of genuine breakthrough that emerge “as it were in a flash,”295 Žižek insists that Schelling remains philosophically inferior to his great rival: he ultimately fails to conceive the radicality implicit in the self-positing act of separating Grund from existence and tries to cover up the non-coincidence in being it indicates by making the two distinct from one another only by being founded within absolute indifference, which itself, as Schelling says, “is not a product of opposites, nor are they contained in it implicite; rather it is a being of its own, separated from all oppositions, on which all oppositions are broken, which is nothing other than their very non-being, and which therefore has no predicate except predicatelessness.”296 The eruptive logic of the Grund, intrinsic to the notion of ontological catastrophe, is thereby completely lost: “from this neither-nor, or from this indifference, duality [...] immediately breaks forth, and without indifference, i.e., without an unground, there would be no twofoldness of the principles,”297 in such a way that “the [Schellingian] Absolute is primarily the 'absolute indifference' providing the neutral medium for the coexistence of the polar opposites” of the Real and the Ideal.298 As such, it provides a way out of the abyss of freedom as a kind of metaphysical violence: the act of unconscious decision, the vanishing mediator that mediates between the real and ideal poles, “'repressed' by the formal envelope of the ‘obscurantist’ Schelling,” becomes relegated to a mere secondary position in a theosophic drama that subsumes it.299 Although Hegel's mature ontology as outlined in the Encyclopedia suffers, Although Hegel's mature ontology as outlined in the Encyclopedia suffers, in Žižek's view, from a similar deficiency by articulating the complete return of the Idea to itself in a manner that attempts to get rid of the psychotic The Abyss of Unconscious Decision 195 The Abyss of Unconscious Decision 195 In other words, the tautology indicated by the category of “and” is, in fact, revelatory of a monism bursting at the seams: to say that Grund and existence are 196 Chapter 9 Chapter 9 196 identified is to say that Grund can only attain to existence, can only subsist in the modality of existence, insofar as it has erased itself, withdrawn from the scene. In this way it makes itself identical to the latter through the institution of its very difference from it, a difference that at some level is always a self-difference, a self-sabotaging, for there is no outside.302 We come across a “self-enclosed” immanence that has produced a transcendence that persists in its heart of hearts in the paradoxical mode of the double feature of inclusion/exclusion, internal/external, presence/absence, so characteristic of Lacan's descriptions of the Real, but in such a way that what is excluded, external, absent, has a power over that from which it has emerged. identified is to say that Grund can only attain to existence, can only subsist in the modality of existence, insofar as it has erased itself, withdrawn from the scene. In this way it makes itself identical to the latter through the institution of its very difference from it, a difference that at some level is always a self-difference, a self-sabotaging, for there is no outside.302 We come across a “self-enclosed” immanence that has produced a transcendence that persists in its heart of hearts in the paradoxical mode of the double feature of inclusion/exclusion, internal/external, presence/absence, so characteristic of Lacan's descriptions of the Real, but in such a way that what is excluded, external, absent, has a power over that from which it has emerged. Negativity means that the indivisibility and power of substance is shattered from within, because it is colonized by its own parasites like a madman is terrorized by his own hallucinations, but with the added effect that there is no need to posit a state of “originary health” of which the devouring restlessness of the negative cannot be predicated to explain the dynamism inherent to reality. The Abyss of Unconscious Decision 197 German Idealism whose lineage culminates in the advent of psychoanalysis. For Žižek, this brief period of breakthrough was quickly left behind by the Schelling3 of the philosophy of mythology and revelation, where we see a return to a pre-modern “essentialism,” traces of which he claims are already hinted at in the third draft of the Weltalter, where Schelling posits another principle of synthesis external to the movement of contraction and expansion within which freedom and determinism are grounded as opposites. It is because of these tendencies (which explain why Žižek qualifies the revolutionary character of Schelling's philosophy by describing him as the father of “New Age obscurantism” just as much as the father of contemporary philosophy of finitude)304 that Žižek so quickly changes tone in the second chapter of The Indivisible Remainder. Yet while consistent with his overall interpretation of Schelling, this emphatic shift is simultaneously ambiguous insofar as Žižek does not distinguish which Schelling he is arguing against or justify how he is able to read the second draft of the Weltalter as an ephemeral rupture in Schelling's thought “which goes farthest in the direction of Freedom.”305 Given that the only way for freedom to exist according to Žižek is through the space opened up by the caustic collapse of being, and that both Hegel's ontology and Schelling's logic fail in their own fashion to come to terms with this insight,306 even if each encounters it in a significant manner, how exactly is Žižek able to retrieve this concept from the second draft without falling into the pitfalls of Schelling's own thinking, insofar as it is evidently against its letter and spirit even as he presents it? How is he able to maintain that the Weltalter and the Freiheitsschrift are the most sustained confrontation in the entire tradition with the frightening origins of subjectivity and its metaphysical implications, that they reveal the most profound penetration into the “perverted” core of the cogito that lurked latent in itself since the very founding gesture of modernity? What specifically interests Žižek in the middle-late work of Schelling is its frantic and uncertain nature. For all its intense, uncontrollable passion, for all its feverish outpouring, it does not get off the ground. Schelling's masterpiece, the Weltalter, never gets finished. The Abyss of Unconscious Decision 195 Negativity means that the indivisibility and power of substance is shattered from within, because it is colonized by its own parasites like a madman is terrorized by his own hallucinations, but with the added effect that there is no need to posit a state of “originary health” of which the devouring restlessness of the negative cannot be predicated to explain the dynamism inherent to reality. Because of the theosophic structure of the middle-late Schelling, Žižek's own appropriations of concepts such as Grund and Entscheidung in the development of his own metaphysics put him in a delicate situation. The issue at hand is further complicated by Žižek's division of Schelling's philosophy into three distinct and irreconcilable stages, which he finds reflected in the three existent drafts of the Weltalter.303 Schelling1 largely coincides with his quasi-Spinozistic philosophy of absolute indifference, where freedom is completely subsumed under the positive order of being. In the first draft this is seen in terms of the explication of freedom as a logical mode of necessity within the inner articulation of substance, its subsumption within the self-harmonizing genesis of the latter's rational structure and order. In the Schelling2 of the second draft of the Weltalter and the Freiheitsschrift, we see an interesting twist with regard to the concern with how the contraction of material being itself is made possible. By conceiving the act of contraction as ultimately free and self-positing, here Schelling is able to think the will-to-contraction (the No) and the will-to- expansion (the Yes) as identical and therefore internal to the dynamic of freedom, which makes his thinking approach that of Hegelian dialectics and secures his position as the concluding step of the unconscious Grundlogik of Because of the theosophic structure of the middle-late Schelling, The Abyss of Unconscious Decision 197 The Abyss of Unconscious Decision 197 The Abyss of Unconscious Decision 197 Although this is not an outlier in Schelling's long philosophical career—his entire corpus is ridden with rapidly written sketches of systems and allusions to publications on the 198 Chapter 9 198 Chapter 9 In the treatise, Schelling claimed that the “veil of melancholy [Schwermut] that is spread out over all of nature is the The Abyss of Unconscious Decision 199 198 Chapter 9 198 Chapter 9 horizon that never appear—there is an undeniable hesitation saturating this period. The trunk of stillborn, abortive drafts of the first book of the Weltalter found in the library of Munich, of which only three remain today due to the fires that wrought havoc in the city in the aftermath of Allied bombings (but of which there were more than twelve different handwritten versions) is enough to demonstrate that this work haunted Schelling in a manner and with an intensity that others did not, as does the fact that after the 1809 Freiheitsschrift he refrained from publishing another major work, and largely withdrew from the German intellectual scene where he was once the rising star. Aside from scattered lectures—for instance, an 1827 and an 1833 course on the System der Weltalter—he would not return to the public eye with any sustained vigor until he was called to Berlin in 1841. At that time, assuming Hegel's chair ten years after the latter's untimely death, addressing an audience that had eagerly awaited him to break his silence for decades (an audience that included both Engels and Kierkegaard) and whose final reappearance was exacerbated by the uncertain fate of Hegelianism, he gave lectures on the philosophy of mythology and revelation. But the Hörsäle, first overflowing with excited, enthusiastic students of philosophy anxious to hear the words of Hegel’s old rival, were quickly abandoned in utter disappointment, leaving only a handful of devoted students, Fichte's son Immanuel Hermann von Fichte among them. But what explains this sudden change in Schelling's career, this lack of desire to publish, to engage in dialogue, to spread his philosophy over the European landscape that had eagerly welcomed it? Jason Wirth hints at something that comes to mind for anyone who knows Schelling's life story: Yet 1809 marked a turning point in Schelling's zeal to publish. Already Schelling's reputation had been injured by Hegel's unwarranted dismissal of the intellectual intuition as the “night when all cows are black.” [...] More seriously, however, Schelling's wife, Caroline, had become very ill. It is hard to read the Freedom essay, published in May 1809, with its analogy between sickness and evil (sickness is to Being as evil is to human being), without thinking of Caroline. 200 Chapter 9 After Hegel’s critique of the Schellingian absolute indifference308 in The Phenomenology of Spirit, and recognizing the strength of Hegel's dialectics, Schelling is forced to rethink the philosophical foundation of his thought and its attempt to balance transcendental idealism with an organic philosophy of nature in such a way that he can at the same time fight against the perceived threats of Hegel's philosophy. He puts his own prowess to use in articulating his account of the emergence of temporality and finitude that does not succumb to the claustrophobic subsumption of human freedom within the self-mediation of the absolute. Arguing that Hegel can only show the notional necessity of things and never their brute reality, he embarks on a theosophic exploration of the creation of the world that would not only challenge, but hopefully put a stake in the heart of the philosophy of his now adversary and former friend. Yet Schelling's middle-late philosophy is not just a theo-cosmogonic odyssey of the vicissitudes of divine being in its restless search for self-revelation. Following reason by analogy and identifying a structural parallel between God's speaking of the Word by which he becomes a full person and the birth of self-experience out of the eternal darkness that precedes it,309 Schelling's philosophy simultaneously functions on two levels, that of the theo-cosmological and that of the metapsychological, in such a way that Schelling is able to say, at the end of the Freiheitsschrift, that we “have established the first clear concept of personality.”310 It assumes that we can pass from the lowest to the highest, from the known to the unknown, by a careful, methodologically guided introspection, insofar as there is “a system of times [...] for us, of which the human system would be just a copy, a repetition within a narrower sphere.”311 What is so intriguing about Schelling's attempt to describe the emergence of finitude from eternity and human subjectivity out of nature is the conceptual restlessness evident in the existent drafts of the project, each of which oscillates around an insurmountable deadlock located in the movement from the Past to the Present: that is, how the divine and human subject could emerge from the non-experiential void that precedes them. The Abyss of Unconscious Decision 199 199 profound and indestructible melancholy of all life.” Caroline died on September 7, 1809. Schelling was devastated. In a letter written less than a month after Caroline's death, Schelling claimed that “I now need friends who are not strangers to the real seriousness of pain and who feel that the single right and happy [glücklich] state of the soul is the divine mourning [Traurigkeit] in which all earthly pain is immersed.” A year later, Schelling began work on Die Weltalter, a philosophical poem about the rotatory movement of natality and fatality, pain and joy, comedy and tragedy within God, that is, within the whole of Being, itself.307 Žižek allows us to offer a new, interesting, and controversial spin on this enigma. If we follow and reconstruct his implicit methodology, we see that he refuses the claim that Schelling's “retreat” was spurred on by the tragic death of his true love and great muse Caroline in 1809 just before the publication of the Freiheitsschrift, or for that matter even by the overwhelming influence of the Hegelian system that could also be seen as leaving him in a state of philosophical and existential paralysis. What Žižek invites us to argue is that, when one looks at the very structure of the Weltalter drafts themselves, one is confronted with an uncanny struggle of composition that reveals something primordial concerning what its texts give witness to and attempt—but ultimately fail—to bring forth. This painful unrest is not to be reduced to the mere level of personal dissatisfaction, as if there were a conscious recognition by Schelling that his own “masterpiece” could never hold its own as a rival of Hegelian logic, nor could it just boil down to some personal trauma that began to cloud Schelling's own philosophical capacities through a devouring melancholy that made the world during these years an agonizing repetition of bitter grey upon bitter grey. Instead, it provides the key to understanding both the self-deploying historico-dialectical (Lacanian) cause of German Idealism at work in Schelling's thought and ultimately dividing it into the three distinct stages as outlined by Žižek and establishing its true greatness in this fragmented second stage, whereby we can open up a legitimate space in which to reactualize it and let it come to terms with that which, by itself, it was not able to do. 200 Chapter 9 Schelling remains unable fully to explicate the meaning of what Žižek calls the “breach of symmetry,” God's contraction of finitude, and in the same vein the emergence of a free, deciding being (the subject) in a manner The Abyss of Unconscious Decision 201 The Abyss of Unconscious Decision 201 that leaves him at ease. In the first draft the abyss of freedom contracts materiality out of necessity, so that both divine and human freedom are subsumed within the self-unfolding of substance, while the second declares that the very contraction itself is an act of freedom, a self-positing, which not only renders the two opposed principles of the Yes and the No identical in the internal dynamic of freedom, but also (and more disconcertingly) results in a structural homology with sickness and evil as an unpredictable negative reversal of established ordered and wholeness from within. The third merely complicates the picture insofar as it breaks from the “Spinozistic” determinism and closed systematicity of the first and the radical philosophy of freedom with its potentially unsettling implications proclaimed by the second: by synthesizing both polar principles within a point of metaphysical simultaneity within the Godhead that is the Ungrund within which both rest as androgynous pairs, God the Creator's personal freedom is saved from being logically dependent upon a moment of cancer in divine being that upsets the joyful bliss of eternity, and in a similar move, subjectivity is no longer made possible by that which disrupts the smooth placidity of substance. These basic conceptual differences at such a foundational level of inquiry reverberate through the entire movement of the three stages in a meaningful way in each draft: 1. Not only is human freedom actually a misrecognition of blind (Spinozistic) causality, so that the movement of dialectical human temporality is an illusion, all being co-present in the self-harmonizing synchronicity of the system—here we see Schelling struggling to rearticulate his old ideas in a new format—but even the act of contracting finitude is a necessity, so it could not not have happened, deriving from the “primordial Freedom in an absolutely immediate, 'blind,' non-reflected, unaccountable way.”312 2. The fact of finitude in God or of freedom in man proclaims that synchronic substance cannot be a devouring totality, but must be an open not-all plagued with fragility and inconsistency all the way down. The Abyss of Unconscious Decision 203 The absolute yearns for self-disclosure once it has willed it, so that all things in the self-institution of finitude and negativity he stumbled across in the Potenzenlehre of the second draft and already implicit in the eruptive logic of the Grund in the Freiheitsschrift; in both there is an attempt to retreat from the notion of a barred organic whole, a metaphysics of the not-all, as forced upon us by the philosophy of freedom whose first outlines we see already in Kant, substantiating Žižek's claim that both Hegel and Schelling repress the unacknowledged truth, the Grundlogik, of German Idealism.313 By referring to the Godhead as das Überexistierende that summons the blind rotatory movement of drives, the third draft strives to save reality from the irredeemable conflict that it is faced with in the contraction of finitude: instead of the latter being absolutely self-positing as a moment in the movement from the potentiality of freedom in its abyssal eternity to its actualization, God in some strong sense now precedes the Grund of His existence, assuring His protection from the non-coincidence to self announced by the dependence of light on darkness: there is “an activity performed at a safe distance.”314 This becomes even more evident in the late philosophy of mythology, where the entire theoretical edifice becomes grounded on the distinction between the Was and the Daß of God. The transcendence of the Daß from the flux of the Potenzen not only allows for an implicit, yet self-articulating teleological movement intrinsic to the antagonisms of finitude of the latter so that there are stages that unfold throughout nature and then human history that ultimately pave the way for the final revelation of God, but it also makes any appearance of ontological disarray merely a “perceptual illusion” generated by a self-harmonizing scheme deduced by negative philosophy (in Žižek's words, this is Schelling’s regress to pre-modern essentialism). The Abyss of Unconscious Decision 201 The very self-positing of finite being in God 202 Chapter 9 not only precludes all attempts to enclose it within a totalizing whole even within the divine understanding itself, since self- positing is equated with an absolute self-assertion, but (even more devastatingly) the emergence of God as a personal being out of its contracted material Grund is uncertain, insofar as there is no guarantee that He will come to His own by speaking the Word, unlike in the first draft where it is part of divine's substance infinite self-articulation. 3. The problematic ramifications of the second position are foreclosed by making God's free existence as an entity predetermined and guaranteed in advance through the dialectical simultaneity of freedom and determinism in a higher synthetic principle. Thus the creation of the world is an absolutely contingent act following from a volitional arbitrariness expressing God's essence as love, making divine being, rather than a turmoil- ridden drama with an unforeseeable end, a self-maintaining whole. In this sense the third draft symbolizes a kind of retreat from the second; one is even tempted to say an (unconscious) attempt to save oneself from the traumatic, horrifying implications of its philosophy of freedom. The true basis of spontaneity was, as it were, too much for Schelling—he had to pull back. Not only do the existent drafts of the Weltalter give testament to an uncompromising tension, an endless circulation around an inassimilable kernel, both in terms of the relationship of God as person and consciousness to the eternal darkness of their ground, but it also appears as if Schelling's own philosophical career could only continue once it cut off, repressed, this encounter with the Real. In the turn to the Schelling3 of the philosophy of mythology (already hinted at in the third draft of the Weltalter) we can glimpse a recoil from the abyssal basis of freedom as pure self-positing unleashed in the second in the same way that the earlier Realphilosophie of Hegel is later abandoned in order to embrace a consoling triadic dialectics: just as Hegel tries to save the absolute from a diremptive wound that can never be healed, here Schelling attempts to save the essence of God and nature from the insoluble deadlock of drives eventually engendered by The Abyss of Unconscious Decision 203 The Abyss of Unconscious Decision 203 the self-institution of finitude and negativity he stumbled across in the Potenzenlehre of the second draft and already implicit in the eruptive logic of the Grund in the Freiheitsschrift; in both there is an attempt to retreat from the notion of a barred organic whole, a metaphysics of the not-all, as forced upon us by the philosophy of freedom whose first outlines we see already in Kant, substantiating Žižek's claim that both Hegel and Schelling repress the unacknowledged truth, the Grundlogik, of German Idealism.313 By referring to the Godhead as das Überexistierende that summons the blind rotatory movement of drives, the third draft strives to save reality from the irredeemable conflict that it is faced with in the contraction of finitude: instead of the latter being absolutely self-positing as a moment in the movement from the potentiality of freedom in its abyssal eternity to its actualization, God in some strong sense now precedes the Grund of His existence, assuring His protection from the non-coincidence to self announced by the dependence of light on darkness: there is “an activity performed at a safe distance.”314 This becomes even more evident in the late philosophy of mythology, where the entire theoretical edifice becomes grounded on the distinction between the Was and the Daß of God. The transcendence of the Daß from the flux of the Potenzen not only allows for an implicit, yet self-articulating teleological movement intrinsic to the antagonisms of finitude of the latter so that there are stages that unfold throughout nature and then human history that ultimately pave the way for the final revelation of God, but it also makes any appearance of ontological disarray merely a “perceptual illusion” generated by a self-harmonizing scheme deduced by negative philosophy (in Žižek's words, this is Schelling’s regress to pre-modern essentialism). Complementing negative philosophy, Schelling envisages a form of positive philosophy, one of whose primary tasks is to find and explicate these stages in nature and history: that is to say, “to function as a kind of 'transcendental empiricism,' and to 'test' the truth of rational construction in actual life,”315 thereby subsuming the restless negativity at the heart of reality as revealed specifically in the second draft into a mere secondary feature of God's plan within the spectacle of the absolute's own self-development, deflating it to a mere ephemeral phase in an overarching cosmic dance towards revelation. The Abyss of Unconscious Decision 203 Complementing negative philosophy, Schelling envisages a form of positive philosophy, one of whose primary tasks is to find and explicate these stages in nature and history: that is to say, “to function as a kind of 'transcendental empiricism,' and to 'test' the truth of rational construction in actual life,”315 thereby subsuming the restless negativity at the heart of reality as revealed specifically in the second draft into a mere secondary feature of God's plan within the spectacle of the absolute's own self-development, deflating it to a mere ephemeral phase in an overarching cosmic dance towards revelation. The absolute yearns for self-disclosure once it has willed it, so that all things in Chapter 9 204 creation move towards it, even if unknowingly: here, not only is nature's unpredictable catastrophism and widespread murder and extinction316 no longer indicative of a lack of a totalizing principle, but man, denied the right to be an autonomous writer in the drama of divine being, is reduced to a mere actor or puppet who follows a pre-written script (a telos) given by the very structure of the Potenzen themselves for the joy of its author in His distant, cold kingdom of divine eternity, the non-rational Daß of God being, after all, distinct from and existing over and above the Was of God. But in the movement from Schelling2 to Schelling3 we should not just draw our attention to how in the latter there is absolutely no room for subjectivity with the very technical sense of the word that we have been using. What is much more revealing is how concerned Schelling3 is to establish God as that which guarantees the consistency and internal coherence of nature and history, previously jeopardized, for us and God Himself: there is a frantic obsession to demonstrate the existence of the Other of the Other in the Real when the inconsistencies and frailty of order have already been pushed upon us. The third stage in Schelling's thought is nothing but a reaction formation against his radical philosophy of freedom. The Abyss of Unconscious Decision 205 redemption, which would merely just institute a triadic dialectics similar to that of Hegel's mature system—it also brings Schelling's philosophy to a halt, a standstill, as it comes upon metaphysical implications revealed by the split nature of subjectivity. Only ever successfully completing the first, preliminary part of his mytho-poetic ages of the world, his description of the eternal Past, Schelling remains forever unable to proceed; it is this which causes him to fade slowly from the public eye, where he was once a star and the leader of one of the greatest intellectual movements the world has ever witnessed, because he came too close to its true horrifying core. The Abyss of Unconscious Decision 203 Leaving aside the possible theological implications of Žižek's reading of Schelling—which deserve to be evaluated in their own right—due to his outright rejection of theosophy as ultimately nothing but a mytho-poetics of the Symbolic's coming into being, what Žižek's Schelling unearths in his frenzied attempt to develop a philosophical program able to battle Hegelian Absolute Idealism is the not-all nature of the absolute. Trying to give an account of the emergence of finitude and driven on by the alchemical intuition that there is an identity between the highest and the lowest, the self-begetting of God and the self-caused birth of the subject in its freedom, he delves into the nocturnal site out of which the human self is born and experiences the terror of the failure of positive being to ground itself, its “self-lacerating rage” (sich selbst zerreißende Wut), which acts as the vanishing mediator between nature and culture by opening up the possibility for the self-assertion of freedom from within the Real and its vicissitudes. This not only by definition prevents the completion of the project of the Weltalter insofar as it precludes any possibility of the reconciliation promised by the Third Age, the Future wherein Grund and existence become reconciled in The Abyss of Unconscious Decision 205 206 Chapter 9 The “victory” that Žižek perceives in the Weltalter is in its gestures towards philosophical logic underlying the birth of consciousness and its wider metaphysical implications. The strangeness and density of the Weltalter project are not due to its out-of-jointness with the modern tradition, but due to the fact that it comes too close to its disavowed essence. The anxiety-inducing reverberations that shake one's body when one reads the descriptions of “Schelling's grandiose 'Wagnerian' vision of God in the state of a endless 'pleasure in pain,' agonizing and struggling with Himself, affected by an unbearable anxiety, the vision of a 'psychotic,' mad God”318 are so difficult because they make us approach the very kernel not just of our existence as subjects but also the nature of the world at large. This experience of reading its drafts is properly “traumatic” in the meaning Lacan gives to this term drawing upon its etymological origins in ancient Greek: they bring forth a devastating encounter by which the symbolic support of our personality is “wounded” or “pierced,” during which the coherence of our identity is torn apart as it comes upon its truth.319 The effect is twofold. i The “victory” that Žižek perceives in the Weltalter is in its gestures towards philosophical logic underlying the birth of consciousness and its wider metaphysical implications. The strangeness and density of the Weltalter project are not due to its out-of-jointness with the modern tradition, but due to the fact that it comes too close to its disavowed essence. The anxiety-inducing reverberations that shake one's body when one reads the descriptions of “Schelling's grandiose 'Wagnerian' vision of God in the state of a endless 'pleasure in pain,' agonizing and struggling with Himself, affected by an unbearable anxiety, the vision of a 'psychotic,' mad God”318 are so difficult because they make us approach the very kernel not just of our existence as subjects but also the nature of the world at large. This experience of reading its drafts is properly “traumatic” in the meaning Lacan gives to this term drawing upon its etymological origins in ancient Greek: they bring forth a devastating encounter by which the symbolic support of our personality is “wounded” or “pierced,” during which the coherence of our identity is torn apart as it comes upon its truth.319 The effect is twofold. 9.2 That Which Is in Schelling More than Schelling Himself—Žižek Schelling's failure to finish the Weltalter is not due to his lack of conceptual prowess or an earth-shattering, existential strife: it results from an encounter with the Real, an encounter that is philosophically meaningful. Because there is an inassimilable kernel within its symbolic space tearing it apart, its discourse is riddled with harrowing tension: unable to bring this encounter to its complete symbolization, Schelling's Weltalter falls to the ground and is only able to save itself through an unconscious disavowal that takes the form of an insistence on the existence of the Other of the Other. By consequence, it has to be interpreted psychoanalytically so that its truth or cause can finally be integrated into its symbolic space, and its place as the concluding step of German Idealism's formative Grundlogik can be firmly established. If this can be accomplished, then we will be permitted to say that, retroactively, Schelling's project fell to the ground not only in the sense of a purely negative collapse, but also as the condition of the possible opening of a new beginning, following the double connotation of the German zugrunde gehen. The failure of the Weltalter, the fact of its incompletion, turns out to be a triumph of human reason:317 its apparent limitations—the feverish frenzy of the work, the inconsistency brought forth by its conceptual unrest—are actually expressive of an underlying ontological and metaphysical intuition that we can catch a glimpse of through the interstices of its discourse. Žižek's claim is that if we pass through the negative determination at the heart of the work, the non-coincident in-itself we witness can become a for-itself. 206 Chapter 9 Firstly, both Schelling's own inability to finish the project, his heap of lost manuscripts, and the subsequent necessity to “invent” a solution to continue his philosophical career and its neglect in the philosophical community as a whole could thus be seen as nothing but a recoil from the Real of our being as finally explicitly brought into the open within his texts. It is thus Schelling who, in an attempt to give a death-dealing materialist response to Hegel's “pan-logicism,” most fully experiences out of all the representatives of the modern tradition the “incomprehensible basis of reality,” that mysterious X that forever haunts transcendentally constituted reality (the Real as a “kink” in the Symbolic), precedes it (the Real as pre-symbolic immediacy lost due to language), and in some modality conditions its very possibility (the Real [R⁄ ] as auto-disruptive substance [N ≠ N] whose self-laceration creates the space for free experience). In an attempt to articulate the irreducibility of freedom, Schelling plunges headfirst into the paradoxical character of subjectivity—not only into the denaturalized state of unruliness we must posit as prior to the emergence of phenomenal reality and culture, the pulsating cauldron of chaotic, heterogeneous forces that first make possible the gesture of infinite withdrawal into the nocturnal irreal self of The “victory” that Žižek perceives in the Weltalter is in its gestures towards philosophical logic underlying the birth of consciousness and its wider metaphysical implications. The strangeness and density of the Weltalter project are not due to its out-of-jointness with the modern tradition, but due to the fact that it comes too close to its disavowed essence. The anxiety-inducing reverberations that shake one's body when one reads the descriptions of “Schelling's grandiose 'Wagnerian' vision of God in the state of a endless 'pleasure in pain,' agonizing and struggling with Himself, affected by an unbearable anxiety, the vision of a 'psychotic,' mad God”318 are so difficult because they make us approach the very kernel not just of our existence as subjects but also the nature of the world at large. The Abyss of Unconscious Decision 207 act. Secondly, the originality of this narrative of German Idealism that Žižek allows us to construct is its implicit claim that the Weltalter is the text of modernity and that its incompletion, fragmentation, and unpopularity are philosophically rich in meaning, perhaps even necessary reactions to its truth. Not only does this contribute to elevating Schelling to his rightful place in history of philosophy after having long been overshadowed by Hegel, it also (and more importantly) calls us to own up to other elements of our being that may be more difficult to bear, even too difficult to bear, by one of their greatest theorists. Žižek's work on Schelling is so crucial for understanding his philosophy as a whole, not only because it provides us the resources we need to show the coherence of his reading of the modern philosophical tradition, but also because it allows us to demonstrate the profound level of philosophical scrutiny and methodological rigor inherent in his apparent heterodoxy. If the self-development of the former's account of subjectivity is characterized by a recoil from the parallax ontology of transcendental materialism at the core of the cogito's self-positing, the traces of which we can see in the negative contortions of its symbolic space through its entire history, it is only by reconstructing his psychoanalytical interpretation of Schelling as the culmination of German Idealism that we can truly legitimate and assess this claim. 206 Chapter 9 This experience of reading its drafts is properly “traumatic” in the meaning Lacan gives to this term drawing upon its etymological origins in ancient Greek: they bring forth a devastating encounter by which the symbolic support of our personality is “wounded” or “pierced,” during which the coherence of our identity is torn apart as it comes upon its truth.319 The effect is twofold. Firstly, both Schelling's own inability to finish the project, his heap of lost manuscripts, and the subsequent necessity to “invent” a solution to continue his philosophical career and its neglect in the philosophical community as a whole could thus be seen as nothing but a recoil from the Real of our being as finally explicitly brought into the open within his texts. It is thus Schelling who, in an attempt to give a death-dealing materialist response to Hegel's “pan-logicism,” most fully experiences out of all the representatives of the modern tradition the “incomprehensible basis of reality,” that mysterious X that forever haunts transcendentally constituted reality (the Real as a “kink” in the Symbolic), precedes it (the Real as pre-symbolic immediacy lost due to language), and in some modality conditions its very possibility (the Real [R⁄ ] as auto-disruptive substance [N ≠ N] whose self-laceration creates the space for free experience). In an attempt to articulate the irreducibility of freedom, Schelling plunges headfirst into the paradoxical character of subjectivity—not only into the denaturalized state of unruliness we must posit as prior to the emergence of phenomenal reality and culture, the pulsating cauldron of chaotic, heterogeneous forces that first make possible the gesture of infinite withdrawal into the nocturnal irreal self of the world, but the very ambiguity of the latter as an absolute self-positing The Abyss of Unconscious Decision 207 The Abyss of Unconscious Decision 207 Although such a recoil from this traumatic Real may be evident in Descartes’s reification of the subject as a thinking thing, Kant's attempt to ontologize “this I or he or it (the thing) that thinks” or more strongly his inability to delve into obscure foundations of unruliness and diabolic evil, or in Hegel's mature system as a covering up of the madness of the night of the world, it is only by recourse to Schelling that he can retroactively posit such a self-deploying disavowed knowledge that deepens itself through the trajectory of the tradition and that leads to its eventual culmination in psychoanalysis.320 What makes Schelling such an important outlier to this sustained recoil, however, was his ability (for a relatively brief period of time) to completely immerse himself within the frenzy, horror, and pandemonium that are the true origin of subjectivity in such a way that he brings to the fore the entire latent Grundlogik of modern philosophy of the subject preceding him and that is surpassed only by psychoanalysis' thematization of the unconscious and Todestrieb. His own turning away 208 Chapter 9 208 Chapter 9 The Abyss of Unconscious Decision 209 The Four Fundamental Concepts of Psychoanalysis appears to be a tribute to Freud, since the four concepts are taken directly from his work. Just as Lacan at that time calls his institute the “Freudian School,” in his seminar he uses the term “Freudian concepts” just to prove that he is not a dissident. But within this “tribute” he tries to go beyond Freud. Not a beyond Freud which leaves Freud behind; it is a beyond Freud which is nevertheless in Freud. Lacan is looking for something in Freud's work of which Freud himself was unaware. Something which we may call “extimate,” as it is so very intimate that Freud himself was not aware of it. So very intimate that this intimacy is extimate. It is an internal beyond.323 But how is Žižek able to accomplish such a feat, seeing that Schelling himself fought against this? 208 Chapter 9 208 from this insight and the various inconsistencies that lace his texts must be seen as symptomatic of a greater tendency within the movement (and in humans in general) towards the development of defense mechanisms in the face of an inability to integrate the psychoanalytical truth the Real tries to force upon us. If Schelling's own recoil—a return to a pre-modern essentialism—is so severe, it is because he, as the one who experienced the horror of the metaphysical implications of subjectivity most strongly, needed a defense mechanism more radical than the rest. In their “very failure, [the Weltalter] are arguably the acme of German Idealism and, simultaneously, a breakthrough into an unknown domain whose contours became discernible only in the aftermath of German Idealism.”321 Žižek thus sees his philosophy not only as an attempt to reactualize the German Idealist tradition, but also as immanently participating within its own self-deployment. He understands his project as a remodulation of its surface logic by clinically working through what he perceives as its internal tension, so as to construct the self-effacing, transcendental materialism that has been its unconscious truth.322 He is not merely performing an act of hermeneutical retrieval, but (more strongly) taking these ideas into what he considers to be their dialectical “completion” from which they have been hindered by the tradition itself. For Žižek, it is Lacan who gives us the methodological tools we need to “rehabilitate” its fundamental concepts, but which amounts to something more than a mere application of psychoanalytical concepts to various texts in the history of philosophy. The approach is much more provocative. By enacting a kind of therapeutic space within key texts of the middle-late Schelling as the culminating point of German Idealism, the wager of Žižek's philosophy is that, by being open to their own inner movement, he can bring forth that which is in Schelling more than Schelling himself: the extimate, traumatic core lodged deep within his spirit and soul and from which Schelling withdrew into philosophical “paralysis” by integrating (constructing) the originally inassimilable theoretical potential and unsettling metaphysical consequences of the second draft of the Weltalter. The relationship of Žižek to Schelling as the endpoint of German Idealism is therefore structurally identical to that of Lacan to Freud. As Miller puts it: The Abyss of Unconscious Decision 209 9.3 A Mytho-Poetics of Creation and the Seducing Hand of Fantasy The answer lies in the second draft, where Žižek sees a distinctively Hegelia structure that enables him to develop a metapsychological reading of the text, preventing its underlying ontology from succumbing to philosophical commitments that he rejects and identifies as in opposition to the unconscious Grundlogik of German Idealism. In fighting against what he perceives to be the limitations and threat of Hegelian logic, Schelling, in the end, only radicalizes its perverse truth, yet is unable to contain the monster that he unleashes. After developing an astonishing philosophy of freedom, Schelling immediately recoils from its implications by positing a principle of mediation that enables the neutral coexistence of Grund and existence through their mutual grounding in what Schelling in the Freiheitsschrift refers to as the Ungrund, that which in itself is ungrounded and thus simultaneously precedes both and is neither one nor the other. Because Schelling here understands the freedom of unconscious decision (Entscheidung) that separates Grund from existence as a return to the primordial origin of all reality that is the abyss of freedom itself, its conceptual edifice displays a structure of quaternity, which is articulated 210 Chapter 9 10 Chapter 9 210 in his thinking largely through a systemization and reconceptionalization of thinkers like Jakob Böhme and Franz Baader, and is thereby able to sidestep the implication of freedom as a cancerous upsurge of pure self- assertion. But insofar as the second draft displays freedom as a kind of self-positing activity that identifies Grund (the will-to-contraction, the No) and existence (the will-to-expansion, the Yes), Žižek jumps at this slight “slip” in Schelling's text, seeing in this discursive inconsistency a possibility of “formalizing” the conceptual movement of the work by “purifying” it of all extraneous theosophic commitments through traces of Hegelian logic he sees operative within it. It is in this sense that Žižek's philosophy is a hybridism of Schellingian ontology and a Hegelian quadruple dialectics of non-reconciliation. For Žižek, Hegel is the superior logician because he has no need to posit a principle of mediation outside of the internal dynamic of Grund and existence. There is no possible return to (or even initial existence of) a state of “originary” health, as typified by the abyss of freedom as independent from the antagonism of the dual principles, for there is nothing that has not always already succumbed to the restlessness of the negative. The Abyss of Unconscious Decision 211 entire logical process: “[t]here is thus no reversal of negativity into positive greatness—the only greatness is 'negativity' itself.”325 In other words, the negativity of the second is entirely inscribed within the first, arising from within its closed immediacy as a kind of tension or contradiction, and is in this regard not separate or distinct from it insofar as there is nothing but the dialectical register of the first. Yet because the second presents us with an internal limit (it itself has no substance of its own) and is thus by definition non-coincident with the first and its operational principles, it is in the same breath minimally distinguished from it, thus engendering a fissure in the logical self-closure of the field in a movement that makes the entire order inconsistent and ill at ease, which in turn opens up a foothold for the possibility of change.326 For Hegel, it is this deadlock of real internal limit—an inassimilable kernel—that ultimately serves as the springboard for all dialectical change by creating the space necessary for the unpredictable self-founding of a new order, but of itself it only becomes explicit in the aftermath of the third as it overthrows the primacy of the first through the paradoxical causality of a retroactive positing of presuppositions (Setzung der Voraussetzungen). There is something like an internal parasitic logic that re-totalizes the entire dialectical framework, so that instead of witnessing a return to the first, an initially subordinate moment degrades its own genetic conditions into its own subordinate moment by means of the unforeseeable and destructive power of negativity, which now reigns supreme.327 With the self-positing of the third (the second positing itself, counting itself, thus making itself the third by a self-doubling), negativity is finally fully “brought to life,” but in such a way that its dark pre-history in the vicissitudes of the previous stage as a purely negative real limit necessarily vanishes from sight, for the contingency of its self-positing has been immanently overcome. But even if the stark remodulation of the (onto)‍logical field within which it occurs, a remodulation that effectuates itself by establishing itself as the supreme category, covers up its steps like an experienced criminal used to getting away with his crime, in the same breath it sets the stage for a new (relatively closed) immediacy and thus the possibility of new unforeseeable dialectical change. 9.3 A Mytho-Poetics of Creation and the Seducing Hand of Fantasy Although textbook Hegelianism presents the third moment of the logic as a synthesis of two previous incompatible conceptual polarities by means of a cancellation of the falsehood and a preservation of the truth contained in each and thereby bringing them into a higher, more comprehensive dialectical standpoint (the banal reading of the equivocal character of the word “Aufhebung”) Žižek thinks this picture misses the true philosophical innovation that we see in the movement from one stage to another.324 The third moment itself is only the second insofar as it hegemonically usurps the position of the first through positing itself as such—which is, in a way, a mere amplification of an already existing interruption in the immanence of the first's logical field, an amplification that destroys its sway from within, but without ever leaving its fold. The dialectical movement from (i) immediacy → (ii) negation → (iii) negation of negation is superior not only because there is no return to the first (something irreducibly different and operatively new emerges, irreversibly reconfiguring the entire [onto]‍logical apparatus through its self-positing) but also because there is no need to posit something outside the self-movement of negativity to explain the y g g g , p dialectical standpoint (the banal reading of the equivocal character of the word “Aufhebung”) Žižek thinks this picture misses the true philosophical innovation that we see in the movement from one stage to another.324 The third moment itself is only the second insofar as it hegemonically usurps the position of the first through positing itself as such—which is, in a way, a mere amplification of an already existing interruption in the immanence of the first's logical field, an amplification that destroys its sway from within, but without ever leaving its fold. The dialectical movement from (i) immediacy → (ii) negation → (iii) negation of negation is superior not only because there is no return to the first (something irreducibly different and operatively new emerges, irreversibly reconfiguring the entire [onto]‍logical apparatus through its self-positing) but also because there is no need to posit something outside the self-movement of negativity to explain the The Abyss of Unconscious Decision 211 The Abyss of Unconscious Decision 211 But we must recognize that this self-positing always comes too late: its upsurge may posit itself at the logical beginning of the movement as necessary, it may make itself the 212 Chapter 9 primary principle, that to which the movement had always tended, but this only becomes visible after the fact and is thus plagued by a devastating belatedness. There is no way notionally to deduce the act of self-positing because, as free, it impossible to predetermine its arrival or even be aware that it could occur at all.328 The reversal characteristic of the third moment befalls being, for “every dialectical passage or reversal is a passage in which the new figure emerges ex nihilo.”329 What we often miss in the necessity of the dialectical movement and what is even obfuscated by it is the fact that every movement of its self-articulation is, in fact, constituted by a series of a contingent acts brought on by negativity, contingencies that only retroactively gain the status of necessity by making themselves necessary.330 The passage from tension to victory is never ontologically guaranteed—and it is precisely this ambiguity intrinsic to the restlessness of negativity that constitutes the Real of the Weltalter. If it was Hegel who gave the former the most profound philosophical articulation in its raw purity in his Science of Logic, it was Schelling who was the first to stumble upon the full range of its metaphysical implications (to which we shall return).331 What is more, this also lets us explain why Hegel could have stopped dead in his tracks in his mature thinking of a triadic relationship between logic, nature, and spirit despite already having at his disposition such a sophisticated account of a metaphysically contingent retroactive restructuring of being from within. Although the inner movement he describes in the Logic gives perfect expression to the quadruple dialectics already hinted at in the night of the world of his Realphilosophie, the problem is that the categories it describes, like Lacan's mathemes, do not supply us with any horizon of meaning. As such, they must be supplemented with the “concrete” symbolic content of the philosophies of nature and spirit, thus creating space in which Hegel could phantastically recoil from his breakthrough insight into the subject as a self-instituting gap in being and its stark consequences for our understanding of the self and the world. The Abyss of Unconscious Decision 211 Hence why the Weltalter is so crucial for grasping the true importance of subjectivity as a contingent, world-changing event: it gives us mytho-poetic content that comes closer to grasping the matter at hand than Hegel's account of the particular sciences does, which rather obfuscates it (just as many empirical accounts continue to do today when faced with the explosion of the Ideal out of the Real). primary principle, that to which the movement had always tended, but this only becomes visible after the fact and is thus plagued by a devastating belatedness. There is no way notionally to deduce the act of self-positing because, as free, it impossible to predetermine its arrival or even be aware that it could occur at all.328 The reversal characteristic of the third moment befalls being, for “every dialectical passage or reversal is a passage in which the new figure emerges ex nihilo.”329 What we often miss in the necessity of the dialectical movement and what is even obfuscated by it is the fact that every movement of its self-articulation is, in fact, constituted by a series of a contingent acts brought on by negativity, contingencies that only retroactively gain the status of necessity by making themselves necessary.330 The passage from tension to victory is never ontologically guaranteed—and it is precisely this ambiguity intrinsic to the restlessness of negativity that constitutes the Real of the Weltalter. If it was Hegel who gave the former the most profound philosophical articulation in its raw purity in his Science of Logic, it was Schelling who was the first to stumble upon the full range of its metaphysical implications (to which we shall return).331 What is more, this also lets us explain why Hegel could have stopped dead in his tracks in his mature thinking of a triadic relationship between logic, nature, and spirit despite already having at his disposition such a sophisticated account of a metaphysically contingent retroactive restructuring of being from within. Although the inner movement he describes in the Logic gives perfect expression to the quadruple dialectics already hinted at in the night of the world of his Realphilosophie, the problem is that the categories it describes, like Lacan's mathemes, do not supply us with any horizon of meaning. The Abyss of Unconscious Decision 213 Hence Schelling can say at the end of the Freiheitsschrift that nature is the first revelation of God.332 But with the highest creature, man, something new emerges in creation as the principles become separable, which leads to the possibility of the free conquering of the dark principle by the light if humans choose to live their lives in imitation of God's spirit as the perfect unity of Grund and existence and, by consequence, to the possibility of a complete revelation of God to Himself insofar as humans then become the image of The immediate problem facing Žižek here is the fact that all of Schelling's middle-late works present themselves as a theogony, an account of the birth of God, based on the anti-dialectical structure of the quaternity, where the last movement is not the expression of something logically irreducible and operatively new immanently emerging from within a system, but a “return” to the first as a kind of direct contact with and raising up to a higher power of a dormant force that is simultaneously the primordial and unfathomable origin of all things. Each text repeats the same movement, although the drafts of the Weltalter—in contradistinction to the Freiheitsschrift—abruptly end before historical time. As the first, the abyss of God's freedom as the impenetrably dark source of divine and created reality rests in the absolute joy of eternity, a blind existence, where it wills nothing because it is the pure virtuality wherein everything is potentially contained, though does not determinately exist. Yet without distinction and duality, the eternal nature as freedom remains unrevealed and thus lacks the fullness of self-knowledge. In order to achieve this, God (who is here not yet a person) must somehow contract finitude and difference, limit His freedom, if He is to have an Other through which He can reveal Himself, thereby establishing the distance necessary to Himself to become a subject capable of owning freedom as a predicate instead of merely being freedom as a pure virtuality. The Abyss of Unconscious Decision 211 As such, they must be supplemented with the “concrete” symbolic content of the philosophies of nature and spirit, thus creating space in which Hegel could phantastically recoil from his breakthrough insight into the subject as a self-instituting gap in being and its stark consequences for our understanding of the self and the world. Hence why the Weltalter is so crucial for grasping the true importance of subjectivity as a contingent, world-changing event: it gives us mytho-poetic content that comes closer to grasping the matter at hand than Hegel's account of the particular sciences does, which rather obfuscates it (just as many empirical accounts continue to do today when faced with the explosion of the Ideal out of the Real). The Abyss of Unconscious Decision 213 The Abyss of Unconscious Decision 213 The Abyss of Unconscious Decision 213 The Abyss of Unconscious Decision 213 The immediate problem facing Žižek here is the fact that all of Schelling's middle-late works present themselves as a theogony, an account of the birth of God, based on the anti-dialectical structure of the quaternity, where the last movement is not the expression of something logically irreducible and operatively new immanently emerging from within a system, but a “return” to the first as a kind of direct contact with and raising up to a higher power of a dormant force that is simultaneously the primordial and unfathomable origin of all things. Each text repeats the same movement, although the drafts of the Weltalter—in contradistinction to the Freiheitsschrift—abruptly end before historical time. As the first, the abyss of God's freedom as the impenetrably dark source of divine and created reality rests in the absolute joy of eternity, a blind existence, where it wills nothing because it is the pure virtuality wherein everything is potentially contained, though does not determinately exist. Yet without distinction and duality, the eternal nature as freedom remains unrevealed and thus lacks the fullness of self-knowledge. In order to achieve this, God (who is here not yet a person) must somehow contract finitude and difference, limit His freedom, if He is to have an Other through which He can reveal Himself, thereby establishing the distance necessary to Himself to become a subject capable of owning freedom as a predicate instead of merely being freedom as a pure virtuality. He breaches this pure virtuality by instituting the conflict of Grund (the No, the darkness of materiality, the contractive energy that holds all together) and existence (the Yes, the light of spirit, the expansive structures that give order to the rulessness of the Grund) within Him, so that He can beget Himself as a self-conscious being and eventually decide to bestow upon His own Grund the status of an independent and productive being, thus becoming God the Creator and allowing this same conflict constitutive of his inner life to be mirrored in all living things. The Abyss of Unconscious Decision 213 He breaches this pure virtuality by instituting the conflict of Grund (the No, the darkness of materiality, the contractive energy that holds all together) and existence (the Yes, the light of spirit, the expansive structures that give order to the rulessness of the Grund) within Him, so that He can beget Himself as a self-conscious being and eventually decide to bestow upon His own Grund the status of an independent and productive being, thus becoming God the Creator and allowing this same conflict constitutive of his inner life to be mirrored in all living things. Henc Schelling can say at the end of the Freiheitsschrift that nature is the first revelation of God.332 But with the highest creature, man, something new emerges in creation as the principles become separable, which leads to the possibility of the free conquering of the dark principle by the light if humans choose to live their lives in imitation of God's spirit as the perfect unity of Grund and existence and, by consequence, to the possibility of a complete 214 Chapter 9 214 Chapter 9 God (imago dei). With the forward march of history, the goal of creation will be attained when evil is completely vanquished, for this will have meant that we, like God, will have achieved the holy unification of the light and dark principles, insofar as we will have made ourselves subjects capable of owning freedom as a predicate by autonomously choosing the Yes and thus overcoming our separation from God by returning to Him. Material being, which we will have then “divinized” in showing how it is capable of the good in and through us, and personal God will then be reconciled and love—which presents itself as the fourth, the positive counterpart at the end of the system corresponding to the Ungrund as its absolute beginning—will prevail: as nature yearns for the spoken Word, humanity longs for for the destruction of the antagonism of principles in the Future, which proves itself to be a paradoxical “return” to the Past, since once the Present has begun, its beginning is always already lost, so that the only way the Ungrund can reemerge is if the world ends in a Future that is to come. The pure virtuality that contains everything potentially may be irretrievable, but redemption— as love—awaits as a point to which the world tends, for this tendency towards its own annihilation as we know it is part of its metaphysical structure. The inner life of all being thus follows a series of fourfolds that are modelled after the structure intrinsic to the life of God: Ungrun ↓ self-manifest God or complete revelation But how is Schelling able to maintain such a narrative within the second period even in face of a structure that apparently forecloses its very possibility? That which enables Schelling to sustain such a narrative in face of a structure that compromises the dynamic of the quaternity paradoxically coincides with his insight into a parallactic dialectics of restless negativity. The success of Schelling's reaction formation goes hand in hand with the intensity of his plunging into the abyssal origins of subjectivity, since the latter constitutes some kind of primordial trauma whose repression is The Abyss of Unconscious Decision 215 The Abyss of Unconscious Decision 215 simultaneous with the transcendental constitution of reality so that it is only to be expected that such a two-way tension would emerge. Given that the origins of the Symbolic withdraw in their founding gesture, if Schelling is to describe the interior involutions of the Real, and in what sense they could bring forth the conditions of the possibility of an absolutely free subjectivity (either human or divine) Schelling must have recourse to a mytho-poetic medium to talk about that which forever precedes and can never enter the light of consciousness and language. If the subject is “the primordial Big Bang” of experiential reality,333 how are we to grasp what occurred only nanoseconds prior to it? The issue is not the Real-as-excess as that which logically precedes and exceeds symbolization, which spectrally haunts all synthetic constitution, but the very moment in which the Symbolic upsurges; and because such an event is a structurally impossible object of discourse, any discourse that attempts to account for it is by the same token plagued by this very impossibility. Faced with this impasse, we are forced to fall back into mythology to explain that which transcends the bounds of ideality as its core: Does not this step involve “regression” to a version of New Age mythology? The Abyss of Unconscious Decision 215 When, at the key points of their theoretical edifice, Freud and Lacan also resorted to a mythical narrative (Freud's myth of the primordial father in Totem and Taboo, his reference to Plato's myth of androgynous primordial man in Beyond the Pleasure Principle; Lacan's myth of “lamella” in his Four Fundamental Concepts of Psycho-Analysis), they were driven by the same necessity as Schelling: the need for the form of mythical narrative arises when one endeavours to break the circle of the symbolic order and to give an account of its genesis (“origins”) from the Real and its pre-symbolic antagonism.334 Although mythology is rationally justified insofar as we have, as it were, deduced the necessity of the non-deducible and now need a new manner to investigate it, it does not come without its risks: the pre-symbolic act at the very founding gesture of language—the vanishing mediator between nature and culture that cannot be found in either—is that which we can “access” through a kind of self-reflexive speculative fabulation that stages “the birth 216 Chapter 9 216 Chapter 9 from darkness into light,”335 but in such a way that we cannot guarantee a priori that such an endeavour will not be plagued by defence mechanisms, unconscious fantasies and wishes, especially given its essentially traumatic nature. To search for our origins, we must plunge into the abyss, but it can lead us astray: the mytho-poetics of speculative fabulation can just as much help us narrate the pre-history of the Symbolic and the always already lost act that institutes it, the trauma that is the lacerating cut of pure self-positing in its full horror, as it can nourish psychoanalytical defences against it when we approach the ontological catastrophe at our nativity as human subjects. It is exactly here at this juncture of interplay of mytho-poetic fabulation and phantasmic interference within Schelling's second period that Žižek intervenes, using the resources of Lacanian psychoanalysis to traverse its fantasy and thereby restructure its conceptual space so as to bring forth that which is in Schelling so intimately that it is most properly characterized as extimate. But how can Žižek accomplish such a feat? Before we can directly answer this question, it is necessary to summarize the two intertwined levels active in the rational mythology of the Weltalter project in order to locate the unsolved tension from which psychoanalysis offers us a way out. The Abyss of Unconscious Decision 215 Within the stillborn drafts of the Weltalter, Schelling divides the passage from the eternal Past to the Present into three distinct stages. Because Schelling's text simultaneously operates in a theosophic and metapsychological mode, I will quickly summarize Žižek's presentation of each stage. Instead of outlining the various conceptual distinctions and internal differences that occur within the three existent drafts of the Weltalter, I will only be dealing with Žižek's own exegesis, which focuses on the second draft, since as we have seen he largely dismisses the importance of the other two versions. 1. In the absolute beginning prior to God's contraction of material being and the annular rotation of drives, there is a joyous nothingness, a pure potentiality that exists in timeless, inexhaustible rapture. God is not a yet a “He,” but merely an impersonal, anonymous “it”: knowing no conflict, God knows not even itself as radical freedom. For Žižek, in contemporary terms this would be equivalent to the pure void that exists before the vacuum fluctuation declared by quantum cosmology, a The Abyss of Unconscious Decision 217 nothingness that must be declared positively charged because from its (auto)disturbance “something” emerges. What is of utmost importance here is the irreconcilable contrast between this stage and the next: the joyous void of divine non-being is breached by the contraction of finitude and the self-diremption of perfection that it entails. Metapsychologically, this sundering of heavenly symmetry is thus structurally identical to the disruption of the oceanic unity of child and mother that supposedly precedes the Oedipus complex, or the smooth, placid functioning of cyclical nature, which is skewered by the advent of human subjectivity. 2. After the contraction of material being, we have what Žižek calls “Schelling's grandiose 'Wagnerian' vision of God.” Within Schellingian cosmogony, this is so “terrifying” because, instead of the endless joy of divine eternity, we have a God as subject who finds Himself caught within the self-lacerating rage of matter. There is a sense in which God the Almighty is in infinite pain because his freedom has been lost within the torment-ridden movement that is coincident with the moment when the blind rotation of drives falls into an erratic, uncontrollable oscillation. The Abyss of Unconscious Decision 215 As such, God is comparable to a helpless animal stuck in a trap.336 To exemplify this point further, Žižek compares this stage to the unfathomable chaos that occurs after the vacuum fluctuation at the origins of the universe: the contraction of matter into an infinite point of absolute singularity, an incomprehensible upheaval within which the logic of our known physical universe breaks down. In terms of a metapsychology, it can be read as a mytho-poetic description of the ontological short circuit within the instinctual schemata of nature occurring before the eruption of human subjectivity. This moment of ontological standstill coincides with the pre-personal realm of anarchic self-experience seen in the night of the world and expresses the first beginnings of psychotic withdrawal of nature into an irreal self that severs the Innenwelt from Aussenwelt. 218 Chapter 9 3. Finally, we have God who is able to speak the Word and thus overcome the deadlock that he found Himself lodged within by becoming a full-fledged subjectivity. Ejecting the materiality that He had contracted, pushing it out of him like some kind of foreign body, he bestows upon this now excremental product its own independent existence and thus becomes God the Creator. For Žižek, this corresponds in physics to the primordial Big Bang itself: the beginning of our material universe as the self-expansion of the initially infinitely dense point of matter. Metapsychologically, in the Word we see the unconscious decision (Entscheidung) that separates Grund and existence for the first time. The Symbolic erupts as an attempt to discipline the unruliness of the previous stage, which has immanently disrupted the primacy of organism's autopoietic schemata. The schismatic split that characterizes the essence of the psychoanalytical experience and the actual freedom of the cogito is posited as such for the first time as a response to the complete denaturalization that is the zero-level of the subject. In both the theosophy of creation, the physics of the Big Bang, and the metapsychology of the birth of subjectivity, this stage is something unpredictable that founds something new (God the Creator, material reality, the universe of meaning). The existent drafts of the Weltalter find themselves confronted with the same problem as the Freiheitsschrift, that is, the arbitrariness of the beginning: why does God speak the Word? The Abyss of Unconscious Decision 219 have become completely lost to themselves at a now inaccessible point in the vicissitudes of their dark pre-history. What is so terrifying here is that, instead of the light of consciousness and the universe of meaning, there could have been nothing but the agonizing rotation of drives in their frantic disorder, nothing but the ontological mayhem/madness wherein being would be irrevocably laid to waste in its own breakdown and catastrophism. If being awakens in a moment of primordial trauma, how could one be certain that its defense formation (subjectification through the Symbolic) would work? That it would solve its antagonism? If freedom is to be irreducible, it must posit itself entirely by its own activity from within the clutches of unbreakable determinism, but this comes at a price—and it is exactly around this problematic of the breach of symmetry necessary for a suitable “foundation” of freedom and its alarming metaphysical implications that the existent stillborn drafts circulate and ultimately falter, each offering a different spin irreconcilable with the others. For Žižek, however, the psychoanalytical tension of the Weltalter drafts displays a much more complex structure than that of a mere attempt to cover up the dialectical contingency at the heart of the unconscious decision and its wider implications. What intensifies the problem at hand is that the entire investigation has the fundamental structure of fantasy.337 In the articulation of the absolute beginning, we insert ourselves as a pure gaze into the Real that is prior to our own conception, just as if we were to imagine ourselves as spectators in our funerals watching our friends react to our death—and if we read the mytho-poetic introspective analysis that leads Schelling to such a discussion with this in mind, then we must conclude that it too exhibits the psychoanalytical traits associated with such fantasy constructions, complete with all of the problems that go along with them. The Abyss of Unconscious Decision 215 If the act of unconscious decision cannot be deduced according to notional necessity (were it able to be, Schelling would fall back into the perceived threat of Absolute Idealism that he is fighting against) there is no guarantee that God, after contracting material finitude, will be able to assert His freedom by making it a predicate of Himself: that is, there is no guarantee that the subject will be able to liberate itself from the deadlock of the drives within which it finds itself, in other words, their unruliness. Attempting to articulate a radical philosophy of freedom, Schelling in the second draft has no possible recourse to an explanatory principle capable of saving God or human being from possible infinite diremption: he is unable to foreclose the possibility that both could The Abyss of Unconscious Decision 219 The Abyss of Unconscious Decision 219 For both endeavour to describe the impossible: a linguistically ordered field of experience where there is none, a world of thought where no thinking could possibly exist, the first merely in a “subjective” sense (I cannot witness my own funeral because my own personal ego will never exist after my death in order to be able even to see such events unfolding as I may imagine them), but the second in an “objective” sense (not only is there no one there to witness the emergence of a world of signification, since consciousness presupposes someone has already emerged, but thought itself 220 Chapter 9 220 Chapter 9 220 as logos has yet even to appear on the scene). When put in this manner, the question that poses itself is how the second, “objective” fabulation can be saved from the various kinds of illusions that may play themselves out in the first, “subjective” mode of fabulation such as those that occur in our daydreaming. In this respect, it is understandable how Schelling could have fallen into a trap: because we can only retroactively posit the material origins of subjectivity from within the Symbolic and the Imaginary and these origins represent the unthinkable basis of thinking itself,338 the descriptions of this natal darkness can serve as a mere screen upon which we project fantasmatic supplements to satisfy unconscious desires. Not only does the very nature of the investigation jeopardize it (since we can structurally never reach the act that brings consciousness into existence, speculation has reached its limit and must pass into dramatic mytho-poetics) but it could easily be abolished through a reduction to the narcissistic orbit of the Imaginary or succumb to various symbolic levels of defence that would prevent the subject from performing its description, even in a self-reflexive mytho-poetic medium. By protecting us from the traumatic Real of our being, something that we of course all want, these supplements can lead us away from the truth for which we are searching. The Abyss of Unconscious Decision 219 But it is precisely here that the strength of psychoanalysis shows itself: as a discourse about discourse, in short, a discourse whose aim is to understand the inherent limitations plaguing all discourse (itself thus included), it is able to bestow upon such theoretical investigation an extra level of self-reflexivity that could help it from falling into such traps: “the way to avoid this utopian reduction of the subject to the impossible gaze witnessing an alternate reality from which it is absent is not to abandon the topos of alternate reality as such, but to reformulate it so as to avoid the mystification of the theosophic mytho- poetic narrative which pretends to render the genesis of the cosmos.”339 Thus, by focusing on the ambiguity of inserting ourselves as a pure gaze into the absolute beginning that we must retroactively posit, that is, the very impossibility of the endeavour itself to succeed in its task, Žižek is able to find a way to entirely cut off the theosophic character of Schelling's text and read it exclusively as a metapsychological account of the emergence of Symbolic from the meaningless Real. Man's inclusion within the divine theo-cosmogonic drama ultimately proves to be an extraneous feature of the Thus, by focusing on the ambiguity of inserting ourselves as a pure gaze into the absolute beginning that we must retroactively posit, that is, the very impossibility of the endeavour itself to succeed in its task, Žižek is able to find a way to entirely cut off the theosophic character of Schelling's text and read it exclusively as a metapsychological account of the emergence of Symbolic from the meaningless Real. Man's inclusion within the divine theo-cosmogonic drama ultimately proves to be an extraneous feature of the The Abyss of Unconscious Decision 221 operative logic of the Weltalter drafts, a code to be deciphered, many features of which can be ignored as a mere phantasmal projection accidentally adjoining itself to an exploration of the abyssal origins of subjectivity. One of Žižek's most provocative claims is that one can see such a phantasmal projection in the attempts to cover up its most groundbreaking insight: the destructive self-movement of negativity in reality that leads to and is radicalized in the self-positing of subjectivity. The Abyss of Unconscious Decision 219 In this way, we can say that paradoxically “it was [Schelling's] very 'regression' from pure philosophical idealism to the pre-modern theosophical problematic which enabled him to overtake modernity itself.”340 But now the question imposes itself upon us: what is revealed when we purify the odyssey that is the Freiheitsschrift and the Weltalter from its mytho-poetic phantasmagoria? operative logic of the Weltalter drafts, a code to be deciphered, many features of which can be ignored as a mere phantasmal projection accidentally adjoining itself to an exploration of the abyssal origins of subjectivity. One of Žižek's most provocative claims is that one can see such a phantasmal projection in the attempts to cover up its most groundbreaking insight: the destructive self-movement of negativity in reality that leads to and is radicalized in the self-positing of subjectivity. In this way, we can say that paradoxically “it was [Schelling's] very 'regression' from pure philosophical idealism to the pre-modern theosophical problematic which enabled him to overtake modernity itself.”340 But now the question imposes itself upon us: what is revealed when we purify the odyssey that is the Freiheitsschrift and the Weltalter from its mytho-poetic phantasmagoria? Notes 294. See Žižek, The Ticklish Subject, p. 55; and The Parallax View, p. 166. 295. Žižek, The Indivisible Remainder, p. 8. 296. Schelling, Freiheitsschrift, p. 276. 296. Schelling, Freiheitsschrift, p. 276. 297. Ibid., p. 278. 297. Ibid., p. 278. 298. Žižek, The Indivisible Remainder, p. 105. 298. Žižek, The Indivisible Remainder, p. 105. 299. Ibid., p. 92. 299. Ibid., p. 92. 300. Ibid., p. 103. 301. Ibid., p. 106. 301. Ibid., p. 106. 302. Žižek, Less Than Nothing, p. 234. 302. Žižek, Less Than Nothing, p. 234. 303. Žižek, The Indivisible Remainder, pp. 35–39. 303. Žižek, The Indivisible Remainder, pp. 35–39. 304. Schelling, Weltalter II, p. 4. 304. Schelling, Weltalter II, p. 4. 305. Žižek, The Indivisible Remainder, p. 38. 305. Žižek, The Indivisible Remainder, p. 38. 306. Ibid., p. 92. 306. Ibid., p. 92. 307. Wirth, “Translator’s Introduction,” in Weltalter III, pp. ix–x. 308. Hegel, The Phenomenology of Spirit, p. 9. 308. Hegel, The Phenomenology of Spirit, p. 9. 309. Žižek, The Indivisible Remainder, pp. 20–21. 310. Schelling, Freiheitsschrift, p. 281. 310. Schelling, Freiheitsschrift, p. 281. 311. Schelling, Weltalter II, p. 121. 311. Schelling, Weltalter II, p. 121. 312. Žižek, The Indivisible Remainder, p. 38. 313. Ibid., p. 92. 313. Ibid., p. 92. 222 Chapter 9 314. Ibid., p. 37. 315. Ibid., p. 39. 316. Schelling, Weltalter II, p. 121. 317. Žižek, The Abyss of Freedom, pp. 3–4. 318. Žižek, The Indivisible Remainder, p. 24. 319. See Laplanche and Pontalis, The Language of Psychoanalysis, trans. D. Nicholson- Smith (London: Karnac Books, 1988), pp. 465–69; and also Lacan, The Seminar of Jacques Lacan, XI: The Four Fundamental Concepts of Psycho-analysis, 1963–1964, ed. Jacques-Alain Miller, trans Alan Sheridan (London: Vintage, 1998), p. 51. 320. Žižek, “Liberation Hurts”; and The Ticklish Subject, p. 48. 321. Žižek, The Abyss of Freedom, pp. 3–4. 321. Žižek, The Abyss of Freedom, pp. 3–4. 322. Žižek, The Indivisible Remainder, p. 92. 323. Miller, “Context and Concepts,” in Reading Seminar XI: Lacan’s Four Fundamental Concepts of Psychoanalysis, ed. Richard Feldstein, Bruce Fink, and Maire Jaanus (Albany: SUNY Press, 1995), pp. 7–8. 324. Žižek, Less Than Nothing, pp. 300ff. 324. Žižek, Less Than Nothing, pp. 300ff. 325. Ibid., p. 198. Compare with pp. 292ff. and 304. 326. Ibid., pp. 293–94. 326. Ibid., pp. 293–94. 326. Ibid., pp. 293–94. 327. Ibid., p. 234. 328. Ibid., p. 285. 329. Ibid., p. 231. 330. Ibid., p. 213. 331. Ibid., p. 274. 332. Schelling, Freiheitsschrift, p. 284. 333. Žižek, The Ticklish Subject, p. 31. 334. Žižek, The Indivisible Remainder, p. 9. 335. Schelling, Freiheitsschrift, p. 239. 336. Žižek, The Indivisible Remainder, p. 23. 337. Ibid., p. 22. 337. Ibid., p. 22. 338. Žižek, Less Than Nothing, p. 645. 339. Ibid., p. 273. 339. Ibid., p. 273. 340. Žižek, The Indivisible Remainder, p. 8. Chapter 10 Radicalizing the Subject Substance Gasping for Breath, the Metaphysics of Quantum Mechanics, and the Žižekian Unconscious Chapter 10 Radicalizing the Subject Substance Gasping for Breath, the Metaphysics of Quantum Mechanics, and the Žižekian Unconscious Although Žižek's transcendental materialism relies upon Schelling to explicate the origins of subjectivity, Hegel is nevertheless omnipresent in Žižek's work. Perceiving a strictly Hegelian structure of self-relating negativity in the second draft of the Weltalter that identifies Grund and existence in a manner inconsistent with its surface structure of quaternity, Žižek offers a psychoanalytical reconstruction of Schelling's aborted masterpiece by showing how Hegelian dialectics is its unconscious truth. Demonstrating this complex and nuanced hybridism of Hegelian logic and Schellingian ontology at the heart of Žižek's philosophy does not merely help us see its originality and singularity; it also enables us to shed light on in what sense his philosophy is a revisionist metaphysics of the subject. The existence of subjectivity not only attests that nature is at best a fragile not-all whose disruptive wounds are more primordial than its positive being. More drastically than this, the absolute, instead of being a fully subsisting reality that exists by means of a self-explanatory surplus, is at its most basic level only minimally indistinguishable from the void of nothingness, whereby the subject becomes one of the names of the eternal disturbance of this void by means of which there is something rather than nothing. This also leads us into a discussion of the paradoxical causality explicit in the self-begetting 224 Chapter 10 Chapter 10 224 of the Žižekian subject and its repercussions for our understanding of the unconscious. 10.1 From the Psychoanalytical Purification of the Theosophic to Substance Gasping for Breath Žižek does not articulate his own solution to the problematic of how he is able to purify the odyssey that is the Freiheitsschrift and the Weltalter from its mytho-poetic phantasmagoria. The issue at hand is how he can formalize Schelling's philosophy by purifying its theosophic content (the illusion of an originary oceanic bliss and its crucial role in covering up the intrinsically dialectical structure inherent to the Grund by means of a quaternity) by traversing its fantasy. To do this, Žižek relies on the primordiality of the psychoanalytical experience and the irreducibility of negativity in Hegelian logic, the traces of which he sees in the second draft's descriptions of the three stages involved in the movement from the Past to the Present. The resources offered by both allow us to internally reconfigure its conceptual movement by removing this distorting fantasy element through a philosophical reconstruction of the abyssal origins of the subject by tarrying with the Real of Schelling's texts. It is this revamped version of the Weltalter's investigations into the spectral, never-present beginnings of the Symbolic that enables Žižek to draw out the full metaphysical implications of the paradoxical nature of psychoanalytico-Cartesian subjectivity. According to psychoanalytical experience, the zero-level fact in the passage from the Past to the Present has to be the second stage,341 that is, the self-lacerating rage of matter. 10.1 From the Psychoanalytical Purification of the Theosophic to Substance Gasping for Breath Because Žižek identifies the subject with the non-coincidence of substance, its alienation from itself,342 this self-lacerating rage is equivalent to what Lacan refers to as the “organic dehiscence” exhibited in the mirror stage that forms the basis of the ontogenesis of personality.343 But if we are to mytho-poetically explain how such a dismemberment forms the meta-transcendental conditions for the possibility of subjectivity's self-positing, we cannot understand it as a haphazard feature of nature that falls upon it like an alien blow from nowhere—rather, its pure contingency must itself emerge from some kind of immanent, self-effacing possibility always already implicit within it.344 As that which metapsychologically corresponds to the eternal calm of the pure Radicalizing the Subject 225 immanence of substance preceding the deadlock of drives and the struggle to speak the Word, the joyous nothingness of divine eternity is therefore merely a part of the ego's fantasy of desired fullness, a narcissistic and reactive attempt to secure the false status of nature as all in order to protect us from the dark truth implicit in the psychoanalytical experience: that one cannot draw a metaphysical distinction between substance as a nothingness that rejoices in the oceanic bliss of non-experience and the unruly basis of human subjectivity that “disrupts” this unity. For Žižek, the transcendental materialist logic of which we see premonitions in the second draft thus allows us to add precision to the late German Idealist attempts to think substance as subject: the model of subjectivity as a disease within the vital fold of being needs to be slightly modified, for there was never a state of metaphysical harmony and innocence that the going-haywire of human unruliness could have brought into ruin once and for all. 10.1 From the Psychoanalytical Purification of the Theosophic to Substance Gasping for Breath This is exactly why Žižek feels justified in proclaiming the superiority of Hegelian logic and violently remodulating Schelling's middle-late ontology to get rid of its notion of absolute indifference, for in positing an initial state of health, Schelling demonstrates a tendency—a tendency only encouraged by the abyssal void of the Real-as-origin—to construct a rampant fantasy whose imaginary support (certain theosophic details) could protect him from his own insight into the restless movement of negativity in the interior life of being and at the core of the subject's pure act, a fantasy that in itself is filled with holes and inconsistencies as shown by the endless proliferation of stillborn drafts of the Weltalter in an attempt to secure it. That the subject is synonymous with the irrevocable self-sundering of substance does not mean that before the advent of complete denaturalization in our constitutive excess there was no internal obstruction within the former's immanent ebb and flow. If the human being is an irreversible blockage in the vital fold of being, it must represent an amplification of an already existing potentiality in nature. We can see this in various forms—natural disasters, deformed animals, mass extinction, black holes, all of which point to ways in which the originary “harmony” of the world is predicated upon disorder, eruptive disarray, and its inability to sustain itself in perfect symmetry. Knowledge in the Real is never perfect: 226 Chapter 10 6 Chapter 10 The most unsettling aspect of such phenomena is the disturbance caused in what Lacan called “knowledge in the Real”: the “instinctual” knowledge which regulates animal and plant activity. This obscure knowledge can run amok. When winter is too warm, plants and animals misread the temperature as a signal that spring has already begun and so start to behave accordingly, thus not only rendering themselves vulnerable to later onslaughts of cold, but also perturbing the entire rhythm of natural reproduction.345 The most unsettling aspect of such phenomena is the disturbance caused in what Lacan called “knowledge in the Real”: the “instinctual” knowledge which regulates animal and plant activity. This obscure knowledge can run amok. 10.1 From the Psychoanalytical Purification of the Theosophic to Substance Gasping for Breath When winter is too warm, plants and animals misread the temperature as a signal that spring has already begun and so start to behave accordingly, thus not only rendering themselves vulnerable to later onslaughts of cold, but also perturbing the entire rhythm of natural reproduction.345 These examples of destruction in nature must, however, be seen in their raw conceptual materiality. Rather than being justified by Žižek's rhetoric, they point to one of the major achievements of Žižek's dialectics: its ability to think through the philosophical implications of what is normally seen as a mere contingent breakdown of all-present order or a short-lived calamitous outburst of pandemonium in the otherwise smooth flow of things. Žižek refuses such a thesis: for him, contradiction and non-coincidence are to be seen as at the very basis of reality to such an extent that reality's logical closure upon itself is unable to sustain itself and is constitutively torment-ridden with fracture points. Rather than being a totalizing weave that is and thus embraces all things in its soft, calming touch, despite things seeming otherwise in experience (just as turbulent eddies are only possible given the oneness of the ocean that persists through them), substance is dirempt and constantly risks bursting at its seams due to its own internal fragmentation. These wounds in the absolute do not exude the vital blood of substance in such a way that they will, after a brief moment of cosmic fright, heal themselves and once again be subsumed within the economy of a symphonized sexualized dance of lack and plenitude, life and death: substance does not engage in a ritual of bloodletting to gain strength, as if conflict, although internal to the system, only existed so that through its conquering substance could express again and again its infinite, almighty power. On the contrary, the wounds of substance are those places where it touches the void. Torn apart from the inside out, substance in its auto-laceration is in danger of dissipating into nothingness, of no longer being capable of holding itself together. 10.1 From the Psychoanalytical Purification of the Theosophic to Substance Gasping for Breath Here one must think of the fundamental presupposition of Schellingian philosophy: were substance (God) all, were there from the very beginning nothing but Radicalizing the Subject 227 a balanced movement, a pure, all-devouring totality, no subjectivity or experience would be possible.346 In this sense, Žižek is not merely trying to radicalize this insight by reactualizing Schelling's metapsychology so that it does not succumb to its theosophic tendencies, but more primordially trying to make it internally consistent. According to Schelling's own words, if we posit an initial state of health, we cannot explain how subjectivity could emerge. Perfection is only an imaginary, fantasmatic extension of the existential horizon of our broken finitude, that is, a reaction formation, a reaction formation within which we encounter again and again the insupportable negativity that tears apart the world, for after all, the repressed always returns. As the greatest thinker of subjectivity, we begin to grasp why there would be such an immense conflict within Schelling's middle-late period between the irreconcilable extremes of a radical philosophy of freedom and an equally radical philosophy of a theo-cosmogonic drama of divine being seeking self-manifest revelation. They go hand in hand: traumatic insight elicits protective defences, but the latter can never be completely successful. Accordingly, Žižek psychoanalytically modifies Schelling's descriptions of the Past as that elusive X that forever haunts and precedes consciousness so that the real encounter (the horror of substance as subject) can be freed from its imaginary, fantasmatic surface (the theosophic odyssey) and brought into explicit mythologico-symbolization. This has severe consequences. First and foremost, we must remember that at the level of logic, Schelling's mytho-poetic narrative of the Past does not primordially present a chronology of the absolute, even if it is derived from phenomenological experience by the alchemical identification of the highest and lowest and can thus be used to explain the ontogenesis of the individual subject by means of reason by analogy. The “stages” Schelling refers to are purely logical and organized according to priority: the category of linear temporality only emerges with the Present as that which mirrors the structural relations intrinsic to the inner life of God. Consequently, there is no sense in which the joyous nothingness temporally precedes God as subject caught in the throes of the self-lacerating rage of matter. 10.1 From the Psychoanalytical Purification of the Theosophic to Substance Gasping for Breath However, insofar as freedom as a predicate of a subject exists (stage three), the abyss of freedom as pure potentiality—a freedom that is not yet posited as such—must be said to logically precede the rotation of drives prior to full-fledged subjectivity 228 Chapter 10 (stage two). Žižek follows the argument thus far, but then makes a crucial change in an attempt to draw out a truth hidden in Schelling's descriptions of the passage from one moment to the other. Given that there is no temporal separation between the descriptions of the joyous nothingness of non-experience and the infinite, agonizing oscillation of the potencies, or substance and subject, they are, in essence, two sides of the same coin. The “passage” is nothing but a logical conversion: Let us step back for a moment and reformulate the primordial contraction in terms of the passage from a self-contented Will that wants nothing to an actual Will which effectively wants something: the pure potentiality of the primordial Freedom— this blissful tranquillity, this pure enjoyment, of an unassertive, neutral Will which wants nothing—actualizes itself in the guise of a Will which actively, effectively, wants this “nothing”—that is, the annihilation of every positive, determinate content. By means of this purely formal conversion of potentiality into actuality, the blissful peace of primordial Freedom thus changes into pure contraction, into the vortex of “divine madness” which threatens to swallow everything, into the highest affirmation of God's egotism which tolerates nothing outside of itself. 228 Chapter 10 In other words, the blissful peace of primordial Freedom and the all-destructive divine fury which sweeps away every determinate content are one and the same thing, only in a different modality—first in the mode of potentiality, then in the mode of actuality.347 The ultimate paradox of the shift from the bliss of divine eternity, freedom as pure potentiality, to the annular rotation of drives in the Grund that serves as the stepping-stone to freedom as the predicate of a subject is that there is no movement at all—Grund is always already the Ungrund, the “closed” circle of nature is always already the scene of the possible emergence of freedom.348 If we equate freedom with negativity, then we see that the Ungrund is no longer that which neutrally grounds the conflict of the polar principles in order to know itself and reveal itself to itself through them, but rather becomes synonymous with the devastating negativity at the heart of being, the places where it risks touching the void, and that even when it remains non-posited Radicalizing the Subject 229 as such, is always already present within the palpitations of the latter like a cardiac arrest waiting to happen: the ground is always already minimally ungrounded, ridden with tension, bursting at the seams. We see this clearly in the dialectic of the ontogenesis of the subject. Although the movement from (i) the rapture of symmetry that is substance to (ii) the harrowing madness of the drives in the unruliness of the infant appears to be due to the grotesque excess of life that is human being as an unfortunate accident, the matter is more complicated. In the first stage freedom (negativity) is always already there as a logical possibility, but remains for the most part non-posited in the general economy of the dynamics of substance, though it shows its head constantly, so that we just have a relatively closed and blind annular rotation of drives; in the second, however, it finally successfully posits itself in a significant manner (although not as such) as the self-lacerating rage of matter, but in such a way that its rawness risks devouring itself. It needs to be tamed, disciplined, gentrified. 230 Chapter 10 things, a causally closed play of forces caught within a blind necessity, is a pure fantasy: the beginning is not a solid, inert density, but a seething mass of heterogeneous matter lacking overarching symmetry and balanced movement. Matter is not some kind of impenetrable or irreducible “real stuff” that persists beyond conscious representation and follows eternal laws—at its very core, matter constantly dematerializes itself by opening up its flesh to the void of (virtual) non-being to the point where it is no longer matter but something more: that is, a full-fledged subject. The self-operative logic of nature as Grund consequently demonstrates that the immanence of substance is not a permeating weave of positive being, a never-ending sea whose fullness encompasses all: it is plagued constitutively by the possibility of self-fragmentation, since irrevocable zones of (virtual) non-being shattering the ordered reality of the world and its causal closure always threaten to erupt and turn it into a chaotic pandemonium of disordered, anarchic forces combating for rulership—a state that would, in fact, bring substance itself to its untimely end, for a substance that has globally turned “into a dispersed floating of membra disjecta”351 is no longer a substance at all. The self-lacerating rage of matter thus takes on a new meaning. The libidinal frenzy of the unruliness of human nature does not merely things, a causally closed play of forces caught within a blind necessity, is a pure fantasy: the beginning is not a solid, inert density, but a seething mass of heterogeneous matter lacking overarching symmetry and balanced movement. Matter is not some kind of impenetrable or irreducible “real stuff” that persists beyond conscious representation and follows eternal laws—at its very core, matter constantly dematerializes itself by opening up its flesh to the void of (virtual) non-being to the point where it is no longer matter but something more: that is, a full-fledged subject. 228 Chapter 10 The decisive moment comes with (iii) the act of decision through which freedom as subject (inner limit of substance) is converted into freedom as the predicate of a subject (self- relating negativity), an act that is not only notionally non-deducible because it is, in itself, radically free, but also one that withdraws in its very gesture of self-positing. When this occurs, negativity, instead of being a single part in the totality of material being, turns itself into an independent center that hegemonically dominates the whole to which it once belonged, which in turn forces the negativity always already contained in the first moment finally to become fully explicit. Freedom is not in direct contact with the Ungrund as that which neutrally grounds the conflict of the polar principles as indifference, nor is it a resurgence of the primordial abyss of freedom: it is nothing but this movement in which the second (the negativity of being), relating to itself, counting itself, usurps the position of the first (being) and thus institutes a mere formal reconfiguration of the whole.349 As pure negativity, full-fledged self-standing freedom (self-relating negativity) does not emerge out of nowhere within a harmonious play of forces, because its existence attests that there is freedom (negativity) all the way down. As Žižek says, “the only greatness is 'negativity' itself.”350 What the psychoanalytico-Cartesian subject thus shows us is that the idea of extra/pre-subjective nature as the self-harmonizing Grund of all What the psychoanalytico-Cartesian subject thus shows us is that the idea of extra/pre-subjective nature as the self-harmonizing Grund of all 30 Chapter 10 230 Chapter 10 The self-operative logic of nature as Grund consequently demonstrates that the immanence of substance is not a permeating weave of positive being, a never-ending sea whose fullness encompasses all: it is plagued constitutively by the possibility of self-fragmentation, since irrevocable zones of (virtual) non-being shattering the ordered reality of the world and its causal closure always threaten to erupt and turn it into a chaotic pandemonium of disordered, anarchic forces combating for rulership—a state that would, in fact, bring substance itself to its untimely end, for a substance that has globally turned “into a dispersed floating of membra disjecta”351 is no longer a substance at all. The self-lacerating rage of matter thus takes on a new meaning. The libidinal frenzy of the unruliness of human nature does not merely represent a single case of the diseased breakdown of the ontological, but rather the inability of substance to posit itself as all: substance is destined towards auto-destruction; the “ground fails to ground,”352 for it is always on the verge of passing over into hemorrhaging conflict and ravaging antagonism. In this regard, substance is constitutively weak, a precarious all whose fragility is not only always struggling to keep itself together as the contractive energy of all things, but also always capable of the new and the unpredictable at any cosmic moment due to its negativity, for what will be is not necessarily causally contained in what was. Here we see the extremely Hegelian logic that Žižek extracts from Schelling in the second draft: it is the failure of the first moment (the self-actualization of substance and the indivisibility of its causal self-enclosure, its closed annular rotation) that leads to/is the second (the unruliness of human nature, the unbearable short circuit in the rotation of drives), whose vicissitudes in turn set the stage for the third (the self-positing of the subject through this rupture in the fold of being, which opens up an autonomous logical space that attempts to Radicalizing the Subject 231 suture the hole). The essence of the third moment is the self-negation of the previous one, which gives it a fully developed notional self-reflexivity in such a way that by guaranteeing the identity of Grund and existence Žižek is able to foreclose the possibility of a theosophic quaternity from within the conceptual fabric of Schelling's text. 230 Chapter 10 In this sense we can finally comprehend two controversial claims put forth by Žižek: first, that that “the founding gesture [the subject as the vanishing mediator] 'repressed' by the formal envelope of the 'panlogicist' Hegel is the same as the gesture which is 'repressed' by the formal envelope of the 'obscurantist' Schelling,”353 for both reveal in their own way the “unacknowledged” Grundoperation of German Idealism; and, second, that “to articulate clearly the Grundoperation of German Idealism [...] necessitates reference to Lacan; that is to say, our premiss is that the 'royal road' to this Grundoperation involves reading German Idealism through the prism of Lacanian psychoanalytical theory.”354 It is in this respect that Žižek's philosophy is a hybridism of Hegelian logic and Schellingian ontology, for it is a mere attempt at the thematization of its unconscious Grundlogik. The metaphysical basis of freedom is the irremovable possibility of negativity from within any self-totalizing, self-enclosed system. There are always unavoidable ruptures and breaks within the logical fold of the world, but these are not mere contingent features of the otherwise harmonious symphony of the infinity of being: the activity of the first moment always already “possesses” its own failure as exhibited in the constant proliferation of negativities that can never be fully subsumed within its dynamics, indicating that substance is constitutively minimally non-coincident to itself. Monstrosities and ontological abortions are an inescapable effect of substance's functioning, for function now shows itself to be one with dysfunction. In terms of Žižek's “reactualization” of Schelling, it is here that its most textually violent moment is located. It proclaims that the only way to save the Schellingian legacy is to say that nature as a full, rich creative potency inherent in the dark womb of the world is an illusion. Nature was always already a sickly creature, whose collapse coincides with its own conditions of (im)possibility. It is not only that nature never knew a moment of eternal happiness and joy, but that the dull, inarticulate pressure of its own gasping for breath (spirit, we remember, comes from the Latin 232 Chapter 10 spiritus, “breath,” and is related to spirare, “to breathe”) precedes the very positivity of its being. Substance can only be substance—nature can only be nature—insofar as it is always already internally torn apart by a constitutive moment of auto-laceration that is the site of spirit/subject: “incompleteness [is] already in itself a mode of subjectivity, such that subjectivity is always already part of the Absolute, and reality is not even thinkable without subjectivity.”355 If the ontological dislocation attested to by emergence of the subject is always already a part of the absolute, then the passage from nature to culture is a mere logical conversion—it only requires a certain gesture or incitation to be brought to the fore while nothing changes at the level of “positive” being. Full-fledged subjectivity, and hence the symbolic universe of meaning that emerges as a belated response, may be an unpredictable event whose result institutes a new age of the world, but its ontological basis demonstrates that there is no ultimate opposition between us and the world. The idea of a unified, self-penetrating substance only comes après-coup as part of a fantasy that helps the subject protect itself, for without such a fantasmatic support of fullness we risk losing our very subjective consistency in face of the tragic incompletion of the world. The fiction of “Nature” is, in many ways, unavoidable: we unconsciously create it to save ourselves from recognizing the true basis of subjectivity and its stark implications, namely, the fact that the world, constitutively ravaged by the Ungrund, is metaphysically imbalanced and thus not-all: True “anthropomorphism” resides in the notion of nature tacitly assumed by those who oppose man to nature: nature as a circular “return of the same,” as the determinist kingdom of inexorable “natural laws,” or (more in accordance with “New Age” sensitivity) nature as a harmonious, balanced Whole of cosmic forces derailed by man's hubris, his pathological arrogance. What is to be “deconstructed” is this very notion of nature: the features we refer to in order to emphasize man's unique status—the constitutive imbalance, the “out-of-joint,” on account of which man is an “unnatural” creature, “nature sick unto death”—must somehow be at work in nature itself, Radicalizing the Subject 233 although—as Schelling would have put it—in another, lower power (in the mathematical sense of the term).356 But here we must be careful. 232 Chapter 10 Insofar as the subject is an immanent event within the world, that is, insofar as through it being has “gained” the power to look upon itself by internal reflection due to a maximization of this constitutive self-sabotaging tendency within nature and symbolization occurs as a means to deal with this trauma, one cannot conclude that symbolization is a mere defence mechanism constitutive of the human ego. The illusionary world of the Symbolic into which we withdraw to save ourselves from the trauma that is the very essence of our freedom is simultaneous with being's own recoil from itself as it achieves self-disclosure. Awakening into the nightmare that is the psychoanalytical horror of substance as subject in all of its ambiguity, the primordial reaction of the world opening its eyes for the first time is that of hellish panic, a panic whose ultimate fate is the necessity of an ontological passage through madness which we enact as symbolic subjects by (re)constituting reality in an ontologically solipsistic, eternally nocturnal space of signifiers, a universe of meaning. If the self-revelation of being to itself leads to an originary madness, then not only should we understand the proliferation within the latter of self-composing dream-like images, a rhapsody of social and political phantasmagoria that function as the fabric of our own identities, as events in being, but also this very fiction of ontological completion we witness in accounts of “Nature.” Through us, in a moment of fantasy, being sees itself as perfect—or as having been possibly perfect were it not for our intervention—by living out the impossibility of its own illusionary fullness, a fantasy that is a complete perversion of the typical narrative of God reaching full self-consciousness only by means of human activity. In this sense, if the theological implications of quantum mechanics, wherein particles can cheat the universe by coming into and out of existence before they are “symbolically” registered, are that we must “posit a God who is omnipotent, but not omniscient,”357 then Žižek's metaphysics compels us to posit a God (nature, the absolute) that is neither omnipotent nor omniscient, yet somehow persists in being But how? 234 Chapter 10 10.2 Eppur si muove: Ontological Dislocation and the Metaphysics of the Void a collapse of the wave function;361 “normal” laws of linear temporality and causality break down as we encounter particles that retroactively “choose” their paths along virtual chains of possibilities and others that can even cheat the universe, coming in and out of existence without the latter even knowing.362 But this not only drastically challenges any sharp distinction between nature and culture,363 but also attests that the micro-universe of quantum particles is strangely “less” than that of the macro-universe that constructs itself from its vicissitudes, in a way that is remarkably similar to how the Kantian subject can only construct a unified world of appearances from the inconsistent fragments of sensation. In an uncanny logical short circuit, it would appear that not only is there no bottom-up causality at the level of experience (transcendental constitution is more real than what Kant calls “a rhapsody of perception,”364 since it founds a coherent field of reality), but even the most fundamental level of the universe is metaphysically more chaotic and constitutively less ontologically complete than the ordered macro-level physical world that science, as according to classical models, describes. Substance is not a given, but an achievement: it is as if all reality is transcendentally constituted, as if nature itself emerges out of this field of quasi-entities. But just as we had to explain the ontogenesis of unruliness, we must also explain the emergence of these ontologically incomplete realities that serve as the basis for the ordered universe, since they cannot be a brute, arbitrary given. The emphasis of the key philosophical question is thus shifted from how the subject can emerge out of the Real qua substance (thought from being) to how the subject can emerge out of the Real qua void (something from nothing), for the more strictly speaking ontological question of ontogenesis thus proves to be irreducibly entangled with the most fundamental of metaphysical questions. The more we analyze reality the more we find a void, because once we reach the level of quanta our conventional conceptions of the ordered cosmos just stop working. 10.2 Eppur si muove: Ontological Dislocation and the Metaphysics of the Void At this conceptual conjuncture, Žižek takes an additional step in his psychoanalytical reconstruction of German Idealism that simultaneously brings into complete culmination its unconscious, disavowed Grundlogik and leads him into a profoundly new variety of metaphysical thinking that is uniquely his own. If in order to explain the emergence of the subject out of substance we have to proclaim that the latter, rather than being an indivisible oneness that weaves all things into an all-encompassing fabric, is predicated upon a site of auto-laceration, then the subject—as a name for this gap in substance that “sets it in motion”—has in turn to be more fully investigated, for we have not yet seen how ontological dislocation could be a creative force. We cannot stay at the level of substance qua nature: the ordered universe that compromises cosmological, geological, and biological time is so ravaged by negativity that the subject in its various vestiges does not merely indicate the places where substance in its trembling and uneasy auto-articulation risks touching the void, that is to say, is in danger of no longer holding itself together, but also the places where the void threatens to (re-)erupt. In this regard, the dynamic processes of the world are not just dependent upon (potential) disarray, but suggest that substance cannot be the last metaphysical word, since it appears on a second glance that the world itself is a response to something more primordial, something that is constantly trying to show its ugly head: ultimately “there is no Substance, only the Real as the absolute gap, non-identity, and particular phenomena (modes) are Ones, so many attempts to stabilize this gap.”358 Risking anachronism,359 Žižek sees a way to reinterpret the German Idealist attempt to think substance as subject through quantum mechanics that sheds light on this problematic. As quantum mechanics teaches us, any metaphysical investigation into the foundation of substance qua nature (the basic material constituents of reality) has a paradoxical result. 10.2 Eppur si muove: Ontological Dislocation and the Metaphysics of the Void Instead of coming upon a dense field of fully constituted realities that form the ultimate building blocks of the universe, “the more we analyze reality, the more we find a void”:360 rather than encountering entities complete in themselves, we see irreducibly indeterminate states lacking being in any traditional sense and from which “hard” reality can only emerge if there is Radicalizing the Subject 23 a collapse of the wave function;361 “normal” laws of linear temporality and causality break down as we encounter particles that retroactively “choose” their paths along virtual chains of possibilities and others that can even cheat the universe, coming in and out of existence without the latter even knowing.362 But this not only drastically challenges any sharp distinction between nature and culture,363 but also attests that the micro-universe of quantum particles is strangely “less” than that of the macro-universe that constructs itself from its vicissitudes, in a way that is remarkably similar to how the Kantian subject can only construct a unified world of appearances from the inconsistent fragments of sensation. In an uncanny logical short circuit, it would appear that not only is there no bottom-up causality at the level of experience (transcendental constitution is more real than what Kant calls “a rhapsody of perception,”364 since it founds a coherent field of reality), but even the most fundamental level of the universe is metaphysically more chaotic and constitutively less ontologically complete than the ordered macro-level physical world that science, as according to classical models, describes. Substance is not a given, but an achievement: it is as if all reality is transcendentally constituted, as if nature itself emerges out of this field of quasi-entities. But just as we had to explain the ontogenesis of unruliness, we must also explain the emergence of these ontologically incomplete realities that serve as the basis for the ordered universe, since they cannot be a brute, arbitrary given. The emphasis of the key philosophical question is thus shifted from how the subject can emerge out of the Real qua substance (thought from being) to how the subject can emerge out of the Real qua void (something from nothing), for the more strictly speaking ontological question of ontogenesis thus proves to be irreducibly entangled with the most fundamental of metaphysical questions. 236 Chapter 10 is no such zero-level: if we go 'all the way down,' we arrive at the Void.”366 This has a surprising consequence, one whose full metaphysical implications quantum mechanics thus summons us to accept: namely, that the closer we get to the origin of all things, the more ontologically incomplete reality is, the less distinguishable its fundamental constituents are from the void, thus forcing us not merely to proclaim that the void is “the only ultimate reality,”367 but more drastically still that “'all there is' is, precisely, not-All, a distorted fragment which is ultimately a 'metonymy of nothing.'”368 In short, what we experience as hard, full reality is at its core a mere vibration of nothingness lacking any true ontological depth, since there exists a certain radical indistinction between being (a structured physical universe) and the void (a structureless zone without any dense ontological determination in any traditional sense).369 But if the building blocks of the world are nothing but variations upon nothingness, then why do they emerge in the first place? And what prevents the universe from imploding upon itself in some kind of triumphant suicidal gesture, leaving us with nothing but the “eternal peace” of the void? Why something rather than nothing? Žižek hints that the answer is to be found in the very tension between the void and this field of quasi-entities that, the further we push them, the more indistinguishable they appear from nothingness itself—using the Higgs field as an example. Physical systems tend towards a state of lowest energy. In another vein, if we take energy away from a system, we should eventually expect to reach a vacuum state where the total energy count would be zero. Yet certain phenomena tell us that “there has to be something (some substance) that we cannot take away from a given system without raising that system’s energy—this 'something' is called the Higgs field: once this field appears in a vessel that has been pumped empty, and whose temperature has been lowered as much as possible, its energy will be further lowered.”370 Incredibly, once a physical system's energy has been lowered to the point where it it is on the brink of zero, this “something” appears, a “something” that requires less energy than nothing. 10.2 Eppur si muove: Ontological Dislocation and the Metaphysics of the Void Not only do we here recognize that “there simply is no basic level,” that “divisions go on indefinitely,” whereby “the quantum level marks the beginning of the 'blurring' of 'basic' full reality,”365 but also that “[o]ne should thus reject the ‘positive’ ontology that presupposes some zero-level of reality where things 'really happen' and dismisses the higher levels as mere abbreviations, illusory self-perception, and so forth. There 6 Chapter 10 236 Chapter 10 Consequently, “'nothingness (the void, being deprived of all substance) and the lowest level of energy paradoxically no longer coincide, that is, it is 'cheaper' (it costs the system less energy) to persist in 'something' than to dwell in 'nothing,' at the lowest level of tension, or in the void, the dissolution of Radicalizing the Subject 237 all being.”371 But here we should radicalize this paradox by extending it to the ultimate metaphysical level of reality. It is not merely that we cannot bring any physical system to a zero-level of energy without the Higgs field positing itself: what the latter suggests is that nothingness is minimally “inconsistent” with itself and that it is this very “inconsistency” that is responsible for the emergence of something out of nothing. The rapture of void, the bliss of an eternity freed from all tension, does not merely come at too high a price for us as creatures living in this world, but is, strictly speaking, impossible. Metaphysics is thus starkly remodulated: What if we posit that “Things-in-themselves” emerge against the background of the Void of Nothingness, the way this Void is conceived in quantum physics, as not just a negative void, but the portent of all possible reality? This is the only true consistent “transcendental materialism” which is possible after the Kantian transcendental idealism. For a true dialectician, the ultimate mystery is not “Why is there something rather than nothing?” but “Why is there nothing rather than something?”372 Although Žižek makes use of the Higgs field mainly as an image, in Less Than Nothing his expansive engagement with quantum mechanics sheds new light on its precise conceptual role in his own thinking. Not only does this engagement enable him to draw out underlying ontological implications from quantum mechanics' fundamental insights, but it more strongly gives us sufficient resources from which we can extract a more strictly speaking metaphysical argument from his discussion of the Higgs field, which attests to how Žižek potentially supplies quantum mechanics with a wider, non- naturalistic foundation while simultaneously founding a new alternative in metaphysics argumentatively independent from its framework. As that which (theoretically) controls whether forces and particles behave differently, the Higgs field has two modes: it is either “switched off” (inoperative) or “switched on” (operative). 238 Chapter 10 for it to be “switched on” (operative), for if a system is in a state of pure vacuum “the Higgs field still has to spend some energy—nothing comes for free; it is not the zero-point at which the universe is just 'resting in itself' in total release—the nothing has to be sustained by an investment of energy.”373 Because the pure vacuum requires the expenditure of energy, in order to solve this paradox we are forced to substitute it with another by “introduc[ing] a distinction between two vacuums”: for it to be “switched on” (operative), for if a system is in a state of pure vacuum “the Higgs field still has to spend some energy—nothing comes for free; it is not the zero-point at which the universe is just 'resting in itself' in total release—the nothing has to be sustained by an investment of energy.”373 Because the pure vacuum requires the expenditure of energy, in order to solve this paradox we are forced to substitute it with another by “introduc[ing] a distinction between two vacuums”: first, there is the “false” vacuum in which the Higgs field is switched off, i.e., there is pure symmetry with no differentiated particles or forces; this vacuum is “false” because it can only be sustained by a certain amount of energy expenditure. Then, there is the “true” vacuum in which, although the Higgs field is switched on and the symmetry is broken, i.e. there is a certain differentiation of particles and forces, the amount of energy spent is zero. In other words, energetically, the Higgs field is in a state of inactivity, of absolute repose. At the beginning, there is the false vacuum; this vacuum is disturbed and the symmetry is broken because, as with every energetic system, the Higgs field tends towards the minimization of its energy expenditure. This is why “there is something and not nothing”: because, energetically, something is cheaper than nothing.374 What is crucial to note with the Higgs field is that the two vacuums whose existence it posits are not by any means equal: rather than encountering a mere polarity, a two-sided principle that brings together a delicate dance of opposites like light and day, life and death, fullness and lack, into equilibrium, we see a constitutive imbalance. 236 Chapter 10 While in the first, given that the system is in a state of pure vacuum, forces and particles cannot be distinguished, in the second symmetry between particles and forces are broken so that differentiations among them can occur. However, the paradox lies precisely in the following: what is so unique about the Higgs field is that it is favorable 238 Chapter 10 Once we apply this principle cosmologically as a metaphysical principle, instead of having an eternal repetition of creation (breaking of the symmetries) and its destruction (return to the void), reality and its disappearance into the abyss, we come across a “displaced One, a One which is, as it were, retarded with regard to itself, always already 'fallen,' its symmetry always already broken.”375 As such, there is nothing but creation or reality because the “pure” vacuum wherein one would expect absolute repose is “false,” that is, stricto sensu impossible—it structurally must have always already passed over into and Radicalizing the Subject 23 became the “true” vacuum; and although this would appear to make the “false” vacuum theoretically superfluous (it never had, or could, exist) “this tension between the two vacuums [is to] be maintained: the 'false vacuum' cannot simply be dismissed as a mere illusion, leaving only the 'true' vacuum, so that the only true peace is that of incessant activity, of balanced circular motion—the 'true' vacuum itself remains forever a traumatic disturbance.”376 But why? For the precise reason that without this primordial antagonism we could not explain the minimal distinction between the void and its vibrations, between the nothing and the ontologically incomplete realities barely distinguishable from it—in short, how the symmetries between particles and forces could have been broken in the first place. Were we only to have the “true” vacuum, then finitude, materiality, and ultimately experience would be a mere illusion, for there would be no difference between the symmetry of the void and its disturbance, leaving nothing but a nirvana- like principle of nothingness to which all things are reducible. But once we witness the irreducibility of the antagonism within the void itself between its two modes, reality becomes less a seeming that we have to break through than an intrinsic part of the void that necessarily arises as a response to the primordial metaphysical trauma that is the perturbation of the “true” vacuum and whose essence is thus preserved. Even if nothing and the metonymy of nothing is all there is, this presupposes that the latter does not logically collapse into the former although the exact boundary between them is blurred. 238 Chapter 10 Hence, the reason Žižek can say that if “[t]he answer to 'Why is there Something rather than Nothing?' is thus that there is only Nothing, and all processes take place 'from Nothing through Nothing to Nothing,'” then “this nothing is not the Oriental or mystical Void of peace, but the nothingness of a pure gap (antagonism, tension, 'contradiction'), the pure form of dislocation ontologically preceding any dislocated content,”377 thus radically changing our very notion of nothingness itself. The Higgs field thus not only offers us an interesting naturalistic explanation of the world when used in cosmology but more strongly hints The Higgs field thus not only offers us an interesting naturalistic explanation of the world when used in cosmology, but more strongly hints at a new variety of metaphysics. Fleshing out its broad consequences, Žižek is not only able to offer a new account of the emergence of things that is inspired by though independent from quantum mechanics, but can also radicalize his ontology of the subject, thereby weaving metaphysics and 240 Chapter 10 Chapter 10 240 ontology into a dynamic, self-articulating whole. For him, the primordial fact from which metaphysics must begin is the fact that nothingness is. Drawing upon insights gained by contemporary science, however, he says that in order to explain the mere existence of things, we must posit that nothingness necessarily fails “to be,” for any attempt to have a purely vacuous void paradoxically costs more energy than things existing against the background of this void. Here we encounter “the primacy of the inner split,”378 whereby irresolvable conflict is at the origin of all things: the absolute is nothing but this fragile de-substantialized process that “arises” out of the self-splitting of a positively charged void, a split that befalls it from within through its own failure “to be” what it is without that very split. There is no primordial fullness, no positive hard reality that self-unfolds according to a creative principle of actualization;379 there is nothing but a pure ontological dislocation of which we can only say eppur si muove (“and yet it moves”). 238 Chapter 10 But insofar as this gap cannot be mediated with the absolute, it presents itself as “the non-dialectical ground of negativity,” so that “[t]he old metaphysical problem of how to name the nameless abyss pops up here in the context of how to name the primordial gap: contradiction, antagonism, symbolic castration, parallax, diffraction, complementarity, up to difference.”380 But the name that is perhaps best suited to this is the subject. This would inscribe this metaphysics of the void into the legacy of the German Idealist attempt to think substance as subject, while bringing us simultaneously beyond it and into a new sphere, a new materialism.381 If German Idealism has taught us that epistemic ambiguities in idealism (how can the subject overcome its own synthetic mediation of the world and reach the latter an sich?) occur because our division from being is identical with being's own division to itself, quantum mechanics calls us to radicalize this notion of the subject even further and claim that it exhibits the same structure as the split responsible for all things: This perhaps is how one can imagine the zero level of If German Idealism has taught us that epistemic ambiguities in idealism (how can the subject overcome its own synthetic mediation of the world and reach the latter an sich?) occur because our division from being is identical with being's own division to itself, quantum mechanics calls us to radicalize this notion of the subject even further and claim that it exhibits the same structure as the split responsible for all things: This, perhaps, is how one can imagine the zero-level of creation: a red diving line cuts through the thick darkness of the void, and on this line, a fuzzy something appears, the object-cause of desire—perhaps, for some, a woman's naked body (as on the cover of this book). 238 Chapter 10 Does this image not supply the minimal coordinates of the subject-object axis, the Radicalizing the Subject 241 truly primordial axis of evil: the red line which cuts through the darkness is the subject, and the body its object?382 the darkness is the subject, and the body its object?382 If in Žižek's strictly metaphysical thinking the emphasis shifts from how the subject can emerge out of the Real qua substance (thought from being) to how the subject can emerge out of the Real qua void (something from nothing), it is because there is no radical difference between the creation of the world and the creation of the world of meaning. Both exhibit the same catastrophic cutting, so that the primary question becomes that of the originary catastrophe itself rather than just one of its specific “instantiations” in human freedom. The fact that the answer for both (the Real is always already subject) is identical proclaims that there is only the Real of the gap, that the splitting precedes what is split. Žižek's paradoxical conclusion is that if the subject is “the primordial Big Bang,”383 it cannot merely be that of the universe of meaning, but must also be that of all that there is. The great lesson to be drawn from the metaphysical archaeology of the subject is that “[w]hat, ultimately, 'there is' is only the absolute Difference, the self-repelling Gap”:384 although substance qua nature proves itself to be not-all (traces of which we see in German Idealist accounts of negativity and most radically in contemporary science), so that we must posit the logical precedence of the void to which its indivisibility is always already contrasted and thus impossiblized, nevertheless just like substance qua nature this originary void in itself also proves to be split—a split that is not merely a mere catastrophic cut, but a rupturing antagonism imbued with an “energetic” or “energizing” force, a force that is somehow less than nothing. This has two drastic consequences. First, the complex order of the cosmos may at some point in time be reduced to unimaginable chaos (being nothing but a heterogenous play of powers) but never to a nirvana-like nothingness, for before the moment at which an absolute zero would be reached the void would, as it were, break its own symmetry in advance and prevent its own repose. 238 Chapter 10 We cannot escape the fragile and macabre dance of the not-all: non-coincidence is the pulsating heart of all reality, a heart whose diastoles and systoles may whimper under their own weight and threaten to collapse upon themselves at any moment in one great and final apocalyptic turmoil, yet cannot, because this turmoil costs the universe less energy than the pure virtuality of the void. Second, if thinking substance as subject reveals that If in Žižek's strictly metaphysical thinking the emphasis shifts from how the subject can emerge out of the Real qua substance (thought from being) to how the subject can emerge out of the Real qua void (something from nothing), it is because there is no radical difference between the creation of the world and the creation of the world of meaning. Both exhibit the same catastrophic cutting, so that the primary question becomes that of the originary catastrophe itself rather than just one of its specific “instantiations” in human freedom. The fact that the answer for both (the Real is always already subject) is identical proclaims that there is only the Real of the gap, that the splitting precedes what is split. Žižek's paradoxical conclusion is that if the subject is “the primordial Big Bang,”383 it cannot merely be that of the universe of meaning, but must also be that of all that there is. The great lesson to be drawn from the metaphysical archaeology of the subject is that “[w]hat, ultimately, 'there is' is only the absolute Difference, the self-repelling Gap”:384 although substance qua nature proves itself to be not-all (traces of which we see in German Idealist accounts of negativity and most radically in contemporary science), so that we must posit the logical precedence of the void to which its indivisibility is always already contrasted and thus impossiblized, nevertheless just like substance qua nature this originary void in itself also proves to be split—a split that is not merely a mere catastrophic cut, but a rupturing antagonism imbued with an “energetic” or “energizing” force, a force that is somehow less than nothing. This has two drastic consequences. 238 Chapter 10 First, the complex order of the cosmos may at some point in time be reduced to unimaginable chaos (being nothing but a heterogenous play of powers) but never to a nirvana-like nothingness, for before the moment at which an absolute zero would be reached the void would, as it were, break its own symmetry in advance and prevent its own repose. We cannot escape the fragile and macabre dance of the not-all: non-coincidence is the pulsating heart of all reality, a heart whose diastoles and systoles may whimper under their own weight and threaten to collapse upon themselves at any moment in one great and final apocalyptic turmoil, yet cannot, because this turmoil costs the universe less energy than the pure virtuality of the void. Second, if thinking substance as subject reveals that Chapter 10 242 nature itself is anything but a powerful, creative source, a pure affirmation displaying ontological closure à la Spinoza, then the void in being imposed upon us by the metaphysical archaeology of the subject demonstrates something much more radical than the fact that the human subject is not a mere accidental breakdown of the natural order, but actually depends upon an inborn negativity that impossiblizes its attempt at a self-articulating totality from the outset (“we should accept that nature is 'unnatural,' a freak show of contingent disturbances with no inner rhyme or reason”385). It further demonstrates that it is not the human subject that is the ultimate ontological catastrophe, but reality as such: There is nothing, basically. I mean it quite literally. But then how do things emerge? Here I feel a kind of spontaneous affinity with quantum physics, where, you know, the idea there is that [the] universe is a void, but a kind of a positively charged void—and then particular things appear when the balance of the void is disturbed. And I like this idea spontaneously very much, that the fact that it's not just nothing—things are out there—it means that something went terribly wrong, that what we call creation is a kind of a cosmic imbalance, cosmic catastrophe, that things exist by mistake.386 However, if what we call creation, the primordial Big Bang, is some kind of cosmic mishap, then this mishap must in some sense have been unavoidable. Radicalizing the Subject 243 is paradoxically “a deprivation, a gesture of taking away which is in itself a giving, productive, generative, opening up and sustaining the space in which something(s) can appear,”387 even if these something(s) only exist because ontological catastrophe is uncannily less than nothing. is paradoxically “a deprivation, a gesture of taking away which is in itself a giving, productive, generative, opening up and sustaining the space in which something(s) can appear,”387 even if these something(s) only exist because ontological catastrophe is uncannily less than nothing. The veritable horror of substance as subject—ontological catastrophe— revealed through the metaphysical archaeology of the psychoanalytico- Cartesian subject is therefore twofold. At the first level, it indicates that nature was always already a sickly creature, its rhythms always already disordered, unsteady, broken. Within the “passage” from drives to desire, substance to subject, no positive content is added, nothing changes at the level of the Real qua Real. What the subject imposes upon us is the realization of the constitutive contingency that lies at the centre of creation, the fragility of the seemingly ordered cosmos that has arisen before us, so that the immanent causality of nature is seen to be predicated upon its potential internal breakdown. On the second level, the metaphysical archaeology of the subject tells us that if we remain here, we have not gone far enough. If we are truly to understand how the not-all of substance could sustain itself in its own precarious being and, more fundamentally, how it could have emerged in the first place, we must radicalize the subject understood as an underlying dysfunctioning of substance's dynamics. If, as Žižek maintains, experience is revelatory of the fact that reality must be not-all, then it also contains a still deeper truth: reminding us that substance fails to ground itself, that it is riddled with holes, that it opens up the logical space of the substance and its contrast, the void, forcing us to explore the latter. We have thus come full circle: an ontological account of the emergence of the subject (the arising of representation out of being) has morphed into a discussion concerning the ultimate metaphysical structure of the world (the upsurge of “something[s]” out of nothing), because the two questions are seen to be intimately linked, the former automatically leading to the latter. 238 Chapter 10 The ontological catastrophe that is creation is a necessity because nothingness itself fails “to be” nothingness and through its failure never ceases to create “something”; and since this “something” is paradoxically less than nothing, the infinite proliferation of ontologically incomplete quasi-entities is only minimally distinguishable from the purely vacuous void itself. The world does not find its origin in a willed creation, an impersonal emanation from a sphere of consummate being overflowing in itself, or the uncontainable productivity of substance qua nature as absolute power—no, the world comes to be in an originary disaster, a primordial metaphysical cataclysm that is always already occurring, it being impossible to pinpoint a logical moment within which nothingness could have succeeded at “being” itself. Yet the split that is the subject in both its modes should not be considered a loss, but rather a liberation. Ontological catastrophe Radicalizing the Subject 243 Radicalizing the Subject 243 And falling upon this intuition, Žižek asks what if the same structure is at play at both levels, so that the answer to the latter will prove to be similar to that of the former, namely, that the movement from the Real of void to creation is not as initially problematic as it may appear because the Real of void shows itself to be always already tainted by the Real of gap. Ontological catastrophe is the zero-level of reality because Chapter 10 244 there is a necessity of the primordial void of which mystics speak to be disturbed, a necessity that is not due to the notional necessity of right- wing Hegelian theology (in order for the Idea to actualize itself as infinite freedom, it must sacrifice itself in nature so that it can fully return to itself in spirit after arising from its own ashes) or the conditional necessity of the late Schellingian quaternity (if the Ungrund were to become self-manifest to itself, then it would have to arbitrarily limit its primordial freedom in the kabbalistic act of contracting finitude) but a necessity that derives from the impotence of the void “to be” a purely vacuous void, a necessity that is synonymous with the absolute non-coincidence of nothingness to nothingness that is motor of all things. The language of catastrophe is in this sense completely justified, for creation does not present itself as intrinsically beautiful and creative or purposeful, but rather as a monstrous seat of ontological abortions and terrors devoid of sense though from time to time capable of miracles. Rather than the world being given to us by the self- overflowing exuberance of the Good or the personal hand of God, it moves from nothing through nothing to nothing due to the self-repelling gap of the void, the internal split of a positively charged nothingness, which denies all positivity to that which it sets in motion: “[p]erhaps this gap separating the two vacuums is then the ultimate word (or one of them, at least) that we can pronounce on the universe: a kind of primordial ontological dislocation or différance on account of which, no matter how peaceful things may appear sub specie aeternitatis, the universe is out of joint and eppur si muove.”388 10.3 The Act of Uneasy Self-Begetting: Entscheidung and the Paradoxical Self-Positing of Freedom By plunging into the abyssal origins of subjectivity Žižek is not just led to a new, disquieting form of transcendental materialism that offers an original account of the relationship between the Real and the Ideal, with stark implications for our understanding of substance, the emergence of order, and even the beginnings of the cosmos. He is also led to develop a theory of the unconscious that challenges both the traditional Freudian and Lacanian accounts, and even the one developed by Schelling that he draws upon. This theory deserves to be highlighted in some detail because it is revelatory of one of the most difficult and provocative aspects of Žižek's philosophy: Radicalizing the Subject 245 that is, the pure act at the origin of the subject that is simultaneous with the ontological passage through madness that forces upon us the rational necessity of speculative fabulation to embark upon a metaphysical archeology of the subject. Schelling's account of the birth of subjectivity is more than a theory of the meta-transcendental state of affairs that must be in place if subjectivity is to arise out of nature with a triumphant cry, for central to Schelling's ontogenetic narrative is that the former, though providing necessary conditions for the birth of the subject in/out of nature, does not supply its sufficient conditions. To explicate the moment of ontological judgement that institutes the pure difference between Grund and existence and with it the entire universe of human meaning (the Present), Schelling must concern himself with the very moment of unconscious decision (Entscheidung) by which subjectivity paradoxically liberates itself out of the immanent field of being to which it once “belonged” in a moment that is a self-caused immaculate conception insofar as this self-positing, which cannot be deduced according to notional necessity, is an arbitrary act of pure freedom. What is of paramount importance is the root of the term, which in English displays a similar play on words: Entscheidung as Ent-Scheidung, decision as de-scission. Since the German suffix -ung refers to a process, Entscheidung designates a “de-scissioning” at the basis of self-consciousness and language as that which, by creating the Present, banishes the Past into the abyssal dregs of forgotten and inaccessible time. 10.3 The Act of Uneasy Self-Begetting: Entscheidung and the Paradoxical Self-Positing of Freedom What Žižek focuses on is precisely the formal structure of the act itself, its activity of severing the Real into the parallax of the unconscious drives of nature and phenomenal reality, whereby the act itself is primordially repressed as necessary for the dawning of full-fledged subjectivity and becomes an impossible object for any discourse. Recognizing that the Entscheidung itself is that which originarily constitutes the conscious/unconscious distinction, Žižek argues that one of the fundamental breakthroughs of the Weltalter is its demonstration that drives themselves are, strictly speaking, nonconscious: if the very conscious/ unconscious distinction only occurs with the utterance of the Word (there cannot be a ground without a grounded: prior to the grounded, the ground cannot be posited as ground as such, for it is merely a self-subsisting, semi-closed system of materiality, rather than the ground of existence) then it semi-closed system of materiality, rather than the ground of existence) then it 246 Chapter 10 Chapter 10 246 would be philosophically fallacious to call this energetic rotation of energy the unconscious proper or the true foundation of subjectivity, even if it incites (or to put it differently negatively carves up the room for) the latter's founding gesture. The result is that the Žižekian-Schellingian subject of the unconscious is radically non-coincident with both the id-forces of the body in its primary mode (the Real of Triebe can only be unconscious as a secondary effect of the self-positing of the act of de-scission[ing] as such) and the more-than-conscious matrix of the Symbolic (the self-generating play of language and culture only emerges after the founding gesture that marks the beginning of transcendental self-reflexivity). That is, the unconscious is the very gap, the irreconcilable parallax, between both registers: it is synonymous with the subject itself as the impossible in-between that binds together materialism (being) and idealism (thought) in their non- relationality by protruding out of yet being simultaneously spectrally present in both. As an irreducible, self-positing negativity that institutes the realm of culture and eternally separates it from nature, the subject of the unconscious is the true site of freedom. Here lies the challenge to Freud and Lacan, who broadly could be said to have respectively located the unconscious in the biological movement of instinctual energy and the alienating effects of language. 10.3 The Act of Uneasy Self-Begetting: Entscheidung and the Paradoxical Self-Positing of Freedom For Žižek one must presuppose a more primordial level of unconscious activity than that of the biologically closed libidinal energetics within the corpo-Real of the body or that which emerges through the split between the subject of enunciation and the enunciating subject caused by the unpredictable reverberations of meaning within the infinite web of language: namely, an act that exploits the libidinal frenzy of the Real of the human body, the unruliness represented by the Todestrieb, to ground the self-generation and self-proliferation of the automatic machinery of language as a reaction formation. In this sense, Žižek's reactualization of the Schellingian unconscious is an attempt to sublate both the traditional Freudian and Lacanian accounts within a higher dialectical unity by showing their dependence on another, more fundamental conceptual level. His original addition to psychoanalysis is the formation of a conception of “the subject [which] at its most elementary is indeed 'beyond the unconscious': an empty form deprived even of unconscious formation encapsulating a variety of libidinal investments.”389 Radicalizing the Subject 247 Here again we see just how much Žižek's account of the subject is highly reliant upon his “modification” of the Weltalter in a way that challenges his so-called Hegelianism. Here again we see just how much Žižek's account of the subject is highly reliant upon his “modification” of the Weltalter in a way that challenges his so-called Hegelianism. Žižek's controversial wager is that there is something more primordial within Schelling's descriptions of the birth of the subject out of the utter twilight of pre-personal being than an account of the self-transformation of the “unconscious” spirit of nature as it rises towards the openness of self-revelation. In a move similar to psychoanalysis' claim that the material processes of our organs cannot be, strictly speaking, unconscious (physiological stimuli and reflexes are of a radically different nature),390 Žižek levels out the richness of the Schellingian account of nature to a mere material autopoiesis that is unable to explain the true seat of personality. 10.3 The Act of Uneasy Self-Begetting: Entscheidung and the Paradoxical Self-Positing of Freedom This is also why Žižek is so adamant that the unconscious is not to be equated with the set of irrational drives that structurally oppose and yet affect the self-transparency of rationality as we see in various forms of Lebensphilosophie.391 What Schelling's account of decision as the metapsychological event par excellence proclaims is that there is no point of positive juncture between nature as Grund and the subject of the unconscious, insofar as decision takes over/usurps the logically primary position of the Grund through its own self-positing freedom, but in a manner that its self-positing is more fundamental than, and even erases, its dark pre-history: the libidinal-material chaos within nature does not come close to establishing the unconscious proper because the latter is never completely subsumable within the dynamic movement of natural history or laws, for it institutes itself into the fabric of being by means of a self-caused immaculate conception. If freedom is to be truly free, then we must not be able to deduce it according to notional necessity; it is as if, when substance risks touching the void in the painful oscillation of drives constitutive of the unruliness of the human organism, the subject creates itself out of nothing, with all the paradox that entails: In the psychoanalytical perspective, of course, this primordial act of free self-positing cannot but appear as the Real of a fantasy-construction: the status of the primordial act is analogous to that of the Freudian parricide—although it never effectively took place within temporal reality, one has Chapter 10 This recollective interiorization (Er-inner-ung) is the direct effect of the subject's uneasy self-begetting, the aftermath of its self-caused immaculate conception in/out of matter that exploits the immanent negativity of the latter for the institution of its “miracle”:394 this recollection is not a mere remembering of something in cosmic memory, an always already existing but non-actualized self awaiting the dawning of consciousness in the world—the very gesture of interiorization is that which creates what is interiorized, that is to say, the self in which the world withdraws out of its bloody night, in such a way that the recollected self or interiority retroactively posits itself as that which was always at the starting point.395 Even a self-caused immaculate conception in being demonstrates a threatening belatedness, for it is stuck in a dialectical contradiction that cannot be resolved: “'[r]econciliation' between subject and substance means acceptance of this radical lack of any firm foundational point: the subject is not at its own origin, is secondary, dependent upon its substantial presuppositions; but these presuppositions do not have a substantial consistency of their own and are always retroactively posited.”396 The only successful ontogenesis is one that underlines the very impossibility, the upper limit, of ontogenesis as such: what is essential is not a transition (whether it be understood in terms of teleological development or the potential productivity of breakdown, as we see in the night of the world) but the pure difference that is the full-fledged subject (which is responsible for “the Radicalizing the Subject 249 madness of the passage to the Symbolic itself”397) which institutes a “gap which makes impossible any account of the rise of the New in terms of a continuous narrative.”398 But we must be very attentive to the conceptual movement if we are not to lose its radicality. Insofar as the chaotic oscillation of drives we witness in unruliness only becomes the Grund of existence after the act of decision, drives only become unconscious in the aftermath of the act itself. Due to the latter's irreducible self-positing, consciousness and the Real of drives qua unconscious both emerge in one magical brushstroke that retroactively constitutes its own evolutionary past—covers up its contingency—by subsuming the ontogenetic pre-history of the subject as part of its own self-effectuation through the paradoxical causality of the Freudian Nachträglichkeit/Lacanian après-coup/Hegelian positing of presuppositions. Chapter 10 248 Chapter 10 Chapter 10 to presuppose it hypothetically in order to account for the consistency of the temporal process. The paradox of the primordial act is the same as that of Baron Münchhausen pulling himself out of the swamp by lifting himself by the hair—in both cases, the subject is somehow already here prior to existence and then, by way of free act, creates-posits himself, his own being.392 The subject's freely posited withdrawal into self is intrinsically paradoxical, for the very act of self-positing creates the very self that is at the origin of said positing. This is why we cannot escape the Baron Münchhausen dilemma: the subject is miraculously present at its own birth.393 But how? To follow Žižek and draw upon a Hegelian logical category, when full-fledged subjectivity emerges in being it can be said to come into existence by a process of recollecting or interiorizing itself. 250 Chapter 10 Real, we are not warranted to make certain claims we have been forced to make: when the subject asserts itself at the beginning of its own pre-logical genesis (posits its own presupposition), it is not actually giving birth to itself (which implies a poietic production or genesis) in some kind of temporal loop, but directly creates itself at the instant of its own upsurge. The example of Baron Münchhausen's pulling himself up out of the swamp by his own hair expresses the intrinsic difficulty posed to any transcendental materialist account of the subject, for the subject does not exist before the very act of self-positing that it nonetheless paradoxically enacts—hence why it all happens in one magic brushstroke. The subject has no history except at the level of speculative fabulation (rationally justified mythology), for it is only after the subject's self-positing that we can raise the question of its origins: “what escapes our grasp is not the way things were before the arrival of the New, but the very birth of the New, the New as it was 'in itself,' from the perspective of the Old, before it managed to 'posit its presuppositions,'”399 which is why Žižek can say that the true arche-fossil for correlationism is not occurrences in cosmological or geological time before the transcendental, but the very transcendental itself.400 In this sense, the difficulty of the task of speculative fabulation finds itself reflected upon itself: that is to say, doubled. Not only do there exist psychoanalytical defence mechanisms to cover up the abyss of unconscious decision at the “heart” of subjectivity by means of fantasy constructions, but because the only way we have access to the latter is through a mytho-poetic ontogenesis of the subject's dark pre-history in the ontological vicissitudes of being, there is always the tendency to swap the real trauma (the pure contingency of the act as absolute spontaneity) for a fake (the painful oscillation of drives in the human infant that shows nature's sickness unto death in humanity). Even Schelling has a tendency to make us forget that the Grund, “this monstrous apparition with hundreds of hands, this vortex that threatens to swallow everything, is a lure, a defence against the abyss of the pure act,”401 which is why psychoanalysis is so important. Chapter 10 Here, however, we notice the difficulty of the Real-as-origin: since the abyssal beginning of the subject—the act of decision—effaces itself in the simultaneous gesture of converting the Real of drives into the unconscious as a libidinal system and founding consciousness, the birthplace of the subject becomes a mere posit that is in and of itself never accessible in language; and, as that which only shows itself in its very withdrawal (“sous rature”), it is denied the right of even “indirectly” shining through the cracks of the Ideal. It can only be narrated. Although we must posit the ontogenetic condition of desire (a pre-logical antagonism in the Real-as- excess) as that which precedes and in a certain sense renders possible the self-positing of the decision, the latter proves itself to be not only superior by saying “No!” to substance, thereby setting up a new age of the world within which the Past is always already “lost” through symbolization (an act that institutes the hegemony of the Real-as-lack), but also to be ontologically primary, despite being ontogenetically secondary. In a paradoxical movement where the cause-and-effect chain loses its grip, the self-unfolding causality of nature as substance is “torn apart” in the upsurge of freedom as self- relating negativity, which demands primordiality at all costs, even making being infinitely non-coincident to itself. It must be remembered, however, that this movement from Grund to subjectivity, from the breakdown of nature into a writhing mass of heterogeneous forces to the abyss of unconscious decision, must be described at the level of mytho-poetics. At the level of the 250 Chapter 10 Although this secondary trauma must still be considered as having explanatory merit—it is, after all, a necessary condition for the primary trauma—the point is to pierce through one trauma (ontological catastrophe as the immanent breakdown of substance) to arrive at a more primordial one (ontological Radicalizing the Subject 251 catastrophe as the pure difference that severs being in two). The “repressed spectral 'virtual history' is not the 'truth' [...] but the fantasy that fills in the void of the act [...] the secret narrative that tells its story is purely fantasmatic,”402 which is why: Schelling's fundamental move is thus not simply to ground the ontologically structured universe of logos in the horrible vortex of the Real; if we read him carefully, there is a premonition in his work that this terrifying vortex of the pre-ontological Real itself is (accessible to us only in the guise of) a fantasmatic narrative, a lure destined to distract us from the true traumatic cut, that of the abyssal act of Ent-Scheidung.403 This is why Schelling's inability to complete his middle-late project of an ontology of freedom is of such paramount importance to Žižek, for “[t]he repeated failure of his Weltalter drafts signals precisely Schelling's honesty as a thinker—[not only] the fact that he was radical enough to acknowledge the impossibility of grounding the act or decision in a proto-cosmic myth,” but also that he was “compelled to posit an uncanny act of Ent-Scheidung (decision or separation), an act in a way more primordial than the Real of the 'eternal Past' itself.”404 Focusing on the necessary posit of the convulsing labyrinth that is the pre-symbolic Real-as-excess in human unruliness, and plunging ourselves into it, we risk forgetting the abyss of the Real-as-origin that stares us in the face. The painful oscillations of the Grund in unruliness merely defer us from the true terror that is the pure act instituting the Symbolic's infinitely self-reflexive play of signifiers in which we live and which guarantees that it possesses no direct connection to the extra-notional world. Because this act absolutizes the short circuit between the Innenwelt and Aussenwelt, we can only encounter its abyssal origins in their very gesture of withdrawal. 250 Chapter 10 It is not nature, even in its mode of self-erasure (N ≠ N), that is the first revelation,405 but rather, the void within which the Entscheidung spins—and we can only catch glimpses of the latter as the impossible in-between negatively tying together nature and culture, the Real and the Symbolic, and thereby hope to develop a comprehensive theory of subjectivity inclusive of a new, paradoxical form of metaphysics from within the mytho-poetics of the subject's slow, unsteady, and painful “emergence” out of the vicissitudes Chapter 10 252 C 252 of being. But all of this has an interesting implication. If the Symbolic is a self-organizing system that freely constitutes the fabric of experience without any contact with objective reality, then this suggests that, from within its ontologically solipsistic dance of cybernetic ciphering, we can break through the impenetrable dusk of psychosis as we (in a reflexive albeit fabulative gesture) see it as the madness it is in a careful reconstruction of its trauma. Here, however, we encounter one of the most fundamental and perhaps paradoxical conclusions of Žižek's parallax ontology. It must be recalled that the trauma at the birthplace of the human subject is not only coincident with its freedom:406 that is, its withdrawal from one's natural environment into an irreal, virtual self,407 but its exploration necessarily demonstrates the structure of fantasy as we insert ourselves as a pure gaze in the moment of our own birth.408 Not only can materialism only justify itself in “the shadow cast by [self-grounding] idealism's insurmountable incompleteness,”409 but more radically, if we follow the true horror of substance as subject at the core of the latter, then we are forced to conclude that a metaphysics is only possible as a form of “successful” psychotic thinking,410 a thinking that, from within the clutches of idealism as an insurmountable ontological psychosis and using the very energy and internal limitations of this psychosis, manages to succeed in achieving the impossible coincidence of subjective and objective reality: that is to say, in developing a comprehensive metaphysics of the Real. A cure to our correlationist imprisonment being excluded from the outset, philosophy has no hope of offering a therapy. 250 Chapter 10 What is more, if such a form of thinking is to be truly successful, not only must we find a way to overcome the realism-idealism debate within idealism, but we must also do so from the side of realism by writing the great epic of being as the eternal Past to come to terms with the ontological passage through madness at the latter's foundation, a task that requires a self-conscious mythologizing, fictionalization, or retrospective narration and is only achievable by entering the abyss of the spectral Real-as-origin and fending off the thrust of unconscious desires that try to protect us from it in order to draw out its stark metaphysical consequences. Only a scientific, psychoanalytically guided fabulation allows one to catch sight of the vanishing mediator that enacts the withdrawal into the night of the world. But how is a radical Radicalizing the Subject 253 Radicalizing the Subject 253 subjective idealism capable of such a feat? And as what brand of metaphysics should we baptize it? Notes 341. For a discussion of the passage, see chapter 9. 342. Žižek, The Ticklish Subject, pp. 88–89. 343. Lacan, “The Mirror Stage as Formative of the I Function,” Écrits, p. 96/77. 343. Lacan, “The Mirror Stage as Formative of the I Function,” Écrits, p. 96/77. 344. See chapter 4. 345. Žižek, Living in the End Times, pp. 350–51. I capitalize “the Real” for consistency. 346. Schelling, Weltalter III, p. 219. 347. Žižek, The Indivisible Remainder, p. 23. 348. Johnson, Žižek’s Ontology, p. 92. 349. Žižek, Less Than Nothing, p. 197. 350. Ibid., p. 198. Compare with pp. 292ff. and p. 304. 351. Žižek, The Ticklish Subject, p. 33. 351. Žižek, The Ticklish Subject, p. 33. 352. Johnson, Žižek’s Ontology, p. 92. 353. Žižek, The Indivisible Remainder, p. 92. 353. Žižek, The Indivisible Remainder, p. 92. 354. Ibid. 355. Žižek, Less Than Nothing, p. 905. 356. Žižek, The Indivisible Remainder, p. 220. See also p. 223. 357. Žižek, Less Than Nothing, p. 923. 358. Ibid., p. 377. 358. Ibid., p. 377. 359. Ibid., p. 947. 360. Ibid., p. 925. 360. Ibid., p. 925. 361. Ibid., p. 724. 361. Ibid., p. 724. 362. See, ibid., pp. 918–19; and The Indivisible Remainder, pp. 220–31. 363. See chapter 5. 363. See chapter 5. 364. Kant, The Critique of Pure Reason, A 156/B195. 365. Žižek, Less Than Nothing, p. 726. 365. Žižek, Less Than Nothing, p. 726. 366. Ibid., p. 730. 366. Ibid., p. 730. 367. Ibid., p. 726. 367. Ibid., p. 726. 368. Ibid., p. 641. 368. Ibid., p. 641. 369. Ibid., p. 60. 254 Chapter 10 370. Žižek, The Puppet and the Dwarf: The Perverse Core of Christianity (Cambridge: MIT Press, 2003), p. 93. 371. Ibid. 371. Ibid. 372. Žižek, Less Than Nothing, p. 925. 373. Ibid., p. 944. 373. Ibid., p. 944. 374. Ibid., pp. 944–45. 375. Ibid., p. 949. 375. Ibid., p. 949. 376. Ibid., pp. 949–50. 376. Ibid., pp. 949–50. 377. Ibid., p. 38. 377. Ibid., p. 38. 378. Ibid., p. 386. 379. Žižek, Living in the End Times, p. 232. 380. Žižek, Less Than Nothing, p. 950. 381. Ibid., p. 60. 381. Ibid., p. 60. 382. Ibid. 382. Ibid. 383. Žižek, The Ticklish Subject, p. 31. 384. Žižek, Less Than Nothing, p. 378. 385. Ibid., p. 298. 385. Ibid., p. 298. 386. Žižek!, dir. Atra Taylor (Zeitgeist Video, 2007). 386. Žižek!, dir. Atra Taylor (Zeitgeist Video, 2007). 387. Notes Žižek, The Puppet and the Dwarf, p. 93. 388. Žižek, Less Than Nothing, p. 377. 388. Žižek, Less Than Nothing, p. 377. 389. Žižek, Living in the End Times, p. 311. 390. Freud, “Instincts and Their Vicissitudes” (1915), SE, XIV, p. 118. 391. See Žižek, The Indivisible Remainder, pp. 28 and 174. 392. Ibid., p. 19. 393. Ibid. 393. Ibid. 394. Žižek and Daly, Conversations with Žižek, p. 59. 394. Žižek and Daly, Conversations with Žižek, p. 59. 395. Žižek, Less Than Nothing, p. 235. 395. Žižek, Less Than Nothing, p. 235. 96. Žižek, Living in the End Times, p. 229. 396. Žižek, Living in the End Times, p. 229. 397. Žižek, Less Than Nothing, p. 331. Ibid., p. 273. 398. Ibid., p. 273. 399. Ibid. 400. Ibid., p. 644. 400. Ibid., p. 644. 401. Žižek, The Fragile Absolute, p. 70. 401. Žižek, The Fragile Absolute, p. 70. 402. Žižek, Less Than Nothing, p. 273. 402. Žižek, Less Than Nothing, p. 273. 403. Ibid., p. 275 (taken initially from The Fragile Absolute, p. 73). 404. Ibid., p. 274. Radicalizing the Subject 255 Radicalizing the Subject 255 405. Schelling, Freiheitsschrift, p. 284. 406. Žižek, Less Than Nothing, p. 265. 407. Žižek, The Ticklish Subject, p. 35. 408. Žižek, The Indivisible Remainder, p. 22. 409. Johnston, Žižek’s Ontology, p. 19. 410. Here I am playing with Henri Maldiney’s reading of Hegel’s Phenomenology as a “successful depressive thinking” and taking up certain gestures already hinted at in Jean-Christophe Goddard’s interpretation of this passage. Compare Maldiney, Penser l’homme et la folie, p. 27; and Goddard, Mysticisme et folie. Essai sur la sim- plicité (Paris: Desclée de Brouwer, 2002), pp. 83–84. I return to these points in chapter 12. III Overcoming Idealism III Overcoming Idealism III Chapter 11 From Radical Idealism to Critical Metaphysics How Idealists Write Being's Poem Chapter 11 From Radical Idealism to Critical Metaphysics How Idealists Write Being's Poem Žižek's transcendental materialism understands itself as an uncanny variation on the late German Idealist theme of providing an ontogenesis of the subject consistent with transcendental idealism. But being more than a rethinking of its central problems, it presents itself as a highly rigorous psychoanalytical reconstruction of its unconscious Grundlogik, which reveals itself as a disavowed insight into the identification of subjectivity with an immanent rupture in being so devastating that not only does being lose all direct access to itself, but we must actually posit ontological dislocation as the primary metaphysical fact. In so doing, however, it would appear that there is a potentially fatal inconsistency in Žižek as he oscillates between an inconsistent idealism (the Hegelian notional or symbolic Real-as-lack) and a self-sabotaging materialism (the Schellingian pre/extra-symbolic Real- as-excess). What is more, pointing to the immense difficulty of describing the emergent parallax of being and thinking—the ontological passage of madness—from within the ontological solipsism of thinking itself, Žižek argues for the necessity of a form of thinking that would enable being and thinking to be fully reconciled from the side of being itself through the mytho-poetic narrativization of the moment of parallax. If radical idealism can succeed at overcoming itself and writing being's poem, it will prove itself capable of being maximally idealistic and realistic in a single gesture that leaves behind the speculative throes of correlationism by rendering correlation itself Chapter 11 260 immanent to the absolute, thus becoming a critical metaphysics that has much to offer today to the so-called speculative turn. From Radical Idealism to Critical Metaphysics 261 idealism: it is not only that concepts do not need to adequate themselves with objects subsisting in a world that exists despite me; more radically, the free ciphering of the Real by the Symbolic means that signification has nothing to do with objective reality in itself, for signifiers only participate in an endless chain of self-relation that precludes access to the “outside” world. There is no room possible for a realist epistemology within Lacan because the very link between signifier and extra-linguistic object is cut. And although Žižek remains unsatisfied with taking such a theoretical edifice as a given and seeks to understand the ontological/meta-transcendental event that it must imply (thus recognizing the ultimate need for giving an account of the Symbolic if psychoanalysis is to find a proper scientific footing) the constraints that Lacan himself bestows upon discourse make Žižek's project appear intrinsically problematic. Doesn't the Real in itself prior to or outside of language remain essentially unknowable (because it is always already constituted by a subject, therefore produced by it, and never without its taint)? Isn't the pre- or extra-symbolic Real an impossible concept, for only that which can be spoken in language exists? Nevertheless, Žižek offers more than another sophisticated form of structuralist linguistic idealism. If Lacanian psychoanalysis proclaims that the Real can only appear as a lack within the ciphering activity of language, Žižek attempts to break out of the correlationist circle of language by showing that the very inconsistencies of thinking offer a solution to the problem of how we are able to reach the Real even if we are trapped within the play of appearance appearing to itself. Because the Real we encounter in thinking the world is never “simply subjective, [whereby] it would present a case of the hollow playing of the subject with itself, and we would never reach the level of objective reality,”413 thinking's inability to close in on itself shows that the pre/extra-Symbolic must insist within it. However, such a formulation does not directly address the minimal transcendental conditions of the possibility of attaining an access to the Real “it itself” from within the Symbolic. Adrian Johnston's formulation of the paradoxical materialism-idealism relation is highly revealing of the deadlock Žižek faces: “materialism [...] formulates itself vis-à-vis the deadlocks internal to radical transcendental idealism. 11.1 Lacan and the Prison-House of the Symbolic As an account of the subject's own immaculate self-begetting from within the material flux of being as that which institutes the genesis of the Symbolic, Žižek's transcendental materialism is an attempt to explicate the ontological origins of a self-grounding idealism wherein ontology has been rendered seemingly impossible. Insofar as the Symbolic is able to generate meaning without external reference, it refuses all forms of realism: access to a Real outside of the correlation being/language is proclaimed a naïve position to be rejected due to the immense linguistic power that constructs our reality. Yet even if our entrapment within the self-referential, masturbatory play of signifiers is absolute, the ontological solipsism that it entails is precisely that: namely, an ontological solipsism whose very existence suggests that this very inability is somehow internal to the interior play of forces constitutive of being. The issue at hand is that “the transcendental standpoint is in a sense irreducible, for one cannot look 'objectively' at oneself and locate oneself in reality; and the task is to think this impossibility itself as an ontological fact, not only as an epistemological limitation.”411 In this minimalist sense, images and words cannot be seen as mere parasites that contingently latch themselves onto being and disturb its otherwise smooth flow, but must be negatively indicative of some self-sabotaging tendency always already at work in its heart of hearts: “[t]he symbolic order is not a cause which intervenes from the outside, violently derailing the human animal and thus setting in motion its becoming-human.”412 In other words, to account for the very “consistency” of the Symbolic we are called upon to write the great metaphysical epic of that forgotten eternal Past preceding our coming into the world as speaking subjects. Such a conceptual move, however, poses an epistemological problem. Within the Lacanian registers, the Real necessarily appears as a lack. As soon as the Symbolic emerges as a self-replicating, self-evolving differential system that transcendentally constitutes the condition of the possibility of phenomenal reality, any direct contact with the Real qua ontological field is lost. The Lacanian thesis is a variation of structuralist linguistic From Radical Idealism to Critical Metaphysics 261 From Radical Idealism to Critical Metaphysics 261 shadow cast by idealism's insurmountable incompleteness.”414 The problem is the following: even if the immanent “breakdowns” of solipsistic self- enclosure—the internal obstructions of language's psychotic dance— represent the inability of the subject to posit itself as an autocratic all hallucinating its world, they only point to a negative experience of the Real and do not suffice to offer a positive articulation of the ontological qua ontological. Of course ciphering sometimes runs into knots, but this very blockage can only be understood in the Symbolic by the Symbolic and in such a way that we never leave its prison. How could a kink in the Symbolic be revelatory of the pre- or extra-symbolic Real that somehow unpredictably upsurges within its sphere? Even given the paradoxical mode of the double-feature of inclusion/exclusion, internal/external, and presence/absence that defines the Real, it is still, formally speaking, a lack, nothing more than a notional antagonism in its ciphering code. It is unclear how this logical torsion, an internal hindrance in ideality, could serve as a foundation for a new materialist metaphysics, for there is no such thing as an outside or Other to symbolic mediation to which we can have access: “the Lacanian Real—the Thing—is not so much the inert presence that 'curves' the symbolic space (introducing gaps and inconsistencies in it), but, rather, the effect of these gaps and inconsistencies.”415 If it is understood as the alien presence of some extra-ideal presence within synthesis, it can only be understood as such insofar as it is posited as such—or, as Fichte says, insofar as there is a transference of the productive activity of the subject to the not-I, but such a transference, although enabling us to give a consistent account of why representations often fail due to some “alien presence” that obstructs them (the necessity of a realist moment in a critical idealism), never leaves the constraints of a subjective idealism. We appear stuck in the most rampant form of correlationism. Even if the Real can negatively show itself through an internal tension pressurizing the Ideal from within, we can never reach the ontological as such. There is always the correlation language/being wherein being itself is reduced to that which is constructed in the symbolic field of discourse, regardless of there being elements that appear within the latter that it cannot control. From Radical Idealism to Critical Metaphysics 261 On this account, materialism is philosophically tenable only as the spectral inverse of idealism, accompanying it as the Chapter 11 262 shadow cast by idealism's insurmountable incompleteness.”414 The problem is the following: even if the immanent “breakdowns” of solipsistic self- enclosure—the internal obstructions of language's psychotic dance— represent the inability of the subject to posit itself as an autocratic all hallucinating its world, they only point to a negative experience of the Real and do not suffice to offer a positive articulation of the ontological qua ontological. Of course ciphering sometimes runs into knots, but this very blockage can only be understood in the Symbolic by the Symbolic and in such a way that we never leave its prison. How could a kink in the Symbolic be revelatory of the pre- or extra-symbolic Real that somehow unpredictably upsurges within its sphere? Even given the paradoxical mode of the double-feature of inclusion/exclusion, internal/external, and presence/absence that defines the Real, it is still, formally speaking, a lack, nothing more than a notional antagonism in its ciphering code. It is unclear how this logical torsion, an internal hindrance in ideality, could serve as a foundation for a new materialist metaphysics, for there is no such thing as an outside or Other to symbolic mediation to which we can have access: “the Lacanian Real—the Thing—is not so much the inert presence that 'curves' the symbolic space (introducing gaps and inconsistencies in it), but, rather, the effect of these gaps and inconsistencies.”415 If it is understood as the alien presence of some extra-ideal presence within synthesis, it can only be understood as such insofar as it is posited as such—or, as Fichte says, insofar as there is a transference of the productive activity of the subject to the not-I, but such a transference, although enabling us to give a consistent account of why representations often fail due to some “alien presence” that obstructs them (the necessity of a realist moment in a critical idealism), never leaves the constraints of a subjective idealism. We appear stuck in the most rampant form of correlationism. From Radical Idealism to Critical Metaphysics 263 From Radical Idealism to Critical Metaphysics 263 this never leads to any knowledge of the Real in itself. From within the originary psychosis of a self-grounding idealism, there can be no overlapping of subjective and objective reality, for the very emergence of the subject as an ontological lacuna has as its founding gesture an irreconcilable rupture between the Innenwelt and Aussenwelt. Despite this, however, Žižek embarks upon an exploration of how a structuralist self-enclosed system preventing any direct engagement with the Real could have arisen by using the very fact of the Symbolic as a manner of proceeding. If the Symbolic is an ontological solipsism, then the lack of access to being we have must be understood as an event in being. In this sense, the Real-as-lack must ultimately open onto a metaphysics of the Real as that which precedes and exceeds the Symbolic and the Imaginary. But Žižek's Lacanian commitments would seem to render such a move unjustifiable. It is uncertain how he can balance his own radical idealism with his other materialist tendencies—indeed, his attempt to inscribe the former within the latter, to make the epistemic limitations of linguistic idealism synonymous with being's non-coincidence to self, appears to intensify the problem. How can a “negative materialism” based on the infinite conflict between mind and body found a new metaphysics? If we take as our starting point the (non-)relation between system (materialism) and freedom (idealism) as revealed by the psychoanalytical experience, then doesn't the logical category of the disjunctive “and” explicit in their contradiction prohibit us from delving into the retroactively posited past of the Real-as-origin to see how the latter explodes out of being as an event? How can we overcome the withdrawal into the nocturnal self of the world at the very birth of subjectivity so as even to see this withdrawal for what it is? From Radical Idealism to Critical Metaphysics 261 We may forever approach it as the negative magnitude contorting our notional apparatus as produced by the latter's very inconsistency, but 11.2 The Hegelian Real-as-Lack: The Painful March of Ontological Solipsism The problem at hand is how to understand the separate theses that (i) the inconsistency of thinking methodologically justifies an overcoming of the realism-idealism debate from within idealism and (ii) given the fact that the Symbolic is always already minimally outside itself we can embark upon an explication of its origins without ever leaving it. Endeavoring to give Lacanian psychoanalysis the support it needs—a comprehensive metaphysics—it is perhaps to be expected that Žižek falls into such a Chapter 11 264 theoretically difficult position. Lacan argues that we are stuck in language and, accordingly, that access to the ontological is impossible. Yet such a claim, if it cannot account for the ontological genesis of that which in the first place impossiblizes our access to the ontological, risks being merely a dogmatically asserted statement. This tension between the two major theoretical roles that the Real must assume—a notional lack and a symbolically excessive activity that is independent to all forms of correlation or access conditions—explains the constant oscillation in Žižek's position as he wavers between his strictly Lacanian and larger metaphysical and ontological commitments, even if the latter are always mediated by the former. Although most of his comments on Schelling, for instance, bespeak the possibility and necessity of delving into the Real-as-excess,416 understood as the pre-symbolic antagonism that gives rise to the transcendental matrices of the subject (the “orgasm of forces”) he often refers to the purely fantasmatic character of such inquiry due to the impossibility of reaching the absolute beginning of language417 and its necessary mytho- poetic component given its resistance to rational speculation,418 often without acknowledging the problem that this could appear to pose to the endeavor of establishing the ontological grounding of the subject. Given that he identifies “the key question” of philosophy as that of “how thought is possible in a universe of matter,” so that we should focus our efforts on “the very rise of representation or appearing out of the flat stupidity of being” if we are to avoid “a regression to a 'naive' ontology of spheres or levels,”419 the conceptual contours of this problematic deserve to be investigated in full. This is further necessitated by the fact that many fundamental Žižekian concepts directly exhibit this tension-ridden simultaneity of two opposed directions between the Real-as-lack and the Real-as-excess. From Radical Idealism to Critical Metaphysics 265 utilization of the concept—which he presents as a reworking of Kant's theory of unruliness—is then used to displace the mature Hegelian dialectical triad and argue for its inability to explain the passage from nature to culture,422 thereby further intensifying its speculative reach. How can he move from one register to the other? Despite this uncertain wavering between the purely logical and metaphysical or ontological value of Žižek's reading of the night of the world, other categories that Žižek extracts from Hegel show why he, as a rule, would generally have a preference for Hegel over Schelling, even in face of the obvious debt to the latter in his own transcendental materialism. The Hegelian concept “tarrying with the negative,” for instance, intrinsically displays the structure of the Real-as-lack and is thus, strictly speaking, completely compatible with Lacanian epistemology, whereas the categories he finds in Schelling pose a more immediate problem. Emerging out of Hegel's critique and extension of the Kantian thing-in-itself, tarrying with the negative is an attempt to show that the latter, as a theoretical posit, is superfluous. Objects give themselves to consciousness, but it is an illegitimate move to say that, beyond their appearing, there is an inner core of the thing that is hiding, ever out of reach of the transcendental ego's limited synthesizing powers, because even this infinite elsewhere of subject- independent interiority is itself only possible from within the manifold field of phenomenal experience: that is, it is itself an appearance. This becomes most evident in the experience of non-coincidence between our concepts and that which they “represent.” Within the inconsistency of the immanent structure of knowledge exhibited by these types of encounters, the object “in itself” shows itself through the form of a negative determination that burdens experience. We could say that what we come across here is the raw positivity of the object that obstructs our idealization and forces us to adapt to it, but only with the qualification that this positivity is revealed within the shadows cast by idealization's failure in such a way that it can only be brought forth or posited retroactively by means of a modification of the matrix of concepts that constitute our mediation of the world so that the original paradox or blockage disappears. 11.2 The Hegelian Real-as-Lack: The Painful March of Ontological Solipsism His discussions of the night of the world in The Ticklish Subject, for instance, have a tendency to treat the Real as the other side of transcendental imagination, and hence as a logical rather than an ontological concept (Žižek warns us that “it is crucial to 'close the circle': we never exit the circle of imagination”420), although he also bestows upon the notion a clear metaphysical reach by locating the destructive force of understanding preceding its synthesizing From Radical Idealism to Critical Metaphysics 265 From Radical Idealism to Critical Metaphysics 265 In the aftermath of this epistemic remodulation, we see that what we initially experienced as an internal or structural deformation of ideal space was the negative Chapter 11 266 refraction of the object's “true” nature such that the Real, less than a solid thing that obstructs ideality from the outside, is a pure effect. This has two implications: firstly, we do not need to overcome the split between phenomenal appearance and the thing-in-itself because this very split arises from within appearance itself, so appearance is “always more than appearance,” as it were. Secondly, the noumenon as a “transcendence” that gives itself of itself to experience is reduced to a mere defence against the potential horror of the immanent, uncontrollable flux of pure appearance, for if a transcendent object causes our representations, then our representations are guaranteed to constitute a minimally smooth fabric. As an illusion of a place wherein all contradiction is always already resolved in a reality complete unto itself, the noumenon helps bestow a sense of order and unity to the structure of experience even where it does not of itself display any, thereby taking the edge off of ideal fragmentation. From Radical Idealism to Critical Metaphysics 267 through which it (only haphazardly) grasps (fragments of) the latter. The issue is not that thinking cannot comprehend being, but that, since it is inscribed within the latter, thinking can obtain no transcendent gaze upon it by which it can totalize it into a complete system. Thinking is necessarily incomplete—there is an irremovable lacuna in every truth-claim—because it can never get rid of the subjective gaze, a gaze that, while making knowledge finite, simultaneously renders it possible in the first place. Following this train of thought to its logical conclusion, we must assert that not only can we never know the system of the world, but if thinking itself is a part of the world, and there is only one world, there is no god’s-eye point of view from which the world can never turn back upon itself and close itself. The gaze of the subject is a non-suturable gap in being—this is what it means to say that “phenomenalization, appearance, 'illusion,' split, finitude, Understanding, and so on, [...] are inherent to the life of the Absolute itself.”426 Through its own subjective movement, thought comes to realize that there is an outside to thinking posited from within as that which insists and persists as the extimate core of all discourse. That is to say, thought is a constant struggle insofar as its struggle with itself is never a mere masturbatory play, but is simultaneously a struggle with the outside world. From Radical Idealism to Critical Metaphysics 265 The infinite (the perfect ontology of the noumenal extra-subjective being) only emerges as a response to the radical finitude of phenomenal reality (ideal obstruction in our dealings with reality), whereby “every positive figure of the In-itself is a 'positivization' of negativity, a fantasmatic formation we construct to order to fill in the gap of negativity”423: there is only the restlessness of the negative, the incessant internal fracturing of experience due to idealism's intrinsic limitations and inability to posit itself as a complete all, which always leads to symbolic dismemberment, so that noumena, now understood as the negativity of phenomena, their internal inconsistency, “designate the In-itself as it appears to us, embedded in phenomenal reality [...] there is no mysterious gap separating us from the unknown, the unknown is simply unknown, indifferent to being-known.”424 In other words, what is on the other side of the screen of consciousness is not another reality, but “the same reality we find in front of the screen”: Žižek tells us to think of the illusion of a theatre stage, where what is responsible for the illusion is not the machinery backstage to which we have no access while the play is going on, but the very theatre stage itself, for even if the backstage and its mechanics are visible not only do we realize that the secretive reality normally “beyond our grasp” is exactly the same as the one being staged but the deceptive From Radical Idealism to Critical Metaphysics 267 From Radical Idealism to Critical Metaphysics 267 Its operationally and epistemologically closed ciphering of the world is capable of knowledge because the very inside-outside distinction becomes intra-discursive, that is, immanent to thinking itself because it is sustained by the activity of thinking, so that there is a paradoxical coinciding of the purely subjective and the purely objective: “[e]very tension between Notion and reality, every relationship of the Notion to what appears as its irreducible Other encountered in the sensible, extra- notional experience, already is an intra-notional tension i.e., already implies a minimal notional determination of this 'otherness.'”427 Although we are trapped in idealization and the Real-as-lack is our doomed fate, there is no need to mourn the loss of some kind of immediate being in itself, some Oedipal womb of nature from which we have fallen: what Hegel shows is that, from within the level of the logical self-articulation of the reflective notional constructs of thinking themselves and the symbolic space of self-generating meaning they engender, we can “reach” objective reality because there is no significant gap between thinking and being at the level of thinking. The Symbolic is that which opens up access to being: we provide a freely 268 Chapter 11 Chapter 11 268 developed notional construct that creates the norm for an intelligible field, a field that then may fall into inconsistency when said notional construct fails to render its corresponding phenomenal reality consistent, but inasmuch as the failure of said notional construct enables us to modify it we thus can be said to slowly and patiently track truth over time. Once we have seen that “[t]he opposition between idealistic and realistic philosophy is therefore without meaning,”428 we can develop a metaphysics critically rather than dogmatically. This dissolves the worry regarding how we can have access to the world from within the clutches of subjective thinking. “out there” because of the inconsistencies and gaps in the symbolic order.430 With this insight gained Žižek, like Hegel, discovers an epistemological foothold—the productive space of ideal inconsistency (in the quote above Žižek is talking about our access to the Real from within the Symbolic rather than its ontological constitution)—from within which he can find the resources required to develop a new metaphysics. It is less idealism that poses a problem for the latter than the traps that it (unconsciously) creates for us as we attempt to catch a glimpse what “lies beyond” phenomenal reality: the inevitable and necessary symbolic dismemberment of a self- grounding idealism that fails to fully become itself not only creates the possibility of error, but since the first showing of the Real is always an internal deformation of ideal structure (a pure negative form lacking content), it itself risks being riddled with a thick layer of fantasmatic projections and unconscious desires. That which allows idealism to overcome itself also can hinder the speculative process. Yet, despite this, the Real-as- lack and the Real-as-excess do not stand in opposition to one another. Although the former is always epistemologically superior or primary, being the transcendental condition of the possibility of access to the latter, we are nevertheless capable of attaining that which is pre- or extra-Symbolic not only despite but more primordially by dint of the Symbolic's clutches. This is why, on Žižek's reading, Hegel's Science of Logic, while never leaving the matrix of self-thinking thought, can coincide with metaphysics and describe pre-subjective reality in its raw categorial purity and dialectical movement. Ontological solipsism is only apparent, for materialism justifies itself in the cracks of a radical idealism: the very condition of possibility of discourse means that discourse is always more than itself, even if that means that its very possibility coincides with its impossibility. Fichte refers to such a theoretical position—a critical idealism brought to fruition—as a real-idealism or an ideal-realism.431 Although his own position may fail to execute this adequately, it can serve nevertheless as the most consequent description of a true, successful idealism. “out there” because of the inconsistencies and gaps in the symbolic order.430 As Žižek correctly points out, “[t]he irony of the history of philosophy is that the line of philosophers who struggle against the sophistic tradition ends with Hegel, the 'last philosopher,' who, in a way, is also the ultimate sophist, embracing the self- With this insight gained Žižek, like Hegel, discovers an epistemological foothold—the productive space of ideal inconsistency (in the quote above Žižek is talking about our access to the Real from within the Symbolic rather than its ontological constitution)—from within which he can find the resources required to develop a new metaphysics. It is less idealism that poses a problem for the latter than the traps that it (unconsciously) creates for us as we attempt to catch a glimpse what “lies beyond” phenomenal reality: the inevitable and necessary symbolic dismemberment of a self- grounding idealism that fails to fully become itself not only creates the possibility of error, but since the first showing of the Real is always an internal deformation of ideal structure (a pure negative form lacking content), it itself risks being riddled with a thick layer of fantasmatic projections and unconscious desires. That which allows idealism to overcome itself also can hinder the speculative process. Yet, despite this, the Real-as- lack and the Real-as-excess do not stand in opposition to one another. Although the former is always epistemologically superior or primary, being the transcendental condition of the possibility of access to the latter, we are nevertheless capable of attaining that which is pre- or extra-Symbolic not only despite but more primordially by dint of the Symbolic's clutches. This is why, on Žižek's reading, Hegel's Science of Logic, while never leaving the matrix of self-thinking thought, can coincide with metaphysics and describe pre-subjective reality in its raw categorial purity and dialectical movement. Ontological solipsism is only apparent, for materialism justifies itself in the cracks of a radical idealism: the very condition of possibility of discourse means that discourse is always more than itself, even if that means that its very possibility coincides with its impossibility. Fichte refers to such a theoretical position—a critical idealism brought to fruition—as a real-idealism or an ideal-realism.431 Although his own position may fail to execute this adequately, it can serve nevertheless as the most consequent description of a true, successful idealism. From Radical Idealism to Critical Metaphysics 267 To say that the Real is a product of thought is not to lapse into a Berkeleyan form of idealism wherein reality is simply created by the subject: “the Real is not some kind of primordial Being which is lost,” but rather “what we cannot get rid of, what always sticks on as the remainder of the symbolic operation.”429 As an aftereffect of this symbolic operation, it ensures that we have an indirect but methodologically secure entry point into the world by means of the inconsistencies that our notional apparatus generates in the freely determined self-generation of the universe of meaning, inconsistencies that unexpectedly let us develop an objective discourse. But the limits of idealism entail that idealism is always already more than itself—thought itself is utterly incapable of positing itself as a self- enclosed positivity that simply creates its own universe of meaning; it becomes contaminated, as it were, by a constitutive “outside” as soon as it tries to posit itself in its own self-determining freedom, so that it must constantly struggle with this outside. This constitutive failure on behalf of thinking successfully to posit itself as all guarantees that there is a subject- independent reality that we experience and can speculatively describe: There is a Real not because the Symbolic cannot grasp its external Real, but because the Symbolic cannot fully become itself. There is being (reality) because the symbolic system is inconsistent, flawed, for the Real is an impasse of formalization. This thesis must be given its full “idealist” weight: it is not only that reality is too rich, so that every formalization fails to grasp it, stumbles over it; the Real is nothing but an impasse of formalization—there is dense reality From Radical Idealism to Critical Metaphysics 269 From Radical Idealism to Critical Metaphysics 269 “out there” because of the inconsistencies and gaps in the symbolic order.430 “out there” because of the inconsistencies and gaps in the symbolic order.430 As Žižek correctly points out, “[t]he irony of the history of philosophy is that the line of philosophers who struggle against the sophistic tradition ends with Hegel, the 'last philosopher,' who, in a way, is also the ultimate sophist, embracing the self- y p p y y y This is why, on Žižek's reading, Hegel's Science of Logic, while never leaving the matrix of self-thinking thought, can coincide with metaphysics and describe pre-subjective reality in its raw categorial purity and dialectical movement. Ontological solipsism is only apparent, for materialism justifies itself in the cracks of a radical idealism: the very condition of possibility of discourse means that discourse is always more than itself, even if that means that its very possibility coincides with its impossibility. Fichte refers to such a theoretical position—a critical idealism brought to fruition—as a real-idealism or an ideal-realism.431 Although his own position may fail to execute this adequately, it can serve nevertheless as the most consequent description of a true, successful idealism. As Žižek correctly points out, “[t]he irony of the history of philosophy is that the line of philosophers who struggle against the sophistic tradition ends with Hegel, the 'last philosopher,' who, in a way, is also the ultimate sophist, embracing the self- Chapter 11 270 referential play of the Symbolic with no external support of its truth.”432 Hegel can accomplish this prima facie paradoxical feat because he is able to demonstrate that accepting the impossibility of leaving the “correlationist circle” does not fall into a naïve idealism whereby objective reality is reduced to nothing, but rather shows how even the self-referential nature of thinking itself always already depends upon and is entangled with the world, thereby attesting that the split between knowledge in itself and for us exists not because we are separated from the world, but because we are a part of it: “the very limitation of our knowing—its inevitably distorted, inconsistent character—bears witness to our inclusion in reality.”433 In this respect, idealism (reflection, notional constructs, language as such) creates the space of reasons in virtue of which things can present themselves to us as they are in reality in itself. This presenting, however, requires a stage upon which their theatrical appearance can be performed, a stage that produces the illusion of a backstage to which we do not have access. From Radical Idealism to Critical Metaphysics 271 From Radical Idealism to Critical Metaphysics 271 the effects of the Symbolic's self-enclosure is that, in point of fact, it allows things to appear intelligibly through the reflective mediation of language. All we have to do is take the appropriate attitude toward the inner limitations of phenomenal reality as such. Paradoxically, a realist epistemology does not make a realist. This is why the choice between idealism and realism is false for the idealist. “out there” because of the inconsistencies and gaps in the symbolic order.430 Instead of merely separating us from the world, the reflexivity of the Ideal thereby allows objects to have meaning for us as something more than objects to be used by specialized biological or natural needs. We symbolize them, grant them a place in discourse, a discourse whose failures make it seem as if a world out there directly attacks our concepts and theoretical models when, in fact, we never exit discourse at all, for only its self-sustaining matrix can sustain phenomenal reality as a universe of meaning. This signifies, moreover, that to pose the question of being qua being, there must be a difference between us and being, for being qua being can only show itself to a finite thinker for whom there exists a distance within which being can be phenomenalized or phenomenalize itself by means of notional constructs. It is important to note, however, that in order to get being right, we must also be able to get it wrong, the minimal condition of which is satisfied by the ontological madness that is the basic structure of ideality as that which prevents us from having a direct “immersion” in the world and as such opens up the possibility of replicating being within itself; the self-stipulating norms of discourse internally guide the constitution of phenomena while their inconsistency demonstrates that we are capable of objectively describing 272 Chapter 11 of Žižek is his nuanced vision of the world that is able to use idealism's resources not only to overcome its own apparent limitations from within, but also to demonstrate how, if this self-overcoming is successfully executed, our understanding of being is simultaneously remodulated. Idealism and realism, transcendentalism and metaphysics, are not mutually opposed if you think them through in relation to one another, for the former forces us to come to grasp what it means for thinking to exist in an irreducible manner, a fact that has stark consequences for our understanding of the world at large. After a long period in contemporary philosophy where there was a general disdain for speculation, what distinguishes Žižek so radically from others who have also raised the question of the possibility of a new metaphysics (perhaps most notably Deleuze and Badiou) and those who are now active in establishing this possibility is his call for a critical metaphysics and its superiority over a mere return to dogmatic philosophizing as a means of overcoming the heritage of what has recently come to be known since Meillassoux's After Finitude as correlationism.438 For Žižek, the urgent call for a philosophy that can combat the apparent speculative throes of the irreducibility of the correlation in our knowledge of the world (if the intelligibility of any specific empirical truth-claim depends upon the subject for whom such a truth-claim has meaning in the first place, how can we even speak about that which occurred before the existence of such a subject without falling into ontic nonsense?) does not have its origin in paradoxes concerning “ancestral” statements concerning what the universe must have been like prior to humanity, as Meillassoux would like us to believe,439 this having been proven to be an intrinsic possibility opened up by the logical space of the correlation as such, but rather what the universe must be like if something like humanity and its transcendental constituting powers are to arise at all. The issue is how the Real could have come to appear to itself—and although this may appear to risk an anthropomorphization of nature (“[w]e should apply here something like a weak anthropic principle: how should the Real be structured so that it allows for the emergence of subjectivity [...]?”440), one should proceed cautiously. 11.3 A Call for a Critical Metaphysics At this juncture we must underline one important feature of this argument for a self-grounding idealism that intrinsically contains an irreducibly real moment. If the Symbolic freely constitutes phenomenal reality “with no external support of its truth,”434 then this suggests that from within its ontologically solipsistic dance of cybernetic ciphering, we can break through the impenetrable dusk of psychosis as we find, in an innovative theoretical gesture, a secure foothold from which to found a new science of being. But this means that we should not merely overcome radical idealism from within idealism (an epistemological sublation of the correlation): we must also overcome it from the side of being by showing how the ambiguities of idealism are in fact a part of the world's fundamental structure through an account of how being comes to appearance/thinking/phenomenalization (an ontological inscription of the correlation). If we can succeed, then realism and idealism will have become intimately dialectically linked. Not only would this entail a strong theory of thinking insofar as the latter would be inscribed within the fold of being as an irreducible event rather than as a mere illusory feature, but also a profoundly rich metaphysics inclusive of both realism and idealism given that both would now reciprocally ground one another (in realistic and idealistic terms) in a completely self-sufficient and self-reflexive whole. That is to say, we would have a new variation on the late German Idealist theme of the unification of system and freedom: or, in other words, another take on the Kantian heritage and how it radically changes the field of philosophizing. In this respect, whereas much of contemporary philosophy's understanding of idealism fails to take it seriously, often equating it with a form of Berkeleyanism (a tradition that runs from Kant's first critics to Lenin435 and taken up once again by Moore436 and most recently by Meillassoux437), one of the greatest strengths 272 Chapter 11 Žižek's metaphysical archaeology of the psychoanalytical subject is an attempt to think the intersection of the Real and the Symbolic, the coldness of being and the From Radical Idealism to Critical Metaphysics 273 fervor of humanity, because if images and words, and by implication thinking, exist, they must exist in the world. The universe is inclusive, not exclusive, of humanity: a true speculative philosophy should comprehend both the Real in its pure non-correlationality (the nonhuman) and how correlation comes to pass in being (the human). Perhaps unexpectedly, the price we pay for this theoretical gain of re-inscribing humanity into nature, that is, the latter's minimal anthropomorphization, is a simultaneous denaturalization of nature and a dehumanization of humanity. Not only is nature now reduced to “a freak show of contingent disturbances with no inner rhyme or reason,”441 but even if humanity still retains a certain qualitatively distinct status in contradistinction to other things in virtue of its autonomy, what we normally take as the great and sublime achievements of thinking are, in fact, grounded in a mere virtual re-compensation for our traumatic disruption from the Real. What thus makes Žižek's speculative real-idealism/ideal-realism (to borrow that German Idealist leitmotif) so penetrating and deserving of attention today is its ability to combine a profoundly idealist epistemology with a dynamic realist metaphysics in one single gesture, which shows us one path that contemporary metaphysics could take: namely, a critical one. In this regard, if Meillassoux's critique of correlationism is a call to station ourselves after (Kantian) finitude, it must be recalled that this is precisely what Schelling and Hegel did in their own critique of correlationism avant la lettre. Žižek is able to balance the real and ideal poles in such a nuanced way because of his precise and original take on the breakthrough inaugurated by transcendental philosophy, a breakthrough that for him presents itself as the unthought (Lacanian) cause at the heart of German Idealism. 272 Chapter 11 One of his central claims is that if we read Kant closely, we see hints that what truly fascinates him is not how the subject brings forth its own universe of meaning as a new kind of metaphysical agent capable of guaranteeing the universality and necessity of experience/empirical truth, the two being identical for Kant, but “something quite different,” something that sets the stage for the “few decades [of German Idealism that] represent a breathtaking concentration of the intensity of thinking” within which “more happened than in centuries or even millennia of the 'normal' development of human thought.”442 Commenting on Kant's description of the Copernican Chapter 11 From Radical Idealism to Critical Metaphysics 275 relationality and whereby one leads to the other: “[w]e can also see in what way two lacks overlap in this impossible object: the constitutive lack of the subject (what the subject has to lose in order to emerge as the subject of the signifier) and the lack in the Other itself (what has to be excluded from reality so that reality can appear).”447 Although the transcendental (our subjective position) objectively exists in the world, it nevertheless appears to itself in its first mode as merely subjective. To overcome itself and pass over into a metaphysics, it must do so from within; it does so by drawing attention to the fact that, beneath the correlation of (the conscious) subject and reality, “there is the more primordial correlation of the subject (of the unconscious) and its Real/ impossible objectal counterpoint, S-a.”448 Although this may seem to be just another correlation (as Heidegger only institutes the correlation being/Dasein as more originary than that of consciousness/world, doesn't Žižek do something similar?) this is a false appearance. Rather than being a strict correlation in Meillassouxian terms this binary points towards the emergence of correlation within being: “this impossible/Real object is the very mode of inscription of the subject into trans-subjective reality; as such, it is not transcendental but (what Derrida would have called) arche- transcendental, an attempt to circumscribe the 'subject in becoming,' the trans-subjective process of the emergence of the subject.”449 Žižek's wager, one that he shares with the entirety of post-Kantian idealism as a critical metaphysics fighting against any dogmatic breed thereof, is that if we are to truly break free from correlationism, no return to a “naïve” realism is possible. Not only is the latter always open to critique insofar as it could remain correlational in a hidden way (isn't its specific image of reality related to a subject?450), so that the specific nature of the correlation, namely the ideal conditions of the possibility of intelligibility of any theory, should always be thematized before embarking on speculation, but more primordially it fails to give us of a grasp of how thought is situated/ comes to pass in being and, therefore, the very ontological conditions of the possibility of its own status as a theory. Chapter 11 274 revolution (the experiment of seeing “if he might not have greater success if he made the observer revolve and left the stars at rest”443), which Kant identifies with his own transcendentalist position, Žižek notes: The precise German terms (“die Zuschauer sich drehen”—not so much turn around another centre as turn or rotate around themselves) make it clear what interests Kant: the subject loses its substantial stability or identity and is reduced to the pure substanceless void of the self-rotating abyssal vortex called “transcendental apperception.”444 What initially appears as a rampant subjectivism wherein the ego reigns above the world proves on closer inspection to be something infinitely more complex, for the self-grounding field of phenomenal reality only exists because it itself revolves around the positively charged void of I. The breakthrough of transcendental idealism is precisely the discovery of this zone of negativity within the subject, that X that can never be fully appropriated into transcendental constitution and yet somehow engenders its very possibility, whereby it shows itself to be not so much a displacing of the substantial unity of reality from the external world to the conceptual forms of cognitive construction (a subjective Ptolemaic counterrevolution against Galileo’s de-centering of the medieval world of teleology445) as the opening up of a new understanding of being, that is, of the radical ontological incompletion of reality, the breakdown of substance, provoked by the meta-transcendental conditions of thought as such. As the late German Idealist reappropriation of the subject demonstrates, what is truly unique in transcendental idealism is that the real and ideal poles are unsettled in one brush stroke: linked to the Todestrieb that destroys the homeostasis of nature and das Unbehagen in der Kultur that prevents our second nature from becoming a new substantialist order, the “objectal status” of the subject is that which is “no longer” ontological and “not yet” symbolic because it cannot be contained in either register in its pure form;446 it is what Žižek sometimes refers to as the absent centre that, by protruding out of all ontological and symbolic structures, negatively ties them together in its very undecidability. Here, and only here, the real and the ideal poles intersect, so that both are given their equal due because of an unapproachable X, a je ne sais quoi, that relates them in their very non- From Radical Idealism to Critical Metaphysics 275 276 Chapter 11 Real, and thus breaks the circle of transcendental correlationism.”451 In this manner, we should search for how the transcendental hints towards its dark beginnings (the pure act of the Real-as-origin and the unruliness of drives in the Real-as-excess that precedes it), the emphasis being on the ontogenetic process of becoming more than on the specificity of the transcendental constitution of reality for us, so that the fundamental issue is no longer so much how can we attain knowledge of the absolute (this already being accomplished by the first methodological step, as for instance in Hegel) but how does our subjective viewpoint fit into it as something that objectively exists: “[t]he true question is therefore how I (as the site where reality appears to itself) emerge in 'objective' reality (or, more pointedly, how can a universe of meaning arise in the meaningless Real).”452 With this, we have come full circle and hit upon the great merit of Žižek's philosophy. Having already overcome idealism from within, and having opened up the space for a speculative philosophy, he can develop a theory that is capable of being maximally realist and idealist and therefore best suited as a self- explanatory theory of the metaphysical “totality” of the world, insofar as it can simultaneously supply the ontological conditions of the possibility of its own status as a theory and the ideal conditions of possibility of its own intelligibility in one sweeping move, thus making the theory itself extremely self-referential in its structure. In short, the theory displays complete systematic self-enclosure: it explains itself as a theory in both the real and ideal registers in such a manner that both depend upon and mutually ground one another in a self-articulating whole; it has succeeded at developing “a concept of the world or the Real which is capable of accounting for the replication of reality within itself.”453 What Žižek teaches us, a lesson already brought to the fore in late German Idealism but since forgotten, is that radical idealism is not a closure to the absolute. It is rather a new approach towards it, a new way of relating to it—and to see it as such merely requires a parallax shift in perception. 11.4 Being's Poem: Speculative Philosophy and the From Radical Idealism to Critical Metaphysics 275 Concerning the latter, merely explaining subjectivity as a purely contingent emergence amongst others à l Meillassoux bodes no better: “one should locate traces of this contingency in a kind of umbilical cord which links the subject to its pre-subjective 11.4 Being's Poem: Speculative Philosophy and the Mytho-Poetic Parallax Shift If Žižek is right to say that the founding gesture of idealism is an ontological passage through madness, then it would not go far enough if we were to admit that the opposition between realism and idealism has been already From Radical Idealism to Critical Metaphysics 277 resolved at the level of idealism. Something is missing, for the real event that immediately precedes the autarchy of the Ideal, and whose exploration would enable this opposition to be also resolved at the level of realism (thus radicalizing and guaranteeing what Fichte referred to as a real-idealism or ideal-realism), is an impossible object of discourse. Conceding that notional antagonism can indeed be spectrally expressive of objective reality, and thus enable us to speculate about reality within ideality, to fully explain how we can have contact with the world where there is properly speaking no contact at all we must nevertheless acknowledge that we can never even indirectly reach the exact moment at which being begins to exist in the modality of the Ideal. The difficulty is much more severe than that the Real is always already minimally symbolized, since the Real-as-origin expresses two fundamental theoretical problems. First, insofar as its abyss of unconscious decision represents a pure self-instituting difference that unpredictably splits the world into two new logically irreconcilable registers, it cannot be deduced from the auto-movement of the Real; there is no “transition,” but only a self-caused “leap” that forever evades complete conceptual or natural dialectic mediation. Second, because this act withdraws in the very gesture of giving birth to the Symbolic, it lies stricto sensu “beyond” the grasp of the latter and can never appear within it, not even negatively. It is in this precise manner that the subject is neither Real nor Symbolic and is only expressible through a series of paradoxical avatars unable to bestow content upon it (the disjunctive “and,” the “in-between,” the “abyss of freedom,” the “vanishing mediator,” and so on). A philosophical discourse about the subject is thus intrinsically paradoxical because the latter is “a non-provable presupposition, something whose existence cannot be demonstrated but only inferred through the failure of its direct demonstration.”454 In short, it can only be investigated at the level of mytho-poetics. 11.4 Being's Poem: Speculative Philosophy and the Mytho-Poetic Parallax Shift But what intensifies the problematic of such a metaphysical archeology of the subject is that without such a mytho-poetic narrativization of its impossible Past we would be unable fully to explicate how the Symbolic can in fact relate to reality in itself, for without it we cannot perform the parallax shift of “transposing the it can only be investigated at the level of mytho-poetics. But what intensifies the problematic of such a metaphysical archeology of the subject is that without such a mytho-poetic narrativization of its impossible Past we would be unable fully to explicate how the Symbolic can in fact relate to reality in itself, for without it we cannot perform the parallax shift of “transposing the tragic gap that separates the reflecting subject from pre-reflexive Being into this Being itself,” whereby “the problem becomes its own solution: it is our very division from absolute Being which unites us with it, since this division Chapter 11 From Radical Idealism to Critical Metaphysics 279 The system of being and thinking has closed upon itself in one final self-referential gesture. Ž Because the objectal status of the subject escapes any straightforward causal explanation and lies outside of all correlation insofar as it is responsible for its very upsurge, the only way to reach it is by means of a mytho-poetics of speculative fabulation. And given that this realistically non-deducible and idealistically inaccessible zone coincides with that very place in which the subject is inscribed within being as one creature amongst others (and is thus that which would enable us to pass without any immanent obstacle from the real pole to the ideal pole and vice versa) the parallax shift from the negative limitation of knowledge to the positive structure of the absolute itself requires more than mere rational ideal discourse. Only then can we “relate the In-itself to the split in the subject,”457 for “what Lacan calls the objet a, the subject's impossible-Real objectal counterpart, is precisely such an 'imagined' (fantasmatic, virtual) object which never positively existed in reality—it emerges through its loss, it is directly created as a fossil.”458 In this sense, Žižek's philosophy is paradoxical precisely because it attempts to think the unthinkable, that is, the cogito ergo sum as “I am that impossible piece of the Real where I cannot think”459 that uncannily corresponds to that space within which the meaningless Real contingently awakens and opens its eyes for the first time. Chapter 11 278 is immanent to Being.”455 In other words, the real and the ideal sides of overcoming idealism are intimately connected and cannot be discussed in isolation from one another: in order for idealism to completely surmount its own apparent limitations, it must be able to come full circle and show how, from within realism, its own ambiguities are not merely epistemological but also ontological; it must show what it means for thinking to be a part of the world, a manner in which the world relates to itself, no matter how paradoxically. In creating itself by an act of immaculate conception, the Real-as-origin of the pure act at the birth of full-fledged subjectivity retroactively takes over what we come to know as the Real-as-excess of the drives. In a contradictory moment in which cause-and-effect relations are torn apart, the effect becomes greater than and autonomous from its cause, even going so far as to write a virtual possibility into the eternal dregs of the Past that never existed prior to its having been written there through an act of positing of its own presuppositions. The subject creates its own past in the same instance in which it begets itself out of nothing, so that the true “arche-fossils” are not ancestral statements concerning what occurred billions of years ago before the emergence of the thinking subject or life itself, but the objectal status of the subject: what Lacan asserts is precisely the irreducible (constitutive) discord, or non-correlation, between subject and reality: in order for the subject to emerge, the impossible object-that- is-subject must be excluded from reality, since it is this very exclusion which opens up the space for the subject. [...] The true problem of correlationism is not whether we can reach the In-itself the way it is outside of any correlation (or the way the Old is outside its perception from the standpoint of the New); but the true problem is to think the New itself “in becoming.” The fossil is not the Old the way it was in itself, the true fossil is the subject itself in its impossible objectal status—the fossil is myself, the way the terrified cat sees me when it looks at me. This is what truly escapes correlation, not the In-itself of the object, but the subject as object.456 From Radical Idealism to Critical Metaphysics 279 From Radical Idealism to Critical Metaphysics 279 Because the objectal status of the subject escapes any straightforward causal explanation and lies outside of all correlation insofar as it is responsible for its very upsurge, the only way to reach it is by means of a mytho-poetics of speculative fabulation. And given that this realistically non-deducible and idealistically inaccessible zone coincides with that very place in which the subject is inscribed within being as one creature amongst others (and is thus that which would enable us to pass without any immanent obstacle from the real pole to the ideal pole and vice versa) the parallax shift from the negative limitation of knowledge to the positive structure of the absolute itself requires more than mere rational ideal discourse. Only then can we “relate the In-itself to the split in the subject,”457 for “what Lacan calls the objet a, the subject's impossible-Real objectal counterpart, is precisely such an 'imagined' (fantasmatic, virtual) object which never positively existed in reality—it emerges through its loss, it is directly created as a fossil.”458 In this sense, Žižek's philosophy is paradoxical precisely because it attempts to think the unthinkable, that is, the cogito ergo sum as “I am that impossible piece of the Real where I cannot think”459 that uncannily corresponds to that space within which the meaningless Real contingently awakens and opens its eyes for the first time. In so doing, the metaphysical archaeology of the subject it offers endeavors to demonstrate that what the subject “loses” in order to become a subject coincides with what is excluded from reality so that reality can appear to itself, in such a way that the Real is thereby transformed from being a primordial being to which we have lost access due to its symbolization into something of which we cannot shake ourselves, no matter how hard we try, because through a mere formal reversal the gaze of the subject is seen to be the gaze of the world upon itself, the ambiguities and difficulties of the former being always already those of the latter: what we see is that “the narrative [we are telling] is not merely the subject coping with its division from Being, it is simultaneously the story Being is telling itself about itself,” so that realism and idealism are no longer in opposition, but stand in a self-sufficient totality inclusive of both as immanent to the life of substance. From Radical Idealism to Critical Metaphysics 279 In so doing, the metaphysical archaeology of the subject it offers endeavors to demonstrate that what the subject “loses” in order to become a subject coincides with what is excluded from reality so that reality can appear to itself, in such a way that the Real is thereby transformed from being a primordial being to which we have lost access due to its symbolization into something of which we cannot shake ourselves, no matter how hard we try, because through a mere formal reversal the gaze of the subject is seen to be the gaze of the world upon itself, the ambiguities and difficulties of the former being always already those of the latter: what we see is that “the narrative [we are telling] is not merely the subject coping with its division from Being, it is simultaneously the story Being is telling itself about itself,” so that realism and idealism are no longer in opposition, but stand in a self-sufficient totality inclusive of both as immanent to the life of substance. The system of being and thinking has closed upon itself in one final self-referential gesture. In Žižek, we must narrativize the movement from being to thinking if we In Žižek, we must narrativize the movement from being to thinking if we wish to reconcile the two and completely escape the speculative throes of Chapter 11 280 correlationism, since at the level of content there is always a minimal “non- dialectizable” difference of one to the other (a division) that prevents such a move in purely rational discourse. But we must highlight the precise logical structure of this solution if we are to come to terms with the originality, daringness, and potential problems of Žižek's position. Given that the exact real event that instigates being's coming into appearance/thinking/ phenomenalization is the primordial ontological trauma that is the subject as object, we must in a mytho-poetic register (the subject in its objectal status being forever elusive) show that this trauma is not a mere accidental, haphazard occurrence in the personal history of an individual subject, but rather reveals itself as a constitutive, yet disrupting part of a greater trauma within being itself. From Radical Idealism to Critical Metaphysics 281 our apparently purely epistemological limitations are intimately linked to the ontological grounding of our notional apparatus. But is such a mytho- poetic narrativization a sufficient basis for a new speculative philosophy? As we will see in the next chapter, delving into this question leads us to three potentially fatal issues with Žižek's critical metaphysics, all emerging from its fundamental concept of ontological catastrophe as the vanishing meditator between the Real and the Ideal. Does Žižek have the resources necessary to combat them? And if so, at what price? From Radical Idealism to Critical Metaphysics 279 But this (Žižekian) dialectical reconciliation is not a complete sublation of the opposition between realism and idealism, a complete break with the paradoxes of correlation, for the problematic nature of the latter is something that must be accounted for rather than explained away. As with all dialectical movement, “reconciliation is a reconciliation with the irreducibility of the antinomy, and it is in this way that the antinomy loses its antagonistic character,”460 so that in this case, reconciliation only truly occurs when we realize that there is no reconciliation (a complete solution) possible because what we take as our finitude should be inscribed into the thing itself (being) as its non-coincidence to self; and although this reconciliation can only thus come to pass at the level of mythological form, and never at that of content, instead of focusing on the impossibility of idealist representation to capture that which is being described, namely the objectal status of the subject, the solution paradoxically “shifts the focus to how (as Lacan put it) the signifier itself falls into the Real, that is, how the signifying intervention (narrativization) intervenes into the Real, how it brings about the resolution of a real antagonism,”461 thus working against its own impossibility: “the narrative path directly renders the life of Being itself.”462 Though we can never “exit the circle of imagination”463 to capture the abyss of unconscious decision at its real origin, by writing being's poem we can see, due to a mytho- poetic parallax shift that the poem we are writing coincides with the one From Radical Idealism to Critical Metaphysics 281 Notes 411. Žižek, Less Than Nothing, p. 239. 412. Ibid., p. 562. 412. Ibid., p. 562. 412. Ibid., p. 562. 413. Žižek, The Abyss of Freedom, p. 45. 414. Johnston, Žižek’s Ontology, p. 19. 414. Johnston, Žižek’s Ontology, p. 19. 415. Žižek, The Puppet and the Dwarf, p. 66. 416. See Žižek, The Ticklish Subject, p. 55; and The Parallax View, p. 166. 417. For example, Žižek, The Indivisible Remainder, pp. 22–23. 418. See, for instance, ibid., p. 9. 418. See, for instance, ibid., p. 9. 419. Žižek, Less Than Nothing, p. 905. 419. Žižek, Less Than Nothing, p. 905. 420. Žižek, The Ticklish Subject, p. 33. 420. Žižek, The Ticklish Subject, p. 33. 421. Ibid., p. 31. 421. Ibid., p. 31. 422. Ibid., pp. 79ff. 423. Žižek, Less Than Nothing, p. 282. 424. Ibid., p. 283. 424. Ibid., p. 283. 425. Ibid., pp. 374–75. 426. Žižek, The Ticklish Subject, pp. 88–89. 427. Žižek, Tarrying with the Negative, p. 20. 428. Hegel, The Science of Logic, p. 124. 429. Žižek, Less Than Nothing, pp. 645–46. 430. Ibid. 431. Fichte, Science of Knowledge, p. 247. 432. Žižek, Less Than Nothing, pp. 76–77. 433. Ibid., p. 390. 434. Ibid., p. 77. 282 Chapter 11 282 Chapter 11 435. References to such are scattered throughout Lenin’s Materialism and Empirio- Criticism (Peking: Foreign Languages Press, 1972). 436. See Moore, “Refutation of Idealism,” Mind 12 (1903). 437. This is at the heart of Meillassoux’s critique of correlationism via the arche-fossil in After Finitude: An Essay on the Necessity of Contingency, trans. Ray Brassier (New York: Continuum, 2008). 438. For a similar in spirit account of the distinction between critical and dogmatic metaphysics, see Gabriel, Das Absolute und die Welt in Schellings, p. 8. For the basic definition of “correlationism,” see Meillassoux, After Finitude, pp. 4–6. 439. Meillassoux, After Finitude, pp. 9–13. 440. Žižek, Less Than Nothing, p. 905. 440. Žižek, Less Than Nothing, p. 905. 441. Ibid., p. 298. 441. Ibid., p. 298. 442. Ibid., p. 8. 442. Ibid., p. 8. 443. Kant, Critique of Pure Reason, B xvi. 443. Kant, Critique of Pure Reason, B xvi. 444. Žižek, Less Than Nothing, p. 631. 444. Žižek, Less Than Nothing, p. 631. 445. Meillassoux, After Finitude, pp. 118–19. 446. Žižek, The Ticklish Subject, p. 36. 447. Žižek, Less Than Nothing, p. 645. 448. Ibid., p. 642. 448. Ibid., p. 642. 449. Ibid. 449. Ibid. 450. Ibid. 450. Ibid. 451. Ibid., pp. 642–43. 451. Ibid., pp. 642–43. 452. Ibid., p. 924. 452. Ibid., p. 924. 453. Gabriel and Žižek, “Introduction: A Plea for a Return to Post-Kantian Idealism,” in Mythology, Madness and Laughter, p. 13. 454. Žižek, Less Than Nothing, p. 730. 454. Žižek, Less Than Nothing, p. 730. 455. Ibid., p. 15. 455. Ibid., p. 15. 456. Ibid., p. 644. 456. Ibid., p. 644. 457. Ibid. 457. Ibid. 458. Ibid., p. 645. 459. Ibid. 460. Ibid., p. 950. 460. Ibid., p. 950. 461. Ibid., p. 16. 461. Ibid., p. 16. 462. Ibid. 463. Žižek, The Ticklish Subject, p. 33. Chapter 12 The Deadlocks of Ontological Catastrophe The Cases of Naturphilosophie, Anton-Babinski Syndrome, and Tarte à la crème 12.1 The Schellingian Real-as-Excess: Iain Hamilton Grant, Naturphilosophie, and the Interior Involutions of Being But is mytho-poetics the only option here? Although Žižek follows Hegel's defence of radical idealism as capable of a speculative account of extra- notional reality, a potentially fatal problem arises internally within Žižek's own position as soon as we take a closer look at Žižek's other major interlocutor: Schelling. Ultimately unsatisfied with Hegel's mature account of the passage from nature to culture, Žižek turns to Schelling to explicate the genesis of the Symbolic. What Hegel missed was the paradoxical essence of this very movement, a dialectical movement that causes dialectics to collapse upon itself. With the birth of subjectivity, we see a snag in substance that prevents the absolute from self-actualizing itself so that, instead of being the Idea completely returning to itself out of its otherness in nature, which would present us with a reconciliation of nature and spirit in the Idea, nature's complete self-sublation in spirit, culture is actually a mere secondary (“virtual, artificial, symbolic, not substantially natural”464) response to the primordial ontological trauma that lies at the core of the human being. For Žižek, this means that culture is the always failed attempt at reconciliation: the Idea is nothing other than this very act of its own returning to itself, this movement being constitutive of that to which is returned;465 for it to reach its end would herald its death. Representing a recoil into a psychotic, irreal space, not only do the subjective and objective/ mind and world thereby fall into infinite conflict with one another, but it becomes impossible to explain why the Ideal emerges. According to Žižek it is Schelling who, fighting against the perceived threat of Hegelian Absolute Idealism, gives the most detailed account of this immanent self-sundering of being into its real and ideal poles in his account of the Grund as the ever elusive, eternal Past of consciousness, and the pure act of unconscious decision underlying the birth of the universe of human meaning. Schelling's position is paradoxical: from within a solipsistic space (transcendental idealism) he tries to develop a philosophical language capable not only of piercing the primacy of the Real-as-lack and explaining its monstrous pre-history within pre-symbolic antagonism (materialism), a logically prior but directly unreachable modality of the Real that precedes and exceeds the Symbolic, but also of describing the inexplicable self-positing of subjectivity But is mytho-poetics the only option here? Chapter 12 The Deadlocks of Ontological Catastrophe The Cases of Naturphilosophie, Anton-Babinski Syndrome, and Tarte à la crème Žižek's theoretical philosophy aims to be a critical metaphysics capable of simultaneously overcoming radical idealism from within idealism (an epistemological sublation of the correlation) and from within realism (an ontological inscription of the correlation). However, at this juncture three potential problems emerge from various directions. The first direction is that of Schelling's Naturphilosophie. Not only does Schelling proclaim that, insofar as the subject is anything but an ontological catastrophe, thought is inscribed within being in such a way that we have no need to overcome radical idealism from within itself, it also challenges Žižek's own psychoanalytical reactualization of Schelling. The second direction is that of the skeptic, who can invent a thought experiment to demonstrate that it is perhaps impossible to develop any positive metaphysics from within a differential system of signifiers without any external reference. The third direction is that of the very basis of ideality itself understood as a psychotic withdrawal into the night of the world, the overcoming of which demands the seemingly impossible task of developing a paradoxical form of “successful” psychotic thinking capable of penetrating the impenetrable dusk of its own psychosis. How does Žižek stand up to such critiques? Is his radical idealism truly capable of providing us with a comprehensive metaphysics? 284 Chapter 12 The Deadlocks of Ontological Catastrophe 285 (idealism). After all, even if we can be said to have access to both the ancestral past of the Real prior to subjectivity and the Real that surrounds us “outside” of language through the very inconsistency of our notional apparatus, the methodology of which Hegel offers us through an analysis of the dialectic of phenomenal appearance and the very structure of symbolic thought, nevertheless the precise moment in which the subject institutes itself into being poses a problem to such a self-overcoming of radical idealism, because it is a leaping point in the Real into a new age of the world that is always “beyond” the Symbolic as its irretrievable origin. Because Schelling realizes the impossibility of a purely speculative account of the subject's emergence, and thus the necessity of a mytho-poetics, it is he and not Hegel who most adequately realizes the quadruple logic of dialectics at the core of German Idealism. But even with his highly methodological psychoanalytical construction of the unconscious Grundlogik of German Idealism, for anyone who is familiar with Schelling's vast corpus it is unclear how Žižek can appropriate Schelling for his own project without potentially destabilizing his most fundamental theoretical category: that of the ontological catastrophe. But even with his highly methodological psychoanalytical construction of the unconscious Grundlogik of German Idealism, for anyone who is familiar with Schelling's vast corpus it is unclear how Žižek can appropriate Schelling for his own project without potentially destabilizing his most fundamental theoretical category: that of the ontological catastrophe. Although the two major concepts Žižek utilizes—denaturalized unruliness (the dark pre-history of subjectivity in the vicissitudes of being) and the unconscious decision (the separating Ent-Scheidung whose effect is the institution of a metaphysically disjunctive “and”)—are meant to explain how we get entrapped in the Symbolic and the matrix of idealization, Žižek's own writings on Schelling do not seem fully to take into account the possibility that the latter might not be able to be so easily translated into the framework that he sees as basic to the entire tradition. His division of Schelling's thinking into three distinct stages—the Schelling1 of a quasi-Spinozistic philosophy of absolute indifference, Schelling2 of a radical materialist ontology of freedom, and Schelling3 of the philosophy of mythology and revelation—already hints at an irremovable tension. 12.1 The Schellingian Real-as-Excess: Iain Hamilton Grant, Naturphilosophie, and the Interior Involutions of Being Although Žižek follows Hegel's defence of radical idealism as capable of a speculative account of extra- notional reality, a potentially fatal problem arises internally within Žižek's own position as soon as we take a closer look at Žižek's other major interlocutor: Schelling. Ultimately unsatisfied with Hegel's mature account of the passage from nature to culture, Žižek turns to Schelling to explicate the genesis of the Symbolic. What Hegel missed was the paradoxical essence of this very movement, a dialectical movement that causes dialectics to collapse upon itself. With the birth of subjectivity, we see a snag in substance that prevents the absolute from self-actualizing itself so that, instead of being the Idea completely returning to itself out of its otherness in nature, which would present us with a reconciliation of nature and spirit in the Idea, nature's complete self-sublation in spirit, culture is actually a mere secondary (“virtual, artificial, symbolic, not substantially natural”464) response to the primordial ontological trauma that lies at the core of the human being. For Žižek, this means that culture is the always failed attempt at reconciliation: the Idea is nothing other than this very act of its own returning to itself, this movement being constitutive of that to which is returned;465 for it to reach its end would herald its death. Representing a recoil into a psychotic, irreal space, not only do the subjective and objective/ mind and world thereby fall into infinite conflict with one another, but it becomes impossible to explain why the Ideal emerges. According to Žižek it is Schelling who, fighting against the perceived threat of Hegelian Absolute Idealism, gives the most detailed account of this immanent self-sundering of being into its real and ideal poles in his account of the Grund as the ever elusive, eternal Past of consciousness, and the pure act of unconscious decision underlying the birth of the universe of human meaning. Schelling's position is paradoxical: from within a solipsistic space (transcendental idealism) he tries to develop a philosophical language capable not only of piercing the primacy of the Real-as-lack and explaining its monstrous pre-history within pre-symbolic antagonism (materialism), a logically prior but directly unreachable modality of the Real that precedes and exceeds the Symbolic, but also of describing the inexplicable self-positing of subjectivity The Deadlocks of Ontological Catastrophe 285 The Deadlocks of Ontological Catastrophe 285 It is uncertain that the materialism-idealism relationship we see in the Schelling of the Freiheitsschrift and the Weltalter can be read as a mytho-poetics of the birth of a radical transcendental idealism in the manner Žižek suggests. When we look at Schelling's thought, even as Žižek himself presents it, we are tempted to say that, if Hegel is able to show that the absolute opposition between Chapter 12 286 idealist and realist philosophy is without meaning from within idealism, Schelling could be said to do the same from within realism and without needing to pass through the former. This is further supported by the fact that Schelling never gives us a dialectical analysis of phenomenal appearance or the structure of symbolic thought in the way Hegel does and describes his own idealism in the Darstellung as “real” or “objective,” for its principal idea is not to use thought's inconsistencies to find a new starting point for speculative philosophy, but rather to directly investigate the origins of thought itself from its dark nonconscious ground,466 a point repeated by the “higher realism” of the Freiheitsschrift467 and the emergence of ideality in the Stuttgarter Privatvorlesungen out of minerals.468 The argument that Schelling2 is an ephemeral rupture risks succumbing to arbitrariness, since now we find a manner to unify the central problematic of the Naturphilosophie and the “theosophic” philosophy of freedom. Given that one of the key tools for the development of Žižek's own metaphysics is the extracting of an unconscious Grundlogik underlying German Idealism, if Schelling's philosophy turns out to be more continuous than Žižek's analysis lets on, then his psychoanalytical construction of the German Idealist tradition could be jeopardized. For Schelling, we do not just “tarry with the negative.” The X that eludes consciousness, one of the centrepieces of Schelling's thought, is never just a mere formal limit: it is an attempt to express the subject-independent interiority of nature to which we have access despite the mediating activity of consciousness precisely because the subject and its ideational capacities are a part of nature, one way through which nature relates to itself as ground. Schelling refuses to separate the ontological in-itself of precognitive or extra- symbolic reality from the epistemological sphere of idealist representations, arguing that the two must be intimately connected if philosophy is to find a secure basis. The Deadlocks of Ontological Catastrophe 285 If there is an identity between the Real and the Ideal, the problem of their relation to one another is relegated to a metaphysical or naturephilosophical level rather than a strictly epistemic or idealist one. Whereas in the middle-late period this idea of identity is expressed by the notion of the Mitwissenschaft (“co-science”) of creation, it is more clearly for our purposes articulated in the earlier Ideas for a Philosophy of Nature, where Schelling argues for the necessity of a dialectically positive The Deadlocks of Ontological Catastrophe 287 288 Chapter 12 288 from the Latin speculari (to watch over), it is a realism that attempts to, through a scrutinizing surveillance, account for the immanent pulsations of the universe as it transforms bodies of matter into the complex field of living being and eventually thought as ideal self-mediation, so that the Real is not only always excessive to the Ideal, that which can never be brought into it due to nature's raw productivity, but the latter also loses its theoretical primacy insofar as nature in the stirrings of its nocturnal ground itself becomes the true a priori. It is interesting to bring up this aspect of Schelling's philosophy, not just because it could be a weak point in Žižek's psychoanalytical reading, but also because it is a specific manner in which Žižek's metaphysical problematic enters into direct debate with the current speculative turn. In this regard, Iain Hamilton Grant's own transcendental materialism is in complete opposition to that of Žižek, so that contrasting the two allows us simultaneously to bring to the fore both the daring character of the latter's position and its potential internal limitations. Taking up Schelling's diagnosis that modern philosophy exhibits an agonizing deficiency—that nature does not exist for it470—Grant puts forward the argument that this diagnosis is just as sound today as ever: “[i]nsofar therefore as the antithetical couple ‘Plato- Kant’ that lay at the heart of the immediate postkantian context continues to organize metaphysics, contemporary philosophy is importantly and immediately postkantian.”471 Through a careful reconstruction of Schelling's philosophical career with an eye towards its explicit naturephilosophical content and the natural scientific context that surrounded and inspired it, Grant's thesis is that by following its spirit, we may finally find a way to leave behind us the Kantian heritage insofar as Schelling offers us, instead of a two-world metaphysics that results in an “eliminative practicism” (the irreducibility of culture to nature), a “one-world physics capable of the Idea.”472 We cannot cut the world in two, for there is no great divide: even to understand culture as culture, we have to understand its genesis from its ground within nature, for culture itself is originally natural. The Deadlocks of Ontological Catastrophe 287 interactivity between mind and matter if transcendental idealism is to have a proper founding, which goes in the face of both the Hegelian proof of the insignificance of an opposition between idealistic and realistic philosophy and a Žižekian metaphysics of the disjunctive “and”: For what we want is not that Nature should coincide with the laws of our mind by chance (as if through some third intermediary), but that she herself, necessarily and originally, should not only express, but even realize, the laws of our mind, and that she is, and is called, Nature only insofar as she does so. Nature should be Mind made visible, Mind the invisible Nature. Here then in the absolute identity of Mind in us and Nature outside us, the problem of the possibility of a Nature external to us must be resolved. The final goal of our further research is, therefore, this idea of Nature.469 It is exactly this problematic that reverberates throughout the entirety of Schelling's thinking, even when he attempts to outline the tension- ridden oscillations of the Yes and the No, the light and the dark principles, constitutive of the self-operative logic of the Grund. Rather than offering some kind of paradoxical eruptive logic balancing materialism and idealism through their infinite conflict, Schelling's philosophy can very easily be read as a passionate attempt to show that the forces underlying human spiritual-transcendental activity are nothing more than the already existent potencies of nature arisen to a higher “power” through nature's auto- development. If the starting point of Žižek's transcendental materialism is a self-grounding idealism (which shows his distinctive Lacanian-Hegelian presuppositions), Schelling's own transcendental materialism is a self- articulating realism (which shows how Schelling has been influenced by the evolutionary dynamism of natural scientists such as Kielmeyer), the stark point separating them being that whereas in the former materialism is always a spectral materialism developed in the cracks of idealism, in the latter idealism becomes, as it were, a mere conditioned phenomenon. In this strict sense, if Schelling's philosophy is an account of the self-unfolding of the powers of nature according to their inner movement, it is because it is a speculative realism understood in its original etymological meaning: deriving The Deadlocks of Ontological Catastrophe 289 organizations according to its own cryptic inner process. According to Grant, Schelling's revolutionary thesis is that the only way we can explicate ideality is to see it as just one specific expression of nature's productivity, one among many possible products of nature as a priori. There is just a difference of degree and not of type between, say, atoms, chemicals, and free ethical subjects, insofar as there is a natural history of mind to which we must have recourse to explain its apparent autonomy. Far from being an idealism that grounds itself from within the infinite self-reflexivity of thinking “with no external support of its truth,”473 Schelling's idealism is a naturephilosophical investigation into the interior involutions of being, the latter being equally inclusive of thought's transcendental activity as the somatic constitution of physical bodies. Nature is a dark dynamicity that brings philosophy far away from the practico-concrete sphere of an anthropocentric universe into the enigmatic palpating powers that gave birth to it in their antagonism and the forgotten aeons of the abyssal dregs of cosmic time that have preceded us. In this manner, Grant is taking Schelling literally at his word: organizations according to its own cryptic inner process. According to Grant, Schelling's revolutionary thesis is that the only way we can explicate ideality is to see it as just one specific expression of nature's productivity, one among many possible products of nature as a priori. There is just a difference of degree and not of type between, say, atoms, chemicals, and free ethical subjects, insofar as there is a natural history of mind to which we must have recourse to explain its apparent autonomy. Far from being an idealism that grounds itself from within the infinite self-reflexivity of thinking “with no external support of its truth,”473 Schelling's idealism is a naturephilosophical investigation into the interior involutions of being, the latter being equally inclusive of thought's transcendental activity as the somatic constitution of physical bodies. Nature is a dark dynamicity that brings philosophy far away from the practico-concrete sphere of an anthropocentric universe into the enigmatic palpating powers that gave birth to it in their antagonism and the forgotten aeons of the abyssal dregs of cosmic time that have preceded us. 288 Chapter 12 There is no absolute split between the Real and the Ideal, mind and matter, the dead movement of inanimate objects and the life of organic being—there are only the unconditioned, groundless powers of nature as a pulsating all that creates specific bodies and their various, innumerable, and unpredictable The Deadlocks of Ontological Catastrophe 289 The Deadlocks of Ontological Catastrophe 289 In this manner, Grant is taking Schelling literally at his word: A great work of the ancient world stands before us as an incomprehensible whole until we find traces of its manner of growth and gradual development. How much more must this be the case with such a multifariously assembled individual as the earth! What entirely different intricacies and folds must take place here! Even the smallest grain of sand must contain determinations within itself that we cannot exhaust until we have laid out the entire course of creative nature leading up to it. Everything is only the work of time, and it is only through time that each thing receives its particular character and meaning.474 Although Grant and Žižek are reading Schelling in different, largely incommensurable ways—the former by a remarkable reconstruction of Schelling's naturephilosophical and scientific context, the other by a violent psychoanalytical overhauling of the entire German Idealist tradition—the fact that they both fall upon the same figure to elaborate their own materialist position is extraordinarily thought provoking. First and foremost, it suggests that the role of Schelling in Žižek's thinking poses Chapter 12 290 a potential problem for the latter's own transcendental materialist ontology of the subject, and not merely for external reasons: it challenges the latter's very notion of a metaphysics of the disjunctive “and.” The great contribution of Schelling's Naturphilosophie was to enact a complete displacement of the human subject, for it imposes upon us the counterintuitive task of a geology of morals that fights against any complete separation of the human (the realm of free acting) from the natural (the realm of necessity). The stark implications of this, as Grant himself notes, were clearly perceived by Eschenmayer, a Fichtean natural scientist, who after reading the Freiheitsschrift fell into a paroxysm of horror due to its primary soul- wrenching implication: “your essay on human freedom seems to me a complete transformation of ethics into physics, a consumption of the free by the necessary, of feeling by understanding, of the moral by the natural, and above all a complete depotentiation of the higher into the lower order of things.”475 Commenting on this passage, Grant writes: We can imagine Eschenmayer’s shock: why does this work on the subject of freedom contain so much geology? Why is the turba gentium [...], the world-disorder or species- riots, presented as the ground of freedom? [... The Deadlocks of Ontological Catastrophe 291 contemporary philosophies of finitude—he is in many ways completely justified, but in so doing he risks opening himself to the criticism that he misses how Schelling proceeds in this endeavour as well as its larger philosophical consequences, which presents two immediate major problems in his own usage of Schelling. First, if Grant is correct, Schelling rejects from the outset the very idea of a self-enclosed transcendental framework and its concomitant mind-body dualism, the ontological emergence of which is, according to Žižek, the fundamental philosophical obsession of the middle-late Schelling. If the unconscious Grundlogik of German Idealism is constituted by the dynamic of a self-grounding idealism and a spectral materialism grounded in the former, then Schelling's precise place within it would seem to be uncertain. Second, if Žižek's transcendental materialism assumes the birth of the I out of the not-I as an impossible event with no true precursor in the ancestral past of the pulsating fires of the heavens, the sluggishly slow evolution of geological formations, or even the forces of evolution in biological life-forms (none of its apparent brethren come close to its pure difference), in such a way that the self-positing of freedom literally cuts the absolute in two in an inexplicable manner, then we must conclude that Schelling's own metaphysics does not automatically result in a metaphysics wherein substance is split at its core, and thus ontological dislocation is the primordial fact. Contra Fichte, Schelling explicitly claims that there is a continuity between the I and the not-I, that the passage is one of a transition, not a leap. If psychoanalysis is to work in a therapeutic setting, an encounter with the Real can only truly be brought forth if all the intended meanings of the subject have been understood for what they are; otherwise a psychoanalytical interpretation does not work, because it does not hit the cause (which is why Lacanian analysts rarely offer interpretations). Moreover, even if we agree that Žižek is right to contend that the theosophical content of the middle-late Schelling is a mythological component that we can formalize in order to render its content more explicit (which Grant's work also implicitly does, given that he does not explicitly deal with the theological aspect of the Freiheitsschrift or the Weltalter), it is uncertain that he can so easily get rid of the naturephilosophical implications of these texts. The Deadlocks of Ontological Catastrophe 289 B]ecause the consequences of the dependence of transcendental physics on dynamic naturalism impose upon Schelling’s reconditioned transcendentalism the demand that the All be grounded in the “subject of nature itself,” i.e., in the forces. Accordingly, the more disorderly the phenomenon, the darker and more abyssal the ground. This is why the inquiry into human freedom must (a) specify the attachment of this power of infinite evolution to a finite phenomenon (human), and (b) consider the ground of such a freedom as derivative of the “self-operation of the ground” or the “will of the deep” in the geological series: the potentiating series through which such a freedom must (repeatedly) evolve must therefore present the expression of geological potencies in practical intelligence.476 In this respect, when Žižek claims that it is Schelling who gives the most detailed description of the ontogenesis of subjectivity in the philosophical tradition—which makes him the father of dialectical materialism and The Deadlocks of Ontological Catastrophe 291 The Deadlocks of Ontological Catastrophe 291 But the stakes are much higher than those of the philological-textual fidelity of an interpretation in this case: if Schelling's Chapter 12 292 C 292 philosophy presents a framework incompatible with the one Žižek sees in it and his own psychoanalytical reconstruction extensively relies upon this for its own argumentative vitality, Žižek's own development of a metaphysics of the Real by an engagement with Schelling is not only potentially misguided, but could also lose sight of various other resources explicit in Schelling that could be used to rethink the materialism-idealism relationship. Elaborating on Grant's argument, we see that Schelling completely bypasses the problem of the materialism-idealism relationship because idealism is never a purely self-referential play. The whole concept of a “spectral” materialism just has no place here; speculative philosophy has a stricto sensu non-idealistic foundation. As Grant succinctly puts it, what is at stake is the “impersonal coincidence of the transcendentally generated universal and self-generating nature [... and] Schelling’s hypothesis is, in other words, that there is a naturalistic or physicalist ground of philosophy,”477 a ground that does not implicate an eliminative materialism wherein all is reduced to empirically observable bodies—somatism—but in such a way that genuine philosophy “consists in the dynamic elaboration of the identity of nature and Ideas.”478 In this manner, if one reads Žižek's own transcendental materialism alongside that which Grant develops from his own reactualization of Schelling, one is presented with an alternative to Žižek's own metaphysics of the not-all, one not centred in the Ideal as being's irreconcilable self-division, but rather one based upon the fragile productivity of nature as it contingently and continually takes on new forms (and destroys others), a productivity that in no way has man as its summit, but will create new creatures (and monsters) without end because there is no stasis, but only a restless movement of the depths, a beautiful and macabre dance of great delicacy and improvisation whose actors simultaneously whimper and laugh under its weight. Lacanian psychoanalysis prohibits this Schellingian move because it would require that the chain of signification constitutive of human language be not based on an operationally closed system with no natural grounding, but could actually open up onto the world as it is in its own interior involutions because it would be, as it were, one with it. The Deadlocks of Ontological Catastrophe 293 at a conception of 'nature as subject,'” this investigation is never a mere reconstruction within the ideal series of the Real, but is rather a movement of the Real itself, for “such a philosophical system does not therefore seek a fixed point from which to gain leverage on an external world, nor to rise above it, but is itself a 'genetic' [...] movement in and on this world, unconditionally.”479 Schelling expresses this identity most daringly when he says that “to philosophize about nature means to create nature.”480 “Mental” activity is always already a part of nature and therefore a part of its own auto- development by being one of its emergent attributes, but nature, being the truly and absolutely a priori, is unconditional and thereby guarantees that thought is never limited to the mere ideational or physiological constraints of conditioned particulars insofar as it is an expression of nature's productivity rather than embodied in the fixity of one of its products. In this way, for Schelling, we are primordially “connected” with nature as the pre-Symbolic, insofar as the Real and the Ideal remain identified at an essential level, the consequence being that the subject is not a dialectically non-sublatable in-between that exists as the psychotic withdrawal of the world into self. Accordingly, Žižek's reliance upon Schellingian ontology risks problematizing his own position, for if Žižek draws upon Schelling as a partner for the elaboration of the impossible genesis of the transcendental out of an orgasm of forces within the pre-symbolic Real, this immediately draws our attention to other possibilities of understanding the subject that do not present the latter as an irrevocable moment of ontological catastrophe in the flux of material being. This would force us to rethink the very nature of the psychoanalytical experience and the essence of the disjunctive “and” that is central to Žižek's own parallax ontology—and in this manner, not only does its theoretical first principle begin to tremble, but the very primacy of the Lacanian psychoanalysis that serves as its starting point is put into question. The Deadlocks of Ontological Catastrophe 291 Although we philosophize about nature by “following a procedure of successive unconditioning performed by thought-operations about nature,” a process that allows us to “arrive 294 Chapter 12 We can begin to see why Žižek's proclamation that his project is Hegelian, but never Schellingian (despite the fact that the Freiheitsschrift and the Weltalter contain the most vivid description of the emergence of the Symbolic), is multilayered. First and foremost, Žižek takes radical idealism as the only true beginning for philosophy insofar as we can only interact with the world through the medium of thought, making correlationism basic to our experience. If the cracks within ideality epistemologically enable us to develop a spectral materialism, then the irreducibility of the Real-as-lack paradoxically does not prohibit us from having access to being as the Real-as-excess because this very concept thereby becomes internal to our notional apparatus. In this manner, Žižek is quite justified in saying that his project is Hegelian given that—on his own reading of Hegel at least—it strictly speaking shares this identical starting point and draws from it the exact same consequences, a move that in turn allows him to avoid the problem of expressing too strong a reliance on and debt to Schelling, which could potentially bring his own thinking uncomfortably close to everything he denies: the non-Freudian unconscious (in its Bergsonian, Jungian, Deleuzian, etc., forms), “pre-modern” cosmology, or Romantic theories of nature. Yet, this does not by any means solve the ambiguity of the Hegel-Schelling relationship in his thinking, for as we have seen, what is crucial to Žižek's own reading of Schelling is that Schelling's own theoretico-epistemological framework, at least in the second period of his thinking, is unknown to itself the same as that of Hegel. Although Žižek has to do great violence to Schelling to extract a Hegelian dialectical structure of negativity in his texts, nevertheless the interpretation he presents is extremely internally consistent and methodologically sound. If the thoroughness of a position like Grant's is a challenge to Žižek, then Žižek's own reading of Schelling is equally a challenge to Grant's and other canonical and non-canonical interpretations that exist. The game goes both ways—and because the conceptual terrain within which both operate is vastly different, it is not evident how we are to decide upon the favourability of one interpretation over another However one has to underline that this If the thoroughness of a position like Grant's is a challenge to Žižek, then Žižek's own reading of Schelling is equally a challenge to Grant's and other canonical and non-canonical interpretations that exist. The Deadlocks of Ontological Catastrophe 293 It is highly revelatory that to make use of Schelling for his project in the first place Žižek can only focus on two works (the Freiheitsschrift and the second draft of the Weltalter) because of their apparently disavowed Hegelian structure as that which would enable one to extract from them a self-operative logic establishing the primacy of the Real- as lack through the abyss of unconscious decision The Deadlocks of Ontological Catastrophe 295 tradition, but an unconscious truth that can be seen to deploy itself through them. Accordingly, not only is there absolutely no contradiction in saying that Žižek's philosophy is a hybridism of Hegelian logic and Schellingian ontology insofar as it is precisely this hybridism that can retroactively be seen to be the traumatic core at the formative heart of the tradition itself, but reflecting upon the intrinsic ambiguity of the Hegel-Schelling relationship helps us reveal the originality and daringness of Žižek's critical metaphysics. 294 Chapter 12 The game goes both ways—and because the conceptual terrain within which both operate is vastly different, it is not evident how we are to decide upon the favourability of one interpretation over another. However, one has to underline that this oscillation between Schelling and Hegel is not so much an inconsistency or sleight-of-hand gesture on Žižek's part, the reason being that what interests him is less the specific differences between the historical thinkers of the The Deadlocks of Ontological Catastrophe 295 Chapter 12 However, the very inconsistency of our notional apparatus allows us to develop knowledge of reality in itself, for as soon as we “apply” a category to the world and it shows itself as inadequate, we see that the field of appearing is always more than appearance, whereby the noumenal now appears as the self-limitation of the phenomenal. Our experience of the world is not a full-blown hallucination: we can use these experiences of breakdown to our advantage in order to explore a world that only seems to be infinitely “beyond” our reach. The Real-as-excess becomes an intra-discursive category, so that epistemic limitations of knowledge negatively demonstrate our inclusion into and thus capacity of understanding the world at large, rather than our imprisonment in a socially constructed universe of discourse. It is precisely because of this that Žižek says that “the true problem is not how to reach the Real when we are confined to the interplay of the (inconsistent) multitude of appearances, but, more radically, the properly Hegelian one: how does appearance itself emerge from the interplay of the Real?”481 Second, given that we are in some sense a part of the absolute, our failure to reach it has to coincide with a failure of the absolute itself. Žižek's wager is that if we do fail in reaching the absolute in thought, this cannot be due merely to the finitude of our notional apparatus: “[i]f we can think our knowledge of reality (i.e., the way reality appears to us) as radically failed, as radically different from the Absolute, then this gap (between the for-us and the in-itself) must be part of the Absolute itself, so that the very feature that seemed forever to keep us away from the Absolute is the only feature which directly unites us with the Absolute.”482 The point is not to “'overcome' the gap [...] but to take note of how this gap is internal to [the Absolute]”:483 arguing that our inability to overcome our entrapment in the Symbolic and find our place within being is already the very lost object we are looking for, Žižek turns epistemological limit into positive ontological condition by inscribing the limitation of knowledge into the world as an event immanent in the latter: that is, by making it an (auto-)‍limitation of the absolute itself. 12.2 Anton-Babinski Syndrome: Slavoj Žižek's Paradoxical Overcoming of Idealism Although Žižek's appropriation of Schelling could be perceived as highly problematic, insofar as the latter may not so easily fit into the proto- structuralist framework Žižek sees as operative in German Idealism, another problem immediately arises that potentially hits the core of his overcoming of radical idealism. Even if we accept the legitimacy of his reading of Schelling, it is unlikely that Žižek's own Hegelian attempt to show the insignificance of any absolute opposition between idealist and realist philosophies from within idealism would satisfy a realist. If we never leave the clutches of idealism, then any knowledge that we possess would never be able to reach the absolute in its pure non-correlationality to the subject. We are always entrapped in the masturbatory play of signifiers in their incessant sliding. Moreover, the Real-as-excess as what precedes and exceeds consciousness is an explicitly impossible concept. Even Žižek's “materialist” response to idealism has as its fundamental task to bring to the fore this intrinsic impossibility: remodulating the Schellingian act of unconscious decision, its seeks to show how the shift from the Real-as-excess (pre- symbolic antagonism) to the Real-as-lack (symbolic imprisonment) is the ultimate ontological parallax by a speculative fabulation of the always lost and inaccessible moment of the auto-disruption of the noumenal realm. At its best, realist metaphysics appears to be reduced to mythology. Žižek's overcoming of idealism articulates itself in two distinct moments. First, since the Real-as-lack is the logical zero-level of any philosophy due to the insurmountability of the Symbolic, to arrive at some kind of knowledge of an “extra-”notional zone of experience (the Real-as-excess) Chapter 12 Chapter 12 296 would appear a priori foreclosed. However, the very inconsistency of our notional apparatus allows us to develop knowledge of reality in itself, for as soon as we “apply” a category to the world and it shows itself as inadequate, we see that the field of appearing is always more than appearance, whereby the noumenal now appears as the self-limitation of the phenomenal. Our experience of the world is not a full-blown hallucination: we can use these experiences of breakdown to our advantage in order to explore a world that only seems to be infinitely “beyond” our reach. The Real-as-excess becomes an intra-discursive category, so that epistemic limitations of knowledge negatively demonstrate our inclusion into and thus capacity of understanding the world at large, rather than our imprisonment in a socially constructed universe of discourse. It is precisely because of this that Žižek says that “the true problem is not how to reach the Real when we are confined to the interplay of the (inconsistent) multitude of appearances, but, more radically, the properly Hegelian one: how does appearance itself emerge from the interplay of the Real?”481 Second, given that we are in some sense a part of the absolute, our failure to reach it has to coincide with a failure of the absolute itself. Žižek's wager is that if we do fail in reaching the absolute in thought, this cannot be due merely to the finitude of our notional apparatus: “[i]f we can think our knowledge of reality (i.e., the way reality appears to us) as radically failed, as radically different from the Absolute, then this gap (between the for-us and the in-itself) must be part of the Absolute itself, so that the very feature that seemed forever to keep us away from the Absolute is the only feature which directly unites us with the Absolute.”482 The point is not to “'overcome' the gap [...] but to take note of how this gap is internal to [the Absolute]”:483 arguing that our inability to overcome our entrapment in the Symbolic and find our place within being is already the very lost object we are looking for, Žižek turns epistemological limit into positive ontological condition by inscribing the limitation of knowledge would appear a priori foreclosed. The Deadlocks of Ontological Catastrophe 297 reality; moving from one to the other just requires a certain switch of perspectives, a parallax shift, whose very possibility we can only explain by making the very irreconcilable split between idealism and materialism the imperceptible truth of both. Žižek's name for that which can strangely mediate between them because it is neither idealistic nor materialistic yet is included in both as excluded (“include me out!”) is the subject as an insurmountable ontological and symbolic lacuna. Consequently, the Symbolic is always already more than itself because it points to its dark origins in being's passage through madness—and even if the precise moment in which the world withdraws into its nocturnal, irreal self is forever lost in the universe of meaning it brings forth, we are nevertheless justified in mytho-poetically fabulating the act of decision that induces our collective psychosis, because the Symbolic can never do away with its origins. Although the undecidable ambiguity of the Real is a problem Žižek inherits from Lacan, a problem similar to that faced by many forms of idealism, Žižek throughout his writings remains true to his great master's attempt to desubstantialize the Real, but with an important twist, a twist that accentuates the theoretical challenge of his critical metaphysics. What most clearly distinguishes Žižek's project from that of Lacan is his refusal to take our lack of access to the Real as a brute fact and his subsequent endeavour to inscribe it within being. The question arises, however, of whether Žižek's account of this emergent ontological parallax is even philosophically possible, given his epistemological commitments. Since phenomenal reality emerges only after being has sundered itself, whereby our access to the Real must be mediated by transcendental constitution, Žižek's double claim that the internal inconsistency of idealism is that which allows us to overcome it from within and that our inability to reach the thing itself is already that which we are looking for poses two important difficulties to his project that, for many, may not be adequately resolved or seem outright problematic. On the one hand, to switch epistemological limit into positive ontological condition by a mere parallax shift of perspective appears in many ways to be a mere sleight-of-hand argument. Chapter 12 If idealism is some form of ontological solipsism, then it must be revelatory of a zone wherein the absolute is irrevocably non-coincident to itself. In another vein, this means that any radically self-grounding idealism is always already a materialism, the two being nothing more than supplementary views on the same underlying The Deadlocks of Ontological Catastrophe 297 The Deadlocks of Ontological Catastrophe 297 Given that this precise moment where our division from the absolute coincides with the self-division of the absolute (so that the story we are telling about being is simultaneously the story that being is telling to itself) can only be 298 Chapter 12 Chapter 12 298 narrativized at the level of mytho-poetics, which reconciles substance and subject at the level of form and never at the level of content, it is unclear that the claim that the problem its own solution does the argumentative work it purports to do. To put it bluntly, since this precise moment defies any proper speculative explanation, it merely covers up the underlying issue that we are facing: that is, how we could have access to being in the first place. On the other, it is unclear that we can really collapse the distinction between realism and idealism by making the very distinction itself intra-conceptual or intra-discursive insofar as this move fails to sufficiently provide the conditions under which we could develop a truly speculative account of reality in itself that is not always already entrapped within the ambiguities of symbolization. In both cases, a realist would be quick to argue that we have done everything but leave the correlationalist circle, that we are stuck in a constituted world for us. For the former, it could not be said that we have some kind of “access” to reality through the immanent obstructions of the Symbolic as that which indicates the spectral presence of an extra-notional reality posited from within it, for the Real is a mere effect of the Symbolic. Does this go far enough in establishing the groundwork for a new metaphysics? If we call the Real an internal limit or limitation of the Symbolic, we must be careful, because the Real is not so much a limit in the sense of a border that separates two distinct yet commensurable terrains, or a limitation in the sense of a restricting condition coming from an exterior force that one ought to overcome. Rather, the adjective “internal” is of utmost importance here because it stresses that the Real is completely immanent to the Symbolic's very idealizing activity in such a way that there is no outside except an outside that is paradoxically posited as inside. The Deadlocks of Ontological Catastrophe 297 If the Symbolic functions within a psychotic withdrawal from the world, not only does it freely (re)‍constitute reality according to an autonomous, self-referential play or ciphering, but any obstruction that occurs within it would only be due to its freedom. In its first guise, the Real is nothing other than “a purely formal parallax gap or impossibility,” “the rupture or gap which makes the order of discourses always and constitutively inconsistent and non-totalizable,”484 so that even if we are permitted to call this negative encounter with the Real a “positive running up against” the exterior world that operates as if it “touches the Real,”485 in the same breath we must qualify this statement 300 Chapter 12 Lost in visual madness, they can never indirectly see the world shine through the inconsistencies of their hallucination. Here the case of Anton-Babinski syndrome should be evoked as a possible skeptical argument against Žižek's overcoming of radical idealism. A rare medical phenomenon, the syndrome is a symptom of brain damage (usually from a stroke) in the occipital lobe. What is so peculiar is that people who suffer from it, although cortically blind, claim that they can see. In their speech and general behaviour there is often, at first, no sign of blindness—family members and the medical team typically only begin to notice something is amiss when the patient begins to stumble into various physical objects in their path, whether it be tripping over a coffee table in front of him, walking into a wall, or describing things that are not really there. Not only do patients continue to refuse to admit their blindness despite all the inexorable obstructions in the all-out hallucination of their own visible field of experience, but, more primordially, it is clear that no amount of tarrying with the negative offered by the latter's internal short- circuiting would ever enable them to develop a “spectral” vision of the world in itself of which they have been deprived through organic devastation. To deal with the incomprehensible agony caused by such constant disturbances in their psychotically self-sufficient and imagined perception of subjective reality, those who suffer from Anton-Babinski syndrome actually find ways of giving support to its free generation by falsifying their memories, a process that in the medical community is called confabulation. The Deadlocks of Ontological Catastrophe 299 insofar as this can only be brought forth in the aftermath of symbolic distortion, that is, après-coup. This recognition of an indirect confrontation with a constitutive outside internal to the Symbolic is in itself just another symbolization: any “materialism” that could be developed by means of it will always already be entrapped within its ambiguities, so that this “materialism” is nothing but a mere retroactive adjustment of ideality to accommodate for its internal inconsistencies, for it is only from within the Symbolic that we see the Real as the residue of a failed attempt to synthesize an “extra”-notional reality. As Adrian Johnston puts it: It's not that there is no Real that isn't immanent to the Symbolic. Instead, the non-immanent Real is accessible exclusively through the deadlocks and inconsistencies immanent to the Symbolic [...]. The Real-as-presupposed [the Real-as-excess as posited in/by the Symbolic] actually exists “for us” only insofar as it indirectly shines through the cracks in the façade of Imaginary-Symbolic reality, insofar as it is asymptotically approached by the parlêtre along the fault lines of this reality's inner conflicts.486 But “...indirectly shines through...” is a misleading metaphor: nothing breaks through the prison of language. As Žižek says, “we do not touch the Real by way of breaking out of the prison of language and gaining access to the external transcendent referent [...]. We touch the Real-in-itself in our very failure to touch it.”487 This is what Žižek emphasizes when he posits an ontological passage through madness at the beginning of the Symbolic, for once it has occurred there is no contact with the world that is possible. If we take Lacan and Žižek at their word, we can never truly liberate ourselves from the psychotically self-sustaining construction of reality that is the Symbolic's autonomous idealization. Even if the latter does not equate to an omnipotent, non-limited hallucination of our world of experience—a Godlike primary process—nevertheless there is no escape from our collective hallucination of reality: impenetrable in its density, omnipresent in its extension, nothing is left untouched by this tenebrous realm of transcendental phantasmagoria within which we live and breathe as speaking subjects. The light of being is unable to radiate through the holes of the all- encompassing web of the Symbolic. 300 Chapter 12 Here the case of Anton-Babinski syndrome should be evoked as a possible skeptical argument against Žižek's overcoming of radical idealism. A rare medical phenomenon, the syndrome is a symptom of brain damage (usually from a stroke) in the occipital lobe. What is so peculiar is that people who suffer from it, although cortically blind, claim that they can see. In their speech and general behaviour there is often, at first, no sign of blindness—family members and the medical team typically only begin to notice something is amiss when the patient begins to stumble into various physical objects in their path, whether it be tripping over a coffee table in front of him, walking into a wall, or describing things that are not really there. Not only do patients continue to refuse to admit their blindness despite all the inexorable obstructions in the all-out hallucination of their own visible field of experience, but, more primordially, it is clear that no amount of tarrying with the negative offered by the latter's internal short- circuiting would ever enable them to develop a “spectral” vision of the world in itself of which they have been deprived through organic devastation. To deal with the incomprehensible agony caused by such constant disturbances in their psychotically self-sufficient and imagined perception of subjective reality, those who suffer from Anton-Babinski syndrome actually find ways of giving support to its free generation by falsifying their memories, a process that in the medical community is called confabulation. In other words, even if a patient, realizing their condition, were to think that they are actually in the process of developing a sound mental map of the physical universe that is around them through the aid of their mishaps as a means of retroactively readjusting their imaginary field, and this not only with the hope of learning to navigate within it, but also to overcome their blindness by making the absolute opposition between a hallucinated world produced in the void of blindness and a vision of objective reality caused by retina input without meaning within their hallucination, it must be concluded that they could never assure themselves that this “spectral” seeing captures the world nor whether it is not just another hallucination that has been produced to save themselves from the psychological trauma of their own blindness. The Deadlocks of Ontological Catastrophe 301 What complicates this philosophical issue is the fact that in all mytho-poetic narratives where the very “origins” of the Symbolic in the Real are at stake, the event in question that institutes the movement from one to the other “never effectively took place within temporal reality, [although] one has to presuppose it hypothetically in order to account for the consistency of the temporal process.”488 The result is that the event of the decision that violently separates Grund from existence potentially never occurred: it could be nothing but a fantasmatic, retroactive posit But isn't this precisely the same situation we find ourselves in with respect to Žižek's attempt to break the correlationalist circle? The only possibility for an ontological grounding of the psychoanalytico-Cartesian subject being a phantom-like vision of the world building itself within the internal obstructions of the Symbolic's ciphering of the world, it would seem that just as it is impossible for those suffering from Anton-Babinski syndrome to spectrally construct a vision of the world that has been lost to them due to their lack of sight, so it is impossible that one could achieve some kind of paradoxical coincidence of the subjective and the objective capable of positive truth from within the nocturnal night of the world. If the self-overcoming of radical idealism proves insufficient to ground a new speculative philosophy, then the only alternative left is a pure mytho-poetic fabulation of the obscure origins of the Symbolic, the latter's ontological solipsism always already pointing beyond itself to an inaccessible material event that haunts it but that remains forever inaccessible. Unable to sublate the opposition of realism and idealism from within idealism, we could still, by writing being's poem, provide a mythological account of how our division from being is the same as being's division to itself, thereby hinting towards how this opposition is always already reconciled from within realism, making the problem itself moot: we must tell a story that inscribes our failure to reach the absolute in the absolute itself, so that which appears to keep us from the absolute is in actuality the only thing that ties us to it. Our madness is being's own. 300 Chapter 12 In other words, even if a patient, realizing their condition, were to think that they are actually in the process of developing a sound mental map of the physical universe that is around them through the aid of their mishaps as a means of retroactively readjusting their imaginary field, and this not only with the hope of learning to navigate within it, but also to overcome their blindness by making the absolute opposition between a hallucinated world produced in the void of blindness and a vision of objective reality caused by retina input without meaning within their hallucination, it must be concluded that they could never assure themselves that this “spectral” seeing captures the world nor whether it is not just another hallucination that has been produced to save themselves from the psychological trauma of their own blindness. Lost in visual madness, they can never indirectly see the world shine through the inconsistencies of their hallucination. The Deadlocks of Ontological Catastrophe 301 The Deadlocks of Ontological Catastrophe 301 But isn't this precisely the same situation we find ourselves in with respect to Žižek's attempt to break the correlationalist circle? The only possibility for an ontological grounding of the psychoanalytico-Cartesian subject being a phantom-like vision of the world building itself within the internal obstructions of the Symbolic's ciphering of the world, it would seem that just as it is impossible for those suffering from Anton-Babinski syndrome to spectrally construct a vision of the world that has been lost to them due to their lack of sight, so it is impossible that one could achieve some kind of paradoxical coincidence of the subjective and the objective capable of positive truth from within the nocturnal night of the world. If the self-overcoming of radical idealism proves insufficient to ground a new speculative philosophy, then the only alternative left is a pure mytho-poetic fabulation of the obscure origins of the Symbolic, the latter's ontological solipsism always already pointing beyond itself to an inaccessible material event that haunts it but that remains forever inaccessible. Unable to sublate the opposition of realism and idealism from within idealism, we could still, by writing being's poem, provide a mythological account of how our division from being is the same as being's division to itself, thereby hinting towards how this opposition is always already reconciled from within realism, making the problem itself moot: we must tell a story that inscribes our failure to reach the absolute in the absolute itself, so that which appears to keep us from the absolute is in actuality the only thing that ties us to it. Our madness is being's own. But if such a medium of expression presents itself as the rational necessity of a non-rational discourse to explain discourse as such, then just as those suffering from Anton-Babinski syndrome create false memories to guarantee the consistency of their self-sufficient hallucination, so too does all speculative fabulation risk always being nothing more than a confabulation. The Deadlocks of Ontological Catastrophe 301 But if such a medium of expression presents itself as the rational necessity of a non-rational discourse to explain discourse as such, then just as those suffering from Anton-Babinski syndrome create false memories to guarantee the consistency of their self-sufficient hallucination, so too does all speculative fabulation risk always being nothing more than a confabulation. What complicates this philosophical issue is the fact that in all mytho-poetic narratives where the very “origins” of the Symbolic in the Real are at stake, the event in question that institutes the movement from one to the other “never effectively took place within temporal reality, [although] one has to presuppose it hypothetically in order to account for the consistency of the temporal process.”488 The result is that the event of the decision that violently separates Grund from existence potentially Chapter 12 302 necessary for the internal consistency of our universe of meaning, so that the distinction between philosophy and fantasy/defence mechanism risks being blurred. As a consequence, Žižek's Schellingian “obscurantist idealist” manner of “deducing” this act from the pre-Symbolic could only be true insofar as it gestures towards the fundamental horror underlying subjectivity, just as the empirically false memories unearthed by those with false memory syndrome (being seduced, child sexual abuse) often merely reveal an underlying deadlock haunting a patient (that there is no sexual relationship). In this regard, not only is it unclear how we could truly test one mytho- poetic fabulation against another so as to guarantee their scientificity, but whether they have any metaphysical or ontological merit as such. Given that our freedom means that we are forever stuck within a Given that our freedom means that we are forever stuck within a constitutive psychosis, the withdrawal of the world into the eternal darkness of its irreal self, the very category of truth here has been so starkly modified that Žižek's own philosophy risks undercutting the very ground it seeks. The subject is reduced to a mere spinning in the void of freedom, a void whose very emergence appears to render itself inexplicable and problematize any knowledge of the “outside” world. The Deadlocks of Ontological Catastrophe 301 A realist will not only always find the reduction of the thought/being opposition to an intra-conceptual distinction an insufficient basis for a positive knowledge of the ontological and its vicissitudes, but will also reject myth as a speculative science insofar as correlationism has been preserved rather than overcome, for without the prior self-overcoming of idealism, the best mytho-poetics can do in the framework of a radical subjective idealism is to reconcile substance and subject at the level of mere mythological form rather than that of content. If idealism is co-incident with an ontological passage through madness, how could we develop a form of linguistic thinking able to overcome the psychotic withdrawal from objective reality that appears to be its very meta- transcendental condition of possibility to describe its event in being? The Deadlocks of Ontological Catastrophe a transcendental materialist account of its emergence out of the not-I? Or is it not Fichte who, by refusing to fall upon the speculative potential opened up by the Anstoß and sticking to the internal dynamics of subjectification, ultimately has the last laugh in the history of post-Kantianism as a paradoxical attempt to develop a new metaphysics in the wake of idealism? Could he have uncannily predicted this dilemma? Perhaps it is in this precise sense that we should read Fichte's incomprehension of his critics, an incomprehension designed not so much to show his disgust at the childish laughter of established scholars at the apparent absurdity of his position (“Fichte, do you really think that air and light are a priori transcendental conditions of human freedom?”) as to directly express by public ridicule his own laughter at the absurdity of their position (“Established scholars, you really think you can break free of correlationism and develop a speculative philosophy?”): I tell them that I have given here an a priori deduction of air and light. They answer me: “Air and light a priori, just think of it! Ha ha ha! Ha ha ha! Ha ha ha! Come on, laugh along with us! Ha ha ha! Ha ha ha! Ha ha ha! Air and light a priori: tarte à la crème, ha ha ha! Air and light a priori! Tarte à la crème, ha ha ha! Air and light a priori! Tarte à la crème, ha ha ha!” et cetera ad infinitum. Stunned, I look around me. Where did I lose my way? I thought that I had entered the republic of scholars. Have I fallen into a madhouse instead?489 For Fichte, true madness is not the psychotic withdrawal at the founding gesture of subjectivity, but rather rejecting its implications—in short, acting as if it never happened. 12.3 Fichte's Laughter, Henri Maldiney, and the Necessity of a “Successful” Psychotic Thinking Spinning in the void of freedom—isn't this the Fichtean position? Does Žižek truly succeed in overcoming the theoretical impossibility forced upon us by the pure I and develop what Fichte thought was contrary to reason: namely, The Deadlocks of Ontological Catastrophe The Deadlocks of Ontological Catastrophe If the ontological solipsism of the Ideal reduces all reality to a mere image, so that all “is transformed into a fabulous dream, without there being any life the dream is about, without there being a mind which dreams; a dream which hangs together in a dream of itself,”490 rather than bemoaning the loss of being, we should realize the implications of this inexplicable leap into freedom, that is, that the phenomenal world “absolutely creates itself [...] in a genesis out of nothing.”491 Just like the Fichtean transcendental Wissenschaftslehrer is able to come to the realization that life as we know it is nothing but a void doubled in Chapter 12 304 on itself, a dream of a dream, so too the Žižekian transcendental materialist is able from within the throes of originary psychosis to see this psychosis for what it is. But he does not stop there. While the Wissenschaftslehrer proclaims that the only thing left for us to do is to actively create, through the infinity of imagination, the groundless images necessary to fully actualize our freedom in concrete striving, the transcendental materialist pauses for a moment at this insight: if we can see that our life is a dream of a dream, if we can understand psychosis as psychotic, then there must be a minimal level of distance possible, as it were, between us and the transcendental (re)constitution of reality as a collective hallucination—and it is precisely this distance that enables us to thematize the entire process for what it is both in terms of the internal dynamics of subjectification and its wider inscription within being. In short, the theoretical gesture at the heart of Žižek's project is the following: if we can recognize our symbolic entrapment as entrapment, then our idealist psychosis is not only non-coincident with itself, but must in certain instances be “unable” to fully lock us within its cage. The very reason why we even know that there is a free transcendental constructionism fabricating our world of experience in the first place is that this constructionism fails and is unable to absolutely create itself: radical idealism fails to be radical idealism because it is haunted by seemingly non-ideal constraints, so that in this immanent failure it opens up the space for a new form of materialism insofar as it demonstrates that the Symbolic is always already minimally outside itself. The Deadlocks of Ontological Catastrophe In this sense, the Real-as-lack, as that which was apparently at the very root of the realist objection to being able to overcome correlationism from within idealism, is of irreducible importance since it enables us to enact a metaphysical archaeology of the subject, and thus mytho-poetically fabulate a picture of its emergence from a pre-symbolic antagonism that sets the stage for the free idealization of the world. For otherwise, we cannot explicate how we can see psychosis as psychotic in the first place. It is the only possible Archimedean point from which we could be saved from confabulation by a constant tarrying with its traumatic piercing. Yet a speculative fabulation is merely that—a fabulation: recognizing the limits of rational inquiry for describing the exact moment of withdrawal into self at the commencement of the universe of The Deadlocks of Ontological Catastrophe 305 The Deadlocks of Ontological Catastrophe 305 For their very incompatibility here is due to an extremely close proximity,” for given that the phenomenological aims to be the (self-) thematization of the phenomenal, their very distinction risks dissipating into nothing as a certain undecidability emerges.494 The task of the phenomenological science of psychosis is to let psychosis live in its fullness, to show its true meaning. schizophrenia as the object of a science). But this presupposition is, in fact, a Hegelian presupposition: “let oneself be carried by the very nature of things” is a literal repetition of the definition of science in the Preface of The Phenomenology of Spirit, where it is said that, in the dialectical method, we must let ourselves sink into the content at hand, thereby “letting it move spontaneously of its own nature.”493 We must always presuppose an interior life of the object whose essence will then be freely and spiritually internalized/mediated/idealized by the concept. Even if this presupposition appears harmless, it has a strange consequence in this context because of the specific object under investigation by the phenomenologist: “[i]t is not enough to bear witness to the incompatibility between science and psychosis. For their very incompatibility here is due to an extremely close proximity,” for given that the phenomenological aims to be the (self-) thematization of the phenomenal, their very distinction risks dissipating into nothing as a certain undecidability emerges.494 The task of the phenomenological science of psychosis is to let psychosis live in its fullness, to show its true meaning. It is this methodological ambiguity that in turn enables Maldiney to reap a wealth of resources from the experience of psychosis. For Maldiney, the similarity that Hegel's dialectical method in The Phenomenology of Spirit bears to Binswanger's phenomenological approach to schizophrenia and thus, by implication, to his own to psychosis is not just limited to how they define their way of proceeding. Rather, it can also be seen at the very level of the investigation of their objects, so that there exists an extremely close proximity between the two levels of investigation that is constantly in danger of conflating them, but with an important twist. The Deadlocks of Ontological Catastrophe 305 In this sense, there is a distinction to be made between (the) phenomenal experience (of schizophrenia) and (the) phenomenological experience (of Chapter 12 306 schizophrenia as the object of a science). But this presupposition is, in fact, a Hegelian presupposition: “let oneself be carried by the very nature of things” is a literal repetition of the definition of science in the Preface of The Phenomenology of Spirit, where it is said that, in the dialectical method, we must let ourselves sink into the content at hand, thereby “letting it move spontaneously of its own nature.”493 We must always presuppose an interior life of the object whose essence will then be freely and spiritually internalized/mediated/idealized by the concept. Even if this presupposition appears harmless, it has a strange consequence in this context because of the specific object under investigation by the phenomenologist: “[i]t is not enough to bear witness to the incompatibility between science and psychosis. For their very incompatibility here is due to an extremely close proximity,” for given that the phenomenological aims to be the (self-) thematization of the phenomenal, their very distinction risks dissipating into nothing as a certain undecidability emerges.494 The task of the phenomenological science of psychosis is to let psychosis live in its fullness, to show its true meaning. schizophrenia as the object of a science). But this presupposition is, in fact, a Hegelian presupposition: “let oneself be carried by the very nature of things” is a literal repetition of the definition of science in the Preface of The Phenomenology of Spirit, where it is said that, in the dialectical method, we must let ourselves sink into the content at hand, thereby “letting it move spontaneously of its own nature.”493 We must always presuppose an interior life of the object whose essence will then be freely and spiritually internalized/mediated/idealized by the concept. Even if this presupposition appears harmless, it has a strange consequence in this context because of the specific object under investigation by the phenomenologist: “[i]t is not enough to bear witness to the incompatibility between science and psychosis. The Deadlocks of Ontological Catastrophe 305 intrinsic inaccessibility of its object. Even if such a medium is justified by means of the Symbolic's own failure, which shows itself in a parallax shift of perspective as a disruptive ontological occurrence, we must concede that the actual narrative cannot truly articulate the miraculous advent of subjectivity in the Real. By delving into the impossible, the best it gives us is a sideways glance into the always absent origin. The scientific legitimacy of mytho-poetics relies, for its theoretical force, largely on Žižek's solution to the realism-idealism debate from within idealism. To embark upon a mythologico-metaphysical archeology of the subject is to try to come to terms with the unfathomable zone in between the pure Real and the Symbolic that lies paradoxically in both and neither. But to describe the passage from one to the other is stricto sensu impossible because such a passage that can be nothing other than an unpredictable event that arises ex nihilo within the Real itself and which simultaneously is always already withdrawn from the very logical space that could rationally investigate it. But to see this as an impossibility in the Real (“the leaping point”) and not just of the Symbolic (its “origin” in unconscious decision) presupposes that the question has changed from how we can gain access to the Real through the Symbolic to the ambiguous genesis of the latter out of the former. But has Žižek given us an adequate foothold from within which we can escape correlationism and answer this? Henri Maldiney, a little known French phenomenologist who rethinks human transcendence through the experience of psychosis, can give us some useful if controversial resources to draw out the intrinsically paradoxical nature of this inquiry. Discussing the introduction to Ludwig Binswanger's case studies on schizophrenia,492 Maldiney outlines a peculiarity in the former's phenomenological method. Rejecting the possibility of understanding schizophrenia directly, either through positivistic methodology or an immediate experience (what the person says being inadequate to express their illness), one has, as Biswanger notes, to “let oneself be carried by the very nature of things,” that is, presuppose an inner, self-articulating structuration of the phenomenon that will reveal itself through a careful description as that which lets its phenomenological essence mediate itself to us. The Deadlocks of Ontological Catastrophe 305 In the case of a phenomenology of psychosis, by letting psychosis speak the fullness of its essence by letting oneself be carried away by the in-dwelling logic underlying its phenomenality, what is at stake is not merely to understand how a psychotic crisis is a singular event responsible for the existential demise of an individual, but more primordially how this demise is revelatory of a failed transcendence and thus continues to participate in the very ambiguity and enigma at the heart of transcendence itself, even if in an out-of- joint manner, for “[i]ts dramatic testifies (pathei mathos) to that which is irreducible in man.”495 If this is the case, then we can question psychosis The Deadlocks of Ontological Catastrophe 307 The Deadlocks of Ontological Catastrophe 307 But each object giving way to another object, those who suffer 308 Chapter 12 Chapter 12 from depression are forced to persevere in this indefinite path—a circle without beginning and end, a circle in which their thinking is ensnared, has become their only horizon.497 For Maldiney, the primordial lost object that the Phenomenology searches for is “in reality existence” itself:498 that is, human transcendence as a capacity of welcoming the completely unexpected and utterly new as revealed to us in the very flesh of sensation wherein subject and object are constituted after the fact by means of a single movement in the flux of a pure appearing that knows no bounds and no a priori. Maldiney relates this not only to the originary impression (ursprüngliche Empfindung) that Hölderlin identifies at the origin of his poetry, a primordial experience that demands a complete transformation—an asubjective becoming-other—wherein the world emerges at each moment as something never seen before,499 but also to the pure present that is the true place within which subjectification and temporalization take place.500 As a result, the key to understanding transcendence is not ideational-conceptual mediation (the gnosologic: the encyclopedia, the systematic), but rather sensibility (the pathic: the eruption of the unpredictable, being held out into the Open), which leads Maldiney to claim that the present is ecstatic—not in the sense analyzed by Hegel, but as an event that is “an outpouring, a gushing, of the new. All of this is just to name the Originary. It is the originarity of the present which founds at each moment the reality of time; and it is its novelty that renders time irreversible.”501 Because Hegel's critique of sense certainty excludes any access to this primordial self-giving outside of conceptual mediation, it is as if the very source of all unity and stability has been obstructed. The paradox is that, although consciousness has lost its primordial object, which should lead to psychopathology, “the Hegelian Aufhebung reproduces—in its own register—the transcendence of existence; but it can only reproduce it (in terms of a substitute of an Ersatz)—and this is the decisive characteristic— because existence has already been lost.”502 It is in this manner that the Phenomenology exhibits a contradictory existential form of “successful” depressive thinking. The Deadlocks of Ontological Catastrophe 307 in order to bring to the fore the various existentialia operative in the very process of subjectification and temporalization central to human existence: “[w]hether his illness [maladie] is organic or vesanic, for man it is first of all a human trail; and it is only possible to understand the latter if one first of all knows what it means 'to be man.'”496 In this sense, the experience of psychosis as a failed transcendence is beneficial for coming to terms with what a successful transcendence would be, by opening up the room for its (self-)thematization. In other words, if there is a certain ambiguity between the phenomenal (psychosis) and the phenomenological (its scientific essence) in this case, it is because the latter shows us that the former is always a possibility for us, an intimate potential of being human, so that the advent of the Real (in the sense of Maldiney) at the core of the subject is indifferent to the success or failure of the latter. The Phenomenology, however, does not present us with a failed transcendence whose very failure highlights irreducible features of the drama of existence, as in Biswanger's case studies or Maldiney's own work on psychosis. But neither does it present us with a straightforwardly successful transcendence (if such a thing even exists) for the movement of consciousness it depicts is identical to the fundamental structure of depression, so that the question itself emerges as to how the Phenomenology can even arrive at dynamic unity and stability if the consciousness it describes is intrinsically depressive in nature: The principle of Aufhebung, “to abolish and to preserve,” is consistent with the general scheme of depressive existence. Its double meaning agrees with, amongst other things, the double dimension of the depressive dramatic as explained by psychoanalytic theory, according to which the “relation to the object” serves to signify being-in-the-world with the same unilaterality that we see in Hegel. From the psychoanalytical perspective, depression is constituted by an uncertain relation to the primordial object, to which the subject remains attached even though it has been detached from it. Compelled, after the loss of the primordial object, to search for another, in the quest for a new object it is always in search of the lost object. The Deadlocks of Ontological Catastrophe 309 To say that the Phenomenology is a form of successful depressive thinking is thus to say that it has apparently immanently overcome depression without ever leaving its clutches on existence by encountering the the Phenomenology this ambiguity is expanded from the distinction between the phenomenal and the phenomenological to the distinction between the phenomenal and phenomenological and the investigator within which this distinction is enacted, so that the in-dwelling phenomenological essence of the phenomenality they investigate can be brought forth. Since the phenomenal here is conscious experience in its depressive existence, the investigator, by assuming the phenomenological attitude, is performing an ideal the Phenomenology this ambiguity is expanded from the distinction between the phenomenal and the phenomenological to the distinction between the phenomenal and phenomenological and the investigator within which this distinction is enacted, so that the in-dwelling phenomenological essence of the phenomenality they investigate can be brought forth. Since the phenomenal here is conscious experience in its depressive existence, the investigator, by assuming the phenomenological attitude, is performing an ideal reconstruction of his or her own consciousness with said structure (which is simultaneously a self-construction of a depressive consciousness within the concept) into a scientific knowledge that is the system of experience. In this sense, the phenomenological level cannot be isolated from the phenomenal any more than it can in the phenomenology of psychosis, but now with an important precision to be made: the phenomenal experience is here the direct self-experience of the consciousness that goes through the odyssey that is the Phenomenology, but in such a way that it is as if phenomenal experience becomes phenomenological to itself, thematizes itself, in an act of gaining distance from itself, for the investigator who is exploring what it means to be a depressive consciousness is implicated in the very process. In short, there is ultimately no distinction between us and the object of investigation, because we are participating in the very thing that we are investigating. The Deadlocks of Ontological Catastrophe 309 the Phenomenology this ambiguity is expanded from the distinction between the phenomenal and the phenomenological to the distinction between the phenomenal and phenomenological and the investigator within which this distinction is enacted, so that the in-dwelling phenomenological essence of the phenomenality they investigate can be brought forth. Since the phenomenal here is conscious experience in its depressive existence, the investigator, by assuming the phenomenological attitude, is performing an ideal reconstruction of his or her own consciousness with said structure (which is simultaneously a self-construction of a depressive consciousness within the concept) into a scientific knowledge that is the system of experience. In this sense, the phenomenological level cannot be isolated from the phenomenal any more than it can in the phenomenology of psychosis, but now with an important precision to be made: the phenomenal experience is here the direct self-experience of the consciousness that goes through the odyssey that is the Phenomenology, but in such a way that it is as if phenomenal experience becomes phenomenological to itself, thematizes itself, in an act of gaining distance from itself, for the investigator who is exploring what it means to be a depressive consciousness is implicated in the very process. In short, there is ultimately no distinction between us and the object of investigation, because we are participating in the very thing that we are investigating. Accordingly, if the Phenomenology reveals that the fundamental structure of consciousness is a depressive structure, for those who embark upon it it is simultaneously a transformation of the mode of existing in transcendence that is depression into a unique style of living, wherein the same structure we see in the psychopathological state of depression is made into that which bestows upon consciousness a profound and never-ending source of energy while resting all the while depressive. If the phenomenology of psychosis brings to the fore various existentialia operative in the very process of subjectification and temporalization so that we see a successful transcendence in its contours, the Phenomenology offers a therapeutic realization of a similar kind of vitality that the latter provides, but within its very failure. The Deadlocks of Ontological Catastrophe 307 The problem is only intensified when one takes into account the The problem is only intensified when one takes into account the methodological ambiguity Maldiney emphasizes in any phenomenology of psychosis between the phenomenal and the phenomenological, insofar as in The Deadlocks of Ontological Catastrophe 309 310 Chapter 12 Consequently, we see three structural levels of depression in Hegel's Phenomenology: the basal depressive structure of consciousness—the loss of the primordial object—which is the zero-level of its transcendence as such (originary depression or depression0, failed transcendence); the psychopathological response that brings a particular person to an existential standstill, which we see in psychiatry (clinical depression or depression1); and lastly the dialectical system of experience that, although depressive, is somehow successfully so, since even if its trial insists in experience it becomes a positive basis for a newfound energy and dynamism rather than a catalyst for decay (a form of successful depressive thinking, which is not a successful depression and is different in nature from depression1). Following a suggestion taken up by Jean-Christophe Goddard, we can expand the idea as follows: it is not merely that the Phenomenology is a paradoxical form of successful depressive thinking, but more radically, it sketches a form of successful psychotic thinking in general.503 Insofar as the event to which we are exposed by being held out into the Open in the pathos of sensation is the utterly new and unpredictable of which we can say nothing in advance, to give oneself over to it in its pure self-outpouring must itself be a form of originary psychosis, so that we see two possible responses: either from within an originary psychosis inflicting human transcendence as such we learn to be successfully psychotic or we opt for a path that would end in a pathological response to this primordial experience at the core of our being by levelling out its trauma instead of using it as the ground for the very impetus of one's subjective life in the world (a capacity Maldiney refers to as transpassibility). Just as Hegel's Phenomenology could be described as an attempt to develop a form of successful depressive thinking, so too it could be ventured that Žižek's transcendental materialism may be an attempt to develop a form of successful psychotic thinking, but psychosis taken here not in Maldiney's unique understanding of it, but in the technical definition that Žižek instills it with as the world's withdrawal into its nocturnal self. The Deadlocks of Ontological Catastrophe 309 Accordingly, if the Phenomenology reveals that the fundamental structure of consciousness is a depressive structure, for those who embark upon it it is simultaneously a transformation of the mode of existing in transcendence that is depression into a unique style of living, wherein the same structure we see in the psychopathological state of depression is made into that which bestows upon consciousness a profound and never-ending source of energy while resting all the while depressive. If the phenomenology of psychosis brings to the fore various existentialia operative in the very process of subjectification and temporalization so that we see a successful transcendence in its contours, the Phenomenology offers a therapeutic realization of a similar kind of vitality that the latter provides, but within its very failure. To say that the Phenomenology is a form of successful depressive thinking is thus to say that it has apparently immanently overcome depression without ever leaving its clutches on existence by encountering the crisis head-on and coming out strong. The Deadlocks of Ontological Catastrophe 311 spontaneously of its own nature, that is, by reconstructing it ideally and scientifically in the concept through a mytho-poetic medium. But if the entirety of the Symbolic is some kind of virtual recompensation for this loss of objective reality, then this originary psychosis, essential to what it is to be a subject, must be primordially repressed if it is to be successful. We must forget that the very fabric of culture is nothing but the deluded ravings of the asylum, since otherwise we are confronted with the very monstrosity— ontological catastrophe, the passage of being through madness—that it was meant to cover up. This is precisely why the psychoanalytical experience is of irreducible importance for Žižek, for we could only hope to get beyond the various defence mechanisms underlying the constitution of our fantasmatic reality and catch a glimpse of their abyssal origin if this reality were wrought with piercing holes: if in our everyday lives we are completely lost in the transcendentally hallucinated world fabricated by the Symbolic, then only the upsurge of the Real as indicative of the infinite disharmony between mind and body can enable us to gain the necessary distance towards our self-loss in psychosis and thereby render possible its free (albeit mythological) internalization within the concept. But the Real not only opens up the space necessary for speculative fabulation as a faculty for explicating how we got “trapped” in ontological solipsism; it also lets us find a way, from within our constitutive psychosis, to minimally overcome this very entrapment. As that which is irreducible to the autonomous construction of the world of experience, the Real creates a realist moment within idealism itself. With these two elements achieved, we could be said to have developed a form of successful psychotic thinking: that is to say, a thinking that, from within its own psychosis, would not be limited by the latter in the same way that a form of successful depressive thinking would not be limited by its depression and would have gained a vitality similar to successful transcendence, despite its remaining depressive in structure. A f l h i hi ki h i i i i i ll A successful psychotic thinking—such an expression is intrinsically ambiguous and reveals the insurmountable difficulty any radical idealism has to overcome its own limitations. 310 Chapter 12 Taking as its object the thematization of the inner structure of psychosis, which, since “madness signals the unconstrained explosion at the very core of human being,”504 can only be done in and through psychosis, it seeks to use psychosis to pierce through its own impenetrable dusk by letting it move The Deadlocks of Ontological Catastrophe 311 The Deadlocks of Ontological Catastrophe 311 If our starting point is a self-grounding subjective idealism, we can never truly get “behind” it from within the universe of ideality, even if this idealism is wrought with fracture lines, inner tension, and agonizing cracks, for its symbolic (re)constitution of 312 Chapter 12 reality functions with no need of an “external support of its truth.”505 Instead of representing that by which the light of being sneaks in despite the correlationalist prison, these can only indicate a torsion, an immanent implosion, from within ideal self-enclosure. By thematizing these places of non-coincidence we can perhaps come to terms with our entrapment and see it for what it is (namely, an entrapment). Yet it is not clear we can hope for much more if these skeptical reflections hold true: a successful psychotic thinking does not result in any strict overcoming of the epistemological constraints psychosis imposes upon us; it is only from within the Symbolic that we see the Real as the residue of a failed attempt of synthesis, thereby rendering it always already minimally symbolized, idealized, always already necessarily lost, there being nothing but the Symbolic and its self-referential play. The paradoxical overlapping of the purely subjective and the purely objective is precisely that: paradoxical. It does not allow for any encounter of the pure Real within the Symbolic, but is rather cognizant of the fact that, since the Real is an impossible concept, any attempt to describe its extra-notional character must be done from within its clutches. Knowledge of reality in itself becomes reduced to a retroactive readjustment of the Symbolic. We see this most clearly in the very expression itself. If a successful psychotic thinking is not a successful psychosis (a cure) but still exhibits the fundamental structure of psychosis, just as a successful depressive thinking in no way means that depression has been left behind and is always searching for the lost object, what we have attained would be at best the upper limit of the dialectic of appearance as such, which has finally collapsed upon itself in one great final cry giving voice to a contradictory combination of absolute power and utmost impotence. The Deadlocks of Ontological Catastrophe 311 In recognizing the very limit of ideal synthesis, even if we are in a certain sense minimally beyond it (the “realist” moment) we can only admit the limit as an impasse with no beyond or content (even the “real” moment is completely determined by the throes of idealist entrapment). But is this sufficient to establish a new metaphysics? To speak metaphorically, it is as if, on Žižek's account, in radical idealism we see the four walls that surround us as a barrier not due to some window to the outside by which we know this site as the prison that it is (the ontological solipsism of the constitutive reality functions with no need of an “external support of its truth.”505 Instead of representing that by which the light of being sneaks in despite the correlationalist prison, these can only indicate a torsion, an immanent implosion, from within ideal self-enclosure. By thematizing these places of non-coincidence we can perhaps come to terms with our entrapment and see it for what it is (namely, an entrapment). Yet it is not clear we can hope for much more if these skeptical reflections hold true: a successful psychotic thinking does not result in any strict overcoming of the epistemological constraints psychosis imposes upon us; it is only from within the Symbolic that we see the Real as the residue of a failed attempt of synthesis, thereby rendering it always already minimally symbolized, idealized, always already necessarily lost, there being nothing but the Symbolic and its self-referential play. The paradoxical overlapping of the purely subjective and the purely objective is precisely that: paradoxical. It does not allow for any encounter of the pure Real within the Symbolic, but is rather cognizant of the fact that, since the Real is an impossible concept, any attempt to describe its extra-notional character must be done from within its clutches. Knowledge of reality in itself becomes reduced to a retroactive readjustment of the Symbolic. We see this most clearly in the very expression itself. The Deadlocks of Ontological Catastrophe 313 due to their very shaking we feel that we are incarcerated in an infinitely claustrophobic space from which we will never emancipate ourselves. From the cracks in the wall there may seep through faint, trembling voices, but in the painstaking process of reconstructing their mumbled words they risk becoming identical with those voices that we hallucinate in our solitude. And even if we succeed at spectrally envisioning this outside by a careful translation of their garbled noise into structured speech, could we be said finally to have liberated ourselves by making the very distinction between liberty and imprisonment void? If a successful form of psychotic thinking is an intrinsically paradoxical concept, it is perhaps because radical idealism is itself intrinsically paradoxical. To say that we can never reach reality, that we are forever stuck within the human universe of meaning, is not merely problematic to ordinary natural and scientific consciousness because it goes against our basic intuitions, but in a more primordially discomforting way: for it proclaims an originary withdrawal from objective reality at the very foundation of what it is to be human, thus potentially reducing the world of experience to the rampant free play of phantasmagoria. For someone who adheres to such a radical idealism, it would appear that even if the inconsistency of our notional apparatus may always be insufficient for a realist to overcome the ambiguity of a successful psychotic thinking, and thus to find resources to establish a strong realism from within idealism itself, we can still embark upon a speculative fabulation of ontological catastrophe as the necessary condition of the possibility of the subject. If a successful form of psychotic thinking is an intrinsically paradoxical concept, it is perhaps because radical idealism is itself intrinsically paradoxical. To say that we can never reach reality, that we are forever stuck within the human universe of meaning, is not merely problematic to ordinary natural and scientific consciousness because it goes against our basic intuitions, but in a more primordially discomforting way: for it proclaims an originary withdrawal from objective reality at the very foundation of what it is to be human, thus potentially reducing the world of experience to the rampant free play of phantasmagoria. The Deadlocks of Ontological Catastrophe 311 If a successful psychotic thinking is not a successful psychosis (a cure) but still exhibits the fundamental structure of psychosis, just as a successful depressive thinking in no way means that depression has been left behind and is always searching for the lost object, what we have attained would be at best the upper limit of the dialectic of appearance as such, which has finally collapsed upon itself in one great final cry giving voice to a contradictory combination of absolute power and utmost impotence. In recognizing the very limit of ideal synthesis, even if we are in a certain sense minimally beyond it (the “realist” moment) we can only admit the limit as an impasse with no beyond or content (even the “real” moment is completely determined by the throes of idealist entrapment). But is this sufficient to establish a new metaphysics? To speak metaphorically, it is as if, on Žižek's account, in radical idealism we see the four walls that surround us as a barrier not due to some window to the outside by which we know this site as the prison that it is (the ontological solipsism of the constitutive psychosis of the Symbolic) and could envisage an escape, but rather The Deadlocks of Ontological Catastrophe 313 Chapter 12 314 of its object, for we have fallen into a new age of mythology that has been rationally justified. Yet language is always more language (this is the major consequence of the Real), and if ontology and metaphysics have been rendered impossible, insofar as we can see this fact for what it is, we must have the right to investigate it in a non-rational discourse, no matter what the status of idealism's self-overcoming. Although a transcendental materialism developed après-coup in the enclosed terrain of a radical idealism may always leave the realist unsatisfied as to its very possibility, turning epistemological limit into positive ontological condition via a mytho-poetic narrativization of being's self- division does allow us to make minimal ontological claims that go beyond mythological imagery. If the Symbolic does present itself as quasi-full- blown constructionism of reality that cuts all ties with an extra-notional, extra-linguistic outside, then the very founding gesture of the Symbolic's self-containment must be structurally homologous with that of psychosis understood as a withdrawal from social reality into an irreal self, and thus must attest to an originary madness at the very ontogenetic basis of human subjectivity. In this respect, were one to refuse the move to mythology as a means of overcoming radical idealism as a mere confabulation that assures the consistency of its order by the production of false memories, the latter would nevertheless reveal a deadlock haunting subjectivity: the deadlock of ontological catastrophe. If the Symbolic exists, then the world is, at best, a fragmented totality whimpering under its own weight—a totality that, unable to posit itself as all, in the case of at least one creature (man) is forced to withdraw into a nocturnal irreal self due to a primordial moment of metaphysical trauma in its heart of hearts, thereby establishing the primary role of dislocation in ontology. The Deadlocks of Ontological Catastrophe 313 For someone who adheres to such a radical idealism, it would appear that even if the inconsistency of our notional apparatus may always be insufficient for a realist to overcome the ambiguity of a successful psychotic thinking, and thus to find resources to establish a strong realism from within idealism itself, we can still embark upon a speculative fabulation of ontological catastrophe as the necessary condition of the possibility of the subject. For we must nevertheless be able to explicate the fact that we can see our life as a dream of a dream, our ontological psychosis as psychotic, for the Symbolic fails to posit itself as all, and must therefore be always already minimally outside itself. Just as Schelling declares that many things take place before the beginning proper, so Žižek contends that a lot happens in the indemonstrable Real-as-origin haunting the Symbolic. The abyss of unconscious decision vibrates with an immense energy, and when we catch a glimpse of this impossibility in a mytho-poetics of the Symbolic's genesis, we simultaneously see a glimmer in the corner of our eye of why idealism For we must nevertheless be able to explicate the fact that we can see our life as a dream of a dream, our ontological psychosis as psychotic, for the Symbolic fails to posit itself as all, and must therefore be always already minimally outside itself. Just as Schelling declares that many things take place before the beginning proper, so Žižek contends that a lot happens in the indemonstrable Real-as-origin haunting the Symbolic. The abyss of unconscious decision vibrates with an immense energy, and when we catch a glimpse of this impossibility in a mytho-poetics of the Symbolic's genesis, we simultaneously see a glimmer in the corner of our eye of why idealism is such an explosive event in being, despite the ontological solipsism of idealism itself. But such an epiphany is limited by the very impossibility Chapter 12 The Deadlocks of Ontological Catastrophe 315 declares that something must have gone horribly wrong in the life of the absolute: for no God, no divine nature, could have wanted this to be our fate, since for both, our fate is tied up with theirs. Chapter 12 Even if we might not be able to say more without writing a great fabulative epic, many details of which may succumb to confabulation and fantasmatic figures, the mere existence of a radically self-grounding idealism would not only demonstrate that reality split itself into two irreconcilable logical zones insofar as thought must exist, but more primordially that ontological catastrophe, whereby being is infinitely divided from itself and doomed to wander in a world of images that are images of nothing, is irreducible in the transcendental explanation of the conditions of the possibility of experience and the Symbolic. Idealism The Deadlocks of Ontological Catastrophe 315 Notes 464. Žižek, Less Than Nothing, p. 314. 465. Ibid., pp. 234–35. 465. Ibid., pp. 234–35. 465. Ibid., pp. 234–35. 466. Schelling, Darstellung, I, 4, p. 106. 466. Schelling, Darstellung, I, 4, p. 106. 467. Schelling, Freiheitsschrift, p. 232. 467. Schelling, Freiheitsschrift, p. 232. 468. Schelling, Stuttgart Seminars, pp. 216ff. 469. Schelling, Ideas for a Philosophy of Nature, trans. Errol E. Harris and Peter Heath (Cambridge: Cambridge University Press, 1995), pp. 41–42. 470. Schelling, Freiheitsschrift, p. 236. 470. Schelling, Freiheitsschrift, p. 236. 471. Grant, Philosophies of Nature after Schelling, pp. viii–ix. 472. Ibid., p. ix. 472. Ibid., p. ix. 473. Žižek, Less Than Nothing, p. 77. 474. Schelling, Weltalter II, pp. 121–22. 475. Schelling, “Briefwechsel mit Eschenmayer bezüglich der Abhandlung ‘Philosophische Untersuchungen über das Wesen er menschlichen Freiheit’,” Schellings sämmtliche Werke, I, 8, p. 150; translation taken from Iain Hamilton Grant, Philosophies of Nature after Schelling, p. 202. 476. Grant, Philosophies of Nature after Schelling, pp. 202–3. 477. Ibid., p. 2. 477. Ibid., p. 2. 478. Ibid., p. 62. 478. Ibid., p. 62. 479. Ibid., pp. 1–2. 479. Ibid., pp. 1–2. 480. Schelling, First Outline of a System of the Philosophy of Nature, trans. Keith R. Peterson (Albany: State University of New York Press, 2004), p. 14. 481. Žižek, The Parallax View, p. 106. 481. Žižek, The Parallax View, p. 106. 482. Žižek and Woodard, “Interview,” The Speculative Turn: Continental Materialism and Realism, pp. 412–13. 483. Ibid., p. 413. 483. Ibid., p. 413. 484. Ibid., p. 409. 484. Ibid., p. 409. 485. Cf. Johnson, Žižek’s Ontology, p. 148. 485. Cf. Johnson, Žižek’s Ontology, p. 148. 486. Ibid., p. 152. 486. Ibid., p. 152. 487. Žižek, Less Than Nothing, p. 959. 487. Žižek, Less Than Nothing, p. 959. 488. Žižek, The Indivisible Remainder, p. 19. 316 Chapter 12 316 Chapter 12 489. Fichte, Fichte: Early Philosophical Writings, pp. 347–48. Also quoted by Žižek, “Fichte’s Laughter,” in Madness, Mythology and Laughter, p. 160, but with a differ- ent interpretation than what I offer here. 490. Fichte, The Vocation of Man, p. 64. For my discussion, see chapter 6. 491. Fichte, Wissenschaftslehre 1805, pp. 127–28. 491. Fichte, Wissenschaftslehre 1805, pp. 127–28. 492. Maldiney, Penser l’homme et la folie, p. 9. 492. Maldiney, Penser l’homme et la folie, p. 9. 493. Hegel, Phenomenology of Spirit, p. 36; cited by Maldiney, Penser l’homme et la folie, p. 263. 494. Goddard, Mysticisme et folie, p. 83. 494. Goddard, Mysticisme et folie, p. 83. 495. Maldiney, Penser l’homme et la folie, p. 7. 496. Ibid., p. 263. 497. Ibid., p. 27. Ibid., p. 27. Ibid., p. 29. 498. Ibid., p. 29. 498. Ibid., p. 29. 499. 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Levi Bryant, Nick Srnicek, and Graham Materialism and Realism. Ed. Levi Bryant, Nick Srnicek, and Graham Harman. Melbourne: re.press, 2011. 406–15. Harman. Melbourne: re.press, 2011. 406–15. Žižek! Dir. Atra Taylor. DVD. Zeitgeist Video: 2007. Žižek! Dir. Atra Taylor. DVD. Zeitgeist Video: 2007. This book is an original investigation into Slavoj Žižek’s return to German Idealism in the wake of Lacanian psychoanalysis. As is well known, Žižek creates productive friction between these traditions by isolating their mutually compatible notions of the death drive, paving the way for Žižek’s highly original model of the subject. Joseph Carew systematizes the stark metaphysical conse- quences of Žižek’s account. If the emergence of the Symbolic out of the Real marks the advent of a completely self-enclosed struc- tural system, then we must posit the absolute This book is an original investigation into Slavoj Žižek’s return to German Idealism in the wake of Lacanian psychoanalysis. As is well known, Žižek creates productive friction between these traditions by isolating their mutually compatible notions of the death drive, paving the way for Žižek’s highly original model of the subject. Joseph Carew systematizes the stark metaphysical conse- quences of Žižek’s account. If the emergence of the Symbolic out of the Real marks the advent of a completely self-enclosed struc- tural system, then we must posit the absolute as a fragile not-all wrought by negativity and antagonism. OPEN HUMANITIES PRESS Cover design by Katherine Gillieson · Illustration by Tammy Lu OPEN HUMANITIES PRESS Cover design by Katherine Gillieson · Illustration by Tammy Lu Cover design by Katherine Gillieson · Illustration by Tammy Lu
https://openalex.org/W4206509121	https://www.scielo.br/j/rbepop/a/YkpnS7DL6m6tWnnKj5KskXR/?lang=en&format=pdf	English	null	The life table paradox: has Brazil already overcome it?	Revista brasileira de estudos de população	2,021	cc-by	12,130	DOI http://dx.doi.org/10.20947/S0102-3098a0179 ORIGINAL ARTICLE DOI http://dx.doi.org/10.20947/S0102-3098a0179 ORIGINAL ARTICLE All the contents of this journal, except where otherwise noted, is licensed under a Creative Commons Attribution License * Universidade Federal de Pernambuco (UFPE), Recife-PE, Brazil (filipe.costas@ufpe.br; https://orcid.org/0000-0001- 9903-5403). Introduction Period life expectancy at birth and infant mortality are the two main social indicators of public health, development and wellbeing of a population (VAUPEL, 2010; SIMEONI; FROVA; DE CURTIS, 2019; GISBERT, 2020). Historically, reductions in child and infant mortality, especially due to reduction in infectious diseases (MATHERS et al., 2015), promoted major positive effects in increasing life expectancy at birth and in reducing the variation in length of life distribution (SMITS; MODEN, 2009). Dubey, Ram and Ram (2015) affirm that, ideally, life expectancy should be a monotonically decreasing function of age, so its maximum could be attained at age zero. However, according to Canudas-Romo and Becker (2011) this characteristic only started to be observed in the most developed nations in the second half of the twentieth century, more precisely, in 1957, for Icelandic females. i Canudas-Romo and Becker (2011) define an imbalanced life table as a life table where the complete life expectancy at a given age x is lower than the complete life expectancy in a given age y, with x < y. Traditionally, this imbalance is observed between life expectancy at birth and at age one, due to the high rates of infant mortality. According to Rowland (2003), this counterintuitive situation is known as the paradox of the life table, and it is still observed in populations in the middle (or earlier stages) of the demographic transition. Therefore, the world can be divided in countries/regions that have already overcome the paradox and those that have not (CANUDAS-ROMO; BECKER, 2011). Although Brazil has achieved important improvements in health and socioeconomic development over the past decades (BOING et al., 2020), Borges (2017) adverts that life expectancy at birth is still greatly impacted by infant mortality (and, therefore, may provide a poor measure of lifespan), and it is still far from the level of most developed nations (ALVAREZ; ABURTO; CANUDAS-ROMO, 2020). Additionally, Brazil is one of the most unequal nations around the globe (BOING et al., 2020), and persistently marked by social and regional inequalities (BOCCOLINI; SOUZA JUNIOR, 2016). The Brazilian Constitution, in Article 196, establishes health as a universal right and a responsibility of the State, which should guarantee equitable and universal access to healthcare. Therefore, regional and social inequalities in health conditions are unacceptable and should be continuously combated (SIMEONI; FROVA; DE CURTIS, 2019). The life table paradox: has Brazil already overcome it? Filipe Costa de Souza* Ideally, life expectancy should be a decreasing function of age. When this fact is not observed, this situation is known as the life table paradox. This paper investigated the timing (and health metrics at the time) in which Brazil and its Federation Units (FU) overcame (or are expected to overcome) this paradox. The data were gathered from the Brazilian Institute of Geography and Statistics and contained 3,416 sex-specific abridged life tables, from 2000 to 2060. At national level, females and males overcame the paradox in 2016 and 2018, respectively. However, when the FU were examined separately, much heterogeneity was observed. Through the decomposition analysis of the change over time in the difference between life expectancy at birth and at age one, we found that Brazil and most of its FU are expected to have both changes declining over time and the total change is expected to be decreasing and greater than zero. Nevertheless, for some Northeastern states the total change is expected to pass from a positive to a negative value; and for two Northern states the total change is expected to be neither decreasing nor increasing. In a public planning perspective, we understand that achieving balancing in the life tables is a goal to be pursued, especially because having an imbalanced table means that life expectancy at birth is still strongly influenced by high levels of infant mortality. Therefore, this knowledge could help planners to properly define strategies to accelerate the balancing process and revert unequal scenarios. Keywords: Life tables. Life expectancy. Infant mortality. Health inequalities. Regional Inequalities. Decomposition. R. bras. Est. Pop., v.38, 1-23, e0179, 2021 Souza, F.C. The life table paradox: has Brazil already overcome it? The life table paradox: has Brazil already overcome it? Introduction In this context, and inspired by the work of Canudas-Romo and Becker (2011), this article aims at investigating the timing (and health metrics at the moment) in which Brazilian Federation Units overcame (or are expected to overcome) the life table paradox, and at exploring regional and sex health inequalities. To that end, three main health metrics were used in this study: the period life expectancy at birth and at age one, and the infant mortality. In addition, to make this investigation possible, we used official sex-specific abridged life tables, from 2000 to 2060, for Brazil and each one of its 27 Federation Units. R. bras. Est. Pop., v.38, 1-23, e0179, 2021 2 2 Souza, F.C. The life table paradox: has Brazil already overcome it? To understand the balancing process of each life table under study, and how it is expected to behave over time, we performed the Kitagawa’s decomposition analysis. Moreover, to provide an idea of the estimated (and projected) magnitude of the health inequality for males and females in Brazil during the period under investigation, which also allows for a discussion about the convergence/divergence process in health indicators among the Federation Units, we also calculated the gaps between best- and worst-practices in life expectancy (OEPPEN; VAUPEL, 2002) at birth and in infant mortality. Besides the importance of providing new points of view about longitudinal investigations of mortality, by discussing the time of the crossing between life expectancy at birth and at age one, and the role of infant mortality in this process; this investigation could provide relevant information for public planners, once having balanced life tables is a goal to be pursued. This becomes clear, especially for the regions still in the balancing process, because, as stated by Gisbert (2020), even though reductions in mortality levels in any age imply an increase in life expectancy at birth, when those improvements are caused by reduction in mortality at young ages (particularly at the first year of life), they also cause a reduction in the variability (inequality) of the length of life distribution. This type of inequality is sometimes viewed as the worst type of inequality a person could suffer, especially because it is impossible to transfer life time from a person to another, and because all other types of inequalities require the person to be alive (GISBERT, 2020). R. bras. Est. Pop., v.38, 1-23, e0179, 2021 Data According to Shkolnikov (2017, p. 1), “life tables are the oldest and most important tool in demography”, and their projections are also essential for social planning, providing, at least, a first impression of the mortalities trends to come (SOUZA, 2020). For the investigation, we used sex-specific life tables, from 2000 to 2060, for Brazil and each one of its 27 Federation Units, totalizing 3,416 abridged period life tables. The data were gathered freely, on June 16th, 2020, from the official website of the Brazilian Institute of Geography and Statistics − IBGE (https://www.ibge.gov.br/), and were based on the 2013 and the 2018 population projections of Brazil and its Federation Units (IBGE, 2013a, 2018a). The 2013 edition provided all the life tables from 2000 to 2009, while the 2018 edition provided the remaining official tables. The 2013 edition of the population projection incorporated the results of the 2010 Population Census and the most recent information on the components (mortality, fertility and migration) of demographic growth. The mortality component remained the same in the 2018 projection (IBGE, 2018b). In the 2013 projection, three main datasets were consulted to construct the life tables: the Census, the Vital Statistics from IBGE, and the Mortality Information System from Brazilian Ministry of Health (IBGE, 2013b). After analyzing the R. bras. Est. Pop., v.38, 1-23, e0179, 2021 3 3 Souza, F.C. The life table paradox: has Brazil already overcome it? number of deaths captured by each dataset and the expected mortality pattern for the Brazilian population, the data from the Mortality Information System were selected for the construction of the life tables (IBGE, 2013c). To construct the projected life tables, for Brazil and each Federation Unit, a continuous reduction of the mortality levels was assumed, reaching the level of the most long-lived populations in 2100, according to the life tables provided by the U.S. Bureau of the Census Health (IBGE, 2013b). Additionally, to correct underreporting of deaths (above and below age one) direct and indirect demographic methods were used. Data The resulting life tables were abridged for the ages of 0, 1, 5, 10, …, 85 and 90 years and above and contained information such as: the number of persons alive at exact age x, lx; the number of deaths between exact ages x and x + n, ndx; the age-specific death rate between exact ages x and x + n, nmx; the number of person-years lived between exact ages x and x + n (which could also be interpreted as the average number of persons alive between exact ages x and x + n), nLx; the complete period life expectancy at exact age x, ex; among other life table metrics. The 2013 and 2018 Brazilian population projections are fundamental to providing information for public and private planning. Readers interested in understanding how those projections were performed and learning details about the construction of the abridged life tables will benefit from the reading of IBGE (2013b, 2013c, 2018b). Methods Canudas-Romo and Becker (2011) proved that the crossover between e0 and ex occur when e0 = ex = 1/xm0, and, in particular, since imbalanced life tables are typically characterized by the fact that e0 < e1, then the age-specific death rate between exact ages 0 and 1, 1m0, is a fundamental metric in studying the life table paradox. Following Canudas- Romo and Becker (2011, p.118), we also refer to 1m0 as infant mortality (IM). i For Brazil and its 27 Federation Units, initially, it was verified (for males and females) the calendar year in which e0 became (or is expected to become) strictly higher than e1, and the values of e0, e1 and IM on that year. Those metrics allow for the comparison of the moment when each location overcame (or is expected to overcome) the life table paradox, and the analysis of important health indicators, which could help identify regional and sex inequalities (CANUDAS-ROMO; BECKER, 2011). Additionally, for each location, we also computed the gap between the year in which the female life table became (or is expected to become) balanced and the year in which the male life table became (or is expected to become) balanced. This gap (female minus male) was called Year Gap. Following Canudas-Romo and Becker (2011) and Dubey, Ram and Ram (2015) we also performed Kitagawa’s decomposition analysis to examine “the components of the change over time in the difference in life expectancies (at birth minus at age one)” (CANUDAS- ROMO; BECKER, 2011, p. 120). In this perspective, let t1 and t2 be two calendar years, with t2 > t1, and, for a given location, let ex(ti) be the period life expectancy at age x and 4 R. bras. Est. Pop., v.38, 1-23, e0179, 2021 4 The life table paradox: has Brazil already overcome it? Souza, F.C. Souza, F.C. at calendar year ti, with the same logic being applied to 1L0(ti) and to 1d0(ti). Moreover, at any calendar year, l0 is a constant equal to 100,000 (according to the value arbitrated in Brazilian official tables). So, the difference between e0 and e1 over time is decomposed as follows (CANUDAS-ROMO; BECKER, 2011; DUBEY; RAM; RAM, 2015): [e0(t2) e1(t2)] [e0(t1) e1(t1)] 1L0(t2) 1L0(t1) l0 1d0(t1) 1d0(t2) l0 . e1(t1) e1(t2) 2 e1(t1) e1(t2) l0 . Methods 1d0(t1) 1d0(t2) 2 (1) According to the authors, the terms between the first pair of brackets on the right side of the equation represent the change occurring below age one (A), while the terms between the second pair represent the change above age one (B). In this study, we investigated the difference between e0 and e1 per decade, focusing on the trends of each component (A and B) and how they impact the total change over time (A + B). f Finally, to offer an idea of the magnitude of the health inequality experienced (and that is expected to be experienced) for males and females in Brazil over time, and to allow for a debate of the convergence/divergence process in health indicators (VALLIN; MESLÉ, 2004; MESLÉ; VALLIN, 2017) among Brazilian Federation Units, we also computed the male and female differences between the best- and worst-practice life expectancies (best minus worst), BW Gap; and between best- and worst-practice infant mortalities, IM Gap. Our definition of best -and worst- practices is based on Oeppen and Vaupel (2002) and, in this paper, for a given calendar year, the worst-practice life expectancy in Brazil is defined as the lowest life expectancy at birth among Brazilian Federation Units. The best-practice life expectancy, on the other hand, is the highest one. The worst-practice IM is the highest IM among Brazilian Federation Units, while the best-practice IM is the lowest one. R. bras. Est. Pop., v.38, 1-23, e0179, 2021 Results According to the official life tables used in this article, at national level, females were the first to overcome the life table paradox in 2016, while for the males this happened two years later, in 2018. However, as it is well known, Brazil is a very unequal country and, evidently, this is also reflected in the timing in which each of its Federation Units overcame (or is expected to overcome) the paradox. Table 1 presents, for males and females and for Brazil and its Federation Units, a summary of the year when the life tables became (or are expected to become) balanced, i.e., the calendar year in which we start (or we are expected to start) to observe that e0 is greater than e1 and other health indicators at that moment. The Federal District alongside with two Southern states (Santa Catarina and Rio Grande do Sul) were the first ones to have female balanced life tables in Brazil, in 2004. By 2006 and 2012 all states from the South and Southeast regions, respectively, had already overcome the life table paradox for the females. For males, the first state to overcome the paradox was also Rio Grande do Sul in 2006, followed by the Federal District and Santa Catarina in R. bras. Est. Pop., v.38, 1-23, e0179, 2021 5 5 Souza, F.C. The life table paradox: has Brazil already overcome it? 2007. Additionally, by 2013, all states from the South and Southeast regions had already balanced their life tables. On the other hand, until 2019, none of the states from the Midwest (with the exception of females from Mato Grosso do Sul) or from the North regions has already experienced the crossover between life expectancy at birth and at age one, which is also true for most of the Northeastern states, with the exception of Pernambuco and Ceará, for both males and females, and females from Rio Grande do Norte. Moreover, the state of Amapá has no value at Table 1 because until 2060 (the last year with official life table projections for each Federation Unit) the life expectancy at age one (81.57 for females and 76.07 for males) is still expected to be higher than the life expectancy at birth (81.28 for females and 75.81 for males). This fact also prevented us from calculating the expected gap in the timing of balancing between Federation Units. R. bras. Est. Pop., v.38, 1-23, e0179, 2021 Results However, even without the precise expected timing of balancing for Amapá, we can compute the median calendar year of balancing for females and males, which are 2020 and 2022, respectively. TABLE 1 Male and female year, e0, IM, when the life tables became (or are expected to become) balanced, and the Year Gap Brazil and Federation Units Location (acronyms) Male Female Year Gap Year e0 IM Year e0 IM Brazil (BR) 2018 72.74 13.47 2016 79.31 12.32 2 North Rondônia (RO) 2045 72.62 13.65 2041 78.61 12.62 4 Acre (AC) 2029 73.71 13.42 2023 79.24 12.61 6 Amazonas (AM) 2038 72.39 13.75 2025 77.53 12.66 13 Roraima (RR) 2038 73.88 13.53 2049 79.98 12.46 -11 Pará (PA) 2029 70.24 14.01 2020 77.16 12.77 9 Amapá (AP) - - - - - - - Tocantins (TO) 2022 71.88 13.78 2025 78.71 12.47 -3 Northeast Maranhão (MA) 2030 70.39 13.96 2028 77.37 12.68 2 Piauí (PI) 2028 68.56 14.57 2031 78.15 12.68 -3 Ceará (CE) 2019 70.55 13.86 2018 78.26 12.34 1 Rio Grande do Norte (RN) 2023 73.17 13.53 2016 79.77 12.46 7 Paraíba (PB) 2020 70.47 13.82 2022 78.67 12.47 -2 Pernambuco (PE) 2016 69.92 14.20 2014 77.06 12.41 2 Alagoas (AL) 2023 69.21 14.19 2025 79.09 12.64 -2 Sergipe (SE) 2021 69.65 14.18 2020 77.91 12.69 1 Bahia (BA) 2023 70.43 14.10 2026 80.21 12.45 -3 Southeast Minas Gerais (MG) 2013 73.46 13.50 2012 79.17 12.47 1 Espírito Santo (ES) 2010 71.90 13.76 2008 79.32 11.85 2 Rio de Janeiro (RJ) 2013 71.53 13.83 2011 78.23 12.64 2 São Paulo (SP) 2009 72.14 13.66 2006 78.29 12.68 3 (continue) TABLE 1 Male and female year, e0, IM, when the life tables became (or are expected to become) balanced, and the Year Gap Brazil and Federation Units TABLE 1 R. bras. Est. Pop., v.38, 1-23, e0179, 2021 6 The life table paradox: has Brazil already overcome it? Souza, F.C. Results Location (acronyms) Male Female Year Gap Year e0 IM Year e0 IM South Paraná (PR) 2010 71.87 13.47 2006 77.04 12.96 4 Santa Catarina (SC) 2007 72.27 13.59 2004 77.74 12.66 3 Rio Grande do Sul (RS) 2006 71.01 13.94 2004 77.62 12.61 2 Midwest Mato Grosso do Sul (MS) 2021 73.35 13.49 2017 79.47 12.51 4 Mato Grosso (MT) 2032 74.42 13.32 2026 79.91 12.35 6 Goiás (GO) 2022 71.96 13.83 2020 78.20 12.54 2 Federal District (DF) 2007 71.45 13.98 2004 77.76 12.81 3 Source: Author’s calculations based on the Brazilian Institute of Geography and Statistics (https://www.ibge.gov.br/). (continued) It is also interesting to highlight that in the year 2000, the crossing between life expectancy at birth and at age 5 had already occurred in all Federation Units but Alagoas. For this Northeastern state, this situation endured until 2000 for females, where e0 = 68.50 and e5 = 68.60, and until 2003 for males, where e0 = 61.67 and e5 = 61.97. For all Federation Units but Amapá, the balancing of the life tables is expected to occur, for males, in the range of life expectancy at birth of 68.56 (in Piauí) and 74.42 (in Mato Grosso) and, for females, of 77.04 (in Paraná) and 80.21 (in Bahia). Moreover, those ranges tend to be higher since, in 2060, as was pointed out, the male and female life expectancy at birth in Amapá are expected to be 75.81 and 81.28, respectively. In turn, for all Federation Units but Amapá, the level of infant mortality is expected to vary from 13.32 (in Mato Grosso) to 14.57 (in Piauí) deaths per thousand live births, for males, and from 11.85 (in Espírito Santo) to 12.96 (in Paraná), for females. For Amapá, in 2060, the projected infant mortality levels for males and females are 16.62 and 15.81, respectively. Moreover, the correlations between life expectancy at birth and the IM at the timing of the balancing is negative (and statistically significant, p-value < 0.05) for both males (-0.93) and females (-0.55). It is also worth noticing that, for Brazil and all its Federation Units that overcame (or are expected to overcome) the life table paradox by 2060, females had higher life expectancies at birth and lower infant mortality levels than males at the timing of balancing, regardless of who (males or females) achieved (or are expected to achieve) the balancing first. R. bras. Est. Pop., v.38, 1-23, e0179, 2021 Results In fact, from 2000 to 2060, the highest male life expectancy at birth at the time of balancing is lower than 74.5 years, while the lowest female life expectancy at birth at the timing of balancing is higher than 77 years. Figure 1 shows males and females scatterplots of the timing of balancing versus the life expectancy at birth at that time. In each plot, Brazil and its five macroregions are highlighted using different colors to make regional clusters easier to spot. From those plots we can clearly observe regional clusters, especially for males, reinforcing some results already discussed. R. bras. Est. Pop., v.38, 1-23, e0179, 2021 7 7 The life table paradox: has Brazil already overcome it? Souza, F.C. FIGURE 1 Males and females scatterplots of the timing of balancing vs. the life expectancy at birth at that tim Brazil and Federation Units – 2000-2050 Males 68.00 69.00 70.00 71.00 72.00 73.00 74.00 75.00 2000 2005 2010 2015 2020 2025 2030 2035 2040 2045 2050 Brazil North Northeast Southeast South Center-West BR RO AC AM RR PA TO MA PI RN AL CE MG SC RJ PR RS MS MT GO DF Life expectancy at birth SP ES SE PE BA PB 76.50 77.00 77.50 78.00 78.50 79.00 79.50 80.00 80.50 2000 2005 2010 2015 2020 2025 2030 2035 2040 2045 2050 Life expectancy at birth Brazil North Northeast Southeast South Center-West BR RO AC AM RR PA TO MA PI RN AL CE MG SC RJ PR RS MS MT GO DF SP ES SE PE BA PB Females Source: Author’s calculations based on the Brazilian Institute of Geography and Statistics (https://www.ibge.gov.br/). FIGURE 1 Males and females scatterplots of the timing of balancing vs. the life expectancy at birth at that time Brazil and Federation Units – 2000-2050 Males 68.00 69.00 70.00 71.00 72.00 73.00 74.00 75.00 2000 2005 2010 2015 2020 2025 2030 2035 2040 2045 2050 BR RO AC AM RR PA TO MA PI RN AL CE MG SC RJ PR RS MS MT GO DF Life expectancy at birth SP ES SE PE BA PB Males Source: Author’s calculations based on the Brazilian Institute of Geography and Statistics (https://www.ibge.gov.br/). Results Another point that deserves our attention is that in six states (Roraima and Tocantins, from the North, and Piauí, Paraíba, Alagoas and Bahia, from the Northeast), which still are in the process of balancing their life tables, male tables are expected to achieve balancing before the female ones. Moreover, the highest differences are observed in the North region. While in Amazonas females are expected to achieve balancing 13 years before males, in Roraima males are expected to overcome the paradox 11 years before females. f When we investigated the difference between e0 and e1 over time (per decade, from 2000 to 2060) and its components, by the decomposition analysis, it was possible to R. bras. Est. Pop., v.38, 1-23, e0179, 2021 8 8 The life table paradox: has Brazil already overcome it? Souza, F.C. identify three main types of trends: i) Both changes below age one (A) and changes above age one (B) decline over time and, for all periods under study, \|A\| > \|B\| and A + B (the total change over time) is decreasing and greater than zero; ii) Both A and B decline over time, but for the first periods \|A\| > \|B\|, and gradually this situation changes to \|B\| > \|A\|, so A + B is decreasing and passes from a positive to a negative value; iii) Both changes A and B decline over time, and for all periods under study \|A\| > \|B\|, but A + B is neither decreasing nor increasing, and might fluctuate over time or presents a bell shape. Moreover, for all periods under analysis, for both males and females and for Brazil and all its Federation Units, we have that A > 0 and B < 0. Examples of those types can be observed in Table 1 in the Appendix (Roraima (RR) – type iii; Pernambuco (PE) – type ii and São Paulo (SP), Rio Grande do Sul (RS), Distrito Federal (DF) and Brazil (BR) – type i). In all datasets, Roraima and Amapá are the only two states of type iii. In both cases, for males, the total change fluctuates over time, while for females, the total change initially increases, reaching a maximum in 2020 and 2030, respectively, beginning to decline thereafter. The Northeastern states of Pernambuco, Rio Grande do Norte (for both males and females) and Alagoas and Ceará (for males) are the only locations of type ii. R. bras. Est. Pop., v.38, 1-23, e0179, 2021 Results In Pernambuco, for example, for both males and females, the changes above age one are expected to have a greater impact at the total change than the changes below age one from 2040, as can be seen in Table 1 in the Appendix. Thus, after the crossing between e0 and e1 in the 2010s, the difference starts to increase, but in the late 2030s, for males, and early 2040s, for females, the difference between those values is expected to slowly reduce. Finally, Brazil and the rest of its Federation Units are expected to be of type i and, after the crossing, the difference between e0 and e1 is expected to increase. Our last analysis focused on the differences between the best- and worst-practices life expectancies at birth and between the best- and worst-practices infant mortalities. As stated, those metrics could provide an idea of the magnitude of the health inequalities experienced (and that are expected to be experienced) for males and females in Brazil over time, and also provide elements for the discussion about the convergence/divergence process of those health indicators among Brazilian Federation Units. Figure 2 (a) shows, for males and females, the estimated and projected worst- and best-practices life expectancies at birth, from 2000 to 2060. For males, four Northeastern states had the worst results concerning life expectancy at birth (Pernambuco in 2000, Alagoas from 2001 to 2015, Maranhão in 2016 and Piauí from 2017 to 2060). On the other hand, Santa Catarina had the best-practice life expectancy at birth for all the period under analysis. For females, three states had the worst results (Alagoas from 2000 to 2002, Roraima from 2003 to 2025 and Rondônia from 2026 to 2060), and three Federation Units had the best results (Rio Grande do Sul from 2000 to 2001, Federal District from 2002 to 2004 and Santa Catarina from 2005 to 2060). Those findings help us highlight the health inequalities between the North and Northeast regions of Brazil and the South region. R. bras. Est. Pop., v.38, 1-23, e0179, 2021 9 The life table paradox: has Brazil already overcome it? Souza, F.C. Results FIGURE 2 Best- and worst-practices life expectancies at birth (a), and the gap between them (b), for males and females Brazil − 2000-2060 (a) 58 63 68 73 78 83 88 2000 2002 2004 2006 2008 2010 2012 2014 2016 2018 2020 2022 2024 2026 2028 2030 2032 2034 2036 2038 2040 2042 2044 2046 2048 2050 2052 2054 2056 2058 2060 Worst-practice - male Best-practice - male Worst-practice - female Best-practice - female Life expectancy at birth 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.0 11.0 2000 2003 2006 2009 2012 2015 2018 2021 2024 2027 2030 2033 2036 2039 2042 2045 2048 2051 2054 2057 2060 BW Gap - male BW Gap - female Life expectancy Gap - years (b) Source: Author’s calculations based on the Brazilian Institute of Geography and Statistics (https://www.ibge.gov.br/). FIGURE 2 Best- and worst-practices life expectancies at birth (a), and the gap between them (b), for males and females Brazil − 2000-2060 (a) 58 63 68 73 78 83 88 2000 2002 2004 2006 2008 2010 2012 2014 2016 2018 2020 2022 2024 2026 2028 2030 2032 2034 2036 2038 2040 2042 2044 2046 2048 2050 2052 2054 2056 2058 2060 Worst-practice - male Best-practice - male Worst-practice - female Best-practice - female Life expectancy at birth FIGURE 2 Best- and worst-practices life expectancies at birth (a), and the gap between them (b), for males and females Brazil − 2000-2060 (a) Life expectancy at birth 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.0 11.0 2000 2003 2006 2009 2012 2015 2018 2021 2024 2027 2030 2033 2036 2039 2042 2045 2048 2051 2054 2057 2060 BW Gap - male BW Gap - female Life expectancy Gap - years (b) (b) 2000 2003 2006 2009 2012 2015 2018 2021 2024 2027 2030 2033 2036 2039 2042 2045 2048 2051 2054 2057 2060 Source: Author’s calculations based on the Brazilian Institute of Geography and Statistics (https://www.ibge.gov.br/). 10 R. bras. Est. Pop., v.38, 1-23, e0179, 2021 Souza, F.C. The life table paradox: has Brazil already overcome it? In addition, by Figure 2 (b), we can observe cycles of convergence and divergence between the best- and the worst-practice life expectancies at birth, for both males and females. R. bras. Est. Pop., v.38, 1-23, e0179, 2021 Results FIGURE 3 Best- and worst-practices in infant mortality (a), and the gap between them (b), for males and female Brazil − 2000-2060 0 10 20 30 40 50 60 70 80 90 2000 2002 2004 2006 2008 2010 2012 2014 2016 2018 2020 2022 2024 2026 2028 2030 2032 2034 2036 2038 2040 2042 2044 2046 2048 2050 2052 2054 2056 2058 2060 IM worst-practice - Male IM best-practice - Male IM worst-practice - Female IM best-practice - Female Infant mortality (a) FIGURE 3 Best- and worst-practices in infant mortality (a), and the gap between them (b), for males and females Brazil − 2000-2060 0 10 20 30 40 50 60 70 80 90 2000 2002 2004 2006 2008 2010 2012 2014 2016 2018 2020 2022 2024 2026 2028 2030 2032 2034 2036 2038 2040 2042 2044 2046 2048 2050 2052 2054 2056 2058 2060 IM worst-practice - Male IM best-practice - Male IM worst-practice - Female IM best-practice - Female Infant mortality (a) 0 2000 2002 2004 2006 2008 2010 2012 2014 2016 2018 2020 2022 2024 2026 2028 2030 2032 2034 2036 2038 2040 2042 2044 2046 2048 2050 2052 2054 2056 2058 2060 0 10 20 30 40 50 60 70 2000 2002 2004 2006 2008 2010 2012 2014 2016 2018 2020 2022 2024 2026 2028 2030 2032 2034 2036 2038 2040 2042 2044 2046 2048 2050 2052 2054 2056 2058 2060 IM GAP - Female IM GAP - Male Infant motality Gap (b) Source: Author’s calculations based on the Brazilian Institute of Geography and Statistics (https://www.ibge.gov.br/). 0 10 20 30 40 50 60 70 2000 2002 2004 2006 2008 2010 2012 2014 2016 2018 2020 2022 2024 2026 2028 2030 2032 2034 2036 2038 2040 2042 2044 2046 2048 2050 2052 2054 2056 2058 2060 IM GAP - Female IM GAP - Male Infant motality Gap (b) Source: Author’s calculations based on the Brazilian Institute of Geography and Statistics (https://www.ibge.gov.br/). (b) Infant motality Gap Source: Author’s calculations based on the Brazilian Institute of Geography and Statistics (https://www.ibge.gov.br/). Results For males, at those processes of convergence and divergence, the gap (BW Gap) achieved its lowest value in 2001 (8.50 years) and is expected to achieve its highest value in 2034 (10.35 years). For females, the gap achieved its lowest value in 2002 (7.32 years) and is expected to achieve its highest value in 2032 (8.20 years), which indicates a higher inequality among males. Figure 3 (a) shows, for males and females, the estimated and projected worst- and best-practices in infant mortality, from 2000 to 2060. For both males and females, the worst-practices in infant mortality had great improvements from the 2000s to the early 2010s, going, respectively, from 79.79 and 53.86 deaths per thousand live births in 2000 to 25.05 and 23.52 in 2015, which caused a great reduction in the gaps between best- and worst-practices in infant mortality (IM GAP), as can be seen in Figure 3 (b). For males, the reduction in the gap is expected to last until 2020, but from 2021 to 2031 the gap is expected to have a small rise, returning to decrease from 2032. For females, the reduction in the gap lasted until 2013, and from 2014 to 2020 the gap is expected to have a small rise, returning to decrease from 2021. For males, three states had the worst results concerning infant mortality (Alagoas from 2000 to 2010, Maranhão from 2011 to 2015, and Amapá from 2016 to 2060), and for females, two states had the worst results (Alagoas from 2000 to 2012, and Amapá from 2013 to 2060). Those bad indicators from Amapá help explain why it is expected to be the last state to overcome the life table paradox. On the other hand, for males, the Federal District and the states from the South had (and are expected to have) the best-practices (Federal District from 2000 to 2002, Rio Grande do Sul from 2003 to 2007, Santa Catarina from 2008 to 2020, and Paraná from 2021 to 2060), while for females, Espírito Santo joins those four Federation Units (Federal District in 2000, Rio Grande do Sul from 2001 to 2004, Santa Catarina from 2005 to 2009, Espírito Santo from 2010 to 2030 and Paraná from 2031 to 2060). Once again, those findings helped emphasize the regional inequalities in health in Brazil. R. bras. Est. Results Pop., v.38, 1-23, e0179, 2021 11 11 The life table paradox: has Brazil already overcome it? Souza, F.C. FIGURE 3 Best- and worst-practices in infant mortality (a), and the gap between them (b), for males and female Brazil − 2000-2060 0 10 20 30 40 50 60 70 80 90 2000 2002 2004 2006 2008 2010 2012 2014 2016 2018 2020 2022 2024 2026 2028 2030 2032 2034 2036 2038 2040 2042 2044 2046 2048 2050 2052 2054 2056 2058 2060 IM worst-practice - Male IM best-practice - Male IM worst-practice - Female IM best-practice - Female Infant mortality (a) 0 10 20 30 40 50 60 70 2000 2002 2004 2006 2008 2010 2012 2014 2016 2018 2020 2022 2024 2026 2028 2030 2032 2034 2036 2038 2040 2042 2044 2046 2048 2050 2052 2054 2056 2058 2060 IM GAP - Female IM GAP - Male Infant motality Gap (b) Source: Author’s calculations based on the Brazilian Institute of Geography and Statistics (https://www.ibge.gov.br/). Discussions Our results revealed clear discrepancies among Brazilian Federation Units concerning the timing of balancing of their life tables. While the Federal District and all the states from the South and Southeast regions were the first to achieve balancing, most of the R. bras. Est. Pop., v.38, 1-23, e0179, 2021 12 The life table paradox: has Brazil already overcome it? Souza, F.C. states from the Midwest and Northeast and especially the states from the North have not experienced it yet. At national level, females were the first to overcome the life table paradox in 2016, while for the males this happened in 2018. However, when analyzing the Federation Units, the median years of balancing for females and males are expected to be 2020 and 2022, respectively. Moreover, for both males and females, we also observed negative and statistically significant correlations between life expectancy at birth and the IM at the timing of the balancing. Canudas-Romo and Becker (2011), using data from the Human Mortality Database - HMD (https://www.mortality.org/), pointed out that, for industrialized nations, the first observed balanced life tables were those from Iceland (in 1957 for females, and in 1967 for males), and the last ones were those from Russia (in 2000, for females) and from Bulgaria (in 2003, for males). In addition, for all analyzed countries (but Slovenia) the balancing occurred for females first. According to the authors, in Slovenia, the life table paradox was overcome, for both males and females, in 1983. However, at this point it is worth noting that at the HMD the historical series of life tables for Slovenia starts in 1983, and that, for males, even though the 1983 life table was balanced, the 1984 and 1985 tables were not. Therefore, due to lack of information for the years prior to 1983 and the fact that fluctuations in life expectancy were observed before the Slovenian male life tables became effectively balanced, conclusions about Slovenia (and other nations with short historical series, such as Chile) should be taken with caution, since the years before the starting point of the series could also be part of a fluctuation process that culminated in a balanced table. Dubey, Ram and Ram (2015) investigated the timing of crossing between life e0 and e5 and between e0 and e1 for India (a country marked by social inequalities) and some selected states. Discussions They found that, until 2009, for all states in their sample in which both males and females had already experienced the crossing between life e0 and e5, it happened first for males. In that regard, our results indicated that although Brazil and most of its Federation Units experienced (or are expected to experience) balancing for females before males, for six states (Roraima, Tocantins, Piauí, Paraíba, Alagoas and Bahia) from the North and Northeast, the poorest regions in Brazil, males are expected to experience balancing in their life tables before females. In Roraima, particularly, males are expected to overcome the life table paradox 11 years before females. However, irrespective of who (males or females) overcame or are expected to overcome the paradox first, females always presented (or are expected to present) higher levels of life expectancy at birth and lower levels of infant mortality at the timing of the crossing. This fact could be explained since females’ life expectancy at age one reached higher levels than that for males, due to lower mortality levels after age one, which implies that females’ infant mortality needed to reach a lower level than that for males so that the crossing between life expectancies at birth and at age one could happen (CANUDAS-ROMO; BECKER, 2011). Moreover, as explained by Moura et al. R. bras. Est. Pop., v.38, 1-23, e0179, 2021 13 The life table paradox: has Brazil already overcome it? Souza, F.C. (2016), mortality differences between sex are the result of a set of factors such as biological and genetic differences, health behaviors, social behaviors and exposure to external causes of death (criminality and car accidents, for example) among others. By the decomposition analysis, Canudas-Romo and Becker (2011, p.126) were able to classify the changes over time between e0 and e1 in three types: (1) a decline in both components (above and below age one over time); (2) fluctuations in both components over time, causing the fluctuation of the total change; and (3) a bell shape type. Discussions Moreover, in the whole period under investigation (1960s-2000s), their illustrative examples had the ‘total change’ and the ‘below age one’ component greater than zero, but, in some cases, their results also indicated values greater than zero for the ‘above age one’ component.f Our results differ from those of Canudas-Romo and Becker (2011) in two main aspects: For Brazil and all of its Federation Units, the ‘above age one’ component was (and is expected to be) negative; and for some states (Pernambuco, Rio Grande do Norte, Ceará and Alagoas), we expect to observe a decline in both components in such a way that the total change over time will go from a positive to a negative value, i.e., the above age one component will have a greater impact on the total change over time than the below age one component. However, even though we have observed, for these four Northeastern states, a process of reduction of difference between the life expectancy at birth and at age one over the final decades of the projected period, the total changes over time A + B are too low and, therefore, do not allow any conjecture about possible reversion of the balancing process in a long run. In our final analysis, we discussed the relationship between best- and worst-practices in life expectancy at birth and in infant mortality, providing the magnitude of the inequalities in the main metrics under study. The results showed that the worst-practices were observed (and are expected to be observed) in the states of the North or Northeast regions and, on the other hand, the best-practices were observed (and are expected to be observed) in states from the South or Southeast or in the Federal District. Besides that, by analyzing the gaps, it was possible to observe a process of convergence and divergence between the worst- and best-practices, especially for male life expectancy at birth. For infant mortality, the major reductions in the gap happened from the 2000s to the early 2010s, leading to a higher homogenization of IM in the country, as was also observed by França et al. (2017), but regional inequalities are still persistent and relevant. Boing et al. Discussions (2020) noted that, from 1990 to 2010, Brazil made significant improvements in expanding and decentralizing health and sanitation services, in increasing the education level of its population and in implementing relevant social and pro-poor cash transference policies, which had important effects on health indicators such as life expectancy at birth and infant mortality, and on the reduction of regional inequalities. Nevertheless, Russo et al. (2019) indicated that despite improvements in the past decades, Brazil is still far from the IM level already achieved by the developed world. 14 R. bras. Est. Pop., v.38, 1-23, e0179, 2021 14 The life table paradox: has Brazil already overcome it? Souza, F.C. Souza, F.C. Boing et al. (2020) also observed that, since 2015, the country has experienced low economic growth and cuts in public investments, which could slow down (or compromise) the progresses that have been made. Moreover, the COVID-19 pandemic has been imposing economic retraction, aggravating the current situation, and is also expected to have a negative impact on life expectancy. In this regard, Castro et al. (2021), based on the death counts by COVID-19 in Brazil in 2020, estimated that life expectancy at birth has declined 1.98 years for males, and 1.82 years for females. Moreover, the authors also estimated the decline in life expectancy in an alternative scenario, assuming that 90% of the deaths by Severe Acute Respiratory Infection without a confirmed diagnosis were caused by COVID-19. Under this hypothesis, the declines were of 2.56 years for males, and 2.37 years for females. It is also worth mentioning that the official count of deaths by COVID-19 from January to April 2021 has already outnumbered the total amount registered in 2020 (CONSELHO NACIONAL DE SECRETÁRIOS DE SAÚDE, 2021), which will probably lead to even more significant negative impacts on life expectancy. According to França et al. (2017, p.53) the main causes of child mortality (< 5 years old) in 1990 and 2015 were (the rank of each cause in 1990 and 2015, respectively, is also presented): prematurity (1st, 1st), congenital anomalies (5th, 2nd), asphyxia and trauma at birth (4th, 3rd), septicaemia and other neonatal infection (6th, 4th), lower respiratory infections (3rd, 5th), other neonatal disorders (9th, 6th), diarrheal diseases (2nd, 7th), meningitis (8th, 8th) and malnutrition (7th, 9th). In this respect, França et al. (2017), Kale et al. (2019) and Tejada et al. R. bras. Est. Pop., v.38, 1-23, e0179, 2021 Discussions (2019) indicated that most of those deaths could be prevented by simple and timely health measures. Additionally, Kale et al. (2019) highlighted that when child and infant mortalities decrease, most of child deaths become concentrated at the neonatal period (0-27 days). The authors also pointed out that in 2015, 55% of all deaths up to the fifth year happened at the neonatal period. Lansky et al. (2014) went further indicating that early neonatal mortality (0-6 days), especially at the first 24 hours, is the main component of IM.Which, for the authors, shows the importance of good health care during the pregnancy and delivery. In this context, Leal et al. (2020) argued that prenatal care is almost universal for women using the Brazilian Unified Health System (SUS), but if a minimum set of exams and other healthcare procedures were to be included in the analysis, an adequate prenatal care is only offered for a quarter of the women using the SUS, showing that the quality of the services provided is still deficient. It was also found that, compared to the rest of the country, the North and the Northeast regions present a higher frequency of pregnancy among adolescents and women with low economic and educational levels. Additionally, in the North, “the proportion of women with no prenatal care was the same for groups of women, with and without obstetric complications” (LEAL et al., 2020, p.9). For the authors, this result could be partially explained by geographical barriers in accessing health centers and the absence of qualified professionals. Tomasi et al. (2017) developed an indicator of prenatal quality, based on which they found that only 15% of women received an adequate prenatal care, with the worst services R. bras. Est. Pop., v.38, 1-23, e0179, 2021 15 Souza, F.C. The life table paradox: has Brazil already overcome it? provided to younger females, from low-income families, living in small municipalities (and with low Human Development Index) from the North and Midwest regions. Additionally, the convergence/divergence process discussed plays an important role in the debate of epidemiological transition (VALLIN; MESLÉ, 2004; MESLÉ; VALLIN, 2017). In this sense, Borges (2017, p. 3) states that “the epidemiological transition in Brazil has not followed the model experienced by most developed countries”. Discussions Alvarez, Aburto and Canudas-Romo (2020) argue that the epidemiological transition theory was conceived in the developed world and, for this reason, it might fail to explain mortality and health trends in the developing world. Moreover, the authors indicated that Brazil is also heavily affected by amenable diseases and has high rates of violence and homicide, especially among men, which is one of the factors preventing the country from achieving the mortality regime of developed nations. Limitations and research agenda For a better interpretation of the results, and in order to develop or strengthen a research agenda, it is fundamental to clearly identify the main limitations of an academic investigation and to propose future paths of study (SOUZA, 2020). In this regard, we understand that the first issue that must be addressed in this subsection is the use of estimated and projected life tables. Although we have used official life tables provided by the Brazilian Institute of Geography and Statistics, which is undoubtedly a natural choice when studying aspects related to mortality and life expectancy in Brazil, we must be aware that the value of some metrics such as life expectancy at birth and infant mortality are influenced by the methods/hypotheses used to construct the life table (MISSOV; NÉMETH; DANKO, 2016; SHOLNIKIV, 2017). Moreover, as pointed by Wolff, Chong and Auffhammer (2011), social metrics released in a given year must be viewed as an inexact value, which could be revised due to data or methodological updating, for example. Along these lines, although population projections are an important demographic tool and a fundamental one for public planning (OLIVEIRA; SILVA; OLIVEIRA, 2019), unforeseen events, particularly the current COVID-19 pandemic, will surely have a negative impact, at least in the short term, in life-expectancy and might affect regions (CASTRO et al., 2021) and subpopulations in different levels. The AIDS epidemic, for example, caused expressive losses in life-expectancy in Africa during the 1990s (SOUZA; RÊGO, 2018). f Even without the pandemic, there is already evidence of differences in health indicators among Brazilian subpopulations. Hone et al. (2017), for example, stated that black and mixed (pardo) races had higher mortality rates from homicides and external causes, are more affected by infectious diseases and had lower life expectancy. Chiavegatto Filho, Beltrán-Sánchez and Kawachi (2014) also pointed out that black and mixed races had lower educational and income levels when compared to whites, and live in more violent communities, which have a negative impact in perpetuating racial inequalities in Brazil. In R. bras. Est. Pop., v.38, 1-23, e0179, 2021 16 The life table paradox: has Brazil already overcome it? Souza, F.C. this regard, Caldas et al. (2017) complemented that the recent reductions in infant mortality in Brazil had less expressive effects for the indigenous subpopulation. Limitations and research agenda Moreover, the recent episodes of immigration (and refugees) in the 2010s, especially from Haiti and Venezuela (OLIVEIRA; SILVA; OLIVEIRA, 2019), and the fact that immigration disproportionally affects Brazilian regions, also demand attention. Unlike immigrants that move to other countries seeking for better job opportunities, for example, refugees had to leave their nations as a matter of survival (MOKDAD et al., 2016), and because of that, they may arrive in the new country in a situation of social vulnerability. In a recent study, Simeoni, Frova and De Curtis (2019) found high disparities between infant mortality among Italian residents and immigrants, especially for children from mothers coming from Africa. Altogether, those facts call for the monitoring of consequences of the COVID-19 pandemic (and of the social isolation) in health metrics, especially in life expectancy; besides quality data and investigations that address the case of Brazilian subpopulations. if A final point to be highlighted is the difference between period and cohort metrics, particularly regarding life expectancy. The period life expectancy expresses the average length of life of a synthetic cohort, assuming that the age-specific death rates will remain constant over time; while the cohort life expectancy expresses the average length of life of a real cohort of people born at the same calendar year (GOLDSTEIN; WACHTER, 2006; LUY et al., 2019; MODIG; RAU; AHLBOM, 2020). Therefore, even though period life expectancy at birth reflects the mortality conditions of a population in a certain period (LUY et al., 2019), which is indisputably a very important health indicator, it does not reflect the average number of years that a newborn child will be expected to live in a real population. Additionally, period life expectancy may be affected by tempo, heterogeneity and cohort effects (LUY et al., 2019; MODIG; RAU; AHLBOM, 2020). In this regard, Canudas-Romo and Becker (2011) already observed that, since mortality rates typically reduce over time, it is most certain that the timing of balancing of the life tables should be observed earlier if cohort tables were used. This conjecture emphasizes the importance of the construction of official cohort life tables for Brazil and its Federation Units and subpopulations. R. bras. Est. Pop., v.38, 1-23, e0179, 2021 References ALVAREZ, J.-A.; ABURTO, J. M.; CANUDAS-ROMO, V. Latin America convergence and divergence towards the mortality profiles of developed countries. Population Studies, v. 74, n. 1, p. 75-92, 2020. BOCCOLINI, C. S.; SOUZA JUNIOR, P. R. B. Inequities in healthcare utilization: results of Brazil National Health Survey 2013. International Journal for Equity in Health, v. 15, p. 1-8, 2016. BOING, A. F. et al. Narrowing geographic inequality in life expectancy in Brazil: a multilevel analysis between 1991 and 2010. Public Health, v. 180, p. 102-108, 2020. BORGES, G. M. Health transition in Brazil: regional variations and divergence/convergence in mortality. Cadernos de Saúde Pública, v. 33, n. 8, e00080316, 2017. CALDAS, A. D. R. et al. Infant mortality according to color and race based on the 2010 Population Census and National Health Information System in Brazil. Cadernos de Saúde Pública, v. 33, n. 7, .e00046516, 2017. CANUDAS-ROMO, V.; BECKER, S. The crossover between life expectancies at birth and at age one: the imbalance in the life table. Demographic Research, v. 24, p. 113-144, 2011. CASTRO, M. C. et al. Reduction in the 2020 life expectancy in Brazil after COVID-19. medRxiv, 2021. Available at: https://doi.org/10.1101/2021.04.06.21255013. Accessed on: 12 Apr. 2021. CHIAVEGATTO FILHO, A. D. P.; BELTRÁN-SÁNCHEZ, H.; KAWACHI, I. Racial disparities in life expectancy in Brazil: challenges from a multiracial society. American Journal of Public Health, v. 104, n. 11, p. 2156-2162, 2014. CONSELHO NACIONAL DE SECRETÁRIOS DE SAÚDE. Painel CONASS COVID-19. 2021. Available at: https://www.conass.org.br/painelconasscovid19/. Accessed on: 1 May 2021. DUBEY, M.; RAM, U.; RAM, F. Threshold levels of infant and under-five mortality for crossover between life expectancies at ages zero, one and five in India: a decomposition analysis. PLoS ONE, v. 10, n. 12, e0143764, 2015. FRANÇA, E. B. et al. Leading causes of child mortality in Brazil in 1990 and 2015: estimates from the Global Burden of Disease study. Revista Brasileira de Epidemiologia, v. 20, n. 1, p. 46-60, 2017. GISBERT, F. J. G. Distributionally adjusted life expectancy as a life table function. Demographic Research, v. 43, p. 365-400, 2020. GOLDSTEIN, J. R.; WACHTER, K. W. Relationships between period and cohort life expectancy: gaps and lags. Population Studies, v. 60, n. 3, p. 257-269, 2006. HONE, T. et al. Association between expansion of primary healthcare and racial inequalities in mortality amenable to primary care in Brazil: a national longitudinal analysis. PLoS Medicine, v. 14, n. 5, e1002306, 2017. Conclusion Inspired by Canudas-Romo and Becker (2011), this article investigated the timing (and health metrics at the time) in which Brazil and its Federation Units overcame (or are expected to overcome) the life table paradox, also exploring regional and sex health inequalities. From a public planning perspective, we understand that achieving a balance in the life tables is a goal to be pursued, especially because, roughly speaking, having an imbalanced table means that life expectancy at birth is still strongly influenced by high levels of infant mortality. Therefore, knowing the expected time and health metrics at that R. bras. Est. Pop., v.38, 1-23, e0179, 2021 17 Souza, F.C. The life table paradox: has Brazil already overcome it? moment, public planners could properly define, for each sex and region, strategies to accelerate the balancing process and revert unequal scenarios. moment, public planners could properly define, for each sex and region, strategies to accelerate the balancing process and revert unequal scenarios. References IBGE − Instituto Brasileiro de Geografia e Estatística. Projeções da população por sexo e idade − Brasil: 2000-2060 e unidades da federação − 2000-2030. Rio de Janeiro: IBGE, 2013a. Available at: https://www.ibge.gov.br/estatisticas/sociais/populacao/9109-projecao-da-populacao. html?edicao=9116&t=resultados. Accessed on: 16 Jun. 2020. R. bras. Est. Pop., v.38, 1-23, e0179, 2021 18 The life table paradox: has Brazil already overcome it? Souza, F.C. IBGE − Instituto Brasileiro de Geografia e Estatística. Projeções da população: Brasil e unidades da federação. Rio de Janeiro: IBGE, 2013b. 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Health in times of uncertainty in the eastern Mediterranean Region, 1990- 2013: a systematic analysis for the global burden of disease study 2013. Lancet Global Health, v. 4, n. 10, 2016. References MOURA, E. C. et al. Mortality in Brazil according to gender perspective, years 2000 and 2010. Revista Brasileira de Epidemiologia, v. 19, n. 2, p. 326-338, 2016. OEPPEN, J.; VAUPEL, J. W. Broken limits to life expectancy. Science, v. 296, n. 5570, p.1029-1031, 2002. OLIVEIRA, H. N.; SILVA, C. A. M.; OLIVEIRA, A. T. R. Imigração internacional: uma alternativa para os impactos das mudanças demográficas no Brasil? Revista Brasileira de Estudos de População, v. 36, p. 1-31, e0076, 2019. ROWLAND, D. T. Demographic methods and concepts. Oxford: Oxford University Press, 2003. RUSSO, L. X. et al. Primary care physicians and infant mortality: evidence from Brazil. PLoS ONE, v. 14, n. 5, e0217614, 2019. SHKOLNIKOV, V. Methods protocols for the human life-table database. Version 2. 2017. Available at: https://www.lifetable.de/methodology.pdf. Accessed on: 15 Apr. 2021. R. bras. Est. Pop., v.38, 1-23, e0179, 2021 19 Souza, F.C. The life table paradox: has Brazil already overcome it? SIMEONI, S.; FROVA, L.; DE CURTIS, M. Inequalities in infant mortality in Italy. Italian Journal of Pediatrics, v. 45, n. 11, p. 1-7, 2019. SMITHS, J.; MONDEN, C. Length of life inequality around the globe. Social Science & Medicine, v. 68, p. 1114-1123, 2009. SOUZA, F. C. Relationships between best-practice and greatest possible life expectancies. European Journal of Ageing, v. 17, p. 331-339, 2020. SOUZA, F. C.; RÊGO, L. C. Life expectancy and health life expectancy changes between 2000 and 2015: an analysis of 183 World Health Organization member states. Journal of Public Health: From Theory to Practice, v. 26, n. 3, p. 261-269, 2018. TEJADA, C. A. et al. Economic crises, child mortality and the protective role of public health expenditure. Ciências & Saúde Coletiva, v. 24, n. 12, p. 4395-4404, 2019. TOMASI, E. et al. Quality of prenatal services in primary healthcare in Brazil: indicators and social inequalities. Cadernos de Saúde Pública, v. 33, n. 3, e00195815, 2017. VALLIN, J.; MESLÉ, F. Convergences and divergences in mortality; a new approach to health transition. Demographic Research, Special Collection 2, p. 11-44, 2004. VAUPEL, J. W. Biodemography of human ageing. Nature, v. 464, p. 536-542, 2010. WOLFF, H.; CHONG, H.; AUFFHAMMER, M. Classification, detection and consequences of data error: evidence from the Human Development Index. The Economic Journal, v. 121, n. 553, p. 843-870, 2011. About the author Filipe Costa de Souza holds a PhD in Economics from Universidade Federal de Pernambuco (UFPE) and he is currently a professor at the Departamento de Ciências Contábeis e Atuariais, also at UFPE. Contact address Universidade Federal de Pernambuco, Centro de Ciências Sociais Aplicadas, Departamento de Ciências Contábeis e Atuariais Avenida dos Funcionários, s/n, Cidade Universitária 50670-901 – Recife-PE, Brazil Universidade Federal de Pernambuco, Centro de Ciênc de Ciências Contábeis e Atuariais Avenida dos Funcionários, s/n, Cidade Universitária 50670-901 – Recife-PE, Brazil Resumo O paradoxo da tábua de sobrevivência: o Brasil já o superou? O paradoxo da tábua de sobrevivência: o Brasil já o superou? Idealmente, a expectativa de vida deveria ser uma função decrescente da idade. Quando tal fato não é observado, a situação é conhecida como o paradoxo da tábua de mortalidade. Este artigo investigou o momento (e métricas de saúde neste momento) em que o Brasil e suas unidades da federação (UF) superaram (ou são esperados superar) este paradoxo. Os dados foram obtidos do Instituto Brasileiro de Geografia e Estatística e continham 3.416 tábuas de mortalidade abreviadas e específicas por sexo, de 2000 até 2060. No nível nacional, mulheres e homens superaram o paradoxo em 2016 e 2018, respectivamente. Contudo, quando foram R. bras. Est. Pop., v.38, 1-23, e0179, 2021 20 The life table paradox: has Brazil already overcome it? Souza, F.C. examinadas as UFs separadamente, grande heterogeneidade foi observada. Pela análise de decomposição da mudança no tempo da diferença entre a expectativa de vida ao nascer e na idade de um ano, observamos que o Brasil e a maioria de suas UFs esperam ter ambas as mudanças decrescentes ao longo do tempo e a mudança total é esperada ser decrescente e maior do que zero. Entretanto, para alguns estados do Nordeste, a mudança total é esperada passar de um valor positivo para um negativo; e para dois estados do Norte a mudança total esperada não deve ser nem crescente nem decrescente. Em uma perspectiva de planejamento público, entendemos que alcançar o balanceamento das tábuas de mortalidade é um objetivo a ser perseguido, especialmente porque ter uma tábua desbalanceada significa que a expectativa de vida ao nascer ainda é fortemente influenciada pelos altos níveis de mortalidade infantil. Assim, este conhecimento pode ajudar planejadores a definirem estratégias apropriadas para acelerar o processo de balanceamento e reverter cenários de desigualdade. Palavras-chave: Tábua de sobrevivência. Expectativa de vida. Mortalidade infantil. Desigualdades em saúde. Desigualdades regionais. Decomposição. Resumen La paradoja de la tabla de vida: ¿Brasil ya a la superó? La paradoja de la tabla de vida: ¿Brasil ya a la superó? TABLE 1 Estimated and projected differences between e0 and e1 over time and their components, for males and females Selected Federation Units (RR, PE, SP, RS and DF) and Brazil − 2000-2060 Roraima (RR) – Males Roraima (RR) – Females Year = t 2000 2010 2020 2030 2040 2050 2060 2000 2010 2020 2030 2040 2050 2060 e0(t) 62.293 66.898 70.356 72.674 74.110 74.957 75.443 68.981 72.517 75.337 77.465 79.000 80.075 80.811 e1(t) 62.897 67.138 70.516 72.749 74.090 74.843 75.237 69.421 72.885 75.618 77.650 79.086 80.062 80.703 D(t) = e0(t) - e1(t) -0.604 -0.240 -0.161 -0.075 0.020 0.114 0.205 -0.440 -0.368 -0.281 -0.185 -0.086 0.013 0.108 A = Changes below age 1 0.456 0.137 0.120 0.113 0.104 0.095 0.139 0.135 0.128 0.120 0.111 0.102 B = Changes above age 1 -0.092 -0.058 -0.034 -0.019 -0.009 -0.004 -0.068 -0.048 -0.032 -0.021 -0.013 -0.007 A+B = D(t+10) - D(t) 0.364 0.079 0.086 0.094 0.095 0.091 0.072 0.087 0.096 0.100 0.099 0.095 Pernambuco (PE) – Males Pernambuco (PE) Females Appendix La paradoja de la tabla de vida: ¿Brasil ya a la superó? Idealmente, la esperanza de vida debería ser una función decreciente de la edad. Cuando esto no sucede, se conoce al hecho como la paradoja de la tabla de mortalidad. Este artículo investigó el momento —y las medidas de salud en ese momento— en que Brasil y sus unidades de la federación UF) superaron —o se espera que superen— esta paradoja. Los datos fueron tomados del Instituto Brasileño de Geografía y Estadística (IBGE) y contenían 3416 tablas de vida abreviadas oficiales y específicas por sexo, desde 2000 hasta 2060, para Brasil y sus 27 UF. A el ámbito nacional, mujeres y hombres habían superado la paradoja en 2016 y 2018, respectivamente. Sin embargo, cuando las UF se examinaron por separado, se observó una gran heterogeneidad. Mediante el análisis de descomposición del cambio a lo largo del tiempo en la diferencia entre la esperanza de vida al nacer y al primer año de edad, encontramos que se espera que Brasil y la mayoría de sus UF presenten cambios que disminuyan con el tiempo y se espera que el cambio total disminuya y sea mayor que cero. No obstante, para algunos estados del noreste, se espera que el cambio total se oriente desde un valor positivo hacia uno negativo, así como para dos estados del norte el cambio total esperado no debería aumentar ni disminuir. En una perspectiva de planificación pública, entendemos que lograr una tabla de mortalidad equilibrada es un objetivo a perseguir, especialmente porque tener una tabla desequilibrada significa que la esperanza de vida al nacer sigue estando fuertemente influida por los altos niveles de mortalidad infantil. Por lo tanto, este conocimiento puede ayudar a los planificadores a definir estrategias adecuadas para acelerar el proceso de equilibrio y revertir los escenarios de desigualdad. Palabras clave: Tabla de vida. Esperanza de vida. Mortalidad infantil. Desigualdades en salud. Desigualdades regionales. Descomposición. Received for publication in 22/08/2020 Approved for publication in 28/06/2021 Received for publication in 22/08/2020 Approved for publication in 28/06/2021 R. bras. Est. Pop., v.38, 1-23, e0179, 2021 21 The life table paradox: has Brazil already overcome it? Souza, F.C. São Paulo (SP) – Females Pernambuco (PE) – Males R. bras. Est. Pop., v.38, 1-23, e0179, 2021 Appendix TABLE 1 Estimated and projected differences between e0 and e1 over time and their components, for males and females Selected Federation Units (RR, PE, SP, RS and DF) and Brazil − 2000-2060 Roraima (RR) – Males Roraima (RR) – Females Year = t 2000 2010 2020 2030 2040 2050 2060 2000 2010 2020 2030 2040 2050 2060 e0(t) 62.293 66.898 70.356 72.674 74.110 74.957 75.443 68.981 72.517 75.337 77.465 79.000 80.075 80.811 e1(t) 62.897 67.138 70.516 72.749 74.090 74.843 75.237 69.421 72.885 75.618 77.650 79.086 80.062 80.703 D(t) = e0(t) - e1(t) -0.604 -0.240 -0.161 -0.075 0.020 0.114 0.205 -0.440 -0.368 -0.281 -0.185 -0.086 0.013 0.108 A = Changes below age 1 0.456 0.137 0.120 0.113 0.104 0.095 0.139 0.135 0.128 0.120 0.111 0.102 B = Changes above age 1 -0.092 -0.058 -0.034 -0.019 -0.009 -0.004 -0.068 -0.048 -0.032 -0.021 -0.013 -0.007 A+B = D(t+10) - D(t) 0.364 0.079 0.086 0.094 0.095 0.091 0.072 0.087 0.096 0.100 0.099 0.095 Pernambuco (PE) – Males Pernambuco (PE) – Females Year = t 2000 2010 2020 2030 2040 2050 2060 2000 2010 2020 2030 2040 2050 2060 e0(t) 60.201 66.828 71.548 74.302 75.724 76.413 76.736 70.076 75.466 78.916 80.885 81.935 82.474 82.746 e1(t) 62.807 67.258 71.421 74.089 75.505 76.196 76.522 72.030 75.684 78.714 80.630 81.680 82.223 82.497 D(t) = e0(t) - e1(t) -2.607 -0.430 0.127 0.213 0.220 0.217 0.214 -1.954 -0.218 0.202 0.255 0.255 0.251 0.249 A = Changes below age 1 2.350 0.625 0.117 0.021 0.004 0.001 1.839 0.460 0.071 0.010 0.001 0.000 B = Changes above age 1 -0.173 -0.069 -0.030 -0.015 -0.007 -0.003 -0.103 -0.039 -0.018 -0.010 -0.005 -0.002 A+B = D(t+10) - D(t) 2.177 0.557 0.087 0.006 -0.003 -0.003 1.736 0.421 0.052 0.000 -0.004 -0.002 São Paulo (SP) – Males São Paulo (SP) – Females Year = t 2000 2010 2020 2030 2040 2050 2060 2000 2010 2020 2030 2040 2050 2060 e0(t) 67.039 72.600 76.120 78.067 79.065 79.556 79.791 76.056 79.550 81.963 83.540 84.534 85.144 85.515 e1(t) 67.319 72.558 75.853 77.657 78.569 79.011 79.217 76.269 79.423 81.619 83.067 83.987 84.556 84.904 D(t) = e0(t) - e1(t) -0.280 0.042 0.267 0.410 0.496 0.545 0.574 -0.213 0.126 0.344 0.473 0.547 0.588 0.610 A = Changes below age 1 0.406 0.262 0.158 0.092 0.052 0.029 0.381 0.238 0.140 0.079 0.044 0.024 B = Changes above age 1 -0.083 -0.037 -0.015 -0.006 -0.003 -0.001 -0.042 -0.021 -0.010 -0.005 -0.003 -0.002 A+B = D(t+10) - D(t) 0.323 0.225 0.143 0.086 0.050 0.028 0.339 0.217 0.130 0.074 0.041 0.022 Appendix (continue) São Paulo (SP) – Males R. bras. Est. Pop., v.38, 1-23, e0179, 2021 22 The life table paradox: has Brazil already overcome it? Souza, F.C. São Paulo (SP) – Males Brazil (BR) – Males Brazil (BR) – Females Year = t 2000 2010 2020 2030 2040 2050 2060 2000 2010 2020 2030 2040 2050 2060 e0(t) 66.006 70.210 73.259 75.276 76.551 77.370 77.898 73.920 77.605 80.251 82.003 83.115 83.808 84.226 e1(t) 67.243 70.550 73.180 75.009 76.199 76.973 77.478 74.812 77.833 80.113 81.691 82.723 83.374 83.769 D(t) = e0(t) - e1(t) -1.237 -0.340 0.079 0.267 0.352 0.397 0.421 -0.892 -0.228 0.138 0.312 0.392 0.434 0.457 A = Changes below age 1 0.982 0.460 0.208 0.096 0.051 0.028 0.725 0.396 0.189 0.088 0.047 0.025 B = Changes above age 1 -0.085 -0.041 -0.020 -0.011 -0.006 -0.004 -0.061 -0.030 -0.015 -0.008 -0.005 -0.003 A+B = D(t+10) - D(t) 0.897 0.419 0.188 0.085 0.045 0.024 0.664 0.366 0.174 0.080 0.042 0.022 Source: Author’s calculations based on the Brazilian Institute of Geography and Statistics (https://www ibge gov br/) Rio Grande do Sul (RS) – Males Rio Grande do Sul (RS) – Females Year = t 2000 2010 2020 2030 2040 2050 2060 2000 2010 2020 2030 2040 2050 2060 e0(t) 68.623 72.443 75.443 77.651 79.198 80.248 80.944 76.217 79.495 82.037 83.924 85.280 86.235 86.895 e1(t) 68.774 72.338 75.142 77.206 78.651 79.630 80.277 76.296 79.340 81.699 83.451 84.710 85.597 86.211 D(t) = e0(t) - e1(t) -0.152 0.105 0.301 0.445 0.547 0.618 0.667 -0.079 0.155 0.338 0.473 0.570 0.638 0.684 A = Changes below age 1 0.308 0.226 0.161 0.111 0.076 0.051 0.272 0.204 0.148 0.104 0.072 0.049 B = Changes above age 1 -0.051 -0.030 -0.017 -0.009 -0.005 -0.003 -0.037 -0.022 -0.012 -0.007 -0.004 -0.002 A+B = D(t+10) - D(t) 0.257 0.196 0.144 0.102 0.071 0.048 0.235 0.182 0.135 0.097 0.068 0.047 Federal District (DF) – Males Federal District (DF) – Females Year = t 2000 2010 2020 2030 2040 2050 2060 2000 2010 2020 2030 2040 2050 2060 e0(t) 68.457 72.549 75.431 77.300 78.448 79.129 79.526 76.068 79.875 82.376 83.916 84.827 85.353 85.652 e1(t) 68.591 72.495 75.224 76.975 78.036 78.655 79.007 76.141 79.767 82.122 83.552 84.382 84.852 85.112 D(t) = e0(t) - e1(t) -0.134 0.054 0.207 0.325 0.412 0.475 0.519 -0.073 0.109 0.254 0.365 0.445 0.501 0.540 A = Changes below age 1 0.245 0.185 0.135 0.096 0.067 0.046 0.227 0.169 0.122 0.086 0.059 0.041 B = Changes above age 1 -0.057 -0.032 -0.017 -0.009 -0.004 -0.002 -0.045 -0.024 -0.012 -0.006 -0.003 -0.001 A+B = D(t+10) - D(t) 0.188 0.153 0.118 0.087 0.063 0.044 0.181 0.146 0.110 0.080 0.056 0.039 Brazil (BR) – Males Brazil (BR) – Females Year = t 2000 2010 2020 2030 2040 2050 2060 2000 2010 2020 2030 2040 2050 2060 e0(t) 66.006 70.210 73.259 75.276 76.551 77.370 77.898 73.920 77.605 80.251 82.003 83.115 83.808 84.226 e1(t) 67.243 70.550 73.180 75.009 76.199 76.973 77.478 74.812 77.833 80.113 81.691 82.723 83.374 83.769 D(t) = e0(t) - e1(t) -1.237 -0.340 0.079 0.267 0.352 0.397 0.421 -0.892 -0.228 0.138 0.312 0.392 0.434 0.457 A = Changes below age 1 0.982 0.460 0.208 0.096 0.051 0.028 0.725 0.396 0.189 0.088 0.047 0.025 B = Changes above age 1 -0.085 -0.041 -0.020 -0.011 -0.006 -0.004 -0.061 -0.030 -0.015 -0.008 -0.005 -0.003 A+B = D(t+10) - D(t) 0.897 0.419 0.188 0.085 0.045 0.024 0.664 0.366 0.174 0.080 0.042 0.022 Source: Author’s calculations based on the Brazilian Institute of Geography and Statistics (https://www.ibge.gov.br/). São Paulo (SP) – Males (continued) A = Changes below age 1 0.245 0.185 0.135 0.096 0.067 0.046 0.227 0.169 0.122 0.086 0.059 0.041 B = Changes above age 1 -0.057 -0.032 -0.017 -0.009 -0.004 -0.002 -0.045 -0.024 -0.012 -0.006 -0.003 -0.001 A+B = D(t+10) - D(t) 0.188 0.153 0.118 0.087 0.063 0.044 0.181 0.146 0.110 0.080 0.056 0.039 Brazil (BR) – Males Brazil (BR) – Females Year = t 2000 2010 2020 2030 2040 2050 2060 2000 2010 2020 2030 2040 2050 2060 e0(t) 66.006 70.210 73.259 75.276 76.551 77.370 77.898 73.920 77.605 80.251 82.003 83.115 83.808 84.226 e1(t) 67.243 70.550 73.180 75.009 76.199 76.973 77.478 74.812 77.833 80.113 81.691 82.723 83.374 83.769 D(t) = e0(t) - e1(t) -1.237 -0.340 0.079 0.267 0.352 0.397 0.421 -0.892 -0.228 0.138 0.312 0.392 0.434 0.457 A = Changes below age 1 0.982 0.460 0.208 0.096 0.051 0.028 0.725 0.396 0.189 0.088 0.047 0.025 B = Changes above age 1 -0.085 -0.041 -0.020 -0.011 -0.006 -0.004 -0.061 -0.030 -0.015 -0.008 -0.005 -0.003 A+B = D(t+10) - D(t) 0.897 0.419 0.188 0.085 0.045 0.024 0.664 0.366 0.174 0.080 0.042 0.022 Source: Author’s calculations based on the Brazilian Institute of Geography and Statistics (https://www ibge gov br/) Rio Grande do Sul (RS) – Males Rio Grande do Sul (RS) – Females ar = t 2000 2010 2020 2030 2040 2050 2060 2000 2010 2020 2030 2040 2050 2060 (t) 68.623 72.443 75.443 77.651 79.198 80.248 80.944 76.217 79.495 82.037 83.924 85.280 86.235 86.895 (t) 68.774 72.338 75.142 77.206 78.651 79.630 80.277 76.296 79.340 81.699 83.451 84.710 85.597 86.211 t) = e0(t) - e1(t) -0.152 0.105 0.301 0.445 0.547 0.618 0.667 -0.079 0.155 0.338 0.473 0.570 0.638 0.684 = Changes below age 1 0.308 0.226 0.161 0.111 0.076 0.051 0.272 0.204 0.148 0.104 0.072 0.049 = Changes above age 1 -0.051 -0.030 -0.017 -0.009 -0.005 -0.003 -0.037 -0.022 -0.012 -0.007 -0.004 -0.002 B = D(t+10) - D(t) 0.257 0.196 0.144 0.102 0.071 0.048 0.235 0.182 0.135 0.097 0.068 0.047 F d l Di t i t (DF) M l d l i i ( ) l tinued) R. bras. Est. Pop., v.38, 1-23, e0179, 2021 Brazil (BR) – Males R. bras. Est. Pop., v.38, 1-23, e0179, 2021 23
https://openalex.org/W4315704919	https://www.cambridge.org/core/services/aop-cambridge-core/content/view/D79208D48F08957426C14E09678888A5/S1323358022000595a.pdf/div-class-title-image-based-searches-for-pulsar-candidates-using-mwa-vcs-data-div.pdf	English	null	Image-based searches for pulsar candidates using MWA VCS data	Publications of the Astronomical Society of Australia/Publications Astronomical Society of Australia	2,023	cc-by	13,133	Publications of the Astronomical Society of Australia (2023), 40, e003, 12 pages doi:10.1017/pasa.2022.59 Abstract Pulsars have been studied extensively over the last few decades and have proven instrumental in exploring a wide variety of physics. Discovering more pulsars emitting at low radio frequencies is crucial to further our understanding of spectral properties and emission mech- anisms. The Murchison Wideﬁeld Array Voltage Capture System (MWA VCS) has been routinely used to study pulsars at low frequencies and discover new pulsars. The MWA VCS offers the unique opportunity of recording complex voltages from all individual antennas (tiles), which can be off-line beamformed or correlated/imaged at millisecond time resolution. Devising imaged-based methods for ﬁnding pulsar candidates, which can be veriﬁed in beamformed data, can accelerate the complete process and lead to more pulsar detections. Image-based searches for pulsar candidates can reduce the number of tied-array beams required, increasing compute resource efﬁciency. Despite a factor of ∼4 loss in sensitivity, searching for pulsar candidates in images from the MWA VCS, we can explore a larger parameter space, potentially leading to discoveries of pulsars missed by high-frequency surveys such as steep spectrum pulsars, exotic binary systems, or pulsars obscured in high-time resolution time series data by propagation effects. Image-based searches are also essential to probing parts of parameter space inaccessible to traditional beamformed searches with the MWA (e.g. at high dispersion measures). In this paper we describe the innovative approach and capability of dual-processing MWA VCS data, that is forming 1-s visibilities and sky images, ﬁnding pulsar candidates in these images, and verifying by forming tied-array beam. We developed and tested image-based methods of ﬁnding pulsar candidates, which are based on pulsar properties such as steep spectral index, polarisation and variability. The efﬁciency of these methodologies has been veriﬁed on known pulsars, and the main limitations explained in terms of sensitivity and low-frequency spectral turnover of some pulsars. No can- didates were conﬁrmed to be a new pulsar, but this new capability will now be applied to a larger subset of observations to accelerate pulsar discoveries with the MWA and potentially speed up future searches with the SKA-Low. Keywords: instrumentation:interferometers – methods:observational – pulsars:general – techniques:interferometric (Received 16 September 2022; revised 12 December 2022; accepted 12 December 2022) of image-based pulsar candidate searches can potentially reduce the computational resources and processing time when compared to traditional Fourier-domain periodicity searches. aContinuum sources are considered candidates until conﬁrmed by standard pulsar searches. Image-based searches for pulsar candidates using MWA VCS data S. Sett1,2 , N. D. R. Bhat1 , M. Sokolowski1 , and E. Lenc2 1International Centre for Radio Astronomy Research, Curtin University, Bentley, WA 6102, Australia and 2CSIRO Astronomy and Space Science, PO Box 76, Epping, NSW 1710, Australia Corresponding author: S. Sett, Email: 20014515@student.curtin.edu.au. Cite this article: Sett S, Bhat NDR, Sokolowski M and Lenc E. (2023) Image-based searches for pulsar candidates using MWA VCS data. Publications of the Astronomical Society of Australia 40, e003, 1–12. https://doi.org/10.1017/pasa.2022.59 c⃝The Author(s), 2023. Published by Cambridge University Press on behalf of the Astronomical Society of Australia. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution and reproduction, provided the original article is p p l it d 1. Introduction For decades, searching for pulsars have been performed using time domain search techniques which are highly sensitive for detecting periodic pulses. This approach has been extremely suc- cessful and led to majority of pulsar discoveries (Manchester et al. 2005). However, the sensitivity of these techniques is negatively affected by factors such as binary eclipses, orbital motion and scattering. Especially at low radio frequencies, dispersion mea- sure (DM), smearing and multipath scattering become dominant factors resulting in lesser number of detections. At these frequen- cies, traditional pulsar searches are also more time consuming and computationally expensive due to the large number of DM trials required at low frequencies. The necessity for performing accel- eration searches to detect pulsars in tight binaries also increase the processing time. An alternative strategy to search for bright pulsars is to exploit the high-time resolution data of interferome- ters such as the Murchison Wideﬁeld Array (MWA; Tingay et al. 2013; Wayth et al. 2018) to produce continuum images, ﬁnd pulsar candidates in the image domain and verify them using high-time resolution time series formed from the very same data. This kind Many attempts have been made to search for pulsars in radio continuum surveys. Although many of them were unsuccessful, the ﬁrst ever millisecond pulsar discovered, J1939+2134, was ini- tially identiﬁed in radio continuum images as an unusual compact source with a steep spectrum (Backer et al. 1982). Furthermore, image-based methods have been gaining momentum in the recent years. For example, the discovery of a highly polarised, steep spectrum millisecond pulsar in a deep image with the Australian Square Kilometre Array Pathﬁnder telescope (ASKAP; Kaplan et al. 2019) have demonstrated the rising potential of imaging sur- veys to detect new pulsar candidates.a Another recent discovery of a circularly polarised variable radio source in the Large Magellanic Cloud (LMC) shows the potential to detect new pulsars (Wang et al. 2022). The primary difﬁculty in the searching for pulsars in image domain is that the observational data are averaged in time and hinder the ability to extract a pulsar proﬁle from time series. The primary challenge, then, is to distinguish interesting pulsar candidates from other unresolved point radio sources. Such Corresponding author: S. Sett, Email: 20014515@student.curtin.edu.au. Cite this article: Sett S, Bhat NDR, Sokolowski M and Lenc E. (2023) Image-based searches for pulsar candidates using MWA VCS data. Publications of the Astronomical Society of Australia 40, e003, 1–12. https://doi.org/10.1017/pasa.2022.59 Published online by Cambridge University Press 1. Introduction (2016) demonstrates the identiﬁcation of probable pul- sar candidates whose scintillation timescales and bandwidths are comparable to the time and frequency resolution of the instru- ment. Another approach is to search for sources that have a high degree of circular polarisation. Previously, a handful of pulsars have been discovered using a combination of spectral properties and linear polarisation (Navarro et al. 1995), but there has not been large-scale surveys including circular polarisation until Lenc et al. (2018). While some steady circularly polarised emission has been seen from sources like ﬂare stars (Lynch et al. 2017b), chro- mospherically active binaries like cataclysmic variables (Mutel & Morris 1988), the majority of polarised sources seen in large scale surveys are pulsars (Lenc et al. 2018). MWA surveys of lin- early polarised sources and pulsar searches were also conducted by Riseley et al. (2018, 2020). Several other surveys are currently ongoing, such as a search for pulsars associated with polarised point sources using LOFAR, which detected 2 new pulsars (Sobey et al. 2022) and a circular polarisation survey for radio stars with the ASKAP, resulting in 37 pulsar detections, despite them only looking for ﬂare stars (Pritchard et al. 2021). In this paper, we consider archival data collected with the Phase I and II conﬁguration of the MWA which we use to demon- strate image-based methods for pulsar searches. In 2018, the 128- tile array with a maximum baseline of ∼3 km (Phase 1) (Tingay et al. 2013) was upgraded to provide a maximum baseline of 5.3 km with 256 tiles (sets of 4×4 cross-dipole antennas). The Phase II upgrade increased the angular resolution by a factor of ∼2 and the sensitivity by a factor of ∼4 as a result of reduction in the classical and sidelobe confusion (Wayth et al. 2018). Originally envisioned as an imaging telescope, requiring only time-averaged tile cross- correlation products (‘visibilities’), it was eventually upgraded to enable the capture of raw complex voltages from each tile with the development of the Voltage Capture System (VCS; Tremblay et al. 2015). The VCS records high-time and frequency resolution voltage data (100 µs/10 kHz), which provides the opportunity to process the data in different ways and maximise data ﬂexibility. 1. Introduction https://doi.org/10.1017/pasa.2022.59 c⃝The Author(s), 2023. Published by Cambridge University Press on behalf of the Astronomical Society of Australia. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution and reproduction, provided the original article is properly cited. https://doi.org/10.1017/pasa.2022.59 Published online by Cambridge University Press https://doi.org/10.1017/pasa.2022.59 Published online by Cambridge University Press S. Sett et al. 2 S. Sett et al. Table 1. Observations processed and analysed as part of this paper. PI represents MWA Phase I Array and PIIE represents MWA Phase II Extended Array. The main application for the observations are given in the table. The ﬁrst 2 observations were mainly used to establish the criteria and the thresholds required to detect highly signiﬁcant pulsar candidates. The other two observations were used to independently test the methodologies and determine the efﬁciency of the different h d Observation name Observation ID Project code MWA phase Duration (mins) Frequency (MHz) Main use A 1276619416 G0071 PIIE 90 184.96 Formulating method 1 and 2 B 1150234552 G0024 PI 80 184.96 Formulating method 3 C 1148063920 G0024 PI 45 184.96 Independent veriﬁcation of all methods D 1274143152 D0029 PIIE 20 154.24 Independent veriﬁcation of all methods differentiation between pulsars and unresolved radio sources can be done using properties that are characteristic to pulsars. making it complementary both to similar low-frequency tele- scopes in the Northern Hemisphere, such as the Low-Frequency Array (LOFAR; van Haarlem et al. 2013), as well as high- frequency telescopes in the Southern Hemisphere, such as the Parkes Radio Telescope (Murriyang). Pulsar physics is one of the key science drivers for the MWA (Beardsley et al. 2019). Despite being less sensitive than LOFAR, the combination of a large FoV and an extremely radio-quiet environment makes the MWA ideal for undertaking pulsar searches at frequencies below 300 MHz. One such metric is the steepness of the average spectrum. It has been successfully employed to discover new pulsars that were otherwise missed via traditional pulsar searching techniques (Frail et al. 2016). This technique has proved to be highly beneﬁcial in targeted observations (Hyman et al. 2019) as well as large sky sur- veys (Frail et al. 2018). In addition to spectral index methods, Dai et al. 1. Introduction Since pulsar ﬂux densities at low frequencies can vary signiﬁcantly from day to day, being able to ﬁnd pulsar candidates in images formed from the MWA VCS data, and vet them by beamform- ing the very same data is a very powerful technique unique to the MWA. In this paper, we present methodologies for ﬁnding pulsar can- didates in the MWA ofﬂine-correlated observations, test their efﬁciency by detecting known pulsars, and making an attempt to detect new pulsar candidates. Section 2 describes the instrument and the observation details. Section 3 describes the data analysis and the metrics applied to the acquired data. Section 4 summarises the results of the data analysis and discusses the implications for the MWA and other low-frequency telescopes such as the MWA. The maximum MWA VCS observation duration used here is ∼90 min and the central frequency ranges from 154 to 185 MHz. Table 1 shows the observations that are processed and analysed as part of this paper. Along with the observation IDs and cor- responding project code, details of the telescope conﬁguration at the time of the observation, duration of the observation, the cen- tral frequency of the observation and the main application of the observation in this paper are mentioned in the table. 3.1 Steep spectrum Individual 1s images were averaged together to produce mean full Stokes images which were then used for further analysis. A block diagram show- ing the above described steps of the imaging pipeline is given in Figure 1. α = log Sa Sb log νa νb . (1) (1) For our methodology, we have used the Rapid ASKAP Continuum survey (RACS; McConnell et al. 2020; Hale et al. 2021) as our counterpart catalogue to calculate the spectral index. The RACS survey is the ﬁrst large sky survey covering the southern sky below declinations +41◦using ASKAP (Hotan et al. 2014). The central frequency of the survey is 887.5 MHz and is one of the deepest radio surveys of the southern sky at these frequencies. The ﬂux densities of the sources from RACS in combination with the ﬂux densities from our images can be used to calculate spec- tral indices of the sources detected in both images or upper limits on spectral indexes for the sources detected only in MWA image. Since pulsars are known for their steep spectra, it is a compelling case to search for radio pulsations of sources exhibiting a steep spectral index. The results of applying a spectral index cutoff to the sources in the images are described in Section 4.4.1. Given the angular resolution difference between MWA and ASKAP, cross- matching with RACS is also a good way to eliminate any other extended sources which may have been pre-selected due to lower spatial resolution of the MWA (∼a few arcmin). A cutout of a mean Stokes I formed from observation A is shown in Figure 2. It shows a region of the Galactic Plane passing through the centre of the image along with a supernova remnant which causes signiﬁcant increase in the noise in that region. The other sources are a combination of stars, active galactic nuclei, known pulsars and other radio sources. The mean rms of this image is 5 mJy beam−1 and increases by almost a factor of 4 near the Galactic Plane and the edge of the image. Almost 9 000 radio sources are detected at 5σ threshold in the whole image, where σ is the mean standard deviation of the noise. These sources are then used in the methodologies described in Section 3. The radio source-ﬁnding and extraction software, AEGEAN (Hancock et al. 3.1 Steep spectrum Pulsars are known steep spectrum radio sources, with an aver- age spectral index of −1.6 ± 0.2 (Jankowski et al. 2018). The ﬁrst millisecond pulsar, PSR J1939+2134 was discovered due to the steep spectral properties of the continuum source, 4C 21.53W (Backer et al. 1982). The ﬁrst discovery of a pulsar, PSR J1824- 2452A in a globular cluster was made possible due to its steep spectrum (Lyne et al. 1987). Since then, there have been a hand- ful of target searches aiming to detect steep spectrum sources as pulsars, for example Damico et al. (1985), Kaplan et al. (2000) and Crawford, Kaspi, & Bell (2000) targeted 18, 16 and 92 sources, respectively, but found no pulsations. While the surveys men- tioned above were sensitive enough to detect the associated pulsars with high signiﬁcance, the available observing frequencies could have affected the detection results for some of the targets. With the advancement in computing resources, such surveys can be re-conducted using techniques such as acceleration searches to detect exotic systems and may be more fruitful. At radio frequen- cies, the spectral behaviour of many pulsars can be described by a power law, where the spectral index calculated from ﬂux density measurements Sa and Sb, at two different frequencies νa and νb, Figure 1. Block diagram of the imaging pipeline, which shows the different steps taken to get the resulting Stokes image. It follows from obtaining the raw voltages, ofﬂine cor- relating and producing measurement sets. WSClean is then used to create the images and apply calibration, ultimately producing Stokes images for every timestamp. These images are then averaged to form the mean Stokes images. set format (McMullin et al. 2007) and applies calibration in the conversion process. It also eliminates the channels affected by radio frequency interference (RFI) by using the in-built software AOFlagger (Offringa, de Bruyn, & Zaroubi 2012). Calibration solutions for the observation are downloaded from the MWA All- Sky Virtual Observatory (MWA ASVO; Sokolowski et al. 2020). Images in instrumental polarisation (XX, YY, XY, YX) are formed using WSCLEAN (Offringa et al. 2014) with a Briggs weighting of −1. These images were then converted to Stokes I, Q, U and V images using the MWA ‘fully’ embedded element beam model (Sokolowski et al. 2017). The image sizes were 8 192×8 192 pixels or 4 096×4 096 pixels, depending on the observation. 3.1 Steep spectrum 2012; Hancock, Trott, & Hurley-Walker 2018) was then used to identify sources from the mean Stokes I and V images and create a catalogue of sources above a 5σ threshold which are then analysed to produce a list of promising pulsar candidates. The individual 1s images were also averaged together to produce light curves at various time resolutions such as 300, 60 and 30s. This can be helpful in detecting sources that are variable in time and hence can be promising pulsar candidates. 2.2 Data processing The MWA is a low-frequency Square Kilometre Array (SKA) precursor telescope, operating in the frequency range of 70-300 MHz, located at the Murchison Radio-astronomy Observatory (MRO) in Western Australia. It is the only radio telescope oper- ating at these frequencies able to access the entire southern sky, The raw complex voltages from the MWA antennas (i.e. ‘tiles’) are ofﬂine correlated using xGPU software correlator (Clark, LaPlante, & Greenhill 2013) to produce visibilities at 1-s time resolution. These are then processed with COTTER (Offringa et al. 2015), which converts data into the CASA measurement https://doi.org/10.1017/pasa.2022.59 Published online by Cambridge University Press https://doi.org/10.1017/pasa.2022.59 Published online by Cambridge University Press Publications of the Astronomical Society of Australia 3 Figure 1. Block diagram of the imaging pipeline, which shows the different steps taken to get the resulting Stokes image. It follows from obtaining the raw voltages, ofﬂine cor- relating and producing measurement sets. WSClean is then used to create the images and apply calibration, ultimately producing Stokes images for every timestamp. These images are then averaged to form the mean Stokes images. 3. Methodologies developed to detect pulsar candidates In the following subsections we describe the methodologies that we have developed and implemented in order to assess the efﬁ- ciency on known pulsars and also attempt to discover pulsar candidates. https://doi.org/10.1017/pasa.2022.59 Published online by Cambridge University Press 3.2 Circular polarisation Until Lenc et al. (2018), most of the all-sky surveys have been conducted using total intensity, that is, Stokes I. As only a frac- tion of the sources present in the total intensity image emit in circular polarisation, it is useful in lowering the classical confu- sion limit (Lenc et al. 2017). Greater sensitivity can be obtained for instruments such as the MWA which is confusion limited in total https://doi.org/10.1017/pasa.2022.59 Published online by Cambridge University Press 4 4 S. Sett et al. Figure 2. Stokes I mean image cutout of Observation A. It shows the Galactic Plane in the centre of the image. The average rms in the image is ∼5 mJy beam−1, with a factor of 4 increase near the Galactic Plane and edge of the image. We can also see a variety of extended sources, such as supernova remnants as well as point sources which are a combination of already known pulsars and other radio sources. Figure 2. Stokes I mean image cutout of Observation A. It shows the Galactic Plane in the centre of the image. The average rms in the image is ∼5 mJy beam−1, with a factor of 4 increase near the Galactic Plane and edge of the image. We can also see a variety of extended sources, such as supernova remnants as well as point sources which are a combination of already known pulsars and other radio sources. intensity (Tingay et al. 2013). This is extremely helpful for sources that are highly circularly polarised; with a trade-off in sensitivity for sources that are not. The advantage of this is that most sources are generally weak in circular polarisation and hence will not con- tribute to side-lobe confusion, as a result reducing the need for deconvolution, making image processing easier. A smaller number of sources compared to Stokes I images also reduces the number of candidate pulsars signiﬁcantly. This method has demonstrated its potential to detect pulsars by the discovery of a highly polarised millisecond pulsar by ASKAP (Kaplan et al. 2019), which would be otherwise missed by traditional searches due to its wide proﬁle and high DM. We implement the method of looking for sources that are highly polarised in Stokes V images in an attempt to detect any potential pulsar candidates. 3.2 Circular polarisation This method will be highly effective in detecting sources that may be below the noise threshold in Stokes I images but are highly circularly polarised (>7%) (Lenc et al. 2018, 2017) and hence very bright in Stokes V images. Further details of this method are discussed in Section 4.4.2. It focuses on how the ﬂux density of the sources vary with time. For example, the ASKAP Variables and Slow Transients (VAST) Pilot project (Banyer, Murphy, & VAST Collaboration 2012) and the LOFAR Transients key science project (Swinbank et al. 2015) have used this methodology to look for transients. Producing light curves essentially requires a prioritised ﬁtting for the sources for all the images and construct a light curve from the measured ﬂux den- sity values. This method can be efﬁcient tool for detecting pulsars that may have high degrees of variability on timescale of seconds. The application of this method to the images obtained from our observation is discussed in detail in Section 4.4.3. 4. Veriﬁcation of the methodologies on the MWA data The methodologies described above have been developed and tested on observation A and B (refer to Table 1 for details). On successful completion of the testing, they have been independently applied to observations C and D (from Table 1) to verify their efﬁciency in detecting possible pulsar candidates. y g We use Stokes I and V images which are of particular inter- est in the analysis of observation A. The images are 8 192 × 8 192 pixels, with pixel size ∼3 arcmin pixel−1, resulting in 40 × 40 degree images. The rms noise levels in the image for observa- tion A are around 5 mJy beam−1 (Stokes I) and 3 mJy beam−1 (Stokes V) in the centre of both the images. The sources are extracted using AEGEAN (Hancock et al. 2012, 2018) with a mean cutoff of 5σ, where σ is the local root-men-square noise level near the source. The mean Stokes I image is complex with over https://doi.org/10.1017/pasa.2022.59 Published online by Cambridge University Press 4.1 Noise characterisation Along with the source catalogues for the Stokes images, AEGEAN also creates maps of the rms noise (σ) in each image. The σ values vary across the image due to a combination of several factors. This includes adjacency to bright sources and unmodelled extended emissions, which are prevalent near the Galactic Plane. The mean σ value across the whole ﬁeld of observation is 5 mJy beam−1 while it changes to 20 mJy beam−1 near the Galactic Plane. There is also a signiﬁcant increase near the edge of the images (∼30 mJy beam−1) where the beam response of the MWA decreases. Due to this varia- tion of the noise across the whole image, the ﬂux density detection thresholds for the sources should be deﬁned locally and depen- dent on the location of the source in the image. For example, a 40 mJy source is easily detectable with a 5σ signiﬁcance away from the Galactic Plane. However, the same source will not be bright enough to be detected at the edge of the image where the threshold is signiﬁcantly higher. Figure 3. The above ﬁgure shows the parameter space that is exclusively accessible to image-based pulsar search techniques, shaded in green. The black dots indicate the image-based pulsar detections and the orange dots represent the detection of pulsars via traditional techniques. The blue dashed line shows the mean ﬂux density threshold of observation A for detection of sources above 5 σ (25 mJy). The red dotted line is the DM threshold (250 pc cm−3) of traditional search techniques for MWA frequencies beyond which the sensitivity decreases signiﬁcantly. 3.3 Time variability One of the easiest ways to conﬁrm if a promising candidate is a pulsar or not is to perform a periodicity search and fold the data at the best-ﬁt period and DM. However, at lower frequencies, sources may suffer from signiﬁcant scattering and dispersion lowers our sensitivity of periodicity searches using high-time resolution data. Having images of the ﬁeld at short timescales for the whole observation opens up a new avenue to detect pulsar candidates. https://doi.org/10.1017/pasa.2022.59 Published online by Cambridge University Press https://doi.org/10.1017/pasa.2022.59 Published online by Cambridge University Press Publications of the Astronomical Society of Australia 5 Figure 3. The above ﬁgure shows the parameter space that is exclusively accessible to image-based pulsar search techniques, shaded in green. The black dots indicate the image-based pulsar detections and the orange dots represent the detection of pulsars via traditional techniques. The blue dashed line shows the mean ﬂux density threshold of observation A for detection of sources above 5 σ (25 mJy). The red dotted line is the DM threshold (250 pc cm−3) of traditional search techniques for MWA frequencies beyond which the sensitivity decreases signiﬁcantly. 9 000 sources including diffuse emission and several extended structures as shown in Figure 2. These sources are then used to cross-match with a pulsar catalogue as well as choose promising pulsar candidates, the details of which are discussed in the sections below. 4.2 Comparison with the ATNF catalogue non-detection of the other pulsar, PSR J1822−1400, could be very steep spectral index of −2.25 (Manchester et al. 2005, ATNF catalogue). The Australian Telescope National Facility (ATNF) pulsar cata- logue (Manchester et al. 2005) contains ∼3 000 pulsars. Around 700 pulsars are present in the 3 dB (half power point) beam of this observation. We expect to detect 42 of them at 5σ threshold when we take into account the local σ around the sources. Out of the 42 expected detections, we were able to detect 21 pulsars in imaging which is reasonable considering the more complex environment and the realistic details of the data and processing. Three of the pulsar non-detections can be attributed to their spectral turnover at low frequency as highlighted by Jankowski et al. (2018). The other 18 non-detections were due to the location of these pulsars near the Galactic centre or the edge of the image. The 21 detected pulsars were also folded with PRESTO (Ransom 2001) in order to verify their detections using standard methods. Only 11 out of 21 were detected via PRESTO. This can be explained by the DM cutoff of ∼250 pc cm−3 for the MWA beyond which the detection sensitivity is highly reduced when dealing with time series data. The pulsars detected via imaging (black circles) and traditional search methods (orange circles) are shown in Figure 3. The blue dotted line indicates the ﬂux density threshold for image-based searches for this observation, the red dashed line indicates the approximate DM cut-off for traditional searches with the MWA. The green shaded region highlights where image-based searches can be more successful than traditional searches. In the ﬁgure, we can see that even though there are more pulsars detected via tra- ditional searches, image-based searches probe a parameter space that is inaccessible to traditional searches. 4.3.2 Cross-matching all sources On cross-matching our catalogue with the RACS all-sky catalogue, we ﬁnd ∼8 000 sources that are present in both the catalogues and 342 sources that are present only in MWA catalogue at 5σ conﬁ- dence level. These 342 sources are of interest to us as potentially intriguing sources for follow up. We further reduce this num- ber by excluding extended sources as pulsars are generally point sources. The ratio of the integrated ﬂux over source in the image (SI) to peak ﬂux of the source in the image (SP), both calculated by AEGEAN, is used to select the point sources such that sources where SI/SP > 1.5 are considered to be extended. This compact- ness criterion is based on the similar criterion in the GaLactic and Extragalactic All-sky Murchison Wideﬁeld Array (GLEAM) cat- alogue (Hurley-Walker et al. 2017) and is applied to the sources present in both catalogues as well as the sources present in MWA catalogue only. The ratio as a function of the signal to noise ratio (SNR) of the sources calculated using the source catalogue is shown in Figure 4. The blue points (∼6 700) are the excluded extended sources and the red points (∼1 600) are the compact sources taken into consideration for further analysis on applica- tion of the compactness criterion. This subset of sources is used for further analysis as described in Section 4.4. 4.3.1 Cross-matching known pulsars 2022), the steeper cutoff is more likely to ﬁnd those that were missed by previous surveys at higher frequencies while also reducing false positives from other source types, for example steep spectra AGN. This cut-off may be relaxed in future (allowing shallower power laws) if additional robust ﬁl- tering criteria are developed in order to reduce the number of false positives. (1) for the sources present in both MWA and RACS catalogues. Furthermore, for the sources that are present only in the MWA Stokes I images, an upper limit on the spectral index is calculated using the local σ from the RACS noise map (5σ for RACS). A recent study of spectral indices of 441 radio pulsars suggests the mean of the spectral index distribution is −1.8 for 79% of pulsars following a simple power law spectrum (Jankowski et al. 2018). Taking this into account along with error margins, a spectral index cutoff of −1.2 is selected, such that sources steeper than −1.2 are considered as steep spectrum sources and used for further anal- ysis. While 29% of the known pulsar population exhibit spectra ﬂatter than −1.2 (Swainston et al. 2022), the steeper cutoff is more likely to ﬁnd those that were missed by previous surveys at higher frequencies while also reducing false positives from other source types, for example steep spectra AGN. This cut-off may be relaxed in future (allowing shallower power laws) if additional robust ﬁl- tering criteria are developed in order to reduce the number of false positives. Figure 6. Pulsars that are detected in imaging and the ones that satisfy the criterion of steep spectrum. The blue dashed line shows the spectral index cutoff and the red dashed line signiﬁes the compactness cutoff applied to the sources. The green region shows the parameter space that the combination of the compactness and spectral steepness criteria can probe in imaging domain. The distribution of the spectral indexes of the sources and the applied cutoff are shown in Figure 5. Application of the criterion resulted in a subset of ∼300 sources that are steep spectrum. Out of these sources, 19 are already known pulsars. Figure 6 shows the pulsars that satisfy the criterion of steep spectrum sources. It also shows the two pulsars that were excluded due to criterion not being satisﬁed. One is in a supernova remnant and was excluded on application of the compactness criterion. 4.3.1 Cross-matching known pulsars Out of these sources, 19 are already known pulsars. Figure 6 shows the pulsars that satisfy the criterion of steep spectrum sources. It also shows the two pulsars that were excluded due to criterion not being satisﬁed. One is in a supernova remnant and was excluded Figure 5. Distribution of the spectral index of the sources detected in the MWA image of observation A. It can be seen that most of the sources have spectral index between −1 and 1. The blue dotted line shows the spectral index cutoff threshold used in this analysis. Figure 6. Pulsars that are detected in imaging and the ones that satisfy the criterion of steep spectrum. The blue dashed line shows the spectral index cutoff and the red dashed line signiﬁes the compactness cutoff applied to the sources. The green region shows the parameter space that the combination of the compactness and spectral steepness criteria can probe in imaging domain. 4.4.2 Circular polarisation A catalogue of sources from the Stokes V image is generated by running AEGEAN on the Stokes V inverted image and the origi- nal Stokes V image. The combination of the catalogues from both the processes is used as the Stokes V source catalogue. The second Figure 4. Ratio of integrated ﬂux to peak ﬂux (SI/SP) as a function of SNR of the sources. The criterion for the source to be a point source was chosen to be the ratio SI/SP < 1.5. Based on this criterion all the point sources are shown in red (∼1 600) and the extended sources are in blue (∼6 700). Figure 4. Ratio of integrated ﬂux to peak ﬂux (SI/SP) as a function of SNR of the sources. The criterion for the source to be a point source was chosen to be the ratio SI/SP < 1.5. Based on this criterion all the point sources are shown in red (∼1 600) and the extended sources are in blue (∼6 700). Figure 5. Distribution of the spectral index of the sources detected in the MWA image of observation A. It can be seen that most of the sources have spectral index between −1 and 1. The blue dotted line shows the spectral index cutoff threshold used in this analysis. Figure 5. Distribution of the spectral index of the sources detected in the MWA image of observation A. 4.3.1 Cross-matching known pulsars The other pulsar, PSR J1834-0010 has a spectral index of −0.9 which is not steep enough to pass the criterion. The ∼300 sources that are considered steep according to our criterion were checked for pulsar-like emission by forming a tied-array beam on each of the sources and perform- ing a PRESTO (Ransom 2001) based search up to a DM of 500 pc cm−3 with minimum DM steps of 0.02 pc cm−3 and maximum of 0.5 pc cm−3. No pulsations from these sources were found in the beamformed data. However, as mentioned before the sensitiv- ity of MWA is signiﬁcantly reduced at DMs above 250 pc cm−3. Therefore, deeper searches with a higher DM range with a high- frequency instrument may enable identiﬁcation of pulses from these sources. 4.3.1 Cross-matching known pulsars It can be seen that most of the sources have spectral index between −1 and 1. The blue dotted line shows the spectral index cutoff threshold used in this analysis. Figure 5. Distribution of the spectral index of the sources detected in the MWA image of observation A. It can be seen that most of the sources have spectral index between −1 and 1. The blue dotted line shows the spectral index cutoff threshold used in this analysis. Figure 5. Distribution of the spectral index of the sources detected in the MWA image of observation A. It can be seen that most of the sources have spectral index between −1 and 1. The blue dotted line shows the spectral index cutoff threshold used in this analysis. Figure 4. Ratio of integrated ﬂux to peak ﬂux (SI/SP) as a function of SNR of the sources. The criterion for the source to be a point source was chosen to be the ratio SI/SP < 1.5. Based on this criterion all the point sources are shown in red (∼1 600) and the extended sources are in blue (∼6 700). Figure 6. Pulsars that are detected in imaging and the ones that satisfy the criterion of steep spectrum. The blue dashed line shows the spectral index cutoff and the red dashed line signiﬁes the compactness cutoff applied to the sources. The green region shows the parameter space that the combination of the compactness and spectral steepness criteria can probe in imaging domain. (1) for the sources present in both MWA and RACS catalogues. Furthermore, for the sources that are present only in the MWA Stokes I images, an upper limit on the spectral index is calculated using the local σ from the RACS noise map (5σ for RACS). A recent study of spectral indices of 441 radio pulsars suggests the mean of the spectral index distribution is −1.8 for 79% of pulsars following a simple power law spectrum (Jankowski et al. 2018). Taking this into account along with error margins, a spectral index cutoff of −1.2 is selected, such that sources steeper than −1.2 are considered as steep spectrum sources and used for further anal- ysis. While 29% of the known pulsar population exhibit spectra ﬂatter than −1.2 (Swainston et al. https://doi.org/10.1017/pasa.2022.59 Published online by Cambridge University Press 4.3.1 Cross-matching known pulsars 4.3.1 Cross-matching known pulsars Observation A (refer to Table 1) is predominantly used for the examination of the ﬁrst criterion. On successful veriﬁcation of the criterion, it is then independently applied on observations C and D. The process begins by ﬁrst selecting sources with a steep spectrum, by calculating the spectral index using Equation Out of the 21 pulsars detected in the test MWA image (Observation A), 19 of them were detected in a Stokes I RACS image. Of the 2 pulsars not present, one is associated with a supernova remnant, SNR G21.5−0.9. A possible reason for the https://doi.org/10.1017/pasa.2022.59 Published online by Cambridge University Press 6 S. Sett et al. 6 Figure 4. Ratio of integrated ﬂux to peak ﬂux (SI/SP) as a function of SNR of the sources. The criterion for the source to be a point source was chosen to be the ratio SI/SP < 1.5. Based on this criterion all the point sources are shown in red (∼1 600) and the extended sources are in blue (∼6 700). (1) for the sources present in both MWA and RACS catalogues. Furthermore, for the sources that are present only in the MWA Stokes I images, an upper limit on the spectral index is calculated using the local σ from the RACS noise map (5σ for RACS). A recent study of spectral indices of 441 radio pulsars suggests the mean of the spectral index distribution is −1.8 for 79% of pulsars following a simple power law spectrum (Jankowski et al. 2018). Taking this into account along with error margins, a spectral index cutoff of −1.2 is selected, such that sources steeper than −1.2 are considered as steep spectrum sources and used for further anal- ysis. While 29% of the known pulsar population exhibit spectra ﬂatter than −1.2 (Swainston et al. 2022), the steeper cutoff is more likely to ﬁnd those that were missed by previous surveys at higher frequencies while also reducing false positives from other source types, for example steep spectra AGN. This cut-off may be relaxed in future (allowing shallower power laws) if additional robust ﬁl- tering criteria are developed in order to reduce the number of false positives. The distribution of the spectral indexes of the sources and the applied cutoff are shown in Figure 5. Application of the criterion resulted in a subset of ∼300 sources that are steep spectrum. 4.4.2 Circular polarisation In order to reduce the storage and CPU requirements, these sources are further subjected to a threshold of 20%, denoted by the red dashed line, for modulation index. The light curves for only sources (∼250 sources) satisfying both threshold are saved for further assessment. These light curves are then visually investigated to determine its ranking in terms of variability and searched for pulses. Figure 7. Distribution of fractional polarisation of the sources after the removal of non-physical leakage around the sources. The blue dashed line shows a fractional polarisation threshold of 7% from the existing literature. The 4 sources with \|V/I\| > 0.07 are known to be circularly polarised pulsars according to the European Pulsar Network (EPN) database. Figure 8. Correlation between χ 2 and modulation index for 30 s timescale for obser- vation B. The blue dashed line indicates the χ 2 threshold of 1 (for this case). In order to reduce the storage and CPU requirements, these sources are further subjected to a threshold of 20%, denoted by the red dashed line, for modulation index. The light curves for only sources (∼250 sources) satisfying both threshold are saved for further assessment. These light curves are then visually investigated to determine its ranking in terms of variability and searched for pulses. Figure 8. Correlation between χ 2 and modulation index for 30 s timescale for obser- vation B. The blue dashed line indicates the χ 2 threshold of 1 (for this case). In order to reduce the storage and CPU requirements, these sources are further subjected to a threshold of 20%, denoted by the red dashed line, for modulation index. The light curves for only sources (∼250 sources) satisfying both threshold are saved for further assessment. These light curves are then visually investigated to determine its ranking in terms of variability and searched for pulses. Figure 7. Distribution of fractional polarisation of the sources after the removal of non-physical leakage around the sources. The blue dashed line shows a fractional polarisation threshold of 7% from the existing literature. The 4 sources with \|V/I\| > 0.07 are known to be circularly polarised pulsars according to the European Pulsar Network (EPN) database. measured Stokes V ﬂux density to suppress any residual leakage resulting in a more robust estimate of the source SNR and frac- tional polarisation. 4.4.2 Circular polarisation A distribution of fractional polarisation, V/I, of sources in the image for observation A is shown in Figure 7. The acceptable threshold for fractional polarisation according to literature (Lenc et al. 2018, 2017) is ∼7%. Application of the threshold reveals 4 sources that satisfy this criterion. According to the European Pulsar Network (EPN) database, 9 pulsars in this ﬁeld are circularly polarised. These 4 sources that satisfy our threshold with V/I > 0.07, are a subset of the 9 pulsars that are cir- cularly polarised. Out of the 5 non-detections, one pulsar, PSR J1823-1115, shows a sign reversal in the mean pulse proﬁle of Stokes V polarisation and the other 4 (highest absolute value of 15 mJy beam−1) are below our detection threshold in Stokes V image (∼35 mJy beam−1). No other interesting pulsar candidates were detected in this image using this method. Further investi- gation in better removing the polarisation leakage is required in order to detect potentially interesting pulsar candidates. surrounding. The statistical quantities described below are for a speciﬁc image pixel (function of (x,y) position of the image), but this dependence was dropped for brevity. For each pixel, the reduced χ2 (signiﬁcance) statistic is deﬁned as χ2 = n i=1 (Si −¯S)2 σ 2 i , (2) (2) where n is the number of images, Si is the ﬂux density of a pixel in image i, σ 2 i is the variance within the images (Bell et al. 2016). ¯S is the weighted mean ﬂux density deﬁned as ¯S = n i=1 Si σ 2 i n i=1 1 σ 2 i (3) (3) In combination with the χ2 test, we also calculate the modula- tion index of the source/pixel that is used to quantify the degree of variability of the source when compared to its surrounding sources. The modulation index (m) is deﬁned as 4.4.2 Circular polarisation A catalogue of sources from the Stokes V image is generated by running AEGEAN on the Stokes V inverted image and the origi- nal Stokes V image. The combination of the catalogues from both the processes is used as the Stokes V source catalogue. The second criterion of circular polarisation uses the sources from Stokes I and V images of observation A to select potential pulsar candidates based on their circular polarisation. Unfortunately, the positive detection of pulsar candidates is hindered by instrumental leak- age, even when leveraging the most advanced MWA primary beam model used for this analysis (Sokolowski et al. 2017). Lenc et al. (2017) showed that such polarisation leakage can be mitigated in drift-scan observations by modelling the leakage pattern across the beam and then subtracting it. A similar approach has been taken in this analysis to deal with the leakage for the observations. For observation A, the Stokes V sources are crossmatched with Stokes I catalogue sources and the median leakage in 5◦radius around each source is then calculated. This is then subtracted from the https://doi.org/10.1017/pasa.2022.59 Published online by Cambridge University Press Publications of the Astronomical Society of Australia 7 Figure 7. Distribution of fractional polarisation of the sources after the removal of non-physical leakage around the sources. The blue dashed line shows a fractional polarisation threshold of 7% from the existing literature. The 4 sources with \|V/I\| > 0.07 are known to be circularly polarised pulsars according to the European Pulsar Network (EPN) database. Figure 8. Correlation between χ 2 and modulation index for 30 s timescale for obser- vation B. The blue dashed line indicates the χ 2 threshold of 1 (for this case). In order to reduce the storage and CPU requirements, these sources are further subjected to a threshold of 20%, denoted by the red dashed line, for modulation index. The light curves for only sources (∼250 sources) satisfying both threshold are saved for further assessment. These light curves are then visually investigated to determine its ranking in terms of variability and searched for pulses. Figure 8. Correlation between χ 2 and modulation index for 30 s timescale for obser- vation B. The blue dashed line indicates the χ 2 threshold of 1 (for this case). 4.4.3 Variability The previously deﬁned characteristics are not exclusive to pul- sars and may be selecting other type of sources for example, ﬂare stars and M-dwarfs (Lynch et al. 2017b; Lynch, Murphy, & Kaplan 2017a). Even though the luminosities of pulsars are relatively sta- ble when averaged over long timescales (Taylor 1991; Matsakis, Taylor, & Eubanks 1997), the received ﬂux density can be mod- ulated by propagation effects such as refractive and diffractive scintillation, for example Armstrong, Rickett, & Spangler (1995). While diffractive scintillation causes variability on timescales of tens of minutes, refractive scintillation constitutes variations on the timescales of weeks to months. Depending on the cadence of the observations using the MWA, we explore the variability timescales of 300, 60 and 30s, probing diffractive interstellar scin- tillation speciﬁcally. This is done by generating light curves of the possible variable sources. These sources are selected based on vari- ability statistics (Equations (2), (3), (4)) such as how signiﬁcant the variability is (signiﬁcance) and the percentage of variability of (modulation) the pixel (or the source) when compared to its m (in %) = σS ¯S (4) (4) where σS is the standard deviation of the ﬂux density of the pixel and ¯S is the mean ﬂux density of the pixel (Bell et al. 2016). The correlation between χ2 and modulation index as shown in Figure 8 is used to determine speciﬁc threshold for the different timescales for every observation. A threshold is chosen such that the number of candidates to be checked via beamforming does not exceed 1 000. For the observations analysed as part of this paper, the thresholds are similar given that it is from the same instrument with similar noise characteristic of the image. For observations which are signiﬁcantly different, this threshold has to be deter- mined such that the candidate sources to beamform on is not extremely high. where σS is the standard deviation of the ﬂux density of the pixel and ¯S is the mean ﬂux density of the pixel (Bell et al. 2016). The correlation between χ2 and modulation index as shown in Figure 8 is used to determine speciﬁc threshold for the different timescales for every observation. A threshold is chosen such that the number of candidates to be checked via beamforming does not exceed 1 000. https://doi.org/10.1017/pasa.2022.59 Published online by Cambridge University Press 4.4.3 Variability For the observations analysed as part of this paper, the thresholds are similar given that it is from the same instrument with similar noise characteristic of the image. For observations which are signiﬁcantly different, this threshold has to be deter- mined such that the candidate sources to beamform on is not extremely high. A test of the above described formalism was done on obser- vation B (see Table 1) consisting of two known pulsars, PSR https://doi.org/10.1017/pasa.2022.59 Published online by Cambridge University Press S. Sett et al. S. Sett et al. 8 Figure 9. Light curves for PSR J0034-0721 (blue) and PSR J0034-0534 (green) for 300 s timescale averaged images for the whole observation. For this test, our χ 2 threshold is 1 and the threshold on modulation index (m) is 20%. The blue light curve has a χ 2 of 1.6 and has a modulation index of 30%, whereas the green curve is approximately constant and has a χ 2 of 0.8 and modulation index of 10%. While the pixel corresponding to the blue curve will be easily selected as a candidate on application of our criterion, the pixel for the green curve will not satisfy our threshold. This supports the variability of J0034-0721 as stated in the literature and indicates that our criterion is indeed useful for detection of true pulsar candidates. arcmin pixel−1 resolution, resulting in 35o × 35o images. The σ for this observation was 30 mJy beam−1 (Stokes I) and 10 mJy beam−1 (Stokes V) and increases almost by a factor of 3 at the edge of the image. AEGEAN (Hancock et al. 2012, 2018) was used with a threshold of 5σ to extract sources from mean image result- ing in ∼1 100 sources in Stokes I image. On the application of the compactness criterion, the extended sources are discarded from further analysis, reducing the number of sources to ∼800. On per- forming a similar comparison to the ATNF catalogue as described in Section 4.2, a total of 335 pulsars were present in the 3 dB (half power point) beam. We expect to detect 52 pulsars at 5- σ level, after inspecting the local standard deviation of the noise near the pulsars. 28 out of the 52 pulsars were detected in imaging. Four of the non-detected pulsars were associated with supernova remnants and hence were not in the analysis when the compact- ness criterion is applied. 4.4.3 Variability The same analysis was then extended to the whole image, using the same thresh- old, which resulted in ∼250 candidates. A careful inspection of the light curves reveal one variable source whose pixel position is coincident with that of the variable pulsar PSR J0034-0721. Furthermore, a distinct variability on timescale of 300 s is observed for J0034-0721 when compared to the light curve of the pixel posi- tion corresponding to that of J0034-0534 as shown in Figure 9. The χ2 for this pixel is 1.6 with a modulation index of 30%, satisfying our threshold of χ2. This demonstrates that any source which is at least as variable as J0034-0721 should be easily detected using the criterion of light curve extraction. The variability procedure is then applied to observations C and D as described in Section 4.5. Out of the compact sources present in our Stokes I catalogue, ∼500 are present in RACS all-sky catalogue. Around ∼350 sources are present only in MWA catalogue and are of high signiﬁcance to us. The spectral index of the sources present in both catalogues is calculated using Equation (1). The upper limit of the spectral index is calculated for the sources present in only MWA catalogue. The spectral index threshold of −1.2 as established in Section 4.4 is applied to both the subsets of sources. A total of ∼100 sources have a steep spectrum. 27 of the 100 sources are already known pulsars. The one excluded pulsar has a spectral index of −1.0 and hence does not satisfy our criterion. The remaining steep spectrum sources were beamformed and searched for pulsar-like signal. None were conﬁrmed as pulsar candidates. The second criterion was applied to the Stokes I and V cat- alogues sources in the similar way as described in Section 4.4.2 after the subtraction of instrumental leakage. When this crite- rion for selecting polarised sources as deﬁned in Section 4.4 was applied, 2 sources are independently detected above the threshold, which can be associated with 2 pulsars. However, the EPN database shows that 4 pulsars should be detected in the Stokes V image. The non-detection of the other 2 pulsars can be attributed to the ﬂux density of the pulsars (highest absolute value is 25 mJy beam−1) being below the noise in the Stokes V image (∼40 mJy beam−1). No other polarised sources above the threshold were detected in this observation. 4.4.3 Variability One possible reason for the remaining non-detections was the pulsar ﬂux density being lower than the mean ﬂux density threshold and hence not bright enough for it to be detected in imaging. Another possible reason for the non- detection may be the turnover of pulsars, which is more dominant at lower frequencies. On application of the pulsar searching and folding algorithm in PRESTO on the 28 imaging detections, we were able to successfully detect 23 of them. The 5 non-detections can be attributed to the high DM of the pulsars, which signif- icantly reduces the detection sensitivity of periodic searches at MWA frequencies. Figure 9. Light curves for PSR J0034-0721 (blue) and PSR J0034-0534 (green) for 300 s timescale averaged images for the whole observation. For this test, our χ 2 threshold is 1 and the threshold on modulation index (m) is 20%. The blue light curve has a χ 2 of 1.6 and has a modulation index of 30%, whereas the green curve is approximately constant and has a χ 2 of 0.8 and modulation index of 10%. While the pixel corresponding to the blue curve will be easily selected as a candidate on application of our criterion, the pixel for the green curve will not satisfy our threshold. This supports the variability of J0034-0721 as stated in the literature and indicates that our criterion is indeed useful for detection of true pulsar candidates. Out of the 28 pulsars detected in imaging, we are able to detect 27 in RACS Stokes I image. The possible reason for the non-detection of PSR J1935+2154 could be its association with a supernova remnant, G57.2+0 (Kothes et al. 2018). J0034-0521 and PSR J0034-0721, the latter of which is variable on short timescales (McSweeney et al. 2017). This test was a sanity check, performed to determine if the variable pulsar would satisfy our criterion without any prior information as one of the vari- able candidates with high signiﬁcance. As an initial test case, a χ2 threshold of 1, was applied to the observation, resulting in a list of ∼5 candidate pixels in a small window around the test pulsar, PSR J0034-0721. Three out of the 5 candidate pixels corresponded to that of the pulsar. The other 2 candidates, when searched using PRESTO did not yield any pulsar-like signals. 4.5 Application of the methodologies to other observations Two observations are used to independently test the formulated methodologies and corresponding criteria to determine the suc- cess rate of detecting pulsar candidates. In this section, we describe the results obtained when the test is performed on the observa- tions C and D. In order to select sources which are variable in nature and could possibly be interesting pulsar candidates, we generated mean images for timescales of 300, 60 and 30 s. The χ2 vs modulation index maps for three above mentioned timescales is used to set a threshold for χ2 such that we produced light curves for only 4.4.3 Variability A reduction of Stokes V leakage will improve the sensitivity of this criterion. 4.5.2 Observation D Stokes I and V images were again produced following the pro- cedure laid out in Section 2.2. The images are 8 192 × 8 192 pixels with 0.3 arcmin pixel−1 resolution, creating images that are ∼40o × 40o. The noise in this image varies from 15 mJy beam−1 at the centre of the images to 30 mJy beam−1 at the edge of the image in Stokes I. For mean Stokes V image, ∼2 mJy beam−1 at the centre of the image and increases by almost a factor of 5 at the edges of the image. The source catalogue for the mean images was produced using AEGEAN and consists of ∼5 000 sources. The removal of the extended sources from the catalogue resulted in ∼4 000 sources. According to the ATNF pulsar catalogue there are 12 pulsars in the ﬁeld. Ten out of 12 are detected in imaging. The 2 pulsars are not detected due to the local noise of the pulsars being high as both the pulsars are at the edge of the image. The variability criterion was then applied at the timescale of 300, 60 and 30 s. A χ2 of 1 and modulation index threshold of 20% was enforced, resulting in ∼1 000 candidates. All candidates were searched using standard pulsar techniques, but none of the candidates displayed pulsar-like emission. The independent application of the criterion to the two obser- vations helped in understanding the efﬁciency of the method- ologies in selecting pulsar candidates. It also provided us with a context of the number of sources that one may expect from the different criterion and that increasing thresholds could be one pos- sible way of reducing the number of candidates. Table 2 shows the expected and actual pulsar detections when the methodologies are applied to the four observations. The potential reasons for vari- ous non-detections of known pulsars are stated in the Sections 4.4 and 4.5. Table 3 shows the initial number of candidates for the four methodologies when applied to the four observations. It is clear that the circular polarisation criterion produces signiﬁcantly fewer candidates, which is possibly driven by our lack of full understand- ing of the polarisation leakage in the MWA images. More effort in understanding this issue and formulating a more robust thresh- old is required for greater success of the criterion. 4.5.1 Observation C Processing of this observation was performed as described in Section 2.2 to produce Stokes I and V images. The images pro- duced for this observation were 4 096 × 4 096 pixel with 0.5 https://doi.org/10.1017/pasa.2022.59 Published online by Cambridge University Press https://doi.org/10.1017/pasa.2022.59 Published online by Cambridge University Press 9 Publications of the Astronomical Society of Australia Table 2. Table shows the expected vs actual pulsar detections on application of the methodologies described in the paper. The last columns of observation A, C and D are empty as there are no variable pulsars in the ﬁeld and hence the criterion of variability is not applicable for these observations. Pulsars Spectral index Polarisation Variability Obs name Expected Actual Expected Actual Expected Actual Expected Actual A 42 21 42 18 9 4 N/A N/A B 10 8 10 7 2 1 1 1 C 52 28 52 27 4 2 N/A N/A D 12 10 12 5 5 3 N/A N/A Table 3. Table shows the total number of candidates when the four methodologies have been applied to the 4 observations. The last column shows the ﬁnal candidates after combining the criteria like spectral index and circular polarisation and spectral index and variability. OBS name Observation ID Spectral index Circular polarisation Variability Total number Candidates after of candidates combining criteria A 1276619416 300 1 150 451 7 B 1150234552 150 1 250 401 4 C 1148063920 100 2 550 652 5 D 1274143152 250 5 1000 1255 4 he total number of candidates when the four methodologies have been applied to the 4 observations. The last column shows th combining the criteria like spectral index and circular polarisation and spectral index and variability. Table 3. Table shows the total number of candidates when the four methodologies have been applied to the 4 observations. The ﬁnal candidates after combining the criteria like spectral index and circular polarisation and spectral index and variability. candidates that are most probable to be pulsar candidates. For this observation, a χ2 threshold of 1, and modulation index threshold of 20% is applied, resulting in ∼550 candidates. A careful scrutiny of the resulting light curves was performed and a list of sources and their corresponding positions was created, however, no inter- esting targets were conﬁrmed as pulsar candidates. sources. After the removal of artefacts and leakage, we apply the fractional polarisation criterion and detect 5 real circularly polarised sources. https://doi.org/10.1017/pasa.2022.59 Published online by Cambridge University Press 4.5.1 Observation C Out of the 5 circularly polarised sources, Three of them are already known pulsars. The two other sources have been searched via traditional searches but no pulsations have been found. Five pulsars in this observation are expected to be circu- larly polarised based on EPN database. The 2 non-detected pulsars (highest absolute value is 10 mJy beam−1) are below the detection threshold for the Stokes V image for this observation (∼32 mJy beam−1). 5.3 Criterion 3—Variability Another aspect of pulsar candidate selection tested is by taking advantage of the variability of the sources in different timescales. This method mainly focuses on detecting pulsar candidates that may be missed by traditional searches due to their nulling behaviour or scintillation. We were able to detect the variable pul- sar, PSR J0034 −0721 in the test observation as a pulsar candidate with high signiﬁcance. Extracting light curves for every single pixel of the image will result in ∼1 million candidates, which is a large number of candidates per image to follow up. In order to limit the number of candidates to a few hundreds, we apply a thresh- old of χ2 and modulation index as described in Section 4.4 by analysing the correlation between χ2 and modulation index for every timescale we process. Even with those reductions in place, it is still not feasible to follow up on all the candidates with other tele- scopes. The beneﬁt of leveraging the MWA VCS data means that all of them can be searched using products from the same tele- scope and observation. The other alternative is to check if these 5.2 Criterion 2—circular polarisation We also examined the Stokes I and V images to detect candidates that may be circularly polarised. Around 50% of the circularly polarised pulsars in the observation were detected as candidates without any prior knowledge about their polarisation. However, we were not able to detect any potential pulsar candidate with high signiﬁcance via this method when the fractional polarisation threshold was applied. Compared to total intensity, the number of candidates resulting from the application of this criterion is lower by almost an order of magnitude. Selection of an appropri- ate threshold for candidates to be considered circularly polarised is beneﬁcial to detect genuine Stoke V polarised sources and pul- sar candidates. Moreover, any candidate that is circularly polarised but not a pulsar is likely to be objects of scientiﬁc interest. For dipole based instruments such as the MWA, the instrumental leakage, where emission from Stokes I leak into other Stokes parameters has to be better understood. This leakage signiﬁcantly affects the detection of circularly polarised pulsars and increases the number of false detections. Even with the improvements in calibration and the model of the primary beam, errors are still be present due to the imperfections of the model and the MWA dipoles itself. In Section 4.4 we describe the process of ﬁtting a surface to the fractional polarisation of the sources for the obser- vations, in order to account for the leakage and detect true pulsar candidates. However, further reduction of the leakage will be nec- essary to reduce the threshold and increase sensitivity of this criterion. Another advantage of image-based pulsar candidate searches over traditional pulsars searches is the notably reduced time and resources that image-based searches take to produce a list of pulsar candidates. If we perform a periodicity search for every pixel in our image, that is 10 million pixels, it would take 40 million CPU hours to obtain pulsar candidates. However, on applying the methodolo- gies, we reduce the number of follow-up processing steps to a more tractable ∼500 per observation. 4.5.2 Observation D On the other hand, variability criterion produces a high number of candidates which may result in considerable amount of false positives. One must be careful when determining the threshold for χ2 and mod- ulation index in order to produce a greater number of true pulsar Around ∼3 800 sources are present in both RACS and MWA catalogue. The spectral index for the sources present in both the catalogues and the upper limit of the spectral index for sources present in only MWA catalogue is calculated using Equation (1). Application of the spectral index threshold men- tioned in Section 4.4 results in a total of ∼250 sources that have steep spectral index. Out of 250 sources, 5 are known pulsars. The rest of the sources are beamformed and searched using the MWA pulsar search pipeline to detect any signal. We were not able to conﬁrm any candidates as pulsars. The sources from the Stokes I and V catalogues were then crossmatched and the fractional polarisation is calculated for the S. Sett et al. 10 of false detections of background radio sources is essential for suc- cess of this criterion. In this paper, we have used a spectral index threshold of −1.2 for selection of sources. By doing so, we were able to detect ∼90% of the pulsars detected via imaging in all the observations along with an average of ∼200 new pulsar candidates that are steep spectrum for each observation. Doing similar anal- ysis with a higher threshold, for example, α = −3, would result in signiﬁcantly less candidates but at the expense of detecting fewer known pulsars. Ideally, a threshold that is able to redetect the max- imum number of pulsars as well as generate a manageable number of candidates is preferred, hence, our threshold of α < −1.2, seems appropriate for the future processing of much larger set of MWA observations. candidates. Lastly, the spectral index criterion, seems to generate a manageable number of candidates that can be followed up using periodicity or single-pulse searches while being computationally inexpensive. 5. Discussion Even though there are lots of pulsars in the ﬁeld of view of the observations, the number of pulsar detection in imaging is affected by the sensitivity of the images, reducing the number of pulsar detections in the imaging space. We also performed a compari- son of the number of pulsars that were detected in imaging space and the detections of the same pulsars when searched for periodic signals using PRESTO. We can clearly deduce that the high DM pulsars are not detected when we perform a periodic search. For example, in observation A, PSR J1835-0643, was detected at ∼5σ in Stokes I image, but was not detected when a periodic search was performed due to its high DM of ∼473 pc cm−3. Another reason for the non-detection of the pulsars in periodic searches is the effect of scintillation and scattering. For example, PSR J1833- 0338, in observation A Stokes I image was detected at ∼10σ via imaging as well as periodic searches. The expected detection sig- niﬁcance for the pulsar is ∼30σ for periodic searches based on its ﬂux density. Due to the scattering timescale of the pulsar (∼0.7 s) being close to its period (∼0.6 s), it suffers from scattering which degrades the detection sensitivity of the pulsar when searching using PRESTO and was hence was detected at a lower signiﬁcance in periodic searches. On the other hand, there are pulsars such as PSR J1801-0357 that are detected by periodicity searches and not in image-based searches due to its ﬂux density being lower than the ﬂux density detection threshold for imaging. Overall, image- based searches target pulsar candidates that may have been missed by traditional searches due to scattering or high DM. Any such pulsar candidates need to be followed up with high-frequency telescopes such as the Parkes radio telescope (Murriyang) or uGMRT. https://doi.org/10.1017/pasa.2022.59 Published online by Cambridge University Press References Armstrong, J. W., Rickett, B. J., & Spangler S. R. 1995, ApJ, 443, 209 Backer, D. C., Kulkarni, S. R., Heiles, C., Davis, M. M., & Goss, W. M. 1982, Natur, 300, 615 Banyer, J., Murphy, T., & VAST Collaboration, 2012, in Astronomical Society of the Paciﬁc Conference Series, Vol. 461, Astronomical Data Analysis Software and Systems XXI, ed. P. Ballester, D. Egret, & N. P. F. Lorente, 725 (arXiv:1201.3130) Beardsley, A. P., et al. 2019, PASA, 36, e050 Bell, M. E., et al. 2016, MNRAS, 461, 908 Beardsley, A. P., et al. 2019, PASA, 36, e050 5.1 Criterion 1—steep spectrum The ﬁrst criterion focuses on candidates that have spectral index < −1.2. We were able to successfully detect most of the pulsars in the Stokes I images which satisfy our criterion. However, we did not detect any new pulsar candidates via this method. The steep spectrum sources detected in this criterion may be other cat- egories of extragalactic sources, such as extended emissions from radio halos and relics in merging galaxy clusters and do not have a pulsar-like signal and hence not detected via PRESTO searches. It may happen that sources that are not detected as pulsars via PRESTO-based searches are indeed pulsars but not detected due to high scattering and hence cannot be conﬁrmed by using only the MWA data. Such candidates needs to be observed with tele- scopes operating at higher frequencies. Overall, selecting an ideal upper limit of the spectral index that increases the fraction of pul- sars detected without any prior bias while minimising the fraction https://doi.org/10.1017/pasa.2022.59 Published online by Cambridge University Press Publications of the Astronomical Society of Australia 11 candidates satisfy the other two criteria, and ranking the candi- dates according to the number of criteria satisﬁed. This set of candidates (∼10) can then be followed up with other telescopes. Unfortunately, we were not able to detect any pulsar-like candi- dates. One of the reasons for the non-detections could be that the sources are variable on longer timescales than the ones that are chosen for the observations. It may also be that the variable sources are not pulsars but part of other variable populations such as ﬂare stars. The lack of detection could also be related to the constraints on our periodicity search using PRESTO at MWA frequencies. It should be noted an improved understanding of imaging artefacts produced while imaging with the MWA is required in order to dis- tinguish between true variable sources and sources that appear to be variable but are in fact spurious detections. Limited. This work was further supported by resources provided by the Pawsey Supercomputing Centre with funding from the Australian Government and the Government of Western Australia. Part of this research has made use of the EPN Database of Pulsar Proﬁles maintained by the University of Manchester. S.S. acknowledge the support from Australian Government Research Training Program Scholarship. 6. Conclusions and future work F., Bailes, M., Barr, E. D., Johnston, Jankowski, F., van Straten, W., Keane, E. F., Bailes, M., Barr S., & Kerr, M. 2018, MNRAS, 473, 4436 Kaplan, D. L., Cordes, J. M., & Condon, J. J. 2000, ApJS, 126, 37 Kaplan, D. L., et al. 2019, ApJ, 884, 96 Kothes, R., Sun, X., Gaensler, B., & Reich, W. 2018, ApJ, 852, 54 Lenc, E., et al. 2017, PASA, 34, e040 S., & Kerr, M. 2018, MNRAS, 473, 4436 S., & Kerr, M. 2018, MNRAS, 473, 4436 Kaplan, D. L., Cordes, J. M., & Condon, J. J. 2000, ApJS, 126, 37 y y Another way to reduce the number of candidates can be opti- misation of the thresholds for all the criteria. This is an ongoing project and will be informed by processing a larger number of MWA VCS observations. Overall, it is demonstrated that we were able to detect a large fraction (>50%) of the pulsars expected to be found as well as a selection of new pulsar candidates for the observations. It is also important to understand the instrumen- tal beam response of the instrument being used as any errors in the beam model will result in leakage. For example, effects such as ‘beam squint’ affect radio telescopes with off-axis feeds such as the (E)VLA (Uson & Cotton 2008), and may also be relevant to the future low-frequency telescopes such as SKA-Low. This is useful in informing pulsar search methodologies with the MWA, and sug- gests that the SKA-Low will be a powerful telescope to ﬁnd pulsar candidates in continuum images. Kaplan, D. L., Cordes, J. M., & Condon, J. J. 2000, Ap Kaplan, D. L., Cordes, J. M., & Condon, J. J. 2000, ApJS, 126, 37 Kaplan, D. L., et al. 2019, ApJ, 884, 96 Kothes, R., Sun, X., Gaensler, B., & Reich, W. 2018, ApJ, 852, 54 Kaplan, D. L., et al. 2019, ApJ, 884, 96 p pJ Kothes, R., Sun, X., Gaensler, B., & Reich, W. 2018, ApJ, 852, 54 Kothes, R., Sun, X., Gaensler, B., & Reich, W. 2018, ApJ, Lenc, E., et al. 2017, PASA, 34, e040 Lenc, E., Murphy, T., Lynch, C. R., Kaplan, D. L., & Zhang, S. N. 2018, MNRAS, 478, 2835 Lynch, C., Murphy T., & Kaplan D. L. 2017a, MWA targeted campaign of nearby, ﬂaring M dwarf stars, MWA Proposal id.2017A-02 Lynch, C. R., Lenc, E., Kaplan, D. 6. Conclusions and future work Bell, M. E., et al. 2016, MNRAS, 461, 908 Bell, M. E., et al. 2016, MNRAS, 461, 908 Clark, M. A., LaPlante, P. C., & Greenhill, L. J. 2013, IJHPCA, 27, 178 Overall, the three methods have been successfully curated and tested on the observations given in Table 1. We have a detec- tion efﬁciency of 50% for both method I (steep spectrum) and II (circular polarisation), while method III (variability) was tested on a small number of test sources all of which were success- fully retrieved, after the relevant pulsars for each observation were taken into account. A thorough analysis of the candidates resulting from the application of the above methodologies to the observa- tions was performed. All candidates were searched using MWA beamformed data, however we were not able to detect any pul- sar signal. The larger number of initial candidates (∼100) were reduced by combining the different methodologies to produce a list of 20 candidates, which can then be followed up with higher frequency telescopes. Care must be taken while combining the criteria as we may be missing interesting sources that may not necessarily satisfy more than one criteria. Crawford, F., Kaspi, V. M., & Bell, J. F. 2000, AJ, 119, 2376 Dai, S., Johnston, S., Bell, M. E., Coles, W. 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The success of this pilot project demonstrates the potential of using image-based methodologies to detect pulsar candidates at low fre- quencies and improve the efﬁciency of future pulsar detections with the MWA as well as the SKA-Low. McSweeney, S. J., Bhat, N. D. R., Tremblay, S. E., Deshpande, A. A., & Ord, S. M. 2017, ApJ, 836, 224 Mutel, R. L., & Morris, D. H. 1988, Properties of stellar magneto- spheres deduced from radio observations of close binaries, 283, doi: 10.1007/978-94-009-2951-7_44 Navarro, J., de Bruyn, A. G., Frail, D. A., Kulkarni, S. R., & Lyne, A. G. 1995, ApJ, 455, L55 Acknowledgement. This scientiﬁc work makes use of the Murchison Radio- astronomy Observatory, operated by CSIRO. We acknowledge the Wajarri Yamatji people as the traditional owners of the Observatory site. Support for the operation of the MWA is provided by the Australian Government (NCRIS), under a contract to Curtin University administered by Astronomy Australia Offringa, A. R., de Bruyn, A. 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https://openalex.org/W2171617774	https://cadmus.eui.eu/bitstream/1814/6446/3/LAW%202006-45.pdf	English	null	State Sovereignty, Popular Sovereignty and Individual Sovereignty: From Constitutional Nationalism to Multilevel Constitutionalism in International Economic Law?	Social Science Research Network	2,006	public-domain	19,721	EUI WORKING PAPERS LAW No. 2006/45 EUI WORKING PAPERS LAW No. 2006/45 EUROPEAN UNIVERSITY INSTITUTE DEPARTMENT OF LAW State Sovereignty, Popular Sovereignty and Individual Sovereignty: from Constitutional Nationalism to Multilevel Constitutionalism in International Economic Law? EUI WORKING PAPERS LAW No. 2006/45 State Sovereignty, Popular Sovereignty and Individual Sovereignty: from Constitutional Nationalism to Multilevel Constitutionalism in International Economic Law? ERNST-ULRICH PETERSMANN Department of Law Department of Law Department of Law Department of Law Department of Law Abstract This paper discusses the basic constitutional problem of modern international law since the UN Charter: How can the power-oriented international legal system based on “sovereign equality of states” be reconciled with the universal recognition of “inalienable” human rights deriving from respect for human dignity and popular sovereignty? State representatives, intergovernmental organizations, international judges and non-governmental organizations often express different views on how far the universal recognition of human rights has changed the subjects, structures, general principles, interpretative methods and “object and purpose” of international law (e.g. by the emergence of erga omnes obligations and jus cogens limiting state sovereignty to renounce human rights treaties, to refuse diplomatic protection of individuals abroad, or domestic implementation of international obligations for the benefit of domestic citizens). The paper explains why effective protection of human rights at home and abroad requires multilevel constitutional protection of individual rights as well as multilevel constitutional restraints of national, regional and worldwide governance powers and procedures. While all European states have accepted that the European Convention on Human Rights and EC law have evolved into international constitutional law, the prevailing paradigm for most states outside Europe remains “constitutional nationalism” rather than “multilevel constitutional pluralism.” Consequently, European proposals for reforms of international economic law often aim at “constitutional reforms” (e.g. of worldwide governance institutions) rather than only “administrative reforms”, as they are frequently favoured by non-European governments defending state sovereignty and popular sovereignty within a more power-oriented “international law among states.” State Sovereignty, Popular Sovereignty and Individual Sovereignty: from Constitutional Nationalism to Multilevel Constitutionalism in International Economic Law? ERNST-ULRICH PETERSMANN EUI Working Paper LAW No. 2006/45 This text may be downloaded for personal research purposes only. Any additional reproduction for such purposes, whether in hard copy or electronically, requires the consent of the author(s), editor(s). If cited or quoted, reference should be made to the full name of the author(s), editor(s), the title, the working paper or other series, the year, and the publisher. The author(s)/editor(s) should inform the Law Department of the EUI if the paper is to be published elsewhere, and should also assume responsibility for any consequent obligation(s). ISSN 1725-6739 The author(s)/editor(s) should inform the Law Department of the EUI if the paper is to be published elsewhere, and should also assume responsibility for any consequent obligation(s). ISSN 1725-6739 © 2006 Ernst-Ulrich Petersmann Printed in Italy European University Institute Badia Fiesolana I – 50016 San Domenico di Fiesole (FI) Italy http://www.eui.eu/ http://cadmus.eui.eu/ http://www.eui.eu/ http://cadmus.eui.eu/ * Professor of International and European Law at the European University Institute and Head of its Law Department, Florence, Italy. Previously professor of international and European law at the University of Geneva and its Graduate Institute of International Studies. Chairman of the International Trade Law Committee of the International Law Association, and former legal advisor to the GATT and the WTO (1981-2006). ulrich.petersmann@eui.eu. This contribution has been accepted for publication in: W.Shan/P.Simons/D.Singh (eds), REDEFINING SOVEREIGNTY IN INTERNATIONAL ECONOMIC LAW, Hart Publishers 2007 Keywords WTO; Council of Europe; European Court of Justice; law; fundamental/human rights; international trade; international relations; intergovernmental conferences. 1 See E.U.Petersmann, Constitutionalism, International Law and ‘We the Peoples of the United Nations’, in: H.J.Cremer et alii (eds), Tradition und Weltoffenheit des Rechts. Festschrift für H.Steinberger (Berlin: Springer Publishers, 2002), 291 ff. STATE SOVEREIGNTY, POPULAR SOVEREIGNTY AND INDIVIDUAL SOVEREIGNTY: FROM CONSTITUTIONAL NATIONALISM TO MULTILEVEL CONSTITUTIONALISM IN INTERNATIONAL ECONOMIC LAW? Prof. Dr. Ernst-Ulrich Petersmann* 2 These constitutional premises prompted me to argue (e.g. in E.U.Petersmann, Constitutional Functions and Constitutional Problems of International Economic Law, Fribourg: University Press Fribourg, 1991) that precise and unconditional, intergovernmental guarantees of freedom, non- discrimination and rule of law (e.g. in GATT Articles II, III, XI:1) can serve “constitutional functions” enlarging corresponding, constitutional freedoms and other rights of citizens. Ernst-Ulrich Petersmann Ernst-Ulrich Petersmann The claims by rulers to supreme ordering power (“political sovereignty”) must be distinguished from democratic constitutional law as the legal source of sovereign powers (“constitutional sovereignty”), as well as from “democratic sovereignty” and “individual sovereignty” in the sense of the actual capacity of a democratic polity and of individual citizens to self-government. Modern globalization is characterized by increasing economic, political, legal and other limitations of political sovereignty and by the re-allocation of government powers to democratic people, indigenous people, international organizations and individual human beings as legal subjects of inalienable human rights. This dynamic transformation of international relations and of international law entails tensions between the “sovereign equality” of UN member states as one of the constitutional principles of the UN Charter (Art.2), the “right to self- determination of all peoples” as universally recognized in the UN human rights covenants of 1966 (Article 1), and the universal recognition, in numerous UN human rights instruments, of “the inherent dignity and of the equal and inalienable rights of all members of the human family [as] the foundation of freedom, justice and peace in the world” (Preamble of the 1966 UN Covenant on Economic, Social and Cultural Rights). Human rights law, like most democratic constitutions, proceeds from the two premises that (1) human rights are not granted, but only recognized by governments; and (2) citizens constitute governments with limited powers that must be exercised for the protection of the human rights and public interests of their citizens.2 The UN Human Rights Committee has rightly emphasized (e.g. in its General Comments 24 and 26 on the UN Covenant on Civil and Political Rights) that - even if human rights treaties are concluded among states – unilateral reservations and denunciations may be legally invalid and cannot take away “inalienable” human rights of citizens3, just as democratic governments may lack powers to unilaterally abrogate constitutional rights of their citizens. This contribution argues for a “constitutional approach” to international law based on the following principles: (1) Equal individual freedom as first principle of justice: The universal recognition of inalienable human rights requires construing state sovereignty, popular sovereignty and “individual sovereignty” in a mutually coherent manner so as to protect more effectively equal freedoms of individuals and other “principles of justice” across frontiers (below Sections I and IV). g g p g g 3 Cf. Compilation of General Comments and General Recommendations by Human Rights Treaty Bodies, UN doc. HRI/GEN/1, Rev.7 (2004). Introduction and Summary: Need for Redesigning Sovereignty for the Benefit of Citizens and their Constitutional Rights Introduction and Summary: Need for Redesigning Sovereignty for the Benefit of Citizens and their Constitutional Rights Over the past centuries, claims to supreme political and legal power over people (sovereignty) by rulers (e.g. the pope, emperors, monarchs, colonial powers), states and international organizations have become increasingly contested. The Westphalian system of international law among sovereign states - based on internal sovereignty (as defined by constitutional law) and external sovereignty (as defined by state-centered international law) - was power-oriented and lacked democratic legitimacy, as illustrated by colonialism and imperial wars. Also the UN Charter (e.g., Chapter V regulating the Security Council) remains based on power-oriented structures lacking input-legitimacy (e.g., in terms of respect for human rights and democracy) as well as output-legitimacy (e.g., in terms of protection of ‘democratic peace’). Even though proclaimed on behalf of “We the peoples of the United Nations determined … to reaffirm faith in fundamental human rights” (Preamble of the UN Charter), the intergovernmental structures of UN law have proven to be incapable of realizing the human rights objectives of UN law. The ineffectiveness of so many UN guarantees of human rights in so many countries undermine also the democratic legitimacy and effectiveness of UN law.1 Ernst-Ulrich Petersmann (2) Human rights require multilevel constitutional protection: Historical experience confirms the constitutional claim that legal guarantees of equal freedoms among individuals and among republican states cannot remain effective over time without multilevel constitutionalism constituting, limiting and legitimizing governance powers and protecting human rights at national, international and transnational levels of human interactions in a mutually coherent manner (below Section II). (2) 2 2 State Sovereignty, Popular Sovereignty and Individual Sovereignty (3) (3) Human rights require the “constitutionalization” of international law: Constitutional approaches to international law have practical consequences for the interpretation and progressive development also of international economic law. For instance, respect for individual and democratic freedom entails diversity and regulatory competition – among individuals as well as among private and public, national, regional and worldwide organizations (e.g., competing worldwide and regional trade liberalization and trade regulation) - which must be respected and promoted by “cosmopolitan principles”, including human rights, “participatory” and “deliberative democracy” so as to promote inclusive decision-making, as well as other “legitimating principles” (such as subsidiarity) and “prioritizing commitments” (such as avoidance of serious harm, satisfaction of urgent needs, cf. Section III). (4) The needed international “social market economy” must be based on “cosmopolitan democracy”: The more remote regional and worldwide governance institutions operate from their citizens, the less effective procedural citizen rights for democratic participation, representation and parliamentary control risk becoming; and the more important are substantive, constitutional rights and their judicial protection through interrelated networks of national and international rule-making, administration and courts. Rights-based multilevel constitutionalism requires more comprehensive processes of weighing and balancing rights and obligations of governments and of individuals in the interpretation and application of international economic law by governments and courts (Section IV). (5) (5) The “transformation power” of economic constitutionalism depends on “struggles for human rights”: The increasing complexity of globalization makes decentralized rules and adjustments protecting individual freedom, democracy and state sovereignty ever more important. Constitutional democracies have strong economic, political and legal reasons for extending rights-based constitutionalism to multilevel economic governance so as to safeguard constitutional democracy and set incentives for the peaceful transformation of non-democratic and less-developed countries. 5 Cf. J.Goldsmith/E.A.Posner, The Limits of International Law (Oxford University Press, 2005). On the “formalism” of the general international law rules coordinating the fragmented bilateral, regional and worldwide treaty systems see Martti Koskenniemi, Fragmentation of International Law: Difficulties Arising from the Diversification and Expansion of International Law, UN document A/CN.4/L.682 of 4 April 2006. 6 See M. Ignatieff, American Exceptionalism and Human Rights, (Cambridge MA: Harvard UP, 2005); J. Rubenfeld, “The Two World Orders”, in: G. Nolte (ed), European and US Constitutionalism, (Cambridge: CUP, 2005), at 280-296. 8 See the broad interpretation of UN human rights as international ius cogens and constitutional limitation of intergovernmental powers by the EC Court of First Instance in Cases T-315/01 (Kadi v Council and Commission) and T-306/01 (Yusuf v Council and Commission), judgments of 21 September 2005, in: Common Market Law Reports 2005, at 1334. 7 On the different conceptions and principles of universal justice (e.g human rights), and of corrective and distributive justice in particular cases (e.g. preferential treatment of poor countries), see : E.U. Petersmann, “Theories of Justice, Human Rights and the Constitution of International Markets, in: Symposium: The Emerging Transnational Constitution”, (2003) 37 Loyola Law Review 407-460. 4 On the pursuit of “order” rather than “justice” in international relations see: R. Foot, J.L. Gaddis & A. Hurrel (eds), Order and Justice in International Relations, (Oxford: OUP, 2003); J. Thomson, Justice and World Order, (Tokyo: United Nations Press, 1992). Ernst-Ulrich Petersmann The difference between American and European approaches lies not in the European recourse to “normative”, “soft” and “civilian power” (which are also used in US foreign policies), but in the European preference for multilevel constitutional restraints on multilevel economic governance rather than for the focus by the US, as well as by other continental nation states, on constitutional nationalism (Section V). The “transformation power” of economic constitutionalism depends on “struggles for human rights”: The increasing complexity of globalization makes decentralized rules and adjustments protecting individual freedom, democracy and state sovereignty ever more important. Constitutional democracies have strong economic, political and legal reasons for extending rights-based constitutionalism to multilevel economic governance so as to safeguard constitutional democracy and set incentives for the peaceful transformation of non-democratic and less-developed countries. The difference between American and European approaches lies not in the European recourse to “normative”, “soft” and “civilian power” (which are also used in US foreign policies), but in the European preference for multilevel constitutional restraints on multilevel economic governance rather than for the focus by the US, as well as by other continental nation states, on constitutional nationalism (Section V). 3 Ernst-Ulrich Petersmann Ernst-Ulrich Petersmann 9 On the ECJ jurisprudence that respect for fundamental rights is a “condition of the legality of Community acts”, see ECJ Opinion 2/92 on accession by the EC to the ECHR, ECR 1996 I-1759; Case C-84/95, Bosphorus ECR 1996 I-3953. On the constitutional functions of the ECHR see the judgment of the ECtHR of 30 June 2005 (Application No 6422/2002, Bosphorus v Ireland) which confirmed, inter alia, “that a Contracting Party is responsible under Article 1 of the Convention for all acts and omissions of its organs regardless of whether the act or omission in question was a consequence of domestic law or of the necessity to comply with international legal obligations” (para. 153). I. International Law among States, among Peoples and among Individuals? Limitation of State Sovereignty by Constitutional, Popular and Individual Sovereignty I. International Law among States, among Peoples and among Individuals? Limitation of State Sovereignty by Constitutional, Popular and Individual Sovereignty The Westphalian system of international law among sovereign states conceived international law as reciprocal limitations on state power aimed at protecting international order rather than human rights and justice.4 Also the state-centred UN legal system protects neither human rights nor democratic peace effectively. Even constitutional democracies (like the US) view compliance with international law often as a national policy choice depending more on the respective costs and benefits than on rule of international law and its culture of legal formalism.5 The universal recognition of human rights and of supranational organizations entails a progressive broadening of the international community to individuals and other non-state actors. Traditional principles of international law (such as sovereign equality of states, non-intervention into domestic affairs, consensus-based rule-making) are increasingly qualified in the law of worldwide and regional organizations. Also civil societies, parliaments and democracies increasingly challenge the legitimacy of power-oriented rules of international law (e.g. the sovereignty of failed states that tolerate genocide and international terrorism); democratic governments insist on their constitutional powers to adopt policy measures even if they are inconsistent with power-oriented international law rules.6 If law is perceived as a struggle for individual constitutional rights and human rights, such challenges to power politics disguised as international law are to be welcomed. Without stronger constitutional safeguards for individual freedom as “first principle of justice” as well as of constitutional protection of other human rights and democratic peace, UN law is bound to remain ineffective in many areas.7 The more the human rights obligations of every UN member state evolve into ius cogens, the more it becomes necessary to construe the “sovereign equality” of states, the limited powers of intergovernmental organizations and the “right to self- determination of all peoples” in conformity with the human rights of their citizens.8 The European Court of Justice (ECJ) and the European Court of Human Rights (ECtHR) emphasize that human rights constitutionally restrain also the limited powers of 4 State Sovereignty, Popular Sovereignty and Individual Sovereignty intergovernmental organizations; they interpret and enforce the fundamental rights guarantees of the European Convention on Human Rights (ECHR) as a “constitutional instrument of European public order”. 10 See E.-U. Petersmann, “Constitutionalism and WTO Law – From a State-Centred Approach towards a Human Rights Approach in International Economic Law”, in: D.L.M. Kennedy & J.D. Southwick (eds), The Political Economy of International Trade Law (Cambridge: CUP, 2002), at 32-67; idem, E.U.Petersmann, Human Rights, Markets and Economic Welfare: Constitutional Functions of the Emerging UN Human Rights Constitution, in: F.M. Abbott, C. Breining-Kaufmann & T. Cottier (eds), International Trade and Human Rights. Foundations and Conceptual Issues, (Ann Arbor: Michigan UP, 2006), 29-68. More generally see: Allen Buchanan, Justice, Legitimacy and Self-Determination. Moral Foundations for International Law (Oxford University Press 2004). I. International Law among States, among Peoples and among Individuals? Limitation of State Sovereignty by Constitutional, Popular and Individual Sovereignty 9 The emerging “multilevel human rights constitution”, based on national, regional and worldwide guarantees of human rights and of democratic self-determination of peoples, calls for construing state sovereignty, popular sovereignty and “individual sovereignty” in a mutually coherent manner so as to protect maximum human liberty and other human rights as basic principles of justice. From such a human rights perspective, the international legal system – including international economic law - must serve human rights and democratic self-government as the proper basis of sovereignty.10 Citizens and democratic people must be recognized and legally protected as subjects of international law. Just as decolonization and democracy resulted from struggles for the liberation of suppressed people, so will the needed democratization of the power-oriented international legal system not come about without bottom-up struggles for the defense of cosmopolitan human rights against power politics, including border discrimination reducing domestic consumer welfare and impeding a mutually beneficial division of labor among citizens across frontiers. The UN High Commissioner for Human Rights has, in a number of reports on the human rights dimensions of the law of the World Trade Organization (WTO), endorsed a human rights approach to international trade law.11 Similarly, WTO Director-General P. Lamy has called for “cosmopolitics” and “cosmopolitan constituencies” in support of global public goods such as a liberal trading system.12 Yet, UN human rights law does not protect freedom of profession, private property, open markets, freedom of trade and other legal preconditions for a mutually welfare-enhancing, international division of 10 See E.-U. Petersmann, “Constitutionalism and WTO Law – From a State-Centred Approach towards a Human Rights Approach in International Economic Law”, in: D.L.M. Kennedy & J.D. Southwick (eds), The Political Economy of International Trade Law (Cambridge: CUP, 2002), at 32-67; idem, E.U.Petersmann, Human Rights, Markets and Economic Welfare: Constitutional Functions of the Emerging UN Human Rights Constitution, in: F.M. Abbott, C. Breining-Kaufmann & T. Cottier (eds), International Trade and Human Rights. Foundations and Conceptual Issues, (Ann Arbor: Michigan UP, 2006), 29-68. More generally see: Allen Buchanan, Justice, Legitimacy and Self-Determination. Moral Foundations for International Law (Oxford University Press 2004). 11 See E.-U. Petersmann, “The Human Rights Approach to International Trade Advocated by the UN High Commissioner for Human Rights: Is it Relevant for WTO Law and Policy?” in: Journal of International Economic Law (JIEL) 2004, 605-628. 12 Cf. S.Charnovitz, The WTO and Cosmopolitcs, in : E.U.Petersmann (ed), Reforming the World Trading System. 13 See the criticism of UN human rights law and policies by E.-U. Petersmann, “Human Rights and International Trade Law – Defining and Connecting the Two Fields”, in: T. Cottier, J. Pauwelyn & E. Bürgi (eds), Human Rights and International Trade, (Oxford: OUP, 2005), at 29-94. 14 Cf. B.Kingsbury, N.Krisch & R.Stewart, The Emergence of Global Administrative Law, in: Law and Contemporary Problems 68 (2005), at 15 ff; C.Joerges/E.U.Petersmann (eds), Constitutionalism, Multilevel Trade Governance and Social Regulation (Oxford: Hart Publishing, 2006). II. Multilevel Economic Governance Requires Multilevel Constitutionalism Protecting Individual Rights and Democratic Governance Modern globalisation and the universal recognition of human rights are transforming the intergovernmental “society of states” into a cosmopolitan community of citizens with complex layers and networks of private and public, national and international governance and legal regulation. I. International Law among States, among Peoples and among Individuals? Limitation of State Sovereignty by Constitutional, Popular and Individual Sovereignty Legitimacy, Efficiency and Democratic Governance (Oxford University Press 2005), 437-446 ; P. Lamy, Towards World Democracy (London : Policy Network, 2005). According to David Held, “cosmopolitanism can be taken as the moral and political outlook that offers the best prospects of overcoming the problems and limits of classic and liberal sovereignty”, D.Held, Law of States, Law of Peoples: Three Models of Sovereignty, in: Legal Theory 8 (2002), 1-44, at 24. 5 Ernst-Ulrich Petersmann labor.13 Nor has the UN Conference on Trade and Development (UNCTAD) acted as a leader for a liberal (i.e. liberty-based) international trade order committed to the protection of human rights. labor.13 Nor has the UN Conference on Trade and Development (UNCTAD) acted as a leader for a liberal (i.e. liberty-based) international trade order committed to the protection of human rights. The worldwide liberalization and regulation of welfare-reducing trade barriers continues to be based on the General Agreement on Tariffs and Trade (GATT) and WTO law. The 149 WTO Members emphasize that the WTO rules and policies must remain “member-driven” and outside the UN system. WTO Members have not responded to the UN proposals for a “human rights approach to international trade”; they continue to leave the clarification of the interrelationships between UN law, WTO law and human rights to the WTO dispute settlement system. The “member-driven” character of the WTO entails that WTO rules and policies tend to be “producer-driven” for the benefit of powerful “rent-seeking interest groups” (such as textiles, agricultural and steel lobbies, periodically elected trade politicians) to the detriment of general consumer welfare and citizen rights. As WTO law does not refer to human rights, democratic self-government and social justice, WTO rules remain contested inside constitutional democracies and by civil society. 13 See the criticism of UN human rights law and policies by E.-U. Petersmann, “Human Rights an International Trade Law – Defining and Connecting the Two Fields”, in: T. Cottier, J. Pauwelyn & E 15 See, for example, E.U.Petersmann, Multilevel Trade Governance Requires Multilevel Constitutionalism, in: C.Joerges/E.U.Petersmann (note 14), chpt. 1; D.M.Johnston, World Constitutionalism in the Theory of International Law, in: R.S.J.Macdonald/D.M.Johnston (eds), Towards World Constitutionalism. Issues in the Legal Ordering of the World Community (Leiden: Nijhoff Publishers, 2005), 3-30; Robert Uerpmann-Wittzack, The Constitutional Role of Multilateral Diverse Forms of Multilevel Economic Governance Five basic types of international economic regulation (e.g. of exchange rates, investments, production, trade, competition, consumption, goods, services, social and environmental standards, transnational movements of capital, persons and communications) can be distinguished14 and interact in manifold ways that often lack transparency: (1) international treaties and intergovernmental organizations at worldwide or regional levels (such as the WTO and the more than 250 regional trade agreements), which increasingly protect also private rights (e.g. in the EC) and (1) international treaties and intergovernmental organizations at worldwide or (1) international treaties and intergovernmental organizations at worldwide or regional levels (such as the WTO and the more than 250 regional trade agreements), which increasingly protect also private rights (e.g. in the EC) and 6 State Sovereignty, Popular Sovereignty and Individual Sovereignty private judicial remedies (e.g. in the World Bank’s International Center for the Settlement of Investment Disputes); private judicial remedies (e.g. in the World Bank’s International Center for the Settlement of Investment Disputes); (2) informal intergovernmental networks among domestic regulatory agencies (such as the Basel Committee of national bank regulators, the International Competition Network among national competition authorities); (2) informal intergovernmental networks among domestic regulatory agencies (such as the Basel Committee of national bank regulators, the International Competition Network among national competition authorities); (2) informal intergovernmental networks among domestic regulatory agencies (such as the Basel Committee of national bank regulators, the International Competition Network among national competition authorities); (3) national authorities implementing international economic rules and policies subject to international regulation and constitutional restraints; (3) national authorities implementing international economic rules and policies subject to international regulation and constitutional restraints; (3) national authorities implementing international economic rules and policies subject to international regulation and constitutional restraints; (4) hybrid public-private “regulatory partnerships”, such as the worldwide administration of website addresses by the private Internet Corporation for Assigned Names and Numbers (ICANN), which is subject to the regulatory supervision by the United States and closely cooperates with the World Intellectual Property Organization (e.g. concerning the peaceful settlement of domain name disputes by the WIPO arbitration procedures); and (4) hybrid public-private “regulatory partnerships”, such as the worldwide administration of website addresses by the private Internet Corporation for Assigned Names and Numbers (ICANN), which is subject to the regulatory supervision by the United States and closely cooperates with the World Intellectual Property Organization (e.g. Diverse Forms of Multilevel Economic Governance concerning the peaceful settlement of domain name disputes by the WIPO arbitration procedures); and (5) private regulatory bodies such as the International Standardisation Organisation (ISO) for the international harmonisation of standards that are also used as a legal basis for intergovernmental trade regulation (e.g. in the WTO Agreement on Technical Barriers to Trade), or the International Chamber of Commerce (ICC) whose private rules and commercial arbitration are closely connected with national and intergovernmental regulatory systems (see, for instance, the private “Independent Review Procedures” administered jointly by the ICC and the WTO in order to determine the compliance by public and private parties with the WTO Agreement on Preshipment Inspection). 18 On the “constitutional functions” of international organizations as a “fourth branch of government” see E.U.Petersmann, ‘Constitutionalism and International Organizations’, in: (1996) 17 Northwestern Journal of International Law & Business, at 398, 415 et seq. 16 Cf. N. Blokker and H. G. Schermers, International Institutional Law, 4th ed (Boston, Mass: Martinu Nijhoff, 2003). Treaty Systems, in: A. von Bogdandy & J. Bast (eds), Principles of European Constitutional Law, (Oxford: Hart Publishers, 2006), 147-181. 16 Cf. N. Blokker and H. G. Schermers, International Institutional Law, 4th ed (Boston, Mass: Martinus Nijhoff, 2003). 17 Cf. J. Klabbers, ‘Constitutionalism Lite’ (2004) 1 IOLR 31, at 45. On the general recognition of multilevel constitutionalism in Europe see C. Joerges and E. U. Petersmann (eds), Constitutionalism, Multilevel Trade Governance and Social Regulation (Oxford: Hart, 2006). 18 On the “constitutional functions” of international organizations as a “fourth branch of government” see E.U.Petersmann, ‘Constitutionalism and International Organizations’, in: (1996) 17 Northwestern Journal of International Law & Business, at 398, 415 et seq. Governance Most states have adopted constitutions that constitute polities and government powers, subject governments to constitutional restraints, and commit government policies to the promotion of human rights and other constitutional objectives. Globalization demonstrates that national constitutions alone can neither protect human rights across frontiers nor secure the collective supply of global public goods (like international peace, rule of law and a healthy environment). National constitutions turn out to be “incomplete constitutional safeguards”; in a globally interdependent world where ever more citizens pursue their happiness by consuming foreign goods and services or travelling abroad, national constitutions can no longer realize many of their objectives without complementary “international constitutional safeguards” protecting constitutional rights, and limiting abuses of power, in transnational and international relations.15 7 7 17 Cf. J. Klabbers, ‘Constitutionalism Lite’ (2004) 1 IOLR 31, at 45. On the general recognition of multilevel constitutionalism in Europe see C. Joerges and E. U. Petersmann (eds), Constitutionalism, Multilevel Trade Governance and Social Regulation (Oxford: Hart, 2006). Ernst-Ulrich Petersmann The more governments cooperate internationally for the collective supply of ‘international public goods’, the more multilevel governance in international organizations is leading to multilevel legal restraints on national policy powers. Since the Constitution (sic) Establishing the ILO of 1919, also many other constituent agreements of international organizations - such as the World Health Organization (WHO) and the UN Educational, Scientific and Cultural Organization (UNESCO) - are named ‘constitutions’ in view of the fact that, e.g., they: The more governments cooperate internationally for the collective supply of ‘international public goods’, the more multilevel governance in international organizations is leading to multilevel legal restraints on national policy powers. Since the Constitution (sic) Establishing the ILO of 1919, also many other constituent agreements of international organizations - such as the World Health Organization (WHO) and the UN Educational, Scientific and Cultural Organization (UNESCO) - are named ‘constitutions’ in view of the fact that, e.g., they: (1) constitute a new legal order with legal primacy over that of the member states; (2) create new legal subjects and hierarchically structured institutions with limited governance powers; (3) provide for institutional checks and balances (e.g. among rule-making, administrative and dispute settlement bodies in the WTO); (4) legally limit the rights of member states (e.g. regarding withdrawal, amendment procedures, dispute settlement procedures); (5) provide for the collective supply of ‘public goods’ that – as in the case of the above-mentioned treaty constitutions (of the ILO, WHO and UNESCO) – are partly defined in terms of human rights (such as core labour rights, the human rights to health and education); and often (6) operate as ‘living constitutions’ whose functions – albeit limited in scope and membership – increasingly evolve in response to changing needs for international cooperation. (1) constitute a new legal order with legal primacy over that of the member states; (1) constitute a new legal order with legal primacy over that of the member states; (2) create new legal subjects and hierarchically structured institutions with limited governance powers; (3) provide for institutional checks and balances (e.g. among rule-making, administrative and dispute settlement bodies in the WTO); (4) legally limit the rights of member states (e.g. 21 See E.U.Petersmann (n 2); J.O. McGinnis & M.L. Movsesian, “The World Trade Constitution”, in: (2000) 114 Harvard Law Review 511-605; P.M. Gerhart, ‘The Two Constitutional Visions of the World Trade Organisation’, in: (2003) 24 University of Pennsylvania Journal of International Economic Law 1-75, contrasts the “inward-looking, economic vision of the WTO” in helping member countries addressing internal political failures with the “external, participatory vision of the WTO” helping WTO members to address concerns raised by policy decisions in other countries. 20 See D.Z. Cass, ‘The Constitutionalization of International Trade Law: Judicial Norm-Generation the Engine of Constitutionalization’, in: European Journal of International Law 2001, at 39-75. Ernst-Ulrich Petersmann regarding withdrawal, amendment procedures, dispute settlement procedures); (5) provide for the collective supply of ‘public goods’ that – as in the case of the above-mentioned treaty constitutions (of the ILO, WHO and UNESCO) – are partly defined in terms of human rights (such as core labour rights, the human rights to health and education); and often (6) operate as ‘living constitutions’ whose functions – albeit limited in scope and membership – increasingly evolve in response to changing needs for international cooperation. (1) constitute a new legal order with legal primacy over that of the member states; (2) create new legal subjects and hierarchically structured institutions with limited governance powers; provide for institutional checks and balances (e.g. among rule-making, administrative and dispute settlement bodies in the WTO); legally limit the rights of member states (e.g. regarding withdrawal, amendment procedures, dispute settlement procedures); provide for the collective supply of ‘public goods’ that – as in the case of the above-mentioned treaty constitutions (of the ILO, WHO and UNESCO) – are partly defined in terms of human rights (such as core labour rights, the human rights to health and education); and often (6) operate as ‘living constitutions’ whose functions – albeit limited in scope and membership – increasingly evolve in response to changing needs for international cooperation. The worldwide ‘treaty constitutions’ differ fundamentally from national constitutions by their limited policy functions and less effective constitutional restraints (e.g. on intergovernmental and national policy powers). State-centered international lawyers therefore prefer to speak of ‘international institutional law’16 or of an intergovernmental ‘constitutionalism lite’. 17 From citizen-oriented economic and constitutional perspectives, however, international organizations are becoming no less necessary for the collective supply of public goods than national organizations. Human rights and their moral value premises (normative individualism) require designing national and international governance as an integrated, multilevel constitutional framework for the protection of citizen rights, democratic self-government and cooperation among free citizens across frontiers.18 International constitutionalism is a functionally limited, but necessary complement to national constitutionalism which, only together, can protect 8 State Sovereignty, Popular Sovereignty and Individual Sovereignty human rights and democratic self-government more effectively across frontiers in a globally integrating world. human rights and democratic self-government more effectively across frontiers in a globally integrating world. 22 See, for example, P.M. Gerhart, “The WTO and Participatory Democracy: The Historical Evidence in: (2004) 37 Vanderbilt Journal of Transnational Law 897-934. J.Trachtman, The WTO Constitution: Toward Tertiary Rules, in European Journal of International Law 2006. f 23 See J.O. McGinnis, ‘The WTO as a Structure of Liberty’, in: (2004) 28 Harvard Journal of Law and Public Policy 81-88. 19 See J.H. Jackson, The World Trade Organisation: Constitution and Jurisprudence, (London: Royal Institute of International Affairs, 1998). 19 See J.H. Jackson, The World Trade Organisation: Constitution and Jurisprudence, (London: Royal Institute of International Affairs, 1998). 20 See D.Z. Cass, ‘The Constitutionalization of International Trade Law: Judicial Norm-Generation as the Engine of Constitutionalization’, in: European Journal of International Law 2001, at 39-75. 21 See E.U.Petersmann (n 2); J.O. McGinnis & M.L. Movsesian, “The World Trade Constitution”, in: (2000) 114 Harvard Law Review 511-605; P.M. Gerhart, ‘The Two Constitutional Visions of the World Trade Organisation’, in: (2003) 24 University of Pennsylvania Journal of International Economic Law 1-75, contrasts the “inward-looking, economic vision of the WTO” in helping member countries addressing internal political failures with the “external, participatory vision of the WTO” helping WTO members to address concerns raised by policy decisions in other countries. 22 See, for example, P.M. Gerhart, “The WTO and Participatory Democracy: The Historical Evidence”, in: (2004) 37 Vanderbilt Journal of Transnational Law 897-934. 23 See J.O. McGinnis, ‘The WTO as a Structure of Liberty’, in: (2004) 28 Harvard Journal of Law and Public Policy 81-88. 24 J.Trachtman, The WTO Constitution: Toward Tertiary Rules, in European Journal of International Law 2006. Constitutional Functions of WTO Law None of the supporters of “international constitutionalism” claims that international “treaty constitutions” constituting and limiting international rule-making, executive and judicial powers for the collective supply of international public goods are, or should become, constitutions in the same sense as national constitutions. In line with the diverse national constitutional traditions, constitutional approaches to multilevel governance differ inevitably. Constitutional Functions of WTO Law For instance, the notion of a “WTO constitution” is increasingly being used in view of (a) the comprehensive rule-making, executive and (quasi-) judicial powers of WTO institutions;19 (a) the comprehensive rule-making, executive and (quasi-) judicial powers of WTO institutions;19 (b) the “constitutionalization” of WTO law resulting from the jurisprudence of the WTO dispute settlement bodies;20 (b) the “constitutionalization” of WTO law resulting from the jurisprudence of the WTO dispute settlement bodies;20 (b) (c) the domestic “constitutional functions” of GATT/WTO rules, for example for protecting constitutional principles (like freedom, non-discrimination, rule of law, proportionality of government restrictions) and domestic democracy (for instance, by limiting the power of protectionist interest groups) for the benefit of transnational cooperation among free citizens;21 (c) (c) the domestic “constitutional functions” of GATT/WTO rules, for example for protecting constitutional principles (like freedom, non-discrimination, rule of law, proportionality of government restrictions) and domestic democracy (for instance, by limiting the power of protectionist interest groups) for the benefit of transnational cooperation among free citizens;21 (d) the international “constitutional functions” of WTO rules, for example, for the promotion of “international participatory democracy” (e.g., by holding governments internationally accountable for the “external effects” of their national trade policies, by enabling countries to participate in the policy-making of other countries) 22 and of the enhancement of “jurisdictional competition among nation states”23 and “the allocation of authority between constitutions”;24 (d) (e) in view of the necessity of “constitutional approaches” for a proper understanding of the law of comprehensive international organizations that use (e) in view of the necessity of “constitutional approaches” for a proper understanding of the law of comprehensive international organizations that use (e) 9 9 Ernst-Ulrich Petersmann constitutional terms, methods and principles for more than 50 years (see, e.g., the “Constitutions” of the ILO, WHO, FAO, EU);25 or (f) (f) in view of the need to interface and coordinate different levels of governance on the national and international level.26 All these constitutional approaches agree that the WTO should not be simply viewed in narrow economic terms (for example, as an institution promoting economic welfare through trade liberalization). 29 See the power-oriented justifications by members of the EC legal services of the frequent EC violations of WTO law (e.g., P.J.Kuijper, WTO Law in the European Court of Justice, in Common Market Law Review 42 (2005), 1313-1341, who criticizes the rule-oriented ‘Kupferberg jurisprudence’ of the ECJ as politically ‘naïve’, at 1320); Kuijper claims, at 1334, that “it is difficult to point out one specific moment at which it can be established beyond doubt that WTO rules have been breached, even after a decision of a panel or report of the Appellate Body”, and “that it is rarely Constitutional Functions of WTO Law WTO rules and policies also pursue political as well as legal objectives that are no less important than the economic benefits from liberal trade; a recent illustration of this are the guarantees of private rights to trade and intellectual property rights, including “rights to import and export”, of private access to independent courts and rule of law in the 2001 WTO Protocol on the accession of China.27 The introduction of open markets and rule of law in China, including a system of independent trade courts (supervised by a chamber of the Chinese Supreme Court specialized in WTO law), illustrates that the WTO Agreement is one of the most revolutionary “transformation agreements” in the history of international law. 25 See, for example, E.U.Petersmann, (note 13); N. Walker, ‘The EU and the WTO: Constitutionalism in a New Key’, in: G. de Búrca & J. Scott (eds), The EU and the WTO: Legal and Constitutional Issues, 2001. 28 See Bogdandy & Bast (note 15). Even though the process of ratification of the TCE ((2004) 47 Official Journal of the EU C 310) has been blocked by two negative referenda in France and the Netherlands, it appears realistic to assume that the basic constitutional principles codified in the TCE will continue to prevail in EU constitutional law. 26 T. Cottier & M. Hertig, The Prospects of 21st Century Constitutionalism, 7 Max Planck Yearbook United Nations Law (2003), at 261. ( ) 27 See WTO document WT/L/432 of 23 November 2001, Part I, Section 2. ( ), 27 See WTO document WT/L/432 of 23 November 2001, Part I, Section 2. Constitutional Nationalism vs Multilevel Constitutionalism The progressive transformation of the intergovernmental EC Treaty rules into constitutional and judicial guarantees of individual rights was largely the result of the struggle of citizens and courts (including the ECJ and the ECtHR) against welfare- reducing abuses of foreign policy powers. In Europe, this struggle for individual freedom and democratic self-government across frontiers has led to the legal and judicial protection of ever more ‘new’ fundamental rights (as codified in the 2000 EU Charter of Fundamental Rights) and ‘constitutional principles’ of EU law (as codified in Part I of the 2004 Treaty Establishing a Constitution for Europe = TCE).28 Their underlying value premises of normative individualism and rule of international law (cf. Article I-2 TCE on the ‘Union’s values’) remain, however, contested by many politicians and their legal advocates – not only outside Europe in treaty regimes dominated by hegemonic countries (like the North American Free Trade Area) but also in the EU’s external relations.29 Most Europeans agree with the US view that popular 10 State Sovereignty, Popular Sovereignty and Individual Sovereignty sovereignty inside democratic nation states remains a precondition for legitimate transnational governance. 30 Yet, the rights-based, cosmopolitan European constitutionalism differs fundamentally from the constitutional nationalism in most democracies outside Europe 31 , especially from the current US focus on national constitutionalism and hegemonic, power-oriented foreign policies.32 Even though the US Constitution confirms the federal insight that sovereignty is divisible, many North- American lawyers remain reluctant to conceive international legal constraints on legislative and executive powers as constitutional rules, for instance because such international rules “do not establish the power they restrain and cannot be understood as emanations of a pouvoir constituant.”33 Constitutional Limits of Judicial Governance WTO Members tend to construe the limitation of the jurisdiction of WTO dispute settlement bodies to “the covered agreements” (Article 7 WTO Dispute Settlement Understanding = DSU) narrowly as permitting only legal claims and defences based on WTO rules. International lawyers and WTO dispute settlement bodies, however, emphasise that the customary methods of treaty interpretation require interpreting WTO rules as part of the international legal system in conformity with general international law rules unless WTO law has excluded recourse to other rules of international law.34 or never possible to speak of a sufficiently serious breach of WTO law” by the political EC institutions justifying the EC’s non-contractual liability for damages pursuant to Article 288 EC Treaty. or never possible to speak of a sufficiently serious breach of WTO law” by the political EC institutions justifying the EC’s non-contractual liability for damages pursuant to Article 288 EC Treaty. 31 For some Anglo-Saxon communitarian lawyers, even Europe cannot be properly described as ‘constitutionalized’ until it embodies a European polity and demos legitimizing its community (cf. J. Weiler, The Constitution of Europe (Cambridge: CUP, 1999)). Other Anglo-Saxon lawyers challenge the recognition of “market freedoms” in European constitutional law on the ground that neither individual economic freedom nor other individual rights are ‘a matter considered essential to constitutionalisation in the received tradition’ of ‘mature constitutional systems, for example in the United States, Canada and Australia’ (D. Z. Cass, The Constitutionalization of the WTO (Oxford: OUP, 2005, at 168, 176, 191). For a criticism of “constitutional nationalism” see my review of the book by Cass in: CMLR 43 (2006), at 890-892, and E.U.Petersmann, Introduction and Summary, in: Joerges/Petersmann (above n. 14). 32 On the US penchant for imperial, unilateral foreign policies, notwithstanding US-leadership in promoting multilateral legal disciplines for other countries after World Wars I and II, see: I. Daalder and J. Lindsay, America Unbound: The Bush Revolution in Foreign Policy (Washington D.C.; Brookings Institution, 2004). On the often skeptical US attitude vis-à-vis international law see: J. F. Murphy, The United States and the Rule of Law in International Affairs (Cambridge: CUP, 2004). 33 Cf. A. von Bogdandy, Constitutionalism in International Law : Comment on a Proposal from Germany, in : Harvard International Law Journal 47 (2006), 223-242, at 240. 34 See. J. 34 See. J. Pauwelyn, Conflict of Norms in Public International Law: How WTO Law Relates to Other Rules of International Law, (Cambridge: CUP, 2003), at 37-38. According to Pauwelyn, “it is for the party claiming that a treaty has ‘contracted out’ of general international law to prove it” (at 213). Many WTO members and WTO lawyers proceed from the contrary presumption that the limitation of the jurisdiction of WTO dispute settlement bodies to “the covered agreements” entail an agreed presumption in favor of exhaustive WTO regulation and confirms that “(i)n international law, every tribunal is a self-contained system (unless otherwise provided)”, as stated by the International Criminal Tribunal for Yugoslavia in its Tadic judgment, International Legal Materials 35 (1996), 32, at para. 11. For an explanation of this narrow perception of the WTO as – in principle – a self- 30 Cf. J.A.Rabkin, Law without Nations? Why Constitutional Government Requires Sovereign States (Princeton University Press, 2005). Ernst-Ulrich Petersmann Ernst-Ulrich Petersmann WTO dispute settlement practice continues to identify an increasing number of general principles of law (for example, on burden of proof, good faith, abuse of rights, due process of law, legal security) and more specific treaty principles for the mutual balancing of rights and obligations under WTO law (for example, principles of legal security, transparency, non-discrimination, necessity, and proportionality).35 Some of these principles (for example, on burden of proof) are applied as general principles of law, whereas others (such as the non-discrimination and necessity principles of WTO law) appear to be construed as WTO-specific “principles underlying this multilateral trading system”, as they have been explicitly acknowledged in the Preamble and in other provisions of the WTO Agreement. The perspective of international judges focusing on the customary methods of international treaty interpretation may differ fundamentally from the perspective of domestic judges confronted with WTO law arguments (focusing, for example, on national and EU constitutional law rather than on WTO obligations of the EC). In the EC and in some other WTO members, the domestic constitutional perspective has occasionally prompted national judges and EC judges to interpret domestic trade rules (for example, the customs union rules in the EC Treaty) as protecting private rights even if the respective trade rules were drafted as intergovernmental rights and obligations and implemented GATT/WTO obligations of the EC (for example, the EC customs union rules implementing GATT Article XXIV). From the European perspective of a common market without a common state, such “empowering functions” of liberal trade rules for the benefit of the private market participants, and their incorporation as an “integral part of the Community legal system” in order to limit governmental trade policy discretion, have prompted me to support such “constitutional interpretations” by EU judges of intergovernmental trade rules for the benefit of EU citizens, and to suggest that EU judges should interpret national and international trade rules as a functional unity inside the EC for the benefit of citizens.36 Yet, such “constitutional interpretations” are not permissible for the international WTO judge who must interpret the intergovernmental WTO rules “in accordance with customary rules of interpretation of public international law”, as prescribed in Article 3.2 DSU. 37 On the “constitutional limits” of WTO jurisprudence see the various contributions (e.g., by R. Howse, W. Davey and E-.U. Petersmann) in: T. Cottier & P. Mavroidis (eds), The Role of the Judge in International Trade Regulation. Experience and Lessons for the WTO, (Ann Arbor: Michigan UP, 2003), at 43-90. 35 On general principles of international law as part of the WTO legal system, and the need for a principle-oriented WTO jurisprudence balancing WTO rights, WTO obligations as well as the numerous WTO exceptions protecting policy autonomy for non-economic regulation, see E.U.Petersmann, WTO dispute settlement practice 1995-2005: Lessons from the past and future problems, in : Y.Taniguchi et alii (eds), The WTO at Ten : Dispute Settlement, Multilateral Negotiations, Regional Integration (Cambridge : CUP 2006). contained legal regime see, for example, J. Neumann, Die Koordination des WTO-Rechts mit anderen völkerrechtichen Ordnungen: Konflikte des materiellen Rechts und Konkurrenzen der Streibeilegung, (Berlin: Duncker & Humblot, 2002). 36 See Petersmann, ‘Application of GATT by the Court of Justice of the EC’, in: (1983) 20 Common Market Law Review 409. Constitutional Limits of Judicial Governance Pauwelyn, Conflict of Norms in Public International Law: How WTO Law Relates to Other Rules of International Law, (Cambridge: CUP, 2003), at 37-38. According to Pauwelyn, “it is for the party claiming that a treaty has ‘contracted out’ of general international law to prove it” (at 213). Many WTO members and WTO lawyers proceed from the contrary presumption that the limitation of the jurisdiction of WTO dispute settlement bodies to “the covered agreements” entail an agreed presumption in favor of exhaustive WTO regulation and confirms that “(i)n international law, every tribunal is a self-contained system (unless otherwise provided)”, as stated by the International Criminal Tribunal for Yugoslavia in its Tadic judgment, International Legal Materials 35 (1996), 32, at para. 11. For an explanation of this narrow perception of the WTO as – in principle – a self- 11 38 See: United States – Sections 301-310 of the Trade Act of 1974, panel report adopted on 27 January 2000, WT/DS152/R, at para.7.72. The Panel makes the following important reservation: “The fact that WTO institutions have not to date construed any obligations as producing direct effect does not necessarily preclude that, in the legal system of any given member state, following internal constitutional principles, some obligations will be found to give rights to individuals. Our statement of fact does not prejudge any decisions by national courts on this issue.” Ernst-Ulrich Petersmann 37 The WTO panel report on Sections 301-310 of the US Trade Act emphasised, for example, that “(n)either the GATT nor the WTO has so far been interpreted by GATT/WTO institutions as a legal order producing direct effect”, i.e., 12 State Sovereignty, Popular Sovereignty and Individual Sovereignty creating rights and obligations not only for WTO member states but also direct individual rights for traders, producers and consumers.38 International law and WTO rules limit, however, also the legislative discretion of national legislatures. The WTO panel report on Mexico’s measures affecting telecommunications services, for instance, concluded that private price-fixing practices remained “anti-competitive practices” prohibited under Mexico’s obligations under the General Agreement on Trade in Services (GATS) even if Mexican legislation required such uniform pricing practices.39 Due to the different jurisdiction, applicable laws and judicial competences of national and international courts, their “constitutional interpretations” may differ at national and international levels. “Constitutionalisation” by means of jurisprudence must respect constitutional limits which may not apply to “constitutionalisation” by national and intergovernmental rule-making. Yet, as the WTO guarantees of freedom, non- discrimination and rule of international law go far beyond the autonomous guarantees in the domestic laws of most states, the legitimacy of WTO jurisprudence derives not only from “legal formalism” 40 but also from protecting individual freedom, non- discriminatory conditions of competition and rule of law across national frontiers for the benefit of welfare-increasing cooperation among citizens. 40 S.Picciotto, The WTO’s Appellate Body : Legal Formalism as a Legitimation of Global Governance, in : Governance : An International Journal of Policy, Administration and Institutions, 18 (2005), 477 ff. III. Practical Policy Relevance of Constitutional Approaches Compared with traditionally power-oriented foreign policy approaches (e.g. in the context of GATT 1947), multilevel constitutionalism may require different policy approaches and judicial approaches, for example in the following areas of multilevel trade governance. 39 Mexico – Measures Affecting Telecommunications Services, Panel report (WT/DS204/R of 2 April 2004) adopted in June 2004, at paras. 7.244-245. 41 On the problems of “constitutional interpretation” see J.E.Alvarez, International Organizations as Law-Makers (Oxford University Press 2005), chapter 2. 42 Cf. E.U.Petersmann, Justice as Conflict Resolution: Proliferation, Fragmentation and Decentralization of Dispute Settlement in International Trade, in: University of Pennsylvania Journal of International Economic Law 27 (2006), 273-367. 43 Cf. E.U.Petersmann, The WTO and Regional Trade Agreements as Competing Fora for Constitutional Reforms, in: L.Bartels/F.Ortino (eds), Regional Trade Agreements and the WTO Legal System (Oxford University Press, 2006), chapter 11. Constitutional Rules for Conflict Resolution The fragmented, decentralized nature of international relations among 200 sovereign states makes normative conflicts between national, bilateral, regional and worldwide rules inevitable. The Vienna Convention on the Law of Treaties (VCLT) and other general international law rules offer formal legal principles for preventing and resolving such conflicts (e.g. on the basis of lex specialis, lex posterior, rules on inter-se agreements, ius cogens, erga omnes obligations). Constitutionalism requires respecting individual and democratic diversity and viewing regulatory diversity, regulatory competition and regulatory conflicts positively.42 For instance, the WTO negotiations on stricter substantive WTO disciplines for regional trade agreements (RTAs) are bound to fail as long as RTAs (such as the more than 60 RTAs concluded after the failure on the 2003 WTO Ministerial Conference to advance the Doha Round negotiations in the WTO) are favored not only as “competing trade liberalization”, but also as competing fora for regional trade regulation that does not appear acceptable in the consensus-based, worldwide WTO negotiations.43 Constitutional Interpretation The interpretation of the constituent agreements of international organizations, and of international guarantees of private freedoms and rule of law (such as the ‘market freedoms’ guaranteed in the EC Treaty), may justify “constitutional” and “dynamic- evolutionary interpretations” different from those of the more “static interpretation” of 13 Ernst-Ulrich Petersmann other contractual elements of international treaties.41 For instance, the customary rules of international treaty interpretation and WTO jurisprudence admit that the “contractual” dimensions of WTO law may require different interpretative approaches (e.g. for interpreting GATT and GATS schedules of commitments, WTO “non- violation complaints” aimed at maintaining the “balance” of reciprocal tariff commitments) than their “constitutional” dimensions (e.g. in case of judicial clarification of the inherent powers of WTO dispute settlement bodies, or of the powers of WTO Members under the WTO exceptions protecting “public morals” and “public order”). The universal recognition of human rights entails "constitutional principles" and ius cogens rules that may require rights-based interpretations going beyond the original meaning of treaty terms at the time of the conclusion of intergovernmental treaties. 44 On ‘development as freedom’ and substantive ‘opportunity to achieve’, see A. Sen, Rationality and Freedom (Cambridge, MA: HUP, 2002), ch 17 on ‘markets and freedoms’; E. U. Petersmann (ed), Developing Countries in the Doha Round, (Florence: EUI, 2005), 3-18. See also, F. A. Hayek, The Constitution of Liberty (Chicago, IL: University of Chicago Press, 1960), 35: ‘Economic considerations are merely those by which we reconcile and adjust our different purposes, none of which, in the last resort, are economic (except those of the miser or the man for whom making money has become an end in itself).’ Constitutional Functions of WTO Rules Liberal international trade rules differ from most other areas of international law insofar as their international guarantees of freedom, non-discrimination and rule of law tend to go beyond the autonomous guarantees of freedom and non-discrimination in national and regional laws. International treaties constituting and limiting multilevel governance are a necessary precondition for the collective supply of global public goods (like a liberal, rules-based world trading system) which cannot be secured through power- 14 State Sovereignty, Popular Sovereignty and Individual Sovereignty oriented foreign policies focusing only on national interests. The inherent tendencies of freedom and competition to destroy themselves (“paradox of freedom”) require internationally agreed “constitutional restraints” on abuses of foreign policy powers in order to secure rule of law in international relations (e.g. by means of the WTO dispute settlement system), open markets (e.g. by means of WTO guarantees of non- discriminatory conditions of competition), respect for human rights and social justice (as reflected and protected in the numerous “exceptions” from WTO rules). Constitutionalism offers criteria for designing the law of international organizations (e.g. the necessary “checks and balances” between rule-making, executive and adjudicative powers). Human Rights Approaches to International Economic Law From a rights-based constitutional perspective (as, e.g., in EU constitutional law), national and international constitutionalism should be perceived as complementary instruments for the protection of the constitutional rights and human rights of citizens. Just as economists (from Adam Smith to Amartya Sen) perceive market economies and economic growth as instruments for enabling and promoting individual freedom as the ultimate goal of economic life and the most efficient means of realizing human welfare, so should national and international law (e.g. the WTO objective of “sustainable development”) be seen as instruments for the protection of human rights and democratic self-government of peoples. 44 The rights-based premises of constitutionalism require empowering individuals and civil society (e.g. non- governmental organizations) as legal subjects of international economic law. Multilevel constitutionalism can promote welfare-enhancing synergies between national and international constitutionalism; power-oriented foreign policies and border discrimination tend to undermine such synergies (e.g. by preventing EC and US courts from applying and enforcing self-imposed WTO guarantees of freedom and rule of law in domestic courts). 46 See J. Habermas, Between Facts and Norms – Contributions to a Discourse Theory of Law and Democracy, (Cambridge MA: MIT Press, 1996), at 315-328, arguing that democratic self-government requires that only those rules may claim democratic legitimacy that can meet with the assent of all rational citizens in a discursive process equally open to all possibly affected persons. Public discourse and collaborative reasoning will enhance problem-solving and mutual learning. On the relationships between democracy and rule of law, see also E.-U. Petersmann, “European and International Constitutional Law: Time for Promoting Cosmopolitan Democracy in the WTO”, in: G. de Búrca & J. Scott (eds), The EU and the WTO, (Oxford: Hart Publishing, 2001), at 81-110. 45 See the contributions by K. Dicke & J. Frowein in: D. Kretzmer & E. Klein (eds), The Concept of Human Dignity in Human Rights Discourse, (The Hague: Kluwer, 2002), at 111 et seq.; E.-U. Petersmann, Human Rights, Markets and Economic Welfare (note 10), at 30 ff. On “individual sovereignty” see also UN Secretary-General Kofi Annan, ‘Two Concepts of Sovereignty’, The Economist, 18 September 1999, and E.U.Petersmann, “From State Sovereignty to the Sovereignty of Citizens’ in the International Relations Law of the EU?”, in: N. Walker (ed), Sovereignty in Transition, (Oxford: Hart, 2003), at 145-166. Individual Empowerment and Responsibility Human rights call for promoting individual rights and accountability in the transnational division of labour among producers, investors, traders and consumers. According to the Universal Declaration of Human Rights (Article 1 UDHR), “(a)ll human beings are born free and equal in dignity and rights. They are endowed with reason and conscience and should act towards one another in a spirit of brotherhood”. UN human rights conventions recognise in their Preambles “that these rights derive from the inherent dignity of the human person”. Respect for human dignity and human 15 Ernst-Ulrich Petersmann rights, i.e., normative individualism - and not just state sovereignty, “the will of the people” (Article 21 of the UDHR) and their “right of self-determination” (Article 1 of the 1966 UN human rights covenants) – are of particular importance for empowering individuals in the international division of labour. The references, in Article 1 UDHR, to “reason and conscience” lend support to the view that the protection of human dignity as the basic objective of all human rights requires respect for “individual sovereignty” (for example, in the sense of moral, rational, personal, and legal autonomy), responsibility and a real capacity for personal self-development.45 Human dignity as a moral “right to have rights” requires individuals to be treated as legal subjects rather than as the mere objects of authoritarian government policies, especially in the regulation of transnational economic activities of private citizens. The authoritarian state-centred practices of treating citizens as mere objects even in those fields of international law which regulate mutually beneficial private co-operation across frontiers (such as WTO law), need to be challenged in the light of the human rights objective of empowering citizens and protecting human rights across national borders. From a human rights perspective, the legitimacy of intergovernmental organisations (such as the WTO) depends less on “output legitimacy” (for example, in terms of the promotion of economic growth) than on “input legitimacy” in terms of respect for human rights, fundamental freedoms, democratic procedures and rule of law for the benefit of citizens, even if the statutory law of the organisation does not explicitly refer to the universal human rights obligations of all states today. Just as democratic polities must be legally constituted by human rights and a political constitution, so must a social world economy be based on and protected by human rights and a citizen-based “economic constitution.” State Sovereignty, Popular Sovereignty and Individual Sovereignty WTO and of many other international organizations do not adequately empower, inform and include citizens and their representative institutions in their collective decision-making procedures so as to ensure “cosmopolitics” for the benefit of all citizens and their individual and democratic self-determination. Cosmopolitanism requires decentralizing decision-making and promoting mutually beneficial cooperation across frontiers based on (1) equal individual rights (subject to democratic diversity) so as to enhance the real capacity of informed, individual and democratic self-determination; (2) stronger, institutionalized accountability mechanisms for the consequences of individual and social actions; (3) inclusive decision-making taking account of each person’s equal rights, arguments and interests as closely as possible to the citizens concerned; (4) protection of “exit” and alternative choices at subsidiary levels of decision- making (e.g. RTAs as alternatives to consensus-based WTO agreements); and (5) avoidance of serious harm and prioritizing of scarce resources on the amelioration of urgent need.47 (4) protection of “exit” and alternative choices at subsidiary levels of decision- making (e.g. RTAs as alternatives to consensus-based WTO agreements); and (5) avoidance of serious harm and prioritizing of scarce resources on the amelioration of urgent need.47 Social Market Economy and Social Justice Contrary to the views of the American legal philosopher John Rawls and to the prevailing “Washington consensus”, individual rights to distributive and social justice and corresponding government obligations should be recognized not only inside constitutional democracies but also in international law and transnational relations, albeit subject to limitations and conditionality.48 For instance, the emerging “Geneva consensus” (Pascal Lamy) in the Doha Development Round negotiations acknowledges that market access commitments by less-developed countries may need to be complemented by international adjustment and capacity-building assistance in order to avoid serious harm, promote the social acceptability of trade competition and help to meet the adjustment costs and human rights obligations in less-developed countries. 47 For a similar list of “constitutive”, “legitimating” and “prioritizing cosmopolitan principles” see Held (note 12), at 24 ff. 48 Cf. Petersmann, Theories of Justice (note 7). 48 Cf. Petersmann, Theories of Justice (note 7). 47 For a similar list of “constitutive”, “legitimating” and “prioritizing cosmopolitan principles” see He (note 12), at 24 ff. 48 Cf. Petersmann, Theories of Justice (note 7). Deliberative Cosmopolitan Democracy Whereas national democracies are constitutionally defined by, and on behalf of, “the people”, the influence of popular and parliamentary democracy on the law and policies of intergovernmental organizations remains inevitably limited. Respect for human rights, private and democratic self-government and social justice require more transparent, participatory and deliberative forms of transnational “cosmopolitan democracy” so that citizens can better comprehend, influence and scrutinise multilevel governance for their mutual benefit.46 The purely intergovernmental structures of the 16 State Sovereignty, Popular Sovereignty and Individual Sovereignty IV. Cosmopolitan Democracy Depends on Multilevel Constitutional and Judicial Safeguards of Human Rights In a globally interdependent world, constitutional democracy cannot remain effective unless local and national constitutional rights and institutions are supplemented by international constitutional guarantees protecting the diversity of democratic polities 17 Ernst-Ulrich Petersmann and mutually beneficial cooperation of free citizens across frontiers. Proposals for stronger, multilevel constitutional restraints on foreign policies for the protection of equal citizen rights aim at strengthening constitutional democracies by enabling citizens, civil society, parliaments and governments to cooperate more effectively in the collective supply of international public goods. In Europe, such international guarantees of human rights and of liberal trade have proven of crucial importance for peaceful economic and legal integration of all 46 member states of the Council of Europe based on common constitutional rules. Inside constitutional democracies and regional integration law, the legal protection of human rights – including economic, social and cultural (ESC) rights - legitimately differs depending on the diverse constitutional traditions, development experiences and priorities of the countries concerned. On the worldwide level, the ratification of the 1966 UN Covenant on ESC Rights by more than 120 countries has – in the words of the chairperson of the UN Committee on ESC Rights – so far failed to change the widely held perception of “violations of ESC rights… as inevitabilities, as a ‘normal’ state of affairs in an unfair world where goods, resources and power are divided in a grossly inequitable manner.”49 Also UN Secretary-General Kofi Annan, in his final address to world leaders assembled in the UN General Assembly on 19 September 2006, admitted that the intergovernmental structures of UN law do not effectively empower individuals to protect themselves against abuses of government powers and to create the economic resources needed for the enjoyment of human rights; the power-oriented international legal system is widely perceived as “unjust, discriminatory and irresponsible” and has failed to effectively respond to the three global challenges to the United Nations – “an unjust world economy, world disorder and widespread contempt for human rights and the rule of law” –, resulting in divisions that “threaten the very notion of an international community, upon which the UN stands.”50 European law remains unique in granting effective legal and judicial remedies empowering individuals against national rulers and intergovernmental organizations. 49 Virginia B.Dandan, Foreword to : S.Leckie/Anne Gallagher (eds), Economic, Social and Cultural Rights. A Legal Resource Guide (Philadelphia : University of Pennsylvania Press, 2006), at ix. 50 The speech of Kofi Annan is reproduced in UN document GA/105000 of 19 September 2006. 51 On these dual functions of human rights and other constitutional rights as rules, as well as principles for optimizing rules depending on what is factually and legally possible in the particular circumstances, see Robert Alexy, A Theory of Constitutional Rights (Oxford University Press 2002), chapter 3. Article I-9, para.3 of the TCE acknowledges these dual functions in the following terms: “Fundamental rights, as guaranteed by the European Convention for the Protection of Human Rights and Fundamental Freedoms and as they result from the constitutional traditions common to the Member States, shall constitute general principles of the Union’s law.” IV. Cosmopolitan Democracy Depends on Multilevel Constitutional and Judicial Safeguards of Human Rights The UN Covenant on ESC rights, for instance, does not protect freedom of profession, private property, open markets, freedom of trade and other legal preconditions for empowering individuals to create the economic welfare – including a mutually beneficial, international division of labour - needed for the enjoyment of human rights. UN human rights bodies have no powers to prevent or effectively sanction the widespread violations of human rights by UN member states. Most UN agencies and the WTO do not pursue effective rights-based development strategies. Empowerment of individuals to use law for securing their ESC rights must be anchored in national constitutional laws in order to offer effective remedies at the local levels where people work and live. Liberal (i.e. liberty-based) constitutional theory offers the most important moral and legal foundation for empowering people across frontiers. 18 State Sovereignty, Popular Sovereignty and Individual Sovereignty A clarification of the constitutional foundation of human rights is the necessary first step in challenging the persistent neglect of ESC rights by many governments and most international organizations. Even inside Europe, the interaction between constitutional law and human rights legitimately differs among countries and among the regional organizations in Europe (such as the EC, the European Free Trade Area, the Council of Europe). The elaboration of the European Charter of Fundamental Rights and of the Treaty Establishing a Constitution for Europe (TCE) confirms that social and intergovernmental agreement on multilevel constitutional safeguards of human rights depends on clarifying the underlying value premises, such as the need to reconcile state sovereignty, popular sovereignty and individual sovereignty through multilevel constitutionalism (discussed above in Sections I to III). The following section addresses three propositions for extending rights-based, multilevel constitutionalism to an international “social market economy” and “cosmopolitan democracy”: First, the synergies between constitutional rights and human rights need to be strengthened by grounding both on respect for human dignity and human liberty rights in conformity with modern theories of justice. Second, the more distant regional and worldwide governance institutions operate from their citizens, the less effective procedural citizen rights for democratic participation, representation and control risk becoming in multilevel economic governance. IV. Cosmopolitan Democracy Depends on Multilevel Constitutional and Judicial Safeguards of Human Rights A social international market economy, and a complementary cosmopolitan democracy, must be founded on stronger substantive, constitutional rights empowering citizens to engage in mutually beneficial private cooperation across frontiers and regulating the international division of labour through integrted networks of national and international rule- making, administration and courts. Third, rights-based multilevel constitutionalism must be progressively extended bottom-up and requires more comprehensive processes of weighing and balancing rights and obligations of governments and of individuals in the interpretation and application of international economic law by governments and courts at national and international levels. 53 According to Immanuel Kant, “freedom constitutes man’s worth”, and “freedom (independence from the constraints of another’s will) insofar as it is compatible with the freedom of everyone else in accordance with a universal law, is the sole and original right that belongs to each human being by virtue of his humanity” (cf. I.Kant, The Metaphysics of Morals, in: I.Kant, Political Writings (ed. by H.Reiss), 1977, at 136). According to Kant, equality and all other human rights derive from this universal birthright of freedom. Justice requires, according to Kant, exercising one’s freedom in accordance with the moral “categorical imperative” that individuals and a just constitution must allow the “greatest freedom” for each individual along with “the most precise specification and preservation of the limits of this freedom so that it can coexist with the freedom of others”. Kant defines law (“Recht”) as “the sum total of those conditions within which the will of one person can be reconciled with the will of another in accordance with a universal law of freedom”; Kant follows from his moral “categorical imperative” that “every action which by itself or by its maxim enables the freedom of each individual’s will to co-exist with the freedom of everyone else in accordance with a universal law is right” (Kant, at 133). 52 On differences between Kantian and Rawlsian principles of justice see Petersmann (note 7 above). Rights-Based Constitutionalism: The ius cogens core of human rights Human rights and other constitutional rights serve complementary functions: they empower individuals by actionable rights and serve as “balancing principles” for “optimizing” rights and obligations depending on the particular circumstances. 51 Rights-based constitutionalism, and the human rights guarantees in democratic constitutions, differ from republicanism (e.g. in ancient Greece, classical Rome, the 19 Ernst-Ulrich Petersmann Ernst-Ulrich Petersmann Italian Renaissance republics) by their contractual justification (rather than e.g. Aristotelian justifications) of democratic polities and of constitutional constraints on government powers in order to secure man’s freedom. Modern theories of justice (from I.Kant to J.Rawls) recognize maximum equal legal freedom of all human beings as the “first principle of justice”. 52 Human rights theories and rights-based, modern constitutional theories - notably since the American and French revolutions in the 18th century and the post-war, rights-based constitutional law in Europe – are rooted much more in normative individualism than it is the case in many other countries, whose constitutional traditions remain influenced by historical power struggles for “mixed constitutions” and by constitutional privileges of monarchs, Houses of Lords, churches or of other previous rulers (e.g. communist parties) limiting equal individual rights. My own publications have construed the recognition – in German and European constitutional law and UN human rights instruments - of human dignity as the source of inalienable human rights in accordance with the Kantian concept of human dignity (e.g. as the moral and rational capacity of human beings of exercising one’s freedom of choice in a “just” manner respecting the equal autonomy and maximum equal freedom of all others). 53 According to Kant, respect for human dignity and justice require treating human beings as ends in themselves, respecting their moral choices, and protecting maximum equal freedom of individuals through ever more precise, national, international and cosmopolitan constitutional rules. 54 As everybody “may seek his happiness in whatever way he sees fit, so long as he does not infringe upon the freedom of others to pursue a similar end which can be reconciled with the freedom of everyone p p j ( ) 53 According to Immanuel Kant, “freedom constitutes man’s worth”, and “freedom (independence from the constraints of another’s will) insofar as it is compatible with the freedom of everyone else in accordance with a universal law, is the sole and original right that belongs to each human being by virtue of his humanity” (cf. I.Kant, The Metaphysics of Morals, in: I.Kant, Political Writings (ed. by H.Reiss), 1977, at 136). According to Kant, equality and all other human rights derive from this universal birthright of freedom. Ernst-Ulrich Petersmann Justice requires, according to Kant, exercising one’s freedom in accordance with the moral “categorical imperative” that individuals and a just constitution must allow the “greatest freedom” for each individual along with “the most precise specification and preservation of the limits of this freedom so that it can coexist with the freedom of others”. Kant defines law (“Recht”) as “the sum total of those conditions within which the will of one person can be reconciled with the will of another in accordance with a universal law of freedom”; Kant follows from his moral “categorical imperative” that “every action which by itself or by its maxim enables the freedom of each individual’s will to co-exist with the freedom of everyone else in accordance with a universal law is right” (Kant, at 133). 54 54 On Kant’s moral “categorical imperatives” for acting in accordance with universal laws (“Act only in accordance with that maxim through which you can at the same time will that it become a universal law”), for respecting human dignity by treating individuals and humanity as ends in themselves (“So act that you use humanity, whether in your own person or that of another, always at the same time as an end, never merely as a means”), and for respecting individual autonomy (“the idea of the will of every rational being as a will giving universal law”) and individual right (“Any action is right if it can coexist with everyone’s freedom according to a universal law”), see also A.W.Wood , Kant’s Ethical Thought, (Cambridge University Press, 1999). On Kant’s social and historical theories of the antagonistic human nature promoting market competition and constitutional “rules of justice”, and on Kant’s moral imperative of transforming the “lawless freedom” in the state of nature into lawful freedom through ever more precise national, international and cosmopolitan constitutional rules see also: E.U. Petersmann, How to Constitutionalize International Law and Foreign Policy for the Benefit of Civil Society?, in: Michigan Journal of International Law 20 (1999), 1-30. 20 State Sovereignty, Popular Sovereignty and Individual Sovereignty else within a workable general law” 55, treating a person as an end-in-herself and respecting her freely chosen objectives require legal protection of a human right to maximum equal freedom in the economic area no less than in other areas of human cooperation. For only the individual himself can know, and decide on, his own ends and personal self-development. 55 Kant, On the Common Saying: This May be True in Theory, but it does not Apply in Practice (note 53, at 74). 56 Paragraph 5 of the Declaration, reproduced in: The UN and Human Rights 1945-1995 (New York: UN 1995), at 450. 57 Ernst-Ulrich Petersmann 59 q p p 59 Article 53 of the VCLT defines a “peremptory norm of general international law” as “a norm accepted and recognized by the international community of States as a whole as a norm from which no derogation is permitted and which can be modified only by a subsequent norm of general international law having the same character.” The ius cogens core of UN human rights is smaller than that of European human rights and constitutional law. In the Kadi and Yusuf cases (note 8), the Court of First Instance examined the consistency of sanctions imposed by the UN Security Council and EC implementing regulations only with regard to the ius cogens core of UN human rights to make use of one’s property, the right to a fair hearing and the right to an effective judicial remedy; the Court claimed that it had no jurisdiction to examine whether the EC implementation of the UN sanctions was also consistent with the broader guarantees of fundamental rights in European Community law. The legal consistency of the listing of alleged terrorist groups with European human rights law was also challenged in the ECtHR which elaborated a concept of presumed compliance of the EC with the ECHR: “the Court finds that the protection of fundamental rights by EC law can be considered to be… “equivalent”… to that of the Convention system” (ECtHR decision of 23 May 2002 on Application No 6422/2002, para. 165); judge Ress rightly emphasized in his concurring opinion that the “concept of a presumption of convention compliance should not be interpreted as excluding a 59 Article 53 of the VCLT defines a “peremptory norm of general international law” as “a norm accepted and recognized by the international community of States as a whole as a norm from which no derogation is permitted and which can be modified only by a subsequent norm of general international law having the same character.” The ius cogens core of UN human rights is smaller than that of European human rights and constitutional law. Ernst-Ulrich Petersmann Hence, human dignity and human liberty are indivisible, as recognized in the 1993 Vienna Declaration adopted by the World Conference on Human Rights: “All human rights are universal, indivisible and interdependent and interrelated.”56 The ECJ has acknowledged that respect for human rights is a condition of the lawfulness of EC acts.57 The ECtHR has likewise recognized in a series of judgments that the human rights guarantees of the ECHR also apply whenever states implement intergovernmental rules adopted in international organizations.58 National and international human rights increasingly limit foreign policy powers even if they are being exercised collectively in intergovernmental organizations. Yet, contrary to the reasoning of the EC Court of First Instance in the Kadi and Yusuf judgments which declined jurisdiction to review UN Security Council resolutions in the light of European human rights, judicial protection of the broader constitutional and human rights guarantees in European constitutional law should not be abandoned in favour of the more limited ius cogens core of UN human rights limiting the powers of the UN Security Council.59 58 See Case Bosphorus v Ireland, ECtHR Grand chamber judgment of 30 June 2005 (note 9): “a Contracting Party is responsible under Article 1 of the Convention for all acts and omissions of its organs regardless of whether the act or omission in question was a consequence of domestic law or of the necessity to comply with international legal obligations”…(para.153). “In … establishing the extent to which State action can be justified by its compliance with obligations flowing from its membership of an international organisation to which it has transferred part of its sovereignty, the Court has recognised that absolving Contracting States completely from their Convention responsibility in the areas covered by such transfer would be incompatible with the purpose and object of the Convention”… (para.154). “State action taken in compliance with such legal obligations is justified as long as the relevant organisation is considered to protect fundamental rights, as regards both the substantive guarantees offered and the mechanisms controlling their observance, in a manner which can be considered at least equivalent to that for which the Convention provides” (para.155). 57 See above note 9. 58 See Case Bosphorus v Ireland, ECtHR Grand chamber judgment of 30 June 2005 (note 9): “a Contracting Party is responsible under Article 1 of the Convention for all acts and omissions of its organs regardless of whether the act or omission in question was a consequence of domestic law or of the necessity to comply with international legal obligations”…(para.153). “In … establishing the extent to which State action can be justified by its compliance with obligations flowing from its membership of an international organisation to which it has transferred part of its sovereignty, the Court has recognised that absolving Contracting States completely from their Convention responsibility in the areas covered by such transfer would be incompatible with the purpose and object of the Convention”… (para.154). “State action taken in compliance with such legal obligations is justified as long as the relevant organisation is considered to protect fundamental rights, as regards both the substantive guarantees offered and the mechanisms controlling their observance, in a manner which can be considered at least equivalent to that for which the Convention provides” (para.155). 56 Paragraph 5 of the Declaration, reproduced in: The UN and Human Rights 1945-1995 (New Yor UN 1995), at 450. 57 55 Kant, On the Common Saying: This May be True in Theory, but it does not Apply in Practice (no 53, at 74). Constitutional Protection against Arbitrary Interference in Individual Freedom: A General Right to Liberty? A general constitutional right to liberty (as recognized in Article 2:1 of the German Basic Law) protects the legal autonomy of citizens by means of individual rights and legal protection against arbitrary interference in “negative liberties” (i.e. legal freedom of action), including against restriction of individual freedom resulting from multilevel governance and from international rules adopted in distant intergovernmental organizations.60 The constitutional commitment to respect for human dignity (e.g. as recognized in the German Basic Law, the EU Charter of Fundamental Rights, UN human rights instruments), and the constitutional guarantees of civil, political, economic, social and cultural human rights, recognize that negative liberty is a necessary but insufficient condition of human dignity; it must be complemented by specific “positive liberties” in order to effectively empower individuals and their personal self-development. 61 Comprehensive constitutional guarantees of individual access to courts (as e.g. in German law) protect individual liberty against arbitrary legislative and administrative restrictions by offering judicial review based on principles of proportionality and balancing of all constitutional rights and principles.62 Of course, constitutional guarantees of a general right to liberty and of fundamental “market freedoms” (as protected in EC law) are not recognized in many constitutional democracies which never suffered from dictatorship (as in Germany) and never secured international democratic peace by multilevel constitutional law (as among the 25 EC member states), including legal and judicial guarantees of international “free movement of persons, services, goods and capital, and freedom of establishment” as “fundamental freedoms” (Article I-4 of the 2004 TCE). It has been argued, for example, that the common law, while protecting specific liberties, does not give any weight to liberty in general;63 and that in “mature constitutional systems, for example in the United States, Canada and Australia”, neither individual economic freedom nor other individual rights are “a matter considered essential to constitutionalization in the received tradition of 61 The German Constitutional Court recognizes a human right to respect and protection of human dignity and focuses “on an understanding of the human being as an intellectual and moral creature capable of freely determining and developing itself. The Basic Law conceives of this freedom not as that of an isolated and autonomous individual, but as that of an individual related and bound to society” (Bundesverfassungsgericht Entscheidungen Vol. 45, 187, at 227). 63 Cf. T.T.S.Allan, Law, Liberty and Justice (Oxford : Clarendon Press, 1993), at 135-143. Ernst-Ulrich Petersmann In the Kadi and Yusuf cases (note 8), the Court of First Instance examined the consistency of sanctions imposed by the UN Security Council and EC implementing regulations only with regard to the ius cogens core of UN human rights to make use of one’s property, the right to a fair hearing and the right to an effective judicial remedy; the Court claimed that it had no jurisdiction to examine whether the EC implementation of the UN sanctions was also consistent with the broader guarantees of fundamental rights in European Community law. The legal consistency of the listing of alleged terrorist groups with European human rights law was also challenged in the ECtHR which elaborated a concept of presumed compliance of the EC with the ECHR: “the Court finds that the protection of fundamental rights by EC law can be considered to be… “equivalent”… to that of the Convention system” (ECtHR decision of 23 May 2002 on Application No 6422/2002, para. 165); judge Ress rightly emphasized in his concurring opinion that the “concept of a presumption of convention compliance should not be interpreted as excluding a 21 Ernst-Ulrich Petersmann Ernst-Ulrich Petersmann 62 See: Alexy (note 51), chapter 7. 60 On the constitutional protection in Germany of a general right to liberty, complemented by specific constitutional liberty rights and other civil, political, economic, social and cultural rights, see Alexy (note 51), notably chapter 7. On the constitutional protection of human rights against multilevel governance interferences in the EC see, e.g., the Yusuf case (note 8 above) concerning decisions of the UN Security Council freezing the bank accounts of alleged terrorists. See: Alexy (note 51), chapter 7. 63 Cf. T.T.S.Allan, Law, Liberty and Justice (Oxford : Clarendon Press, 1993), at 135-143. 61 The German Constitutional Court recognizes a human right to respect and protection of human dignity and focuses “on an understanding of the human being as an intellectual and moral creature capable of freely determining and developing itself. The Basic Law conceives of this freedom not as that of an isolated and autonomous individual, but as that of an individual related and bound to society” (Bundesverfassungsgericht Entscheidungen Vol. 45, 187, at 227). This reconciliation of the principle of negative liberty with the social interdependence of the individual is explained by the Court in the following terms: “the individual must submit himself to those limits on his freedom of action which the legislature sets in order to maintain and support social co-existence within the limits of what is generally acceptable according to the relevant subject-matter, and so long as the independence of the person remains guaranteed” (Bundesverfassungsgericht Entscheidungen Vol. 4, 7, at 16). 63 Cf. T.T.S.Allan, Law, Liberty and Justice (Oxford : Clarendon Press, 1993), at 135-143. 62 See: Alexy (note 51), chapter 7. case-by-case review by this Court of whether there was really a breach of the Convention” (para.2 the Opinion). Constitutional Protection against Arbitrary Interference in Individual Freedom: This reconciliation of the principle of negative liberty with the social interdependence of the individual is explained by the Court in the following terms: “the individual must submit himself to those limits on his freedom of action which the legislature sets in order to maintain and support social co-existence within the limits of what is generally acceptable according to the relevant subject-matter, and so long as the independence of the person remains guaranteed” (Bundesverfassungsgericht Entscheidungen Vol. 4, 7, at 16). 22 State Sovereignty, Popular Sovereignty and Individual Sovereignty constitutionalization.”64 US constitutional law protects individual economic freedom and a common market, inter alia, by constitutional requirements of a legal basis for governmental restrictions of individual liberty, rather than by a general individual right to liberty. 65 As US constitutional, antitrust and economic law effectively protect a common market inside the US, most US lawyers see no need for the kind of judicial review by US courts of national governmental restrictions of economic freedom as it is practiced, e.g., by the German Constitutional Court, the EC Court and by the WTO Appellate Body. From the perspective of citizens, constitutional protection of a general right to liberty may offer more effective legal and judicial remedies vis-à-vis multilevel governance in distant international organizations. For instance, it extends the substantive scope of constitutional rights protection and requires the justification of all governmental restrictions of individual liberty. As human and constitutional rights also express objective, constitutional principles that must be taken into account in the whole legal order, a general right to liberty may influence the burden of proof in the balancing of competing constitutional rights, obligations and principles. 66 Even though national constitutional traditions may legitimately differ from country to country, there are important arguments for basing legal and judicial remedies against abuses of multilevel governance powers on the principle that multilevel governance restrictions of individual freedom require constitutional justification and judicial remedies. The WTO dispute settlement rules and practices – such as that WTO Members have a right to complain under the WTO dispute settlement system without proving a “legal interest”67, and that violations of WTO rules are presumed to “nullify” treaty benefits (e.g. reasonable expectations of non-discriminatory conditions of trade competition)68 – are based on principles similar to those of constitutional protection of a right to liberty. 67 In the WTO dispute over the European Communities import restrictions on bananas, the European Communities claimed that the United States had no 'legal right or interest' to sue as it did not export any bananas (Panel Report, EC – Bananas III, WT/DS27/R/USA, para. II.21). The Appellate Body confirmed the finding by the Panel that the DSU did not require a 'legal interest to sue' (Appellate Body Report, EC – Bananas III, WT/DS27/AB/R, para. 132). 66 For a discussion of the German Constitutional Court case-law on the general right to liberty as protecting “human freedom of action in the widest sense”, subject to the broad constitutional limitation clause, see Alexy (note 51), chapter 7. In view of Article 93:1 German Basic Law on “constitutional complaints, which can be raised by anyone on the grounds that their constitutional rights … have been infringed by a public authority”, the German Constitutional Court has held: “Everyone can allege by way of constitutional complaint that a law limiting his freedom of action does not belong to the constitutional order because it infringes (either procedurally or substantively) individual provisions of the Constitution or general constitutional principles and thus that it infringes his constitutional right under Article 2:1 Basic Law” (Bundesverfassungsgericht Entscheidungen Vol.6, 32, at 41). y p p 68 Cf. E.U.Petersmann, The GATT/WTO Dispute Settlement System (Deventer: Kluwer 1997), 142 ff. Deborah Z. Cass (note 31), at 168, 176, 191. 65 Cf. F.L.Morrison/R.E.Hudec, Judicial Protection of Individual Rights under the Foreign Trade Laws of the United States, in: M.Hilf/E.U.Petersmann (eds), National Constitutions and International Economic Law (Deventer: Kluwer 1993), 91-133, at 92 f. Deborah Z. Cass (note 31), at 168, 176, 191. 65 Cf. F.L.Morrison/R.E.Hudec, Judicial Protection of Individual Rights under the Foreign Trade Laws of the United States, in: M.Hilf/E.U.Petersmann (eds), National Constitutions and International Economic Law (Deventer: Kluwer 1993), 91-133, at 92 f. 66 For a discussion of the German Constitutional Court case-law on the general right to liberty as protecting “human freedom of action in the widest sense”, subject to the broad constitutional limitation clause, see Alexy (note 51), chapter 7. In view of Article 93:1 German Basic Law on “constitutional complaints, which can be raised by anyone on the grounds that their constitutional rights … have been infringed by a public authority”, the German Constitutional Court has held: “Everyone can allege by way of constitutional complaint that a law limiting his freedom of action does not belong to the constitutional order because it infringes (either procedurally or substantively) individual provisions of the Constitution or general constitutional principles and thus that it infringes his constitutional right under Article 2:1 Basic Law” (Bundesverfassungsgericht Entscheidungen Vol.6, 32, at 41). 67 In the WTO dispute over the European Communities import restrictions on bananas, the European Communities claimed that the United States had no 'legal right or interest' to sue as it did not export any bananas (Panel Report, EC – Bananas III, WT/DS27/R/USA, para. II.21). The Appellate Body confirmed the finding by the Panel that the DSU did not require a 'legal interest to sue' (Appellate Body Report, EC – Bananas III, WT/DS27/AB/R, para. 132). 68 Cf. E.U.Petersmann, The GATT/WTO Dispute Settlement System (Deventer: Kluwer 1997), 142 ff. 64 Deborah Z. Cass (note 31), at 168, 176, 191. 69 P.Alston and D.Cass have imputed to me the absurd views “that the WTO agreements consist of a series of human rights” (Cass, note 31, at 146); “that the WTO has direct effect” (at 147); that “any state attempts to deny that possibility are not legally binding” (at 148); and that my support for a “human rights approach to international trade” is “fixated on only one category of human rights, namely individual economic rights” (at172). Alston and Cass overlook that my arguments for protecting freedom of trade are based on constitutional rights to liberty (e.g. in German and EC law) and on the EC legal obligations to respect the GATT and WTO guarantees of freedom of trade. My proposal to interpret – inside the EC - the “human right to liberty” (e.g. in Article II-66 EU Charter of Fundamental Rights, Article 3 UDHR) more broadly than it is done in common law countries follows from my Kantian definition of the “first principle of justice” (cf. Petersmann, note 7); it aims at avoiding gaps in human rights law (e.g. the lack of UN human rights provisions protecting freedom of profession and freedom of contract). I have opposed a “human right to free trade” as running counter to the “indivisibility” of human rights and to the necessary balancing of liberty rights with all other human rights and constitutional rights. Constitutional Protection against Arbitrary Interference in Individual Freedom: The progressive evolution of the core of inalienable human rights into ius cogens, and the increasing recognition of the dual nature of human rights – i.e. as individual constitutional rights as well as objective constitutional principles for the mutual balancing and “optimizing” of other constitutional rights and obligations (e.g. by means 23 Ernst-Ulrich Petersmann of proportionality requirements) - support the claim that intergovernmental guarantees of private freedom might constitute not only intergovernmental rights, but may also justify legal and judicial protection of private freedoms based on domestic constitutional rights to liberty and constitutional requirements to exercise governance powers in “strict observance of international law” (Article I-3, para.4 TCE). Such interpretations of constitutional rights to freedom (e.g. in German and EC constitutional law) in conformity with self-imposed international guarantees of freedom (such as GATT’s customs union rules and their incorporation into the EC Treaty) have nothing to do with the proposition of a human right to free trade.69 g 70 See Petersmann (note 18). 71 On the emerging “human right to democratic governance”, see, for example, T.M. Franck, The Emerging Right to Democratic Governance, (1992) 86 American Journal of International Law (AJIL), 46-91; G.H. Fox & B.R. Roth (eds), Democratic Governance and International Law, (Oxford: OUP, 2000); E. Stein, ‘International Integration and Democracy: No Love at First Sight’, (2001) 95 AJIL 489-532. On the recognition and promotion of human rights by international organizations, see: “Interdependence between democracy and human rights”, Report of the Office of the High Commissioner for Human Rights, E/CN.4/2004/54 of 17 February 2004. Procedural and Participatory Citizen Rights vis-à-vis Multilevel Governance From a human rights perspective, individuals and their elected representatives constitute not only “peoples” and democratic states, but – through the intermediary of their state agents – also intergovernmental organisations as a necessary “fourth branch of governance”.70 None of these various forms of collective governance institutions has a constitutional mandate to disregard or violate human rights. The legitimacy of international organisations (such as the UN, the WTO and the EU) depends on respect for, and promotion of human rights. Yet, at international levels - far away from the local communities in which citizens live and co-operate, and far away also from national parliaments - intergovernmental rules and procedures inevitably suffer from “democratic deficits” compared to local and national democratic processes and parliamentary decision-making. It remains an important task to strengthen the democratic accountability of multilevel governance as well as legal and judicial remedies against abuses of foreign policy powers. UN human rights instruments recognize that human rights need to be protected and mutually balanced through democratic legislation, and that “everyone has the right to take part in the government of his country, directly or through freely chosen representatives” (Article 21:1 UDHR); “(t)he will of the people shall be the basis of the authority of governments; this will shall be expressed in periodic and genuine elections which shall be by universal and equal suffrage and shall be held by secret vote or by equivalent free voting procedures” (Article 21:3 UDHR). These core guarantees of a 24 24 State Sovereignty, Popular Sovereignty and Individual Sovereignty human right to democratic governance71 focus on national rather than international governance; but UN human rights instruments also recognize a human right “to a social and international order in which the rights and freedoms set forth in this Declaration can be fully realised” (Article 28 UDHR). Just as European constitutional law commits EU member states to respect for “representative democracy” (Article I-46 TCE) and “participatory democracy” (Article I-47 TCE) at national and European levels, so does the realization of UN human rights require stronger safeguards for participatory, representative and deliberative democracy in multilevel trade governance. g y 72 See the comparative study of the control of trade policies by 11 parliaments in : The Role of Parliaments in Scrutinising and Influencing Trade Policy, European Parliament (PE 370-166v01-00) December 2005 (the study suggests that, with the exception of the Swiss Parliament and US Congress, most parliaments do not appear to control trade policy and WTO rule-making effectively). Procedural and Participatory Citizen Rights vis-à-vis Multilevel Governance Democratic control by citizens and by national parliaments of international bureaucratic bargaining in distant, intergovernmental organisations is – for numerous reasons - far more difficult than in domestic political processes.72 For example, national parliaments and civil society groups often complain of the “information asymmetries” which result from intergovernmental rule-making by bureaucratic networks in non-transparent, intergovernmental organisations without effective parliamentary participation and effective democratic control by public opinion and civil society (for example, insufficiently “inclusive” decision-making without participatory and/or consultative rights of non-governmental groups that may be affected by intergovernmental decisions). Such democratic concerns increase if intergovernmental negotiations (for example, in the WTO) are strongly influenced by powerful interest groups (for example, agricultural, textile and steel lobbies); take place behind closed doors without adequate “deliberative democracy”; do not refer to human rights in the international negotiations and balancing processes; and may lead to comprehensive “package deals” (like the 1994 WTO Agreement) which can hardly be re-opened at the request of a single national parliament, or at the request of a few WTO members in the consensus- based WTO decision-making processes, once the international negotiations have been closed. Substantive Citizen Rights in Multilevel Economic Governance Human rights and democratic procedures can be combined in many ways which legitimately differ among countries. Yet, the more remote governance institutions operate far away from the citizens affected by intergovernmental regulation (e.g. in the EU, UN and the WTO), the less effective procedural citizen rights for democratic participation and representation risk becoming, and the more important are the legal and judicial protection of substantive rights (like the “market freedoms” and other fundamental rights protected by the EC Court). It is therefore to be welcomed that, even 25 Ernst-Ulrich Petersmann though the EC and EU Treaties continue to be drafted primarily in terms of rights and obligations of governments, the Union is explicitly though the EC and EU Treaties continue to be drafted primarily in terms of rights and obligations of governments, the Union is explicitly • “founded on the values of respect for human dignity, freedom, democracy, equality, the rule of law and respect for human rights”, without reference to sovereignty;73 and • the EU Charter of Fundamental Rights protects general and specific dignity rights (Title I), liberty rights (Title II), equality rights (Title III), solidarity rights (Title IV), citizen rights (Title V) and “rights to justice” (Title VI) that go beyond those in national constitutions and in UN law.74 As it was to be expected from the perspective of rights-based constitutional theory, European integration law and multilevel governance in the EU have proven to be most successful in those areas (like European competition law and common market law) where individual citizens and national courts were empowered to act as self-interested guardians of the rule of law and could directly enforce the legal obligations of EC institutions and national governments through domestic courts. Vice versa, those areas of Community law where EC citizens and national courts were prevented from directly enforcing the international obligations of the EC and of EC governments (like multilevel trade governance in the WTO) have remained characterized by frequent violations of the rule of law to the detriment of consumer welfare and the individual rights of EC citizens. 76 See Kuijper (note 29 above). At the request of the political EC institutions, the EC Court has refrained from reviewing the legality of EC acts in the light of GATT law for more than 30 years, even if the EC violations had been formally established in legally binding GATT and WTO dispute settlement rulings. Notwithstanding the longstanding EC jurisprudence that GATT and WTO obligations form an “integral part of the Community legal system” binding on all EC institutions and judicially enforceable vis-à-vis EC member states, the Court consistently denies “direct effects” of GATT and WTO rules as well as judicial protection of EC citizens and EC member states against violations of WTO rules by EC institutions. The EC Court’s arguments against “direct applicability” rely on obvious misinterpretations of WTO rules (cf. Case T-69/00, FIAMM, December 2005, nyr: “applicants are wrong in inferring from Articles 21 and 22 of the DSU an obligation on the WTO Member to comply, within a specified period, with the recommendations and rulings of the WTO bodies”) as well as on political arguments (like lack of international reciprocity, need to protect the 74 The text of this Charter, first proclaimed by the European Parliament, the EU Commission and the EU Council in December 2000 (Official Journal of the EC, C 364/1-22 of 18 December 2000), has become Part II of the TCE (note 28). 75 Case T-115/94, Opel Austria, C.M.L.R. 1997, 733. 75 Case T-115/94, Opel Austria, C.M.L.R. 1997, 733. 73 Article I-2 Treaty TCE (above note 27), which corresponds to Article 2 of the EU Treaty. Substantive Citizen Rights in Multilevel Economic Governance Due to the pressures by the political EC organs and the judicial self-restraint by the ECJ, there has been only one single judgment by the EC Court establishing a violation by the EC of its international legal obligations since 195875, compared with more than 35 GATT and WTO dispute settlement findings of violations of GATT and WTO obligations by the EC and EC member states. The legal advocates of the EC Commission justify the frequent violations of the EC’s WTO guarantees of freedom - even if EC violations of WTO rules had been formally established in legally binding WTO dispute settlement rulings and the “reasonable period of time” for implementing such rulings had long expired - by power-oriented, Machiavellian arguments that judicial protection by the ECJ of the EC citizens’ reliance on respect for the rule of international law would be “naïve.”76 26 State Sovereignty, Popular Sovereignty and Individual Sovereignty In the field of the EU’s common foreign and security policy, legal and judicial remedies are even more limited in the EU (cf. Article 46 EU Treaty) as well as in worldwide governance institutions. The more trade and foreign policies operate by “internal” restrictions of EU citizens (such as their freedom to buy foreign bananas, beef, genetically modified food), and the more European courts exercise judicial deference and – as in the recent Kadi and Yusuf cases – abdicate their judicial task of reviewing EC restrictions of individual freedom for their compliance with all EC obligations77, the less rule of law can be maintained inside the EU. Without rule of law, also democracy becomes illusionary in the EU’s multilevel governance.78 The legally incoherent UN and WTO jurisprudence of the ECJ amounts to a “political question doctrine” which arbitrarily disregards the EC’s WTO obligations and WTO dispute settlement rulings but gives legal primacy to UN resolutions as “supreme law, a supreme law offering virtually no guarantees of judicial review, at any level” 79, without subjecting the domestic implementation of such UN resolutions and WTO obligations to effective judicial review inside the EC. 77 Cf. Kuijper (note 29) who claims that the ECJ should focus on the political “law in action” rather than the WTO “law in the books” (e.g. at 1332-34). In the Kadi and Yusuf judgments, the Court of First Instance held that the Court of First Instance has no jurisdiction to review the legal consistency of UN Security Council decisions freezing the bank accounts of alleged terrorists with EU fundamental rights guarantees (cf. Case T-306/01 (note 8 above), para. 272. 78 Cf. P.Eeckhout (note 77), at 20: “In CFSP matters there is both a democratic and judicial deficit”. “scope of manoeuvre” of the political EC bodies) that had been rejected as legally irrelevant by the EC Court in its Kupferberg judgment (Case 104/81, ECR 1982, 3659). The EC legal services support this judicial self-restraint vis-à-vis WTO law and welcome that also “the Nakajima doctrine has never really been applied … because this doctrine … (is) untenable on grounds of legal logic” (Kuijper, at 1340). In the Kadi and Yusuf judgments, the Court of First Instance claimed that UN Charter obligations prevail not only over other international legal obligations (cf. Article 103 UN Charter) but also over EU human rights law; this submission to UN law has been rightly criticized as amounting to “judicial abdication”, cf. P.Eeckhout, Does Europe’s Constitution Stop at the Water’s Edge? Law and Policy in the EU’s External Relations (Leuven: Europa Law Publishing, 2005): “The judicial abdication by the CFI means that, insofar as the resolutions are implemented by EC regulations, there can be no review by either the EU Courts or national courts” ... “Community law … becomes an instrument for turning UN resolutions into supreme law, a supreme law offering virtually no guarantees of judicial review, at any level”. “scope of manoeuvre” of the political EC bodies) that had been rejected as legally irrelevant by the EC Court in its Kupferberg judgment (Case 104/81, ECR 1982, 3659). The EC legal services support this judicial self-restraint vis-à-vis WTO law and welcome that also “the Nakajima doctrine has never really been applied … because this doctrine … (is) untenable on grounds of legal logic” (Kuijper, at 1340). In the Kadi and Yusuf judgments, the Court of First Instance claimed that UN Charter obligations prevail not only over other international legal obligations (cf. Article 103 UN Charter) but also over EU human rights law; this submission to UN law has been rightly criticized as amounting to “judicial abdication”, cf. P.Eeckhout, Does Europe’s Constitution Stop at the Water’s Edge? Law and Policy in the EU’s External Relations (Leuven: Europa Law Publishing, 2005): “The judicial abdication by the CFI means that, insofar as the resolutions are implemented by EC regulations, there can be no review by either the EU Courts or national courts” ... “Community law … becomes an instrument for turning UN resolutions into supreme law, a supreme law offering virtually no guarantees of judicial review, at any level”. 79 Eeckhout (note 77), at 26. 79 Eeckhout (note 77), at 26. g g p 78 Cf. P.Eeckhout (note 77), at 20: “In CFSP matters there is both a democratic and judicial deficit”. 79 Eeckhout (note 77) at 26 Cf. P.Eeckhout (note 77), at 20: In CFSP matters there is both a democratic and judicial deficit . Eeckhout (note 77), at 26. 80 This term was used already in the Panel report on US-Section 301 (WT/DS152/R), para.7.22. The Appellate Body has recently confirmed in EC-Chicken Cuts: “Interpretation pursuant to the customary rules codified in Article 31 of the Vienna Convention is ultimately a holistic exercise that should not be mechanically subdivided into rigid components” (WT/DS269,286/AB/R, para. 176, adopted in September 2005). 83 Such struggles inevitably entail social antagonism (which Kant described as the ‘unsocial sociability’ of rational individuals) and ‘an uncomfortable condition of permanent revolution’ (cf. A. W. Wood, Kant’s Ethical Thought (Cambridge: CUP, 1999), at 333) for the progressive extension of rights- based constitutionalism. 82 For a discussion of Kant’s constitutional and cosmopolitan legal theories, and for a criticism of th lack of constitutional theory in public international law doctrine, see Petersmann (note 54). 81 See above notes 8-11 and 13 and related text. 81 See above notes 8-11 and 13 and related text. Substantive Citizen Rights in Multilevel Economic Governance Conclusion: Need for More Comprehensive ‘Balancing’ and Judicial Protection of Public and Private Rights and Obligations As more and more countries adopt democratic constitutions and (inter)national legal and judicial guarantees limiting the powers of the rulers, the need for legislative, administrative and judicial “balancing” of rights and obligations, as well as of public and private interests, on the basis of “fair procedures” and “constitutional principles” (such as non-discrimination, necessity, proportionality) is increasingly recognized. This is particularly so in constitutional systems (like German and EC law) with broad liberty rights and constitutional requirements that restrictions of liberty must be constitutionally justifiable. National constitutional courts as well as international human rights courts acknowledge that human rights and other “fundamental rights” constitute rules as well as principles for reconciling (“optimizing”) competing principles and rules 27 Ernst-Ulrich Petersmann relative to what is legally and factually possible in particular circumstances. Similarly, international economic courts (like the ECJ and the WTO Appellate Body) emphasize the need for “holistic” treaty interpretation balancing intergovernmental rights and obligations with broader constitutional principles; the requirement, in Article 31 VCLT, that "a Treaty shall be interpreted in good faith in accordance with the ordinary meaning to be given to the terms of the treaty in their context and in the light of its object and purpose", constitutes "one holistic rule"80, whose different elements (text, context, object and purpose) may be difficult to separate. The comprehensive UN reports analysing interrelationships and potential conflicts between human rights and international trade law suggest that human rights law and international and European trade law appear to be flexible enough in order to be interpreted and applied in mutually coherent ways.81 84 Cass (note 31), at x; see notably Part III of her book (“Against Constitutionalization”) which agrees with the “anti-constitutionalization critique”, for instance that the “WTO undermines decisions of democratically authorized national constituencies” (at 212). Notwithstanding seven chapters favoring national over multilevel constitutionalism, her Chapter 8 concludes that “trading democracy, not merely trading constitutionalization, should be the key to WTO constitutionalization in this century” (at 242). For Europeans familiar with the reality of European constitutional law and other “international treaty constitutions”, it appears anachronistic to deny the emerging international constitutional law. The most important insight of Cass’ book remains, as admitted at the end, that her “received account” of Anglo-Saxon constitutionalism has, indeed, “been revealed as neither descriptively adequate nor normatively appealing” (p.240). p y q 85 Cf. Rubenfeld (note 6), at 283, 293. V. Struggles for Human Rights: American Constitutional Nationalism versus European Multilevel Constitutionalism V. Struggles for Human Rights: American Constitutional Nationalism versus European Multilevel Constitutionalism The moral and democratic importance of complementing constitutional nationalism by multilevel constitutionalism for limiting abuses of foreign policy powers and protecting human rights more effectively in international law can be appreciated best by recalling the Kantian origins of multilevel constitutionalism. According to Kantian legal philosophy, the moral ‘categorical imperative’ of protecting human dignity and maximum equal freedoms of individuals through a ‘universal law of freedom’ requires national, international as well as cosmopolitan constitutional rights protecting individual freedom against abuses of power in all human relations (i.e. inside states, in intergovernmental relations, as well as in cosmopolitan relations among individuals and foreign governments). 82 The inevitable struggles for progressively extending such constitutional liberty rights also depend on democratic discourse in a communicating public that must transcend intergovernmental diplomacy and may eventually turn into a “cosmopolitan constituency” for the collective supply of global public goods (including multilevel constitutional guarantees of human rights).83 From a Kantian and European perspective, international law must be evaluated from the standpoint of human rights and must be transformed into an instrument for the constitutional protection of broadly 28 State Sovereignty, Popular Sovereignty and Individual Sovereignty defined human rights. The perspective of non-European countries, however, is often very different. defined human rights. The perspective of non-European countries, however, is often very different. American Constitutional Nationalism Distrusts International Law The post-war US leadership for a liberal international economic order was based on hegemonic US leadership and national constitutionalism (e.g. strong distrust of the US Congress and US courts vis-à-vis international law, congressional insistence on powers to adopt measures in violation of international law). Many Anglo-Saxon lawyers and “realist” politicians argue “against constitutionalization” of international relations and claim, for example, “that the WTO is not constitutionalized, and nor, according to any current meanings of the term, should it be.”84 The European openness to international law is criticized as a democratic deficiency by some US lawyers who praise the US’ unilateralism and resistance against international influences as living up to the ideal of democratic self-determination; the post-war US support for internationalism and multilateralism was “for the rest of the world, not for us”, even though America’s commitment to internationalism in economic affairs is recognized as serving US interests.85 The widespread criticism by American, Australian and Canadian lawyers (like P. Alston, D.Z.Cass, R.Howse) of multilevel constitutionalism often reflects process-based (rather than rights-based) conceptions of parliamentary democracy and communitarian (rather than cosmopolitan) traditions. Many developing countries, even if they criticize the North-American focus on civil and political rather than economic and social human rights and argue in favour of development-oriented reforms of international law, likewise emphasize the classical international law principle of state sovereignty rather than the need for multilevel constitutional restraints of national policy powers. Multilevel European Constitutionalism is Committed to “Strict Observance of International Law” (Article I-3 TCE) Cf. Rubenfeld (note 6), at 283, 293. Multilevel European Constitutionalism is Committed to “Strict Observance of International Law” (Article I-3 TCE) Europeans are more inclined to infer from the European integration experience that multilevel governance requires rights-based, multilevel constitutionalism for adjusting and preserving individual and democratic self-government in an interdependent world. Just as the EU remains democratically legitimate only as a legal community with limited powers committed to “strict observance of international law” (Art. I-3 TCE), so must international law overcome its obvious “constitutional deficits” by constitutional 29 Ernst-Ulrich Petersmann reforms. Whereas Americans tend to view international courts as a potential threat to national democracy, such “counter-majoritarian difficulties” are less perceived in the European context where “judicial governance” by the EC Court and the European Court of Human Rights inevitably affects European democracies and their changing, democratic majorities. This contribution has defended the European views that: • Globalization demonstrates that ever more constitutional objectives can no longer be achieved without multilevel governance and multilevel constitutionalism promoting the collective supply of international public goods. • Globalization demonstrates that ever more constitutional objectives can no longer be achieved without multilevel governance and multilevel constitutionalism promoting the collective supply of international public goods. • In order to remain legitimate and effective, multilevel governance - such as intergovernmental rule-making, administration and judicial governance in worldwide institutions (such as the WTO and UN agencies) and in regional regimes – must respect and protect human rights and remain democratically accountable; this cannot be achieved without stronger multilevel constitutional restraints. European integration illustrates that international law and international organisations for the collective supply of international public goods can also enlarge citizen rights, enhance the legitimacy of multi-level governance, limit abuses of multi-level governance, and facilitate parliamentary accountability of foreign policies beyond what is possible through national constitutionalism. • Human rights and democratic self-government require redesigning “constitutional sovereignty” in a manner reconciling state sovereignty, popular sovereignty and individual sovereignty based on respect for human dignity and human rights. The existing forms of worldwide governance and constitutional nationalism remain highly ineffective in terms of protection and promotion of universal human rights, international rule of law, democratic peace and a healthy environment. y g 88 Yet, also in Africa and in the Americas, an increasing number of RTAs explicitly refer to human rights as an objective or fundamental principle of economic integration, or implicitly limit membership to countries committed to protection of human rights; cf. the contributions by S. F. Musungu, ‘International Trade and Human Rights in Africa: A Comment on Conceptual Linkages’, and by F. J. Garcia, Integrating Trade and Human Rights in the Americas, to F. M. Abbott, C. Breining-Kaufmann and T. Cottier (note 10), chpts. 15 and 16. 87 Since the 1990s, it became official European Union (EU) policy to include ‘human rights clauses’ in all new trade and cooperation agreements with third countries, cf. L. Bartels, Human Rights Conditionality in the European Union’s International Agreements (Oxford: OUP, 2005). 86 Cf. H.Sjursen, What kind of power? in: Journal of European Public Policy 13 (2006), 169-181. Multilevel Constitutionalism as a ‘Transformation Policy” and ‘Struggle for Human Rights’ The more than 2’400 years of historical experiences with republicanism confirm that rights-based constitutionalism offers the most effective safeguards for protecting the public interests of citizens. The differences between American and European approaches to multilevel governance lie not in the European recourse to “normative”, “soft” and “civilian power” (which are also used in US foreign policies), but in the European preference for multilevel constitutional restraints on multilevel economic governance rather than for state-centred approaches to international law and constitutional nationalism. The American and European approaches compete in worldwide treaty negotiations (e.g. US rejection of the UN Convention on the International Criminal Court, the UNESCO Convention on Cultural Diversity, the UN Biodiversity Convention) as well as in the competing recourse to regionalism, bilateralism and unilateralism as policy alternatives whenever international public goods cannot be supplied effectively at worldwide levels (e.g. in the Doha Round negotiations in the WTO). 30 State Sovereignty, Popular Sovereignty and Individual Sovereignty State Sovereignty, Popular Sovereignty and Individual Sovereignty At the regional level, the EU has succeeded in using its normative and economic “power of attraction” for extending its multilevel constitutionalism to 25 EU member states; the rights-based free trade rules and basic human rights commitments of EC law have been further extended to more than 20 additional countries in Europe and the Mediterranean. This peaceful transformation of ever more countries in Europe – based on EU accession, association and cooperation agreements – was made possible by the constitutional and economic benefits of EU law rather than by unilateral “power” of the EU to make other countries do what they would not otherwise do.86 With regard to countries outside Europe, the EC’s cooperation agreements (e.g. with Russia and the African, Caribbean and Pacific countries) and “human rights conditionality” 87 have been much less successful in promoting multilevel constitutionalism. The more than 200 regional trade agreements concluded in the Americas, Africa and Asia were often more influenced by the national interests of “regional hegemons” (such as the US in North America, Argentina and Brazil in MERCOSUR, South Africa in the Southern African integration agreements, China in Asia) and tend to avoid multilevel constitutionalism (such as supranational regulatory agencies and courts).88 “Continental hegemons” (like Brazil, China, India, Russia, South Africa) are likely to follow more the state-centred foreign policy approaches of the US than Europe’s multilevel constitutionalism. The future legal and constitutional structures of international economic law remain uncertain. The history of constitutionalism suggests that the needed “constitutionalization” of international economic law depends on “struggles for human rights” by citizens, civil society, parliaments and courts against the never-ending abuses of private and public power by self-interested rulers.- 31
https://openalex.org/W4381683281	https://journals.ufs.ac.za/index.php/trp/article/download/6090/4741	English	null	Estimation and Web-GIS geovisualisation of a suitable solid waste disposal site: Case study of New City, Harare	Town and regional planning/Stads- en streekbeplanninq	2,023	cc-by	11,756	Ms Diana Dahwa, Department of Geography, Geospatial Sciences and Earth Observation, University of Zimbabwe, Harare, Zimbabwe. Email: ddahwataku@gmail.com Mr Aldridge Nyasha Mazhindu (corresponding author), Department of Geography, Geospatial Sciences and Earth Observation, University of Zimbabwe, Harare, Zimbabwe. Email: amazhindu@gis.uz.ac.zw, ORCID: https://orcid.org/0000-0002-1118-4373 Ms Kudzai Chirenje, Department of Geography, Geospatial Sciences and Earth Observation, University of Zimbabwe, Harare, Zimbabwe. E-mail: kudzaic1@gmail.com Review article DOI: https://doi.org/10.38140/trp.v82i.6090 DOI: https://doi.org/10.38140/trp.v82i.6090 Received: August 2022 Peer reviewed and revised: January 2022 Published: June 2023 The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article Received: August 2022 Peer reviewed and revised: January 2022 Published: June 2023 The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article Received: August 2022 Peer reviewed and revised: January 2022 Published: June 2023 The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article Received: August 2022 Peer reviewed and revised: January 2022 Published: June 2023 The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article BERAMING EN WEB-GIS GEOVISUALISERING VAN ‘N GESKIKTE STORTINGSTERREIN VIR VASTE AFVAL: GEVALLESTUDIE VAN NEW CITY, HARARE How to cite: Dahwa, D., Mazhindu, A.N. & Chirenje, K.C. 2022. Estimation and Web-GIS geovisualisation of a suitable solid waste disposal site: Case study of New City, Harare. Town and Regional Planning, no. 82, pp. 110-123. © Creative Commons With Attribution (CC-BY) Published by the UFS http://journals.ufs.ac.za/index.php/trp How to cite: Dahwa, D., Mazhindu, A.N. & Chirenje, K.C. 2022. Estimation and Web-GIS geovisualisation of a suitable solid waste disposal site: Case study of New City, Harare. Town and Regional Planning, no. 82, pp. 110-123. © Creative Commons With Attribution (CC-BY) Published by the UFS http://journals.ufs.ac.za/index.php/trp Met die steeds toenemende menslike bevolking is daar ’n behoefte om nuwe stedelike nedersettings vir menslike bewoning te ontwikkel. Vir hierdie nuwe nedersettings is dit noodsaaklik om verskillende grondgebruiksones, insluitend stortingsterreine vir vaste afval, optimaal te vestig. Die doel van hierdie artikel is om geskikte terreine te bepaal om ’n stortingsterrein in ’n nuwe ontwikkelde stad in Zimbabwe rondom Mt. Hampden, genaamd die Nuwe Stad, te vind. Die Nuwe Stad sal verskeie residensiële, kommersiële en industriële gebiede hê. Dit behels die behoefte aan ’n behoorlike terreinkeuse vir ’n stortingsterrein om die negatiewe sosiale en omgewingseffekte soos besoedeling van waterliggame en verspreiding van siektes soos malaria te verminder. GIS en afstandwaarneming was die belangrikste metodes wat gebruik is om die geskikte gebiede te karteer. Multi-kriteria evaluering en geweegde oorleg analise metodes is gebruik in die stortingsterrein seleksie- proses. Faktore wat gebruik word vir die keuse van stortingsterreine was riviere, nedersettings, paaie, beskermde gebiede en gronde. ’n Geskiktheidskaart is gegenereer wat vyf potensiële terreine toon wat geskik is vir stortingsterrein in die Nuwe Stad. Matig geskikte gebiede beslaan ongeveer 8%. ’n Verdere 73% van die totale grondoppervlakte in die studiegebied is hoogs ongeskik vir ’n stortingsterrein. ’n Intydse Web-GIS- moniteringskoppelvlak is ontwikkel om grondgebruik op die geselekteerde gebied te monitor omdat die Nuwe Stad ’n nuwe gebied is wat ontwikkel word. Die gebruik van ’n Web-GIS- koppelvlak maak data maklik toeganklik vir omgewingsbeplanners, ekoloë, ruimtelike grondbeplanners en ander besluitnemers. Diana Dahwa, Aldridge Mazhindu & Kudzai Chirenje Review article Town and Regional Planning 2023 (82):110-123 \| ISSN 1012-280 \| e-ISSN 2415-0495 KHAKANYO LE WEB-GIS GEOVISUALIZATION EA SEBAKA SE LOKETSENG SA HO LAHLELA LITHŌLE: MOHLALA OA NEW CITY, HARARE Ho sebelisa marang-rang a Web-GIS ho etsa hore linthla li fihlellehe habonolo ho bareri ba tikoloho, litsebi tsa tikoloho, bareri ba mobu le ba bang ba etsang liqeto. Inappropriate disposal of solid waste can be manifested by contamination of surface and groundwater through leachate, a contaminated liquid generated during the decomposition of MSW (Mohammad et al., 2022: 2), soil contamination through direct waste contact (Butt et al., 2022: 4), air pollution by burning of wastes, spreading of diseases by different vectors such as birds, insects and rodents, or uncontrolled release of methane by anaerobic decomposition of waste (Visvanathan, 2006: 6). It is no different in Zimbabwe, where immediate urbanisation in the small towns has brought serious MSW management challenges, as some MSW is dumped at disposal sites that are not properly lined and sited (Nhubu et al., 2021: 2). In Zimbabwe, urban local authorities are responsible for the collection and management of MSW generated within their jurisdictions. However, all the urban local authorities in Zimbabwe are collecting less than 50% of the MSW generated and dumping it at improper dumpsites (Nhubu et al., 2021: 2). As in most of the developing countries, the remainder of the MSW is left and the inhabitants, in most of the small towns, often use unsafe solid waste disposal practices such as open dumping, burning, and burying (Sisay, Gebre & Getahun, 2020: 2). These uncontrolled self-managed waste disposal practices do not guarantee cleanliness and safety. For example, burning one’s trash can give rise to significant localised, negative externalities such as air pollution, depending on how it is burned (Sisay et al., 2020: 3). Regardless of these challenges, hardly anything has been done to harness the latest advances in methodologies available for selecting appropriate sites and managing the solid waste dumping in developing countries with rapid population growth rates (such as Zimbabwe). Furthermore, there is a paucity of research to show the utility of Multi-Criteria Decision Analysis (MCDA) with the aid of Geographical Information Systems (GIS) and Remote Sensing (RS) in estimating landfill sites for developing countries such as Zimbabwe which are still creating new settlements that need landfill sites. KHAKANYO LE WEB-GIS GEOVISUALIZATION EA SEBAKA SE LOKETSENG SA HO LAHLELA LITHŌLE: MOHLALA OA NEW CITY, HARARE predominantly significant in third- world countries, where large amounts of MSW are disposed without taking serious precautions about the environment and people, thereby putting a burden on threatened land, air, and water. For the vast majority of African nations, open dumping is the most common technique used for MSW management, due to the absence of sanitary landfills (Sohoo et al., 2022: 2). appropriate distance from the centre of the city (Muheirwe, Kombe & Kihila, 2022: 2). Human settlements within less than 1 km from these dumping sites are associated with vector-borne diseases such as malaria and yellow fewer caused by mosquitoes (Nta et al., 2020: 3). Ka palo e ntseng e eketseha ea batho, ho hlokahala hore ho thehoe libaka tsa bolulo tse ncha tsa litoropong. Bakeng sa libaka tsena tse ncha tsa bolulo, ho bohlokoa ho fana ka libaka tse fapaneng tsa tšebeliso ea mobu ho kenyeletsoa libaka tsa ho lahlela lithole. Sepheo sa sengoloa sena ke ho fumana libaka tse loketseng tsa ho lahlela lithōle toropong e ncha e tsoetseng pele ea Zimbabwe haufi le Mt.Hampden, e bitsoang New City. New City e tla ba le libaka tse fapaneng tsa bolulo, tsa khoebo le tsa indasteri. Sena se kenyelletsa tlhokeho ea khetho e nepahetseng ea sebaka sa ho lahlela lithōle ho fokotsa litlamorao tse mpe sechabeng le tikolohong joalo ka tšilafalo ea libaka tsa metsi le ho ata ha mafu a kang malaria. GIS le remote sensing e ne e le mekhoa e meholo e sebelisoang ho etsa limmapa tsa libaka tse loketseng. Litekanyetso tse ngata le mekhoa ea tlhahlobo li ile tsa sebelisoa ts’ebetsong ea khetho ea sebaka sa lithōle. Lintho tse neng li sebelisoa ho khetha libaka tsa ho lahlela lithōle e ne e le linōka, libaka tsa bolulo, litsela, libaka tse sirelelitsoeng le mobu. Ho entsoe ‘mapa o loketseng o bonts’ang libaka tse hlano tse lokelehileng ho ka sebelisoa ele libaka tsa ho lahlela lithōle New City. Libaka tse loketseng hantle li akaretsa hoo e ka bang 8%. Karolo e ‘ngoe hape ea 73% ea sebaka sohle sa mobu sebakeng sa boithuto ha se tšoanelehe haholo bakeng sa sebaka sa ho lahlela lithōle. Ntlafatso ea Web- GIS e entsoe ho beha leihlo tšebeliso ea mobu sebakeng se khethiloeng hobane New City ke sebaka se secha se ntseng se ntlafatsoa. Abstract With the ever-increasing human population, there is a need to develop new urban settlements for human habitation. For these new settlements, it is imperative to optimally site different land-use zones, including solid waste disposal sites. The aim of this article is to determine suitable sites for locating a landfill in a new developed city in Zimbabwe around Mt. Hampden, named the New City. The New City will have various residential, commercial, and industrial areas. This entails the need for a proper site selection of a landfill to reduce the negative social and environmental effects such as contamination of water bodies and proliferation of diseases such as malaria. GIS and remote sensing were the major methods used in mapping the suitable areas. Multi-criteria evaluation and weighted overlay analysis methods were used in the landfill site selection process. Factors used for landfill site selection were rivers, settlements, roads, protected areas, and soils. A suitability map was generated, showing five potential sites that are suitable for landfill siting in the New City. Moderately suitable areas cover approximately 8%. A further 73% of the total land area in the study area is highly unsuitable for siting a landfill. A real-time Web- GIS monitoring interface was developed to monitor land use on the selected area, because the New City is a new area under development. Using a Web-GIS interface makes data easily accessible to environment planners, ecologists, spatial land planners, and other decision makers. Keywords: Web-GIS, Analytic Hierarchy Process, GIS, municipal solid waste, land fill, land fill sites, waste disposal, Zimbabwe Town and Regional Planning 2023 (82):110-123 \| ISSN 1012-280 \| e-ISSN 2415-0495 110 Dahwa, Mazhindu & Chirenje 2023 Town and Regional Planning (82):110-123 1. INTRODUCTION Currently, solid waste siting is a challenge in urban centres of the world, resulting in the accelerated rate of illegal disposal sites. The rapid increase in world population, including sub-Saharan Africa (De la Croix & Gobbi, 2022: 2), has added to the accumulation of municipal solid waste (MSW) (Lyons et al., 2009: 438). On a global scale, it is very difficult to dispose of the huge amounts of MSW being produced (Sultana et al., 2021: 1). This is KHAKANYO LE WEB-GIS GEOVISUALIZATION EA SEBAKA SE LOKETSENG SA HO LAHLELA LITHŌLE: MOHLALA OA NEW CITY, HARARE The study thus proposes a methodology for estimating suitable solid waste disposal sites, by using GIS, remote sensing, and multi- criteria evaluation and a Web-GIS interface for the geovisualisation of the suitable sites, in order to minimise the risk of ecological and human health problems for the New City in Zimbabwe. In the vast majority of cities such as Harare in Zimbabwe, health institutions and industries follow their own way of waste removal, while others dispose of it into nearby water bodies (Nhubu et al., 2021: 2). Although most of the developing countries have temporary waste transfer stations, there are no scientifically approved waste dumping sites (Sohoo et al., 2022: 2). The dumping sites are not well planned, and they are open field disposal (no sanitary landfill), are close to roads and to rural settlements (where there are water bodies, and crops), and not at an 2.1 Solid waste management to plastics, metals, glass, paper, food waste, grass, leaves, and other waste (Silva de Souza Lima Cano, Iacovidou & Rutkowski, 2022: 3). Proximity to roads and railways: Most of the studies suggest that the solid waste dumping site should be located within a 1 km buffer from roads and other transportation facilities to avoid odour pollution (Kumar & Deswal, 2022: 4). Solid waste disposal sites should not be placed too far from the roads, in order to decrease the cost of transportations (Yildirim et al., 2022: 6). Solid waste management is referred to as the discipline associated with the control of generation, storage, collection, transfer and transport, processing and recovery, and final disposal of solid wastes in a manner that is in accordance with the best principles of public health, economics, engineering, urban and regional planning, conservation, aesthetics, and other environmental considerations (Abdalqader, 2011: 8). Soil permeability areas that may include liquefaction or subsidence- prone soils, extensive layers of soft soil, anomalous soil characteristics, a high groundwater table, or subsurface cavities should be avoided (Rodwell & Mucina, 1998). The soil map characteristics that are considered most in the selection of a suitable landfill site are permeability and porosity. The constraint on the soil factor is that a site cannot be located on soils that are very permeable and porous (Jothimani et al., 2021: 3). To this end, there is a need to introduce solid policies for disposing waste and its management. In fact, sound policies by municipal authorities should translate to efficient solid waste management practices (Muheirwe et al., 2022: 1-2). Groundwater depth: Unscientific and improper management of MSW is one of the direct sources of contamination of groundwater in developing countries (Choudhury et al., 2022: 2-3). MSW leads to a decrease in acidity, with increasing depth for groundwater bodies near MSW sites (Choudhury et al., 2022: 2-3). Considering this, a landfill should not be placed close to areas with groundwater, which is near the earth’s surface. In other studies, areas with a groundwater depth greater than 50m are suitable for a solid waste disposal site (Balew et al., 2022: 12). Solid waste disposal is an important part of a waste-management system, which requires much attention to avoid environmental pollution and health problems (Balew et al., 2022: 3). 2.3 GIS applications for site selection GIS and RS have been used extensively to estimate suitable sites for different phenomena including landfill sites (Majid & Mir, 2021: 1; Mussa & Suryabhagavan, 2019: 1). Thus, GIS and RS both present opportunities for selecting a better solid waste siting technique (Balew et al., 2022: 2). Remotely sensed data has been used widely in introducing different criteria to be used as inputs in the Analytic Hierarchy Process (AHP). In a study to delineate groundwater potential zones in a tropical river basin, remote sensing was used to present the different criteria to be weighed. These criteria included lithology, lineament density, and geomorphology (Achu, Thomas & Reghunath, 2020: 1). In the same study, a GIS, in particular ArcGIS, was used to run the AHP. Proximity to nearby settlements: Solid waste disposal sites should not be placed too close to settlement areas and recreation centres (Yildirim et al., 2022: 5). In fact, findings in Turkey by Şener et al. (2010: 6) showed that the distance between disposal sites and settlement areas must be more than 1 000m and the haul distance between the solid waste disposal site and the main city centre should not exceed 30 km. 2.1 Solid waste management One solid waste management solution is to dispose of solid waste in properly designed and constructed places such as managed landfills, where it is safely contained. Therefore, locating proper sites for solid waste disposal and selecting appropriate landfill sites far from residential areas, environmental resources, and settlements is the main solution for the management of solid waste (Jothimani, Geberslasie & Duraisamy, 2021: 2). 2.1 Solid waste management Waste is a material discharged and discarded from each stage of daily human life activities, which leads to adverse impacts on human health and the environment (Naibbi & Umar, 2017: 2). Solid waste refers 111 Dahwa, Mazhindu & Chirenje 2023 Town and Regional Planning (82):110-123 to plastics, metals, glass, paper, food waste, grass, leaves, and other waste (Silva de Souza Lima Cano, Iacovidou & Rutkowski, 2022: 3). Solid waste management is referred to as the discipline associated with the control of generation, storage, collection, transfer and transport, processing and recovery, and final disposal of solid wastes in a manner that is in accordance with the best principles of public health, economics, engineering, urban and regional planning, conservation, aesthetics, and other environmental considerations (Abdalqader, 2011: 8). great natural resources to most African communities, with the New City locality not being an exemption. Rivers and streams are ecosystems that play an important role in supporting marine life and providing water for both domestic and industrial use; they play a big role in the ecological balance, providing a crucial ecosystem service delivery (Biggs, von Fumetti & Kelly-Quinn, 2016: 3). Furthermore, it is crucial to human activities for the vital role it plays in ecological balance. Taking this into consideration, landfills should not be placed close to these resources, in order to mitigate groundwater pollution (Sohoo et al., 2022: 4). Rivers can be endangered by the landfill because of leachate, thus bringing much pollution to the river (Sohoo et al., 2022: 4). great natural resources to most African communities, with the New City locality not being an exemption. Rivers and streams are ecosystems that play an important role in supporting marine life and providing water for both domestic and industrial use; they play a big role in the ecological balance, providing a crucial ecosystem service delivery (Biggs, von Fumetti & Kelly-Quinn, 2016: 3). Furthermore, it is crucial to human activities for the vital role it plays in ecological balance. Taking this into consideration, landfills should not be placed close to these resources, in order to mitigate groundwater pollution (Sohoo et al., 2022: 4). Rivers can be endangered by the landfill because of leachate, thus bringing much pollution to the river (Sohoo et al., 2022: 4). protected areas (a distance of more than 7 km) (Karimi et al., 2018: 12). 3. STUDY AREA criterions, including proximity to road, buildings, water bodies, airport, and residential areas (Majid & Mir, 2021: 1-9). A solid waste dumping site was also estimated for the town of Logia, Ethiopia, that used MCDA and spatial modelling (Mussa & Suryabhagavan, 2019: 1). Furthermore, Isalou et al. (2012: 1) used integrated fuzzy logic, analytic network, spatial analyst, and 3D analyst to overlap criterion layers and analytical processes for site selection. Findings by Akintorinwa and Okoro (2019: 1) combined electrical resistivity method and GIS-based modelling to generate thematic overburden maps for assigning weights, in order to select a suitable landfill site. site. It is helpful to set appropriate selection criteria for the identification of new solid waste dumping sites through scientific methods. criterions, including proximity to road, buildings, water bodies, airport, and residential areas (Majid & Mir, 2021: 1-9). A solid waste dumping site was also estimated for the town of Logia, Ethiopia, that used MCDA and spatial modelling (Mussa & Suryabhagavan, 2019: 1). Furthermore, Isalou et al. (2012: 1) used integrated fuzzy logic, analytic network, spatial analyst, and 3D analyst to overlap criterion layers and analytical processes for site selection. Findings by Akintorinwa and Okoro (2019: 1) combined electrical resistivity method and GIS-based modelling to generate thematic overburden maps for assigning weights, in order to select a suitable landfill site. Figure 1 shows the location of the area where the study was carried out in Zimbabwe. It is located 40 km west of Harare, the capital city, with approximately an area coverage of 54.23 km2, and it consists of 11 suburbs. The New City is located in the Mashonaland West, Central, and Harare provinces and comprises farms, human settlements, factories, and mines. The area has some established infrastructure, including, among others, roads, electrical lines, human settlements, and schools. In fact, the New City now houses the new parliament of Zimbabwe as it is expected to be the new commercial capital of Zimbabwe. In proposing a suitable landfill site for the New City, there is a need to consider the existing infrastructure. The study area was chosen as it is a new development in Zimbabwe. As it will be a city, there is a need for optimum siting for solid waste disposal. 3. STUDY AREA In as much as these researchers have shown the utility of MCDA and spatial modelling in the estimation of suitable sites, hardly anything has been done to understand the utility of this methodology in estimating suitable landfill sites in Zimbabwe, whose population is ever-growing and a country that still has much free space for urban expansion and, in this case, the New City. Hardly anything has been done to assist decision makers who would use the same information for spatial land planning with a geovisualisation tool, considering that such a tool accessible on any computing device with a web browser will be easily accessible to the different stakeholders in an interactive web viewer (Collado et al., 2022: 2). In fact, a Web-GIS has been successfully implemented for sharing different kinds of information. It has been used, among others, for the geovisualisation of heritage information (Sánchez-Aparicio et al., 2020: 2), and the management of waste collection (Rada, Ragazzi & Fedrizzi, 2013: 2), to name a few. 2.4 Multi-criteria decision analysis GIS-based MCDA is a process for combining and converting spatial data into a final result (Balew et al., 2022: 3). The MCDA processes are decision rules that establish a link between input and output maps. MCDA aims to solve problems, by evaluating competing factors in decision-making (Ghosh et al., 2022: 4). To this end, MCDA has been used in various studies to estimate the optimum locations for various phenomena. It has been successfully used for estimating the optimum sites for photovoltaic power plants (Boumaiza, Sanfilippo & Mohandes, 2022: 51), selection of the most suitable site for applying managed aquifer recharge systems (Papadopoulos, 2022: 1), to name a few studies. 2.2 Site-selection criteria for a solid waste disposal site Land-use types such as grassland, forests, agricultural land, wetland, and bushlands would be considered and assigned an appropriate index of land-use suitability for solid waste disposal sites (Mussa & Suryabhagavan, 2019b: 5). The importance of minimising the association of conflicting land-use (LU) in solid waste disposal siting can be realised by reviewing locally unwanted land-use areas (Mussa & Suryabhagavan, 2019: 6). Surface water vicinity: Surface Land-use types such as grassland, forests, agricultural land, wetland, and bushlands would be considered and assigned an appropriate index of land-use suitability for solid waste disposal sites (Mussa & Suryabhagavan, 2019b: 5). Proximity to cultural and protected sites: These sites include national heritage sites, shrines of religious importance, animal sanctuaries such as game parks, and areas of national interest. To protect these sites, a solid waste disposal site should be sited as far away as possible from these Considering the utility of GIS and remote sensing in site selection, it has been extensively combined with MCDA to also site landfills. For instance, studies on a suitable landfill site in Srinagar City used remotely sensed data and multiple For instance, studies on a suitable landfill site in Srinagar City used remotely sensed data and multiple Surface water vicinity: Surface water, rivers, and streams are 112 Dahwa, Mazhindu & Chirenje 2023 Town and Regional Planning (82):110-123 Dahwa, Mazhindu & Chirenje 2023 Town and Regional Planning (82):110-123 4. MATERIALS AND METHODOLOGY Exploratory research (Casula, Rangarajan & Shields, 2021: 1) was used for the study, with data from both archived and public sources. Figure 1: Study region Source: Author’s compilation, 2021 GIS is widely used for producing, editing, analysing, and visualising spatial data (Church, 2002: 7). This data will thus be easily accessible to environment planners, ecologists, spatial land planners, and other decision makers. Choosing a landfill site using GIS techniques requires many criteria to be used in one system for proper assessment. The selection method should evaluate multiple criteria such as groundwater, soil type, geology, protected areas, and roads (Karimi et al., 2018: 4). In multi-criteria evaluation, many criterion layers are to be processed by a GIS system to select a suitable Figure 1: Study region Source: Author’s compilation, 2021 Figure 1: Study region Source: Author’s compilation, 2021 113 Dahwa, Mazhindu & Chirenje 2023 Town and Regional Planning (82):110-123 The variables for this study were developed, using expert opinions, previous research studies, as well as relevant legislation and regulations. The data includes environmental, social, and physical factors whose layers were generated in DIVA GIS. The research used heights above sea level. For all the data sets generated in a GIS, the World Geodetic System- WGS1984 system was used as the geographic coordinate system. The criteria’s information layers were created with ArcGIS. Data sets and their respective buffers were created on geographical features such as rivers, road networks, and settlements. Each data set, in vector format, was converted to raster data format (vector to raster conversion). The third step dealt with the raster calculator to apply limits (constraints) to each data set map, using ArcGIS tools. Weights were applied to produce the final suitable sites. Figure 2 shows the flowchart followed in the research to derive the suitable estimate areas for the landfill. 4 MATERIALS AND METHODOLO 3 000 m buffer as the most suitable sites (Kumar & Deswal, 2022: 6). because it is important to standardise (normalise) criteria before combining them and, if necessary, to modify them so that all criteria maps are positively linked with appropriateness (Seyedmohammadi et al., 2018: 4). Land-use and land-cover changes: The land-use map was divided into 5 classes. Bare area occupying 3% was given the highest suitability score, followed by the forest, 58.05% and given the second priority, and agriculture, 21%, followed by urban area occupying 1.90% (Sohoo et al., 2022: 7). 4.1.2 Social criteria Proximity to roads: For easy accessibility, landfills must always be situated near road proximity so as not to compromise waste-collection and transportation costs (Jothimani, Geberslasie & Duraisamy, 2021: 4). This notwithstanding, it should not be located too close to the road so as to prevent environmental pollution and aesthetic damage. Using ArcGIS, multi-ring buffer zones of roads with 300 m, 500 m, 1 000 m, and 2 500 m were created (Jothimani et al., 2021: 4). Only the available main and proposed roads were considered, with a 0 to 300m buffer as unsuitable and a 300 to 500 m buffer as most suitable, whereas 500 to 1 000 m buffer as suitable and 1 000 to 2 500 m buffer as less suitable for waste disposal site location. Proximity to rivers: The study area comprises rivers and multiple streams running across it. Hence, in order to avoid water pollution, buffer zones of 500 m, 700 m, and 1 000 m were created around these water surfaces and classified into four categories, as shown in Figure 6. The 0 to 500 m buffer was considered unsuitable; 500 to 700 m buffer as less suitable; the 700 to 1 500 m buffer as suitable, and the 1 500 to Y an & Shields, 2021: 1) was used for the public sources. The variables for this study previous research studies, as well as data includes environmental, social, and erated in DIVA GIS. The research used sets generated in a GIS, the World Geodetic the geographic coordinate system. The d with ArcGIS. Data sets and their respective atures such as rivers, road networks, and mat, was converted to raster data format tep dealt with the raster calculator to apply , using ArcGIS tools. Weights were applied e 2 shows the flowchart followed in the areas for the landfill. 4.1.1 Environmental criteria Suitability of subsurface soil: The soils in the New City were grouped into 3 groups and included Hypoluvic Arenesols (11.02%), Haplic Lixisols (0.07%), and Ferralic Cambisols (60.62%) (Xiao et al., 2016: 4). Their permeability and porosity were used to define the criteria classification of the soil in the New City. Hypoluvic Arenesols have high profile development but are very porous and have high permeability, whereas Ferralic Cambisols have very low permeability and are highly drained (Xiao et al., 2016: 4). The soil groups with the lowest permeability and porosity were assigned the highest suitability score (Dewitte et al., 2013: 5). 4.1 Criteria selection, and standardisation Exploratory research (Casula study, with data from both ar During the site selection process, criteria with the greatest effect on the selection process must all be addressed (Yildirim et al., 2022: 3). For this study, these criteria were grouped into three main categories, namely environmental, social, and physical. The criteria were assessed on different scales, were developed, using expert opini relevant legislation and regulations physical factors whose layers were heights above sea level. For all the System-WGS1984 system was use criteria’s information layers were cr buffers were created on geographic settlements. Each data set, in vecto (vector to raster conversion). The t limits (constraints) to each data set to produce the final suitable sites. F research to derive the suitable estim 4. MATERIALS AND METHODOLOGY The open water surfaces occupying 0.15% of the study area were given zero suitability. Equation 1 Equation 1 4.3.2 Weighted Linear Combination • A binary overly (Boolean logic) (Singh, Jha & Chowdary, 2017: 3)) method, for determining potential site areas, using five constraints; The WLC technique is based on the decision maker’s weighting of each criterion. A total score is calculated for each choice, by multiplying the allocated weight value with a scaled value by the criterion (Hwang, 2011: 3). Each alternative is evaluated separately for each criterion if there are m number of choices and n number of criteria. The weights assigned to each criterion reflect its relevance in relation to the other criteria. Following that, the weighted average score for all options is determined using Equation 2 (Hwang, 2011: 3). • Weighted Linear Combination (WLC) (Adamczyk & Wałdykowski, 2022: 2) method, for selecting candidate sites (alternatives) from potential site areas, using five factors, and • Weighted Linear Combination (WLC) (Adamczyk & Wałdykowski, 2022: 2) method, for selecting candidate sites (alternatives) from potential site areas, using five factors, and • AHP (Ghosh et al., 2022: 2) method, for determining preferred candidate sites from potential sites. • AHP (Ghosh et al., 2022: 2) method, for determining preferred candidate sites from potential sites. 4.1.3 Physical criteria Figure 2: Data acquisition and processing flow chart Source: Author’s compilation 2021 Web map Digitised layers Roads Rivers Settlements Geology Data processing Data acquisition Buffering Rasterise AHP MCDA Reclassification Overlay analysis Weighted overlay Landfill suitability Land Figure 2: Data acquisition and processing flow chart Source: Author’s compilation, 2021 Data acquisition Buffering Proximity to protected areas: Like Asefa et al. (2013: 5), this study considered churches, settlements, and schools as protected areas that need to be located away from landfill sites because of the presence of high population concentration in these areas. Buffer zones of the following classes were created: 0 to 500 m as unsuitable, 500 to 1 000 m as less suitable, 1 000 to 1 500 m as suitable, and 1 500 to 5 000 m as most suitable. Thi th t th it bilit Overlay analysis This means that the suitability of a landfill site increases as the presence of population decreases. Figure 2: Data acquisition and processing flow chart Source: Author’s compilation 2021 Figure 2: Data acquisition and processing flow chart Source: Author’s compilation, 2021 Figure 2: Data acquisition and processing flow chart Source: Author’s compilation 2021 Figure 2: Data acquisition and processing flow chart Source: Author’s compilation, 2021 114 Dahwa, Mazhindu & Chirenje 2023 Town and Regional Planning (82):110-123 4.3.1 Binary overlay methods (Boolean logic) Considering that each site for siting a MSW landfill is either good or not, the result is a binary map, hence the need to use Boolean logic in the study. Boolean Logic is mostly used in the early screening stages or in stages where unusable options for usable alternatives are divided (Adamczyk & Wałdykowski, 2022: 3). The number of classes is determined by whether or not the desired requirement is accurate, and the final output expression is applied to each variable (Wu et al., 2007: 15). These variables were used to specify two classes multiple times. For the targeted location and the output of a new map, these classes were 0 and 1, respectively. Ai = Σ(i,j)w(j)Nj=1 i=1;2;3;… Ai: Weighted total score of alternative (i) a i; j: Score of alternative (i) according to (j) criteria w j: Weight of j criteria Equ 4.3 GIS-based MCDA data processing To find the optimum site for a landfill in the New City, the researchers used MCDA theory approaches in a GIS context. MCDA is a decision-making technique that employs the identification and choosing of criteria to find the best solution based on different weighted factors and considering the decision makers’ expectations (Mateo, 2012: 25). MCDA supports decision-making processes through 4.2 Determination of criteria weights the analysis of multiple inputs at several levels of description, including economic, technical, social, and environmental factors (Adamczyk & Wałdykowski, 2022: 1). It is worth mentioning that there are various methods of implementing MCDA. For this study, the following MCDA approaches were used: Equation 1 defines Boolean logic, which converts relevant information from each input map into binary form (True or False) or (0 and 1): C = {1 if class A > or < X} and C = class A > or <X Equation 1 defines Boolean logic, which converts relevant information from each input map into binary form (True or False) or (0 and 1): C = {1 if class A > or < X} and C = class A > or <X In spatial analysis, problems involve criteria of varying importance to decision makers (see Table 1). Thus, information about the relative importance of the criteria is required. A weight is a value assigned to an assessment criterion that indicates its relevance in comparison to other criteria being considered. The greater weight the criterion has in the total usefulness, the more significant it is (Seyedmohammadi et al., 2018: 5). Weights may be developed using a variety of methods. In extremely simple instances, splitting 1.0 among the criteria might be used to give criteria weights. It becomes difficult to perform weight judgements on the whole data set when more than a few criteria are involved, and several variables apply. After that, the weights are generally adjusted to equal one. Some of the most popular methods for the development of weights (Seyedmohammadi et al., 2018: 5) are ranking, rating, and pairwise comparison. In this study, the criterion weights were evaluated, using the ranking method. C = {1 if class A > or < X} and C = class A > or <X Equation 2 WLC is built on the notion of a weighted average, in which continuous criteria are standardised to a common numeric range and then merged using a weighted average (Mallick et al., 2022: 6). The decision maker gives relative significance weights to each attribute map layer directly. The overall score for each alternative is calculated, Table 1: The fundamental scale of absolute numbers Intensity of importance Definition Explanation 1 Equal importance How activities contribute equally to the objective 3 Moderate importance Experience and judgement slightly favour one activity over another activity 5 Strong importance Experience and judgement strongly favour one activity over another activity 7 Very strong importance An activity is favoured very strongly over another activity 9 Extreme importance The evidence favouring one activity over another is of the highest possible order of affirmation 2,4,6,8 Intermediate values between the two adjacent judgements When compromise is needed Reciprocals of above If activity i has one of the above non-zero numbers assigned to it when compared with activity j, when j has the reciprocal value when compared with i (see equation 2) A reasonable assumption Source: Mogaji et al., 2022: 4 Table 1: The fundamental scale of absolute numbers Table 1: The fundamental scale of absolute numbers Intensity of importance Definition Explanation 1 Equal importance How activities contribute equally to the objective 3 Moderate importance Experience and judgement slightly favour one activity over another activity 5 Strong importance Experience and judgement strongly favour one activity over another activity 7 Very strong importance An activity is favoured very strongly over another activity 9 Extreme importance The evidence favouring one activity over another is of the highest possible order of affirmation 2,4,6,8 Intermediate values between the two adjacent judgements When compromise is needed Reciprocals of above If activity i has one of the above non-zero numbers assigned to it when compared with activity j, when j has the reciprocal value when compared with i (see equation 2) A reasonable assumption Source: Mogaji et al., 2022: 4 Source: Mogaji et al., 2022: 4 115 Dahwa, Mazhindu & Chirenje 2023 Town and Regional Planning (82):110-123 by multiplying the significance weight allocated to each attribute by the scaled value assigned to that characteristic in the alternative, then adding the products across all attributes (Mallick et al., 2022: 6). The scores for all of the options are totalled, and the one with the highest overall score is picked. Equation 2 The approach may be used with any GIS system that supports overlays, and allows the evaluation criterion map layers to be merged to produce the composite map layer, which is the final result (Yin et al., 2020: 7). The weighted linear combination technique combines elements, by assigning a numerical value to each one. Following that, the findings are summed to provide a suitability map. maps, could easily be used by a wide audience. A Web-GIS provides easy- to-use tools for the geovisualisation of any mapped product that will help decision makers in spatial planning, by using technologies with which they are already familiar, in this case any computing device with an internet browser and internet connectivity (Collado et al., 2022: 2). Distributing geospatial information on the internet allows different users with varying levels of literacy to use GIS systems to easily interact with a GIS on a web browser. In fact, a web browser and the internet allow all levels of society to access geospatial information. It provides a media for accessing geo- related information with no location restrictions. The technology has been used for the geovisualisation of different phenomena, including the geovisualiation of transport infrastructure (Mazhindu & Madamombe, 2022: 2). For this study, the results or extracted suitable areas were exported to HTML, Javascript, and CSS, which are web-mapping technologies using a QGIS plugin – QGIS2Web, for geovisualisation on a web-GIS that allows data on landfill site selection to be accessed over the internet. and levels are developed (Andreolli et al., 2022: 3). The upper part of the hierarchy is shown as the main objective of the decision problem. However, all the other criteria contributing to the objective are placed at lower levels and other alternative factors to be evaluated are placed at the bottom (Andreolli et al., 2022: 2). The weights of each criterion are determined through pairwise comparisons generated in semantic judgements. Each alternative is compared pairwise with respect to a specific criterion to obtain the scores (x1... xnn) of alternatives, where x1 is the first alternative to the nth alternative xn. The eigenvectors are obtained after normalising the judgemental matrices. Sisay et al. (2020: 4) used the lambda max technique to obtain criteria weights, by applying the pairwise comparison method. Equation 2 Alternatives are compared pairwise with respect to each criterion to obtain weights (w1...wn), where w1 is the weight of the first alternative to the weight of the nth alternative given by wn. 4.3.3 Analytic hierarchy process method For decision analysis, the AHP method provides the best performing model of decision-making (Mojaver et al., 2022; Andreolli et al., 2022; Noorollahi et al., 2022; Belay et al., 2022), as it has proven over time to be a well-established method to address complex decisions and obtain a priority ranking of alternatives (Mogaji et al., 2022: 4). in the study. Table 3 indicates the criteria chosen for selecting the suitable sites. The AHP allows for the quantitative and/or qualitative evaluation of criteria and factors on the same measurement scale and the recreation of the original decision factor into a hierarchy, in which the hierarchical links between stages 4.4 Web-GIS Application development and results exportation After generating a mapping product, there is a need to ensure that the products, for instance, suitability Table 2: Exclusionary criteria for landfill potential sites suitability and their recommended limits Criteria and reference Weight Set limits and reference Protected land, urban land, roads, open water 7 500 m (Şener, et al., 2010) Land use land cover 4 Less preference to residential and agricultural areas and water zones (areas of low elevation) Soil types 3 High permeability and porosity Source: Jothimani et al., 2021: 3 Table 3: Chosen criteria and respective buffers (km) for the suitable sites’ selections Factor Criteria Roads 500 m Rivers >2 000 m Soil type Lowest permeability and porosity Land use/land cover High suitability score to be assigned to bare areas, followed by forest or natural areas. Agricultural and residential to have less scores Protected areas >4 000 m Source: Jothimani et al 2021: 3 Table 2: Exclusionary criteria for landfill potential sites suitability and their recommended limits Source: Jothimani et al., 2021: 3 116 Dahwa, Mazhindu & Chirenje 2023 Town and Regional Planning (82):110-123 Figure 3: Road classification Figure 3: Road classification 5.1 Roads Table 4: Suitability value and coverage of roads Distance from roads Suitability level Value Area (km2) Area (%) 300 m Unsuitable 1 7.1 29 500 m Most suitable 4 8.3 32.1 1 000 m Suitable 3 9.8 17.6 2 500 m Less suitable 2 13.5 29 Figure 3: Road classification Source: Author’s compilation, 2021 Figure 4: Proximity to rivers binary map Source: Author’s compilation, 2021 Table 5: Suitability value and coverage of rivers Proximity to rivers Suitability level Value Area (km2) Area (%) 500 m Unsuitable 1 216.9 78.9 700 m Less suitable 2 37.1 13.5 1 500 m Suitable 3 9.3 3.4 3 000 m Most suitable 4 11.7 4.3 Figure 5: Protected areas classification Figure 3 shows the suitability map considering distances to roads. The results show that areas represented with a brown colour are most suitable for citing a landfill. Approximately 60% of the area is unsuitable for the landfill site when considering proximity to roads, as shown in Table 4, with central areas such as Bendauch and Komani having the largest areas of unsuitable sites. Areas such as Kinvara and Haydon have a larger spatial extent of most suitable area for a landfill site. Figure 3: Road classification Source: Author’s compilation, 2021 Table 4: Suitability value and coverage of roads Table 4: Suitability value and coverage of roads Distance from roads Suitability level Value Area (km2) Area (%) 300 m Unsuitable 1 7.1 29 500 m Most suitable 4 8.3 32.1 1 000 m Suitable 3 9.8 17.6 2 500 m Less suitable 2 13.5 29 5.2 Proximity to rivers Figure 4 shows the suitability map considering distances to rivers. Results show that areas represented with a green colour and located in the east part of the map extending from Mgutu to Glenara and Komani are most suitable for siting a landfill based on proximity to rivers. Approximately 8% is most suitable and suitable for the landfill site, as shown in Table 5, with central areas such as Bendauch, Selby, Durnbury Park, and Mt. Hampden having the largest areas of unsuitable sites. Areas such as Kinvara and Haydon have some parts of spatial extent of suitable areas for landfill site. Figure 4: Proximity to rivers binary map Source: Author’s compilation, 2021 Figure 4: Proximity to rivers binary map Source: Author’s compilation, 2021 Table 5: Suitability value and coverage of rivers Table 5: Suitability value and coverage of rivers Proximity to rivers Suitability level Value Area (km2) Area (%) 500 m Unsuitable 1 216.9 78.9 700 m Less suitable 2 37.1 13.5 1 500 m Suitable 3 9.3 3.4 3 000 m Most suitable 4 11.7 4.3 5.3 Proximity to protected areas Figure 5 shows the suitability map considering proximity to protected areas (churches, settlements, and schools). Results show that the areas represented with a green colour and located in the east part of the map extending from Mgutu to Glenara and Komani are most suitable for siting a landfill based on proximity to protected areas. In Figure 5, a small area is suitable for the landfill site, with central areas such as Bendauch, Selby, Durnbury Park, and Mt. Hampden having the largest areas of unsuitable sites. Areas such as Kinvara and Haydon have some parts spatial extent of suitable areas for a landfill site. Figure 5: Protected areas classification Source: Author’s compilation, 2021 Figure 5: Protected areas classification Source: Author’s compilation, 2021 Figure 5: Protected areas classification Source: Author’s compilation, 2021 117 Dahwa, Mazhindu & Chirenje 2023 Town and Regional Planning (82):110-123 Figure 7: Land cover/use suitability Source: Author’s compilation, 2021 Figure 6: Soil suitability classification Source: Author’s compilation, 2021 Figure 7: Land cover/use suitability Source: Author’s compilation, 2021 Table 6: Pairwise comparison matrix Proximity to rivers Proximity to roads Proximity to protected areas Land use/ cover Soil type Proximity to rivers 1 3 5 7 9 Proximity to roads 0.33 1 3 3 8 Proximity to protected areas 0.2 0.33 1 3 7 Land use/cover 0.14 0.33 0.33 1 7 Soil type 0.11 0.12 0.14 0.14 1 Table 7: Percentages of eigenvector weight Factor Eigenvector weight Percentage weight Proximity to rivers 0.10 10 Proximity to roads 0.09 9 Proximity to protected areas 0.11 11 Land use/cover 0.51 51 Soil type 0.19 19 Figure 6: Soil suitability classification Source: Author’s compilation, 2021 Figure 6: Soil suitability classification Source: Author’s compilation 2021 5.4 Soil suitability Figure 6 shows suitability classes for soils, with suitable areas occupying the largest area, moderately deep to deep, reddish brown granular clays; formed on mafic rocks, extending from the northwest to the southeast, with a colour code of brown. The most suitable areas were identified in the study area, extending from the northwest to the southeast, with a colour code of blue covering suburbs such as Kinvara, Haydon and part of Selby, Mt. Hampden, Komani, Bendauch, and Durnburry Park. A low suitability area was identified on the edges of Mgutu suburb in a northern direction. Figure 6: Soil suitability classification Source: Author’s compilation, 2021 Figure 6: Soil suitability classification Source: Author’s compilation, 2021 Figure 7: Land cover/use suitability Source: Author’s compilation 2021 5.5 Land cover/use suitability Figure 7 shows suitability classes for land cover and land use, with built-up areas detected mostly in suburbs such as Haydon, Kinvara, Mt. Hampden, thus the southern part of the study area and partially in Komani, Glenara, and Eskbank, thus the eastern parts of the study area. Agricultural activities were detected as dominating nearly every portion of the study area, mostly in the northern and eastern parts of the study area in suburbs such as Mgutu, Glenara, Durnbury Park, and Eskbank. Bare areas were detected in Kinvara and Hydon, however, at smaller areas. 118 Figure 7: Land cover/use suitability Source: Author’s compilation, 2021 Table 6: Pairwise comparison matrix Proximity to rivers Proximity to roads Proximity to protected areas Land use/ cover Soil type Proximity to rivers 1 3 5 7 9 Proximity to roads 0.33 1 3 3 8 Proximity to protected areas 0.2 0.33 1 3 7 Land use/cover 0.14 0.33 0.33 1 7 Soil type 0.11 0.12 0.14 0.14 1 Table 7: Percentages of eigenvector weight Factor Eigenvector weight Percentage weight Proximity to rivers 0.10 10 Proximity to roads 0.09 9 Proximity to protected areas 0.11 11 Land use/cover 0.51 51 Soil type 0.19 19 Figure 8: Suitability map Source: Author’s compilation, 2021 118 Figure 7: Land cover/use suitability Source: Author’s compilation, 2021 Table 6: Pairwise comparison matrix Proximity to rivers Proximity to roads Proximity to protected areas Land use/ cover Soil type Proximity to rivers 1 3 5 7 9 Proximity to roads 0.33 1 3 3 8 Proximity to protected areas 0.2 0.33 1 3 7 Land use/cover 0.14 0.33 0.33 1 7 Soil type 0.11 0.12 0.14 0.14 1 Table 7: Percentages of eigenvector weight Factor Eigenvector weight Percentage weight Proximity to rivers 0.10 10 Proximity to roads 0.09 9 Proximity to protected areas 0.11 11 Land use/cover 0.51 51 Soil type 0.19 19 Figure 8: Suitability map Source: Author’s compilation, 2021 Figure 7: Land cover/use suitability Source: Author’s compilation, 2021 Table 6: Pairwise comparison matrix Table 6: Pairwise comparison matrix Table 7: Percentages of eigenvector weight 5.7 Final suitability map overlaying the extracted suitable areas. The analysis helped limit the process to only select areas that meet the selection criteria, thus eliminating suburbs such as Kinvara, Hydon, and Mt. Hampden, which are located in the southwest, as they show residential infrastructure. Other suburbs such as Mgutu, Bendauch, and Komani are showing either complete or partial agricultural activities. Hence, this eliminates them during the overlay process with the extracted predicted suitable areas. However, other areas such as Oldburry, Eskank, and Durnbury Park have some parts that satisfy the landfill site selection conditions, namely away from protected areas, agricultural activities, rivers, and 500 m from roads. Results in Table 8 and Figure 8 show that approximately 18% (green areas on map) of the New City is highly suitable for siting a landfill. Moderately suitable areas cover approximately 8% (yellow areas on map). A further 73% (red areas on map) of the total land area in the study area is highly unsuitable for siting a landfill. Figure 9 shows the extraction of final suitable areas considering all the criteria merged together. The aim was to observe and measure the spatial extents suitable areas. The largest spatial extent observed was a 10 km2 covering from Durnbury Park to Haydon in a north to south direction. The northern part has moderately suitable areas with a combined spatial extent of 11.9 km2, in Mgutu and Eskbank. The extracted results were overlaid on top of the Google Hybrid Satellite in QGIS for near real-time visualisation of suitable areas, as demonstrated in Figure 9. Table 8: Percentages of final suitability Suitability level Area (%) Low suitability 73.1 Moderate suitability 7.9 High suitability 18.0 Suitability level Area (%) Low suitability 73.1 Moderate suitability 7.9 High suitability 18.0 5.6 Determining suitable map based on weights from Analytical Hierarchy Process The AHP was used to derive the weights for each element. This method consisted of ranking the elements according to their relative importance in the siting process, using the pairwise comparison matrix (see Table 6). From the pairwise comparison matrix, the eigenvector weight was derived in ArcGIS and presented in percentages (see Table 7). Figure 8: Suitability map Source: Author’s compilation, 2021 The eigenvector weights derived from the AHP were attributed to their respective raster layers and with the use of the weighted overlay tool they were all weighted together to produce the suitability map in Figure 8. Figure 8: Suitability map Source: Author’s compilation, 2021 118 Dahwa, Mazhindu & Chirenje 2023 Town and Regional Planning (82):110-123 5.10 Suitable areas and Google hybrid satellite overlay south-eastern part of the study area. Figure 10 also shows a Web-GIS for near real-time change detection and suitable area selection. The results from Boolean integration of reclassified raster layers show that the bigger area in New City is unsuitable for siting a landfill site and only a smaller percentage is suitable according to the predefined criteria. However, it is not always the case to find unsuitable and suitable sites treating each criterion as equally important. All classified raster layers went through weighted overlay technique, where weights were assigned to each reclassified layer and then integrated to produce final suitability maps with areas ranging from least suitable to most suitable sites (Jothimani et al., 2021). Findings show the areas that are suitable according to weighted overlay, using AHP. These findings will greatly assist spatial and town planners for the New City in the siting of an appropriate landfill site and in the process assist in the siting of other land zones such as, for instance, residential areas and the central business district. Figure 10 shows an overlay of the final extracted suitable areas with a near real-time satellite to visualise any changes in the study region. The largest predicted suitable area (10 km2) was predicted on top of already built-up infrastructures, for example Zimbabwe’s new parliament building. Other predicted suitable areas were overlaid and identified to be on top of agricultural activities and residential areas. However, some areas were correctly predicted as they were far away from rivers or streams, protected areas, and agricultural activities, thus the suitable area Oldburry city in the 5.9 Near real-time analysis of any changes in the study area Areas in the western parts such as Durnbury Park, Kinvara, Selby, Mt Hampden, and Haydon and areas in the eastern part such as Oldburry and Eskbank are more suitable for siting a landfill site. Areas such as Bendauchm Komani, and Mgutu have a low suitability. Park to Haydon in a n suitable areas with a c The extracted results near real time visualis The study area was used to demarcate the area of interest, as shown in Figure 10. An analysis was made based on the Google hybrid satellite to identify changes before rth to south direction. T mbined spatial extent ere overlaid on top of t tion of suitable areas Figure 9: Extracted suitable areas and their spatial extents Source: Author’s compilation, 2021 Figure 9: Extracted suitable areas and their spatial extents Source: Author’s compilation, 2021 Figure 9: Extracted suitable areas an Source: Author’s compilation, 2021 Figure 9: Extracted suitable areas and their spatial extents Source: Author’s compilation, 2021 Figure 9: Extracted suitable areas a Source: Author’s compilation, 2021 Figure 9: Extracted suitable areas and their spatial extents Source: Author’s compilation, 2021 119 an c 119 Dahwa, Mazhindu & Chirenje 2023 Town and Regional Planning (82):110-123 5.11 Web-GIS visualisation of suitable area, measurement, and selection Figure 11 shows the selected suitable areas located away from residential areas and agricultural activities, but close to road network for easier transportation within the study area. The web-GIS measuring tool was used to measure the total area for the selected suitable area. The total area was 6.11 km2. Figure 10: An overlay of all extracted suitable areas, with on Google hybrid satellite Source: Author’s compilation, 2021 The results also prove that, given a range of factors as a standard for locating a suitable site, they are not equally important to each other and thus need to be ranked and assigned a certain weight that will be used to produce a final suitability map. The final map that was produced indicated areas that are not suitable, suitable, fairly suitable, and moderately suitable for siting a landfill site. Figure 10: An overlay of all extracted suitable areas, with on Google hybrid satellite Source: Author’s compilation, 2021 Figure 10: An overlay of all extracted suitable areas, with on Google hybrid satellite Source: Author’s compilation, 2021 Figure 11: Web-GIS for suitable area selection after overlay and area measurement Source: Author’s compilation, 2021 The results show that areas of high viability, including Kinvara, Haydon and part of Selby, Mt. Hampden, Komani, Bendauch, and Durnburry Park, have ferralic cambisols type of soils with low diffusion coefficient and are less porous, preventing groundwater pollution through infiltration. According to studies, landfills are most suitable in areas with less permeable soils and less porous soils, which prevent groundwater contamination if water continues to penetrate within the soils, putting public health at risk (Khademalhoseiny, Ahmadi Nadoushan & Radnezhad, 2017: 5). Clarke (2015: 2) postulates that Figure 11: Web-GIS for suitable area selection after overlay and area measurement Source: Author’s compilation, 2021 Figure 11: Web-GIS for suitable area selection after overlay and area measurement Source: Author’s compilation, 2021 Clarke (2015: 2) postulates that landfills create detrimental gases 120 Dahwa, Mazhindu & Chirenje 2023 Town and Regional Planning (82):110-123 Dahwa, Mazhindu & Chirenje 2023 Town and Regional Planning (82):110-123 Dahwa, Mazhindu & Chirenje 2023 Town and Regional Planning (82):110-123 gazetted by the responsible government. Furthermore, the area has artisanal miners and these areas should also be sites to avoid. and leakage, making them unfit to be in proximity to surface waters. Since landfill is characterised by several types of waste, its pollution capabilities are high. 5.11 Web-GIS visualisation of suitable area, measurement, and selection Henceforth, to avoid pollution of rivers and transmission of hazardous chemicals into underground water, which can lead to bio-accumulation of chemicals in the enact species, a landfill must be located far away from streams, rivers, and water reservoirs. Landfills located near water surfaces have resulted in the death of aquatic life and health implications for the people who depend on surface water sources for domestic use. gazetted by the responsible government. Furthermore, the area has artisanal miners and these areas should also be sites to avoid. BELAY, S., GOEDERT, J., WOLDESENBET, A. & ROKOOEI, S. 2022. 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CHEMOSPHERE.2021.131867 MOJAVER, M., HASANZADEH, R., AZDAST, T. & PARK, C.B. 2022. KUMAR, S. & DESWAL, S. 2022. Comparative assessment of Kurukshetra City waste dumping sites using RIAM analysis: A case study. Lecture Notes in Civil Engineering, vol. 154, pp. 31-38. doi. org/10.1007/978-981-16-1993-9_4 KUMAR, S. & DESWAL, S. 2022. Comparative assessment of Kurukshetra City waste dumping sites using RIAM analysis: A case study. Lecture Notes in Civil Engineering, vol. 154, pp. 31-38. doi. org/10.1007/978-981-16-1993-9_4 Comparative study on air gasification of plastic waste and conventional biomass based on coupling of AHP/ TOPSIS multi-criteria decision analysis. Chemosphere, 286, article 131867. https://doi.org/10.1016/J. CHEMOSPHERE.2021.131867 Saharan Africa. Economics and Human Biology, vol. 45, article 101102. doi. org/10.1016/j.ehb.2021.101102 DEWITTE, O. et al. 2013. 7. CONCLUSIONS ADAMCZYK, J. & WAŁDYKOWSKI, P. 2022. Planning for sustainable development of tourism in the Tatra National Park Buffer Zone using the MCDA approach. Miscellanea Geographica, 26(1), pp. 42-51. doi. org/10.2478/mgrsd-2020-0067 The study resulted in optimal sites being identified, analysed, and weighted. A criterion was used based on factors such as water sources, protected areas, soil, road, rivers, and boundary. The study shows that MCDA can be successfully implemented to site a landfill for a developing country such as Zimbabwe that has abundant land for development and an ever- growing population. Furthermore, it is interesting to note that Web-GIS can be successfully implemented to assist decision makers in the management of landfills and MSW. In fact, the geovisualisation of the suitable sites assists decision makers wherever they are, regardless of geographic location, to visualise the suitable areas in relation to features to which they can relate on a map. 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W4286850466.txt	https://zenodo.org/records/5727077/files/Chirita_Le_Role.pdf	fr		Le rôle de la mémoire dans l'exploration du texte dramatique en classe de FLE	Zenodo (CERN European Organization for Nuclear Research)	2,021	cc-by	1,864	CZU: 811 LE RÔLE DE LA MÉMOIRE DANS L’EXPLORATION DU TEXTE DRAMATIQUE EN CLASSE DE FLE Mariana CHIRIȚA Alecu Russo Bălți State University, Moldova marianachirita83@gmail.com Abstract: The article examines the role memory plays in the process of acquisition of French as a foreign language while using drama in the classroom. The suggested activities are designed to help learners improve and train their memory while working with the play Cocorico by Sylvaine Hinglais. The activities can be used with all learners regardless of their language proficiency level. Keywords: memory, drama, FFL classroom. Chez les enfants, la mémoire est très forte; voilà pourquoi elle stimule à l’excès la fantaisie, qui n’est que mémoire, soit dilatée, soit composée. Giambattista Vico [Bagdasar et al., 1995: 333] En analysant l’affirmation de Giambattista Vico, nous pouvons constater que les enfants sont ceux qui jouissent d’une forte mémoire. Dans l’acception de l’auteur, notamment la mémoire forte stimule la fantaisie, qualifiée comme mémoire dilatée ou composée. Par conséquent, pour cultiver la créativité, il faut laisser la fantaisie errer à l’aide d’une mémoire bien entrainée. La définition de la mémoire comporte trois acceptions : «1. Activité biologique et psychique qui permet d’acquérir, de conserver et de restituer des informations. 2. Aptitude à se souvenir. 3. L’esprit, en tant que siège des souvenirs. » [Le petit LAROUSSE illustré : 716] En analysant la définition, nous observons que la mémoire constitue en même temps une activité biologique et psychique de l’être humain, une aptitude de celui-ci de se souvenir de quelque chose, mais aussi un centre des souvenirs. Appartenant au domaine de la psychologie, la mémoire est définie comme « la capacité et le processus psychologique de réflexion de l’expérience antérieure à travers la fixation, reconnaissance et reproduction des images sensorielles, des idées, des états affectifs. 47 Elle réalise la cohérence de la vie intérieure et des liaisons interdépendantes avec la réalité objective. » [Șchiopu et al., 1997: 439] Phénomène complexe, la mémoire assure la diffusion de l’expérience antérieure d’un individu à la suite de la réalisation de trois opérations : fixation, reconnaissance et reproduction de tout ce qu’il voit, à quoi il pense et de ce qu’il sent. U. Șchiopu avance par l’idée que «la mémoire est comme la colonne vertébrale de la personnalité, étant impliquée dans la formation de l’identité.» [Ibidem] Vraiment le rôle de la mémoire dans la construction de la personnalité est incontestable. Or, la présence dans la mémoire des événements, des états affectifs attestés dans le vécu de quelqu’un contribuent à l’éducation de la personne aux valeurs, c’est-à-dire à la construction de la verticalité de celle-ci. Si nous parlons de la typologie de la mémoire, alors dans LAROUSSE on distingue les types suivants: 1. „mémoire immédiate, dont la capacité est limitée; 2. mémoire à long terme, dont la capacité est illimitée; 3. mémoire épisodique, qui concerne les événements ponctuels; 4. mémoire sémantique, relative à des faits, à des connaissances générales; 5. mémoire déclarative (les savoirs); 6. mémoire procédurale (les svoir-faire); 7. mémoire inconsciente (qui influe sur l’activité psychique).” [Le petit LAROUSSE illustré : 716] La psychologue U. Șchiopu précise que la mémoire peut être : • « spontané, • involontaire, • verbale, • actionnelle, • affective, • volontaire, • intentionnelle. » [Ibidem] Quant au processus d’apprentissage, il faut préciser que tous les types de mémoires y sont impliqués. En ce qui concerne la didactique des langues, la mémoire jouit d’un rôle à part. Il s’agit de l’acquisition de différentes structures, éléments et codes, soit du domaine linguistique (phonétique, lexical, grammatical), soit culturel et civilisationnel, qui aident le locuteur à construire correctement son discours. 48 Nos activités visant l’impliquation de la mémoire dans l’exploration du texte dramatique seront conçues autour de la pièce Cocorico [Hinglais et al., 1999: 58-61]. Mais avant de passer au côté pratique, il faut préciser que les ressources déposées dans la mémoire, auxquelles l’apprenant doit faire appel lors du travail avec le texte dramatique, parcourent une longue voie. Il s’agit de quelques étapes, dont: 1. « l’encodage de l’information, ou constitutions des traces, qui largement automatique en MCT, est davantage sous le contrôle du sujet en MLT et est favorisé par la structuration de l’apport; 2. le stockage. Alors que la durée de conservation des informations issues de l’apport est très limitée en MCT, la capacité de stockage permanent est considérée comme illimitée; 3. la récupération, facilitée par des indications contextuelles associées à l’information à rappeler. » [Cuq et al., 2003: 163] De la multitude de typologies de la mémoire présentées dans la littérature de spécialité, nous nous axerons sur l’étude de quelques types de mémoires entrainés dans l’exploration du texte dramatique: 1. la mémoire affective; 2. la mémoire sensorielle; 3. la mémoire verbale; 4. la mémoire actionnelle. La mémoire affective fait appel au vécu affectif de l’apprenant. Elle libère l’imagination. Cette chose est possible grâce à un entrainement assidu. Il est à mentionner que tous les exercices doivent être réalisés en groupe. Si nous nous transférons dans le cadre qui vise l’exploration du texte dramatique, alors il faut citer les suivants types d’exercices qui contribuent au développement de la mémoire affective: • les exercices de décontraction; • l’improvisation. Quant aux exercices de décontraction, A. Cormanski propose le jeu De la tête aux pieds, dans le but de repérer et éliminer toute tension. Les exercices d’improvisation contribuent, à leur tour, à la libération de tout ce qu’on a en soi. Par exemple : • Le jeu De la tête aux pieds [Cormanski, 2005: 17]: Déroulement S’asseoir sur une chaise, mettre le dos des mains à plat sur les genoux, paumes tournées vers le ciel, épaules lâches, jambes écartées. 49  Visage Remuer la bouche en se concentrant sur les commissures des lèvres. Pincer celles-ci en tendant la bouche fermée vers l’avant, puis la relâcher. En relâchant les lèvres, les étudiants émettront un son choisi du titre de la pièce « Cocorico ».  Corps Toujours assis sur la chaise, lever lentement le bras à la verticale, puis l’épaule comme si un fil invisible le tirait par le haut. Quand le fil invisible tire l’épaule en haut, l’étudiant prononce le mot « cocorico ».  Tête Les étudiants déplacent les têtes délicatement de droite à gauche dans un mouvement très lent. Chaque fois qu’ils déplacent la tête, ils prononcent cocorico en ajoutant des syllabes : co-/coco-/cocori/cocorico.  Voix Se tenir debout, jambes écartées les talons proches des pieds de la chaise. S’asseoir sur la chaise et se lever lentement. Répéter le mouvement en prononçant au moment de l’expiration le son [o]. • Improvisation. Cet exercice permet aux étudiants de faire appel à leurs sensations et émotions : Par exemple : Vous allez chez le docteur. Vous ne pouvez pas parler. Tâchez de lui expliquer vos problèmes en utilisant d’autres ressources de communication. Ou Vous avez les mêmes problèmes que les personnages de la pièce. Vous allez chez le docteur. Discutez avec lui. La mémoire sensorielle fait appel aux sensations visuelles, auditives, olfactives ou tactiles. Pour entrainer ce type de mémoire, nous proposons la méditation. Par exemple : - Par rapport aux personnages de la pièce, vous êtes bien portants. - Vous pensez à un dîner avec un menu copieux. - Le menu contient des produits qui vous aident à maintenir la santé du corps et de l’esprit. - Vous sentez l’odeur des plats. Pendant ce type d’exercice, il faut garder le silence. C. GrossetBureau précise que cet exercice sur les perceptions « rend l’élève plus attentif sur à ce qu’il vit dans sa vie quotidienne » [Grosset-Bureau et 50 al., 2000 : 49]. L’auteur propose de suivre plusieurs étapes pendant le jeu : 1. Les étudiants doivent être allongés sur le sol, les bras le long du corps. 2. Les yeux sont fermés et les muscles détendus pendant une ou deux minutes. 3. Ils s’éveillent et s’asseyent doucement. La mémoire verbale est nécessaire pour retenir un texte. Pour qu’un texte soit mieux retenu, il faut l’articuler. Pour bien mettre en oeuvre cet exercice (l’articulation) C. Grosset-Bureau (cf C. GrossetBureau et al., 2000 : 79-81) suggère de suivre 3 étapes : 1. La sonorisation des sons-voyelles. Par exemple : - Identifier les sons-voyelles dans la pièce à étudier ([a], [e], [i], [o], [u]). Articulez-les en vous regardant dans un mirroir. Inspirer puis chanter les sons. - Projeter des syllabes. En inspirant da-, ensuite peu à peu en expirant de-, di-, do-, du-. - Articuler de petites phrases qui présentent des difficultés de prononciation. (LE MARI : Coco... Cocoriri... Cocori...). 2. La sonorisation des sons-consonnes. Par exemple : - Prendre conscience de certaines consonnes, comme g (consonne sonore)/ c (consonne sourde) :ga, go, gu/ca-, co-, cu. 3. L’intonation. Par exemple : Dire la même phrase sur un ton exclamatif, interrogatif : « LA FEMME : [...] Quand il était petit, elle ne le lâchait pas d’une semelle. [...] » Dans la catégorie de la mémoire verbale, nous pouvons aussi ajouter les techniques de lectures comme : - la lecture à haute voix; - la lecture écrémage; - la technique des mots-clés; - la méthode des associations; - la méthode vocale (apprendre le texte par coeur à haute voix, ligne par ligne, sans intonation), etc. La mémoire actionnelle implique le mouvement. Pour mieux retenir le texte et jouer son rôle, les étudiants peuvent : 51 - dire leurs répliques en bougeant, en marchant, en courant, en dansant, etc. - soit - choisir les actions accomplies par les personnages de la pièce et les mettre en oeuvre (par exemple : aller chez le docteur, mener une discussion avec le docteur, s’énerver, etc). En conclusion, nous pouvons affirmer que la mémoire jouit d’une grande importance dans le processus d’exploration du texte dramatique. De la multitude des types de mémoires, impliqués dans ce processus, nous avons choisi de décrire seulement quelques-uns. Bien sûr qu’il y a beaucoup plus d’exercices et des techniques à présenter. On peut aussi intervenir dans le parcours de ceux présentés par nous et de les adapter à son public cible. Quand même, il faut préciser que pour l’étape de la lecture et de la compréhension du texte dramatique, on fait appel plutôt à la mémoire verbale. Pour entrainer le côté de la représentation, on impliquera la mémoire affective, la mémoire sensorielle et la mémoire actionnelle. Bref, les ressources de la mémoire, quelle que soit leur nature, aident les étudiants lors du processus d’exploration du texte dramatique à mieux : - retenir les répliques, - comprendre et construire son personnage, - jouer le spectacle. Bibliographie : 1. Bagdasar N., Bogdan V., Narly C., Antologie filosofică, București: Editura UNIVERSAL DALSI, 1995. - 655 p. 2. Cormanski, Alex, Techniques dramatiques: activités d’expression orale, Paris : Hachette Livres, 2005. – 126 p. 3. Cuq, Jean-Pierre et al., Dictionnaire de didactique du FLE, Paris : CLE International, 2003. – 303 p. 4. Grosset-Bureau, Claude, Christophe Suzanne, L’expression théâtrale au cycle 3, Paris : BORDAS, 2000. - 170 p. 5. Hinglais, Sylvaine, Liberman, Myrtha, Pièces et dialogues pour jouer la langue française, Paris : Retz, 1999. – 206 p. 6. Le petit LAROUSSE illustré, Paris : LAROUSSE, 2013. - 2016 p. 7. Șchiopu, Ursula (coord.), Dicționar de psihologie, București: Editura Babel, 1997. – 740 p. 52
https://openalex.org/W4206033283	https://www.intechopen.com/citation-pdf-url/79793	English	null	Stochastic versus Ray Tracing Wireless Channel Modeling for 5G and V2X Applications: Opportunities and Challenges	IntechOpen eBooks	2,022	cc-by	7,453	Stochastic versus Ray Tracing Wireless Channel Modeling for 5G and V2X Applications: Opportunities and Challenges Sulaiman Tariq, Hussain Al-Rizzo, Md Nazmul Hasan, Nijas Kunju and Said Abushamleh Sulaiman Tariq, Hussain Al-Rizzo, Md Nazmul Hasan, Nijas Kunju and Said Abushamleh Abstract Due to the rapid development of wireless communication applications, the study of Multiple Input Multiple Output (MIMO) communication systems has gained comprehensive research activities since it can significantly increase the channel capacity and link reliability without sacrificing bandwidth and/or transmitted power levels. Researchers tend to evaluate the performance of their MIMO antenna arrays using various channel modeling tools. These channel models are mainly categorized into either deterministic channels based on Ray Tracing (RT) tools or Stochastic Channel Models (SCM). In this chapter, we compare these two categories in terms of the MIMO channel capacity using a complete description of the antennas at the transmitting and receiving ends in terms of 3D polarimetric radiation patterns and scattering parameters. The performance is evaluated for 5G New Radio (NR) Enhanced Mobile Broadband (eMBB) and Ultra-Reliable Low-Latency Communica- tion (URLLC) services and Vehicle-to-Everything (V2X) systems using state-of-the- art commercial SCM and RT tools to provide information regarding the capabilities and limitations of each approach under different channel environments and the Quality of Experience (QoE) for high data rate and low latency content delivery in the 5G NR sub-6GHz mid-band Frequency Range-1 (FR1) N77/N78 bands. Keywords: MIMO, channel capacity, ray tracing channel modeling, stochastic channel modeling, 5G, V2V, V2X Keywords: MIMO, channel capacity, ray tracing channel modeling, stochastic channel modeling, 5G, V2V, V2X Keywords: MIMO, channel capacity, ray tracing channel modeling, stochastic channel modeling, 5G, V2V, V2X 1. Introduction MIMO communication systems have received significant research activities both in industry and academia since the emergence of 3G systems and are currently attracting many developers of 5G and 6G systems [1–3]. Services such as eMBB and URLLC have played an important role in the development of the 5G NR and Intelligent Transport System (ITS) and their network performance in V2X commu- nications which incorporates Vehicle-to-Vehicle (V2V), Vehicle-to-Infrastructure (V2I) and Vehicle-to-Pedestrians (V2P) communication modes. Wireless channel modeling plays a significant role in designing, assessing, and optimizing the 1 Antenna Systems performance of the systems components including the physical layer, networking protocols, and the antenna arrays at the Transmitter (Tx) and Receiver (Rx) using either RT or SCM tools [4–7]. However, very little research has been reported on the differences between these two channel modeling approaches including their strength, limitations and how they consequently affect the evaluation of the MIMO channel capacity for realistic scenarios [8–14]. Comparisons between deterministic and SCM are reported in [8–14]. However, none of them provided sufficient comparison based on a rigorous representation of the antenna arrays in terms of circuit parameters and 3D far-field patterns for both the co- and cross-polarized vector field components. In [8], the difference in the MIMO channel capacity between the SCM COST 259 and a deterministic urban city model simulated using RT for 3G cellular system is reported. However, only omni- directional antennas with no consideration for the mutual coupling are used and the results are only simulated in outdoor scenarios under one SNR. In addition, the testing scenarios are not identical, as for the RT model, they used varying heights for the base station towers, but for the SCM, fixed heights are used. g A comparison of the angles of arrival between 3GPP 3D statistical channel model and a deterministic urban channel model is presented in [9]. However, the MIMO channel capacity and throughput results are not evaluated. The authors in [10] considered a large-scale massive MIMO system to compare the downlink through- put between an outdoor urban deterministic model and the statistical i.i.d. Rayleigh model. However, the i.i.d. Rayleigh model is not suitable to represent urban channel models. In addition, the i.i.d. Rayleigh model uses data generated from the RT software. An evaluation of the MIMO channel capacity is presented in [11] using deter- ministic and stochastic indoor channel models. 1. Introduction TGn C, D and E are used as indoor office SCM and a close representation is created and used in an RT scenario as the deterministic model. The RT channel capacity show close comparison to the results from the TGn stochastic model E. However, in the deterministic channel model, the Mobile Terminal (MT) antennas are placed in only two rooms while ignoring other locations in the building which would subsequently affect the accuracy of the calculated results. Considering the asymmetric distribution of rooms and the small size of the model, the results should be studied for different locations of Tx and a complete distribution of Rx antennas in the entire model’s area. In [12], the difference between RT and SCM for network connectivity is reported. Neither capacity nor throughput results are presented, and all the simulations are for Single-Input Single-Output (SISO) scenarios. A massive MIMO study is presented in [13] comparing RT generated MIMO channel capacity with results from i.i.d. Ray- leigh statistical channel model. Similar to [10], the i.i.d. Rayleigh is not a realistic representation of indoor nor urban channel models. The results were also calculated for only one SNR. Lastly, in [14], the authors presented a survey about different channel modeling approaches and the challenges that accompany them in 5G net- works. However, the paper did not present comparison data nor case studies. The main objective of this chapter to provide a fair comparison between the two channel modeling techniques in terms of the MIMO channel capacity. To this end, several important parameters have to be taken into consideration when evaluating the channel capacity, such as the 3D radiation patterns of transmitting and receiving antennas for both the co- and cross-polarized components, the scattering parame- ters of the antenna arrays under consideration to invoke direct and mutual coupling between the antenna elements, the distribution of the Tx and Rx components using different channel environments (indoor and outdoor) for the 5G NR and the ITS V2X systems. We utilize state-of-art commercial channel modeling tools for our case studies. MIMObit [15] is used for SCM and Wireless InSite [16] is used to 2 Stochastic versus Ray Tracing Wireless Channel Modeling for 5G and V2X Applications… DOI: http://dx.doi.org/10.5772/intechopen.101625 Stochastic versus Ray Tracing Wireless Channel Modeling for 5G and V2X Applications… DOI: http://dx.doi.org/10.5772/intechopen.101625 represent deterministic models. 1. Introduction MIMObit is an electromagnetic propagation simu- lator that utilizes standardized stochastic spatiotemporal channel models and uses rigorous analytical electromagnetic formulation to produce precise antenna-to- antenna channels and evaluate the performance of MIMO systems in different propagation models [15]. Wireless InSite is a 3D RT wireless electromagnetic solver that considers the physical characteristics of the materials in an environment and uses shooting and bouncing rays as electromagnetic waves to track their reflection, diffraction, transmission and scattering through objects and evaluate the received power, capacity and throughput at each point in a study area [16]. p p y g p p y The new contributions of this chapter can be summarized as follows. A compre- hensive research of the capabilities and limitations of stochastic and deterministic channel modeling tools is presented for the first time in different indoor and outdoor channel environments. The effects of the antenna’s 3D radiation patterns and scat- tering parameters on the MIMO channel capacity for 5G and V2X applications are considered using cutting-edge SCM [15] and RT [16] tools. It should be noted that all the simulations included in this chapter are intended for the downlink transmission utilizing the 2.45 GHz ISM band and the 3.7 GHz 5G NR FR1 N77/N78 bands. g The rest of the chapter is organized as follows. In Section 2, we present a case study involving the evaluation of the SISO and MIMO channel capacity for the Two-Ray model using Wireless InSite and MIMObit in order to validate the results against analytical formulation. In Section 3, we study the MIMO channel capacity in an indoor office environment using RT and SCM. Section 4 presents an evaluation of the MIMO channel capacity in an outdoor scenario using RT and SCM. In Section 5, the performance of the two channel modeling tools is presented for a V2X scenario involving a fixed gNodeB base station (gNB) and a moving vehicle. Finally, the chapter is concluded in Section 6. scenario involving a fixed gNodeB base station (gNB) and a moving vehicle. Finally, the chapter is concluded in Section 6. 2. Case study 1: two-ray model Initially, the accuracy of the RT and SCM tools are verified using a simple scenario involving the channel capacity of the classical Two-Ray channel model over a flat Perfect Electrically Conducting (PEC) surface since an analytical solution is already available. Wireless InSite by Remcom is used for the RT approach while MIMObit is used to calculate the channel characteristics for a set of Tx and Rx antennas inserted in a half free-space environment above an infinite PEC surface. To test the various settings and parameters for the proper operation of both software tools, the first case focused on evaluating the SISO channel capacity in a Two-Ray model and compare it to the theoretical calculations. The Two-Ray model consists of a single large flat layer of PEC. A single half-wavelength dipole trans- mitting antenna operating at 2.45 GHz and an identical receiving antenna are placed 30 m apart at a height of 2 m. The model consists of two rays, a Line-of-Sight (LOS) component and a Non-Line-of-Sight (NLOS) component that is reflected over the ground plane. Figure 1 shows the two rays between the Tx antenna and the Rx antenna. To evaluate the received power at the receiver, Eq. (1) is used [17]. Pr ¼ 4Pt λ 4πd 2 GtGr cos 2 ∆∅ 2 (1) (1) where Pr is the received power in W, Pt is the transmitted power in W, λ is the wavelength, d is the distance between the Tx and Rx, Gt and Gr are the gain of the 3 Tx and Rx antennas, respectively, and the phase difference, Δϕ, can be calculated using Eq. (2). Figure 1. Two-ray model. Antenna Systems Antenna Systems Figure 1. Two ray model Figure 1. Two-ray model. g Two-ray model. Tx and Rx antennas, respectively, and the phase difference, Δϕ, can be calculated using Eq. (2). Tx and Rx antennas, respectively, and the phase difference, Δϕ, can be calculated using Eq. (2). Tx and Rx antennas, respectively, and the phase difference, Δϕ, can be calculated using Eq. (2). Δϕ ¼ 2π ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ ht þ hr ð Þ2 þ d2 q ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ ht hr ð Þ2 þ d2 q λ (2) (2) The Shannon capacity formula is used to find the SISO channel capacity [18] as shown in Eq. (3). 2. Case study 1: two-ray model The Shannon capacity formula is used to find the SISO channel capacity [18] as shown in Eq. (3). C ¼ B log 2 1 þ SNR ð Þ (3) (3) where C is the channel capacity in bits/s, B is the channel bandwidth in Hz and SNR is the Signal to Noise Ratio. The thermal noise is assumed to be equal to 100.99 dBm which is the standard used in [15, 16]. The geometry of the model simulated using Wireless InSite is shown in Figure 2. An equal total noise and interference of 100.99 dBm is used for both software tools. The channel capacity obtained from the three approaches are displayed in Table 1. Table 1 shows a close agreement between the SISO channel capacity results obtained from the RT tool, SCM tool and analytical calculations. A difference of Figure 2. g Two-ray model in wireless insite. Model Channel capacity (bps/Hz) Difference % compared to theoretical calculations Theoretical Calculations 22.98 — Wireless InSite 23 0.09% MIMObit 22.87 0.48% Table 1. Two-ray SISO model. 4 4 Stochastic versus Ray Tracing Wireless Channel Modeling for 5G and V2X Applications… DOI: http://dx.doi.org/10.5772/intechopen.101625 Stochastic versus Ray Tracing Wireless Channel Modeling for 5G and V2X Applications… DOI: http://dx.doi.org/10.5772/intechopen.101625 Stochastic versus Ray Tracing Wireless Channel Modeling for 5G and V2X Applications… DOI: http://dx.doi.org/10.5772/intechopen.101625 0.09% is noted between the RT tool and theoretical channel capacity while 0.48% difference is observed between the SCM capacity and the theoretical one. This provides impetus to perform precise channel modeling comparisons when moving to the more realistic indoor and outdoor environments. 0.09% is noted between the RT tool and theoretical channel capacity while 0.48% difference is observed between the SCM capacity and the theoretical one. This provides impetus to perform precise channel modeling comparisons when moving to the more realistic indoor and outdoor environments. To provide further validation over the reliability of the software tools, the next comparison uses the same Two-Ray model, however, this time using a 2x2 MIMO scenario. Two-vertically polarized, half-wavelength dipole antennas with a separa- tion of 2λ between the elements, operating at 2.45 GHz are used as the Tx and Rx antennas, centered at the same locations as the SISO two-ray model, separated by 30 m horizontally at a height of 2.5 m and 1.5 m, respectively. 2. Case study 1: two-ray model A single LOS ray and a single NLOS ray propagate from each Tx antenna element to each Rx antenna element, resulting in a total of eight rays in the model. The MIMO channel capacity can be expressed as shown in Eq. (4) [19]: C ¼ B log 2 I þ SNR Nt HHH (4) (4) where I is the identity matrix, Nt is the number of transmitting antenna ele- ments and H is the channel matrix between the Tx and the Rx. The achieved MIMO channel capacity is 24.92 bps/Hz in RT and 23.01 bps/Hz in SCM with a difference of 7.98% which further validates the operation of these channel modeling tools. 3. Case study 2: indoor channel modeling The second case concerns the evaluation of the MIMO channel capacity in a deterministic indoor channel model and comparison against a stochastic indoor channel model. The indoor floor plan shown in Figure 3 is provided by Remcom. It has a width of 66 m, a length of 35 m and a height of 3 m and consisting of 23 offices, one main lobby and two big office areas containing 22 desks. Wood, con- crete, glass, drywall and metallic materials were used to build the model. It provides a multipath-rich environment which is essential for MIMO applications. A corresponding SCM is created in MIMObit with similar dimensions using TGn 802.11n channel model B which is used to represent indoor office environments with NLOS conditions. A two-element MIMO antenna array is used as a transmitter and two-element half-wavelength dipole arrays are used as the receiving antennas in both models. Figure 3. RT indoor model. Figure 3. RT indoor model. g 3 RT indoor model. 5 Antenna Systems The transmitting antenna array used in both the RT and SCM tools is a modifi- cation of the design reported in [4] which is a miniaturized two-element monopole antenna array decoupled using a frequency selective structure, mounted on a grounded dielectric substrate and fed by two coaxial cables. The array operates at 3.7 GHz with a bandwidth of 160 MHz extending from 3.62 GHz to 3.78 GHz as determined by the S-parameters presented in Figure 4. The antenna has a mini- mum S11 of 30.7 dB and a reduced mutual coupling below 20 dB with a mini- mum of 38 dB over the operating bandwidth. The array achieves orthogonality between the main lobes of the 3D radiation patterns of the antenna elements as shown in Figures 5 and 6, which is favorable for spatial diversity and multiplexing. Each element pattern has a maximum gain of 7.01 dBi at boresight. g g The array is placed 36 m from the west main wall and 6.5 m below the north wall of the floor plan (blue spot in Figure 3) at a height of 2.5 m and is rotated 180o across the length as an Access Point (AP) with an input power of 1 W. 1056 two- element half-wavelength dipole antenna arrays operating at 3.7 GHz are uniformly distributed over the model at a height of 1.5 m and are used as the MT receiving antennas. Figure 6. Figure 6. 2D antenna radiation pattern (θ is the complement of the elevation angle and ∅is the azimuth angle). transmission and diffraction per path is seven, one and two, respectively. These numbers are chosen after a trade-off between simulation time and accuracy. The space between the transmitted rays is chosen as 0.25°. MIMO open-loop scheme with no channel-state information is chosen as the MIMO scheme with no precoding or beamforming using 20 MHz bandwidth. Equal gain combining is used as the combining method operated at the receivers. With the absence of interfer- ence in the channel, each MT will experience a unique multipath from the AP and hence resulting in different MIMO channel capacities as shown in Figure 7. The average capacity over the 1056 locations considered in the simulation is displayed in Figure 9 for different SNRs ranging from 5 to 30 dB as the deterministic channel model MIMO capacity results. transmission and diffraction per path is seven, one and two, respectively. These numbers are chosen after a trade-off between simulation time and accuracy. The space between the transmitted rays is chosen as 0.25°. MIMO open-loop scheme with no channel-state information is chosen as the MIMO scheme with no precoding or beamforming using 20 MHz bandwidth. Equal gain combining is used as the combining method operated at the receivers. With the absence of interfer- ence in the channel, each MT will experience a unique multipath from the AP and hence resulting in different MIMO channel capacities as shown in Figure 7. The average capacity over the 1056 locations considered in the simulation is displayed in Figure 9 for different SNRs ranging from 5 to 30 dB as the deterministic channel model MIMO capacity results. p y A similar scenario is created using MIMObit’s TGn 108.11n model B SCM where the AP antenna is used at the same coordinates. However, only one receiving MT antenna is placed at (10 m, 5 m, 1.5 m) as shown in Figure 8 and the average capacity is computed over a certain number of channel realizations in the time domain. The model is simulated over 1000 channel realizations where the channel environment changes at each realization resulting in a different set of multipath experienced by the signal traveling from the AP to the MT and hence resulting in a different MIMO channel capacity. 3. Case study 2: indoor channel modeling The MT dipole elements are separated by 2λ where λ is the free-space wavelength at the operating frequency. The maximum number of ray reflection, Figure 4. Tx/Rx antenna S-parameters. Figure 5. 3D antenna radiation pattern. 6 Figure 4. Tx/Rx antenna S-parameters. Figure 5. 3D antenna radiation pattern. Figure 5. g 5 3D antenna radiation pattern. 6 Stochastic versus Ray Tracing Wireless Channel Modeling for 5G and V2X Applications… DOI: http://dx.doi.org/10.5772/intechopen.101625 Stochastic versus Ray Tracing Wireless Channel Modeling for 5G and V2X Applications… DOI: http://dx.doi.org/10.5772/intechopen.101625 i 6 DOI: http://dx.doi.org/10.5772/intechopen.10 Figure 6. 2D antenna radiation pattern (θ is the complement of the elevation angle and ∅is the azimuth angle). Figure 6. The number of realizations has been chosen to achieve statistically reliable results. Comparison of the average MIMO channel capacity is shown versus SNR ranging from 5 to 30 dB in Figure 9. p y g g g The MIMO channel capacity in the SCM is higher than the capacity obtained from the deterministic channel model at SNR values larger than 20 dB. A 7 Antenna Systems Figure 7. Indoor model channel capacity. Figure 8. SCM indoor model. Figure 7. Indoor model channel capacity. Figure 8. SCM indoor model. Figure 7. Indoor model channel capacity. Figure 8. SCM indoor model. Figure 9. Indoor MIMO channel capacity. Figure 7. I d d l h l Figure 7. g 7 Indoor model channel capacity. Figure 8. SCM indoor model. g SCM indoor model. Figure 9. Figure 9. Indoor MIMO channel capacity. 8 8 Stochastic versus Ray Tracing Wireless Channel Modeling for 5G and V2X Applications… DOI: http://dx.doi.org/10.5772/intechopen.101625 Stochastic versus Ray Tracing Wireless Channel Modeling for 5G and V2X Applications… DOI: http://dx.doi.org/10.5772/intechopen.101625 maximum difference of 23.9% is observed at a SNR of 30 dB where the capacities are 13.1 and 10.3 bps/Hz from the SCM and RT models, respectively. To further improve the results obtained from the SCM model, a new simulation scheme is developed in which the number of the receiving MT is increased in MIMObit to six of different locations ranging from 3.16 m to 33.02 m away from the AP all at a height of 1.5 m as shown in Figure 10. g g Figure 11 reveals that at a constant SNR, changing the location of the receiving antenna does not have a significant impact on the MIMO channel capacity since each position is simulated 1000 times as the stochastic channel changes resulting in a maximum difference of 3.31% between any two Rx locations at SNR = 15 dB. Therefore, it is concluded that any Rx location within the geometry is valid for the comparison with the MIMO channel capacity generated from the RT tool. Finally, we conclude that our results provide close agreement in the MIMO channel capacity between the SCM and the RT tools particularly at SNRs below 20 dB. The difference slightly increases at higher SNRs because the effect of MIMO is more prominent and. Figure 6. It should be emphasized that the observed 20% difference in results leads to a difference of only 3 bps/Hz which is accepted given that the SCM and RT are based on entirely different analytical formulations and numerical implementation. Due to space limitation, only one indoor model has been Figure 10. SCM indoor model with different Rx positions. Figure 11. Indoor MIMO channel capacity for different Rx positions. Figure 10. SCM indoor model with different Rx positions. Figure 10. SCM indoor model with different Rx positions. Figure 11. Figure 11. Indoor MIMO channel capacity for different Rx positions. g Indoor MIMO channel capacity for different Rx positions. 9 Antenna Systems considered in the RT and SCM channel models. We recommend that future research should include different indoor environments to be compared with different SCM models. Figure 12. RT outdoor channel model. 4. Case study 3: outdoor channel modeling To further investigate the strengths and limitations of RT and SCM tools, the third case study considers the MIMO channel capacity in a realistic outdoor envi- ronment. The same software, MIMObit and Wireless InSite are used to model the stochastic and deterministic outdoor channels, respectively. A 2x2 MIMO antenna array operating in the 5G NR N77/N78 bands is used in the gNB. The 3GPP 3D Urban Macro cell (UMa) channel model is used as the SCM of the urban model. The deterministic urban channel model is imported from Remcom’s example library and is shown in Figure 12. It contains 39 buildings with different structures and heights. The gNB MIMO antenna used in this study is the same two-element array from the indoor study operating at 3.7 GHz and is placed at the edge of the rooftop on a building (the green box in Figure 12) at a height of 126.57 m with 180° tilt about the x-axis so that the antenna pattern’s main-lobes are pointing towards the ground. The MT antennas are two-element dipole arrays operating at 3.7 GHz. 500 MT antennas are placed randomly across the city at different heights ranging from 1.5 to 50 m. The antenna array at the gNB is fed with a total of 1 W power. The maximum number of ray reflection, transmission and diffraction per path is seven, one and two, respectively. The spacing between the transmitted rays is 0.25°. The MIMO open-loop diversity method is chosen for the study with no precoding or beamforming with 20 MHz allocated signal bandwidth Equal Gain Combining is The antenna array at the gNB is fed with a total of 1 W power. The maximum number of ray reflection, transmission and diffraction per path is seven, one and two, respectively. The spacing between the transmitted rays is 0.25°. The MIMO open-loop diversity method is chosen for the study with no precoding or beamforming with 20 MHz allocated signal bandwidth. Equal Gain Combining is used as the combining method operated at the receivers. The average MIMO chan- nel capacity perceived at the MTs is displayed in Figure 13 under different SNR values ranging from 5 to 30 dB. p p y y p g beamforming with 20 MHz allocated signal bandwidth. Equal Gain Combining is used as the combining method operated at the receivers. g RT outdoor channel model. 4. Case study 3: outdoor channel modeling The model is simulated under 1000 instantiations and the average MIMO channel capacity for SNRs ranging from 5 to 30 dB is shown in Figure 13. Similar to the results obtained in the indoor environment case study, the MIMO channel capacity in the SCM is close to the one obtained from RT. The difference in the capacity between the two channel modeling tools is 12.3% at SNR = 5 dB where the SCM achieved 2.41 bps/Hz and the deterministic model achieved 2.13 bps/Hz, 14.9% at SNR = 15 dB where 5.97 bps/Hz and 5.14 bps/Hz capacities are achieved in the stochastic and deterministic models, respectively, and 31.9% at SNR = 30 dB where 13.8 bps/Hz and 10 bps/Hz capacities are achieved in the stochastic and deterministic models, respectively. 4. Case study 3: outdoor channel modeling The average MIMO chan- nel capacity perceived at the MTs is displayed in Figure 13 under different SNR values ranging from 5 to 30 dB. The 3GPP 3D UMa SCM is used to represent the stochastic urban environment in a squarish geometry with a length of 500 m. The 3GPP UMa model is used for cities with gNBs located above roof tops of building [20]. The number of clusters and rays per cluster vary with the model and could reaching up to 23 clusters and 20 rays per cluster [20]. Both LOS and NLOS propagations are considered in this Figure 12. RT outdoor channel model. Figure 12. RT outdoor channel model. 10 Stochastic versus Ray Tracing Wireless Channel Modeling for 5G and V2X Applications… DOI: http://dx.doi.org/10.5772/intechopen.101625 Stochastic versus Ray Tracing Wireless Channel Modeling for 5G and V2X Applications… DOI: http://dx.doi.org/10.5772/intechopen.101625 model. The same gNB antenna operating at 3.7 GHz is placed at a height of 126.57 m and a location/orientation similar to that from the RT software. The receiving MT antenna is a two-element half-wavelength dipole array operating at 3.7 GHz and is placed at the center of the model at a height of 1.5 m. The model is simulated under 1000 instantiations and the average MIMO channel capacity for SNRs ranging from 5 to 30 dB is shown in Figure 13. Si il h l b i d i h i d i d h MIMO Figure 13. Outdoor MIMO channel capacity. Figure 13. Outdoor MIMO channel capacity. model. The same gNB antenna operating at 3.7 GHz is placed at a height of 126.57 m and a location/orientation similar to that from the RT software. The receiving MT antenna is a two-element half-wavelength dipole array operating at 3.7 GHz and is placed at the center of the model at a height of 1.5 m. The model is simulated under 1000 instantiations and the average MIMO channel capacity for SNRs ranging from 5 to 30 dB is shown in Figure 13. model. The same gNB antenna operating at 3.7 GHz is placed at a height of 126.57 m and a location/orientation similar to that from the RT software. The receiving MT antenna is a two-element half-wavelength dipole array operating at 3.7 GHz and is placed at the center of the model at a height of 1.5 m. 5. Channel modeling for V2X applications Traditionally, deterministic channel modeling tools are often used in V2X stud- ies to evaluate the performance of antennas designed to operate in free-space when installed on a mast or a vehicle, Advanced Driver-Assistance Systems (ADAS), and interference among different vehicles [21–24]. Specific models have been recently developed for these particular applications utilizing various geometries to assess V2X communication systems in dynamic scenarios, the review of which is provided in [25, 26]. The IEEE 802.11p and LTE-V standards are widely used for V2X com- munications. However, nowadays, 5G technologies are also being utilized for vehi- cle communications after the massive development in 5G networks and their capabilities in delivering high speed and reliable links between devices and equip- ment. In this section, we compare the capabilities and limitations of SCM and RT tools in assessing the performance of V2X communication systems in the physical (PHY) layer. It should be noted that the RT approach is the method of choice for static indoor and outdoor environments in particular for the initial design and deployment as well as the optimal number of transmitters to ensure coverage for the desired coverage area. Once the network is deployed and operational, the performance will obviously degrade in certain spots due to relative motion between different objects 11 Antenna Systems Antenna Systems in the network which can be resolved by increasing the transmitted power, in the network which can be resolved by increasing the transmitted power, adjusting the tilt of the antenna array or installing additional transmitters as needed. In a realistic simulation involving multiple vehicles moving at different speeds on a highway, multiple trajectories have to be defined for several vehicles involved in the scenario. Each vehicle should be represented with the proper geometry to represent the vehicle type such as, sedans, trucks, motorcycles and SUVs with their respective material properties and included in the RT solution at different positions, the number of which depends on the speed of the fastest vehicle and the data rate of the V2X system in order to relate the sampling in time to the channel coherence time. Obviously, this leads to extremely large computational demands that can only be achieved on highly dedicated cluster computers. b d by g d p , adjusting the tilt of the antenna array or installing additional transmitters as needed. 5. Channel modeling for V2X applications The antenna is placed on the vehicle rooftop, 1.5 m above ground level. The 2 2 MIMO system is simulated with 1 W total transmission power from the gNB. The maximum number of ray reflection, transmission and diffraction per path is seven, one and two, respectively. The spacing between the transmitted rays is 0.25°. The model is simulated as a closed-loop MIMO system with beamforming. The orthogonal radiation patterns of the antenna arrays is optimal for beamforming applications as they provide a narrow beam pointing at the vehicle while it moves, with Maximum Ratio Transmission (MRT) as the precoding scheme. The average MIMO capacity perceived at the vehicle’s antenna array is 14.05 bps/Hz at an average SNR of 40 dB. Figure 14 shows the MIMO channel capacity at each vehicle point along the traversed path in Mbit/sec with 20 MHz bandwidth. p g p In the absence of a vehicular model in the current version of MIMObit, we utilized the 3GPP 3D Urban UMa channel model as the stochastic channel where a gNB is placed at the coordinates (150, 0, 50) as illustrated in Figure 15 with the same MIMO antenna array used in the RT software. Both LOS and NLOS compo- nents are considered. To represent a vehicle movement in the SCM, we developed a new approach in which 20 independent MT antenna arrays are placed at different locations along the path. In this approach, each array is assigned a temporal behav- ior where it turns on momentarily at the time the vehicle reaches that point. For example, assuming the vehicle is moving at 50 km/h, Rx1 turns on at time, t = 0 s, then turns off, Rx2 turns on at t = 1.8 s then turns off, Rx3 turns on at t = 3.6 s then turns off, and so on. The model is simulated as a closed-loop 2x2 MIMO system with beamforming and the average achieved MIMO channel capacity is 15.3 bps/Hz at an average SNR equal to 40. g q A 4.32% difference in the closed-loop beamforming MIMO channel capacity between the two modeling approaches is observed. This is due to that the RT study involves a static environment with no object mobility due to the limitations of the available computational resources and hence there is no time-varying signal distor- tion caused by mobility. 5. Channel modeling for V2X applications In a realistic simulation involving multiple vehicles moving at different speeds on a highway, multiple trajectories have to be defined for several vehicles involved in the scenario. Each vehicle should be represented with the proper geometry to represent the vehicle type such as, sedans, trucks, motorcycles and SUVs with their respective material properties and included in the RT solution at different positions, the number of which depends on the speed of the fastest vehicle and the data rate of the V2X system in order to relate the sampling in time to the channel coherence time. Obviously, this leads to extremely large computational demands that can only be achieved on highly dedicated cluster computers. g y p Instead, the approach we followed to model simple V2X scenarios involves several simplifying assumptions in order to make the computational demands trac- table. The vehicle structure has not been included in the RT approach since a dense outdoor environment is involved. We selected a path as shown in Figure 14 for which the capacity has been evaluated along 410 m of discrete path points each separated by 1 m. p y The same outdoor urban channel model in the previous section is utilized in studying the channel capacity available to vehicles traversing a path of 410 m at a given speed in a city surrounded by concrete buildings while transmitting and receiving signals from a gNB located close to the pathway in a 2 2 closed-loop MIMO system with no interference as shown in Figure 14. The channel parameters obtained from this model can be used in post processing to test different V2X scenarios. The vehicle has a two-element MIMO antenna array operating in the 5G N77/N78 bands at 3.7 GHz. The radiation patterns of the array shown in Figure 14. V2X route MIMO channel capacity. Figure 14. V2X route MIMO channel capacity. g V2X route MIMO channel capacity. 12 Stochastic versus Ray Tracing Wireless Channel Modeling for 5G and V2X Applications… DOI: http://dx.doi.org/10.5772/intechopen.101625 Stochastic versus Ray Tracing Wireless Channel Modeling for 5G and V2X Applications… DOI: http://dx.doi.org/10.5772/intechopen.101625 Stochastic versus Ray Tracing Wireless Channel Modeling for 5G and V2X Applications… DOI: http://dx.doi.org/10.5772/intechopen.101625 Figures 5 and 6 are used in the simulation. The antenna is placed on the vehicle rooftop, 1.5 m above ground level. Figures 5 and 6 are used in the simulation. Figure 15. SCM V2X model. 5. Channel modeling for V2X applications However, the cluster birth-death process in different channel realizations accommodates for the non-static channel behavior of the SCM. Figure 15. SCM V2X model. 13 Antenna Systems Additionally, the temporal characteristics of the Rx antenna defines the vehicle movement in the SCM. Nevertheless, incorporating a large number of vehicles in an RT tool moving in an urban environment with different speeds and trajectories places severe limitations in terms of computational time and resources. 6. Conclusion In the near future, more RT and SCM tools will be developed as the demand and applications of various wireless systems relying on these channel modeling tools constantly grow for the advancement of wireless communications. This chapter provided a case study for the evaluation of the SISO and MIMO channel capacity using the Two-Ray model for validation purpose, studies of the MIMO channel capacity evaluation using RT and SCM tools for indoor and outdoor environments, and a performance evaluation of RT and SCM tools for a V2X communication scenario. In spite of the assumptions made in the RT approach, especially for the V2X applications, there are only minor differences in the MIMO channel capacity between RT and SCM. The SCM is capable of characterizing stationary and non- stationary dynamic V2X communication systems operating at different velocities since it considers the temporal and spatial domains while the deterministic model is capable of representing realistic object geometries. However, in the RT approach, the vehicles’ models can only be included at discrete positions and the simulations have to be performed at each location. RT tools, however, yield more accurate results for link-level simulations of static networks as it can model objects such as buildings, road obstacles, traffic signs, etc. and achieve channel information characterizing precise channel effects such as path loss, shadowing and multipath fading. While SCM tools also model these channel effects, its adaptation for temporal behavior and non-stationary channel models makes it more suitable for link- and system-level simulations analyzing data transmission and communications at the bit level. References arrays. 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In: 2018 IEEE 29th Annual International Symposium on Personal, Indoor and Mobile Radio Communications (PIMRC). Bologna, Italy: IEEE; 2018. Author details Author details Sulaiman Tariq1, Hussain Al-Rizzo1, Md Nazmul Hasan2, Nijas Kunju3 and Said Abushamleh4 1 University of Arkansas, Little Rock, USA 2 University of British Columbia, Vancouver, Canada 3 ANSYS Inc., Bangalore, India 4 Indiana Institute of Technology, Fort Wayne, USA Address all correspondence to: sztariq@ualr.edu © 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2021 The Author(s). Licensee IntechOpen. 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https://openalex.org/W3198390285	https://ora.ox.ac.uk/objects/uuid:8e6aa1ae-0337-4550-afe7-3fe8f95bfef1/files/rc247ds516	English	null	Reactogenicity and immunogenicity after a late second dose or a third dose of ChAdOx1 nCoV-19 in the UK: a substudy of two randomised controlled trials (COV001 and COV002)	Lancet	2,021	cc-by	10,372	Lancet 2021; 398: 981–90 Lancet 2021; 398: 981–90 Published Online September 1, 2021 https://doi.org/10.1016/ S0140-6736(21)01699-8 See Comment page 933 Contributed equally Jenner Institute, Nuffield Department of Medicine (A Flaxman DPhil, D Jenkin MRCP, J Aboagye BSc, S Belij-Rammerstorfer PhD, M Bittaye PhD, F Cappuccini PhD, S Davies BSc, K J Ewer PhD, P M Folegatti MD, J Fowler BA, Prof A V S Hill FRS, A M Minassian DPhil, H Sanders BSc, E Sheehan PhD, H Smith MSc, D Woods BSc, Prof S C Gilbert FMedSci, T Lambe PhD), Oxford Vaccine Methods In this substudy, volunteers aged 18–55 years who were enrolled in the phase 1/2 (COV001) controlled trial in the UK and had received either a single dose or two doses of 5 × 10¹⁰ viral particles were invited back for vaccination. Here we report the reactogenicity and immunogenicity of a delayed second dose (44–45 weeks after first dose) or a third dose of the vaccine (28–38 weeks after second dose). Data from volunteers aged 18–55 years who were enrolled in either the phase 1/2 (COV001) or phase 2/3 (COV002), single-blinded, randomised controlled trials of ChAdOx1 nCoV-19 and who had previously received a single dose or two doses of 5 × 10¹⁰ viral particles are used for comparison purposes. COV001 is registered with ClinicalTrials.gov, NCT04324606, and ISRCTN, 15281137, and COV002 is registered with ClinicalTrials.gov, NCT04400838, and ISRCTN, 15281137, and both are continuing but not recruiting. Findings Between March 11 and 21, 2021, 90 participants were enrolled in the third-dose boost substudy, of whom 80 (89%) were assessable for reactogenicity, 75 (83%) were assessable for evaluation of antibodies, and 15 (17%) were assessable for T-cells responses. The two-dose cohort comprised 321 participants who had reactogenicity data (with prime-boost interval of 8–12 weeks: 267 [83%] of 321; 15–25 weeks: 24 [7%]; or 44–45 weeks: 30 [9%]) and 261 who had immunogenicity data (interval of 8–12 weeks: 115 [44%] of 261; 15–25 weeks: 116 [44%]; and 44–45 weeks: 30 [11%]). 480 participants from the single-dose cohort were assessable for immunogenicity up to 44–45 weeks after vaccination. Antibody titres after a single dose measured approximately 320 days after vaccination remained higher than the titres measured at baseline (geometric mean titre of 66·00 ELISA units [EUs; 95% CI 47·83–91·08] vs 1·75 EUs [1·60–1·93]). 32 participants received a late second dose of vaccine 44–45 weeks after the first dose, of whom 30 were included in immunogenicity and reactogenicity analyses. Articles Articles Reactogenicity and immunogenicity after a late second dose or a third dose of ChAdOx1 nCoV-19 in the UK: a substudy of two randomised controlled trials (COV001 and COV002) Amy Flaxman, Natalie G Marchevsky, Daniel Jenkin, Jeremy Aboagye, Parvinder K Aley, Brian Angus, Sandra Belij-Rammerstorfer, Sagida Bibi, Mustapha Bittaye, Federica Cappuccini, Paola Cicconi, Elizabeth A Clutterbuck, Sophie Davies, Wanwisa Dejnirattisai, Christina Dold, Katie J Ewer, Pedro M Folegatti, Jamie Fowler, Adrian V S Hill, Simon Kerridge, Angela M Minassian, Juthathip Mongkolsapaya, Yama F Mujadidi, Emma Plested, Maheshi N Ramasamy, Hannah Robinson, Helen Sanders, Emma Sheehan, Holly Smith, Matthew D Snape, Rinn Song, Danielle Woods, Gavin Screaton, Sarah C Gilbert, Merryn Voysey, Andrew J Pollard, Teresa Lambe, and the Oxford COVID Vaccine Trial group Summary y Background COVID-19 vaccine supply shortages are causing concerns about compromised immunity in some countries as the interval between the first and second dose becomes longer. Conversely, countries with no supply constraints are considering administering a third dose. We assessed the persistence of immunogenicity after a single dose of ChAdOx1 nCoV-19 (AZD1222), immunity after an extended interval (44–45 weeks) between the first and second dose, and response to a third dose as a booster given 28–38 weeks after the second dose. Paediatrics (N G Marchevsky MSc, P K Aley PhD, B Angus MD, S Bibi PhD, P Cicconi PhD, E A Clutterbuck PhD, C Dold PhD, S Kerridge MSc, Y F Mujadidi MSc, E Plested, M N Ramasamy DPhil, H Robinson DipHE, M D Snape MD, R Song MD, M Voysey DPhil, Prof Sir A J Pollard FMedSci), and Chinese Academy of Medical Science (CAMS) Oxford Institute (T Lambe), University of Oxford, Oxford, UK; NIHR Oxford Biomedical Research Centre, Oxford, UK (N G Marchevsky, S Bibi, E A Clutterbuck, C Dold, M Voysey, Prof Sir A J Pollard); Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK (W Dejnirattisai PhD, J Mongkolsapaya PhD, Prof G Screaton DPhil) Lancet 2021; 398: 981–90 Published Online September 1, 2021 https://doi.org/10.1016/ S0140-6736(21)01699-8 See Comment page 933 *Contributed equally Jenner Institute, Nuffield Department of Medicine (A Flaxman DPhil, D Jenkin MRCP, J Aboagye BSc, S Belij-Rammerstorfer PhD, M Bittaye PhD, F Cappuccini PhD, S Davies BSc, K J Ewer PhD, P M Folegatti MD, J Fowler BA, Prof A V S Hill FRS, A M Minassian DPhil, H Sanders BSc, E Sheehan PhD, H Smith MSc, D Woods BSc, Prof S C Gilbert FMedSci, T Lambe PhD), Oxford Vaccine Group, Department of Paediatrics (N G Marchevsky MSc, P K Aley PhD, B Angus MD, S Bibi PhD, P Cicconi PhD, E A Clutterbuck PhD, C Dold PhD, S Kerridge MSc, Y F Mujadidi MSc, E Plested, M N Ramasamy DPhil, H Robinson DipHE, M D Snape MD, R Song MD, M Voysey DPhil, Prof Sir A J Pollard FMedSci), and Chinese Academy of Medical Science (CAMS) Oxford Institute (T Lambe), University of Oxford, Oxford, UK; NIHR Oxford Biomedical Research Centre, Oxford, UK (N G Marchevsky, S Bibi, E A Clutterbuck, C Dold, M Voysey, Prof Sir A J Pollard); Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK (W Dejnirattisai PhD, J Mongkolsapaya PhD, Prof G Screaton DPhil) Correspondence to: Dr Amy Flaxman, Jenner Institute, Nuffield Department of Medicine, University of Oxford, Headington, Oxford, OX3 7DQ, UK amy.flaxman@ndm.ox.ac.uk For more on COVAX see https://www.who.int/initiatives/ act-accelerator/covax Evidence before this study homologous third dose of BNT162b2, 6 or 12 months after the second dose. Multiple vaccines against SARS-CoV-2 have now been authorised for use in various countries. Most vaccines are given in a two-dose primary schedule, and further doses might be required to maintain protective immunity or control emerging variants. We searched PubMed for research articles published between database inception and June 23, 2021, using the search terms “SARS-CoV-2”, “vaccine”, “clinical trial”, AND (“third dose” OR “late boost”) with no language restrictions. We identified animal studies using combinations of three-dose vaccine delivery in prime-boost schedules. Additionally, we identified three clinical trials of three-dose delivery, including two in solid organ transplant recipients. In the first study in transplant recipients, antibody titres increased after the third dose of either BNT162b2 (Pfizer–BioNTech) or mRNA-1273 (Moderna) vaccines in a third of patients who had negative antibody titres and in all patients who had low-positive antibody titres. In the second study in transplant recipients, prevalence of antibody titres increased from 44% after a second dose to 68% after a third dose. In a phase 1 and 2 trial of a protein subunit vaccine ZF2001, the safety and immunogenicity data support the use of a 25 μg dose in a three-dose schedule. A number of clinical studies are measuring the effect of a third dose of vaccine, including a phase 1 study of 144 participants who received a Articles Biomedical Research Centre, Chinese Academy of Medical Sciences Innovation Fund for Medical Science, Thames Valley and South Midlands NIHR Clinical Research Network, AstraZeneca, and Wellcome. Biomedical Research Centre, Chinese Academy of Medical Sciences Innovation Fund for Medical Science, Thames Valley and South Midlands NIHR Clinical Research Network, AstraZeneca, and Wellcome. Biomedical Research Centre, Chinese Academy of Medical Sciences Innovation Fund for Medical Science, Thames Valley and South Midlands NIHR Clinical Research Network, AstraZeneca, and Wellcome. Copyright © 2021 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4 Introduction the possible risk of breakthrough infection as new variants emerge. The COVID-19 pandemic continues to put a substantial burden on health-care systems and a massive global effort is underway to protect populations through vaccination. COVID-19 vaccine supply shortages in many countries are causing concern about com promised immunity as the interval between the first and second dose extends beyond 12 weeks.1 WHO recommends that the second dose of the ChAdOx1 nCoV-19 vaccine is given 8–12 weeks after the first dose because the clinical trial data provide support for good levels of protection with this interval;2,3 however, many countries cannot obtain sufficient supplies to allow second doses to be administered by 12 weeks. These supply shortages are leading to longer intervals and uncertainty among policy makers about whether protection against COVID-192,3 will be maintained because no data exist on the efficacy of the immunisation schedules with intervals between the first and second dose that extend beyond this limit. ChAdOx1 nCoV-19 (AZD1222), a replication deficient adenoviral vectored vaccine that encodes the SARS-CoV-2 spike protein, is one of the most widely used vaccines globally. More than half a billion doses have been distributed to more than 168 countries across six continents, including provision through the COVAX Facility. Here, we describe tolerability and immune response to a late second dose (44–45 weeks after the first dose) of ChAdOx1 nCoV-19, and after a third dose (28–38 weeks after the second dose). We also report the persistence of antibody and cellular responses at 182 days and for antibodies up to 320 days after first dose of ChAdOx1 nCoV-19. Study design and participants y g In this substudy, we extended the data already collected as part of the UK COV001 and COV002 trials. In these trials, participants were randomly assigned to receive ChAdOx1 nCoV-19 or a meningococcal conjugate vaccine (MenACWY) as a control. Procedures, safety, immune responses, and efficacy before late vaccination and after Conversely, some high-income countries with highly vaccinated populations are considering administration of a third dose of a COVID-19 vaccine because of uncertainty about duration of immunity after the first two doses and Added value of this study We report immune responses to ChAdOx1 nCoV-19 following a second dose after an extended interval between the first and second dose, and after a third dose with an extended interval between the second and third dose. The extended interval between the first two doses (44–45 weeks) resulted in higher antibody titres after the second dose than with a shortened interval. A third dose given 28–38 weeks after the primary series increased the antibody titres to above those after a second dose with a shortened interval. Reactogenicity was lower after the second or third dose than after the first dose. Lancet 2021; 398: 981–90 Antibody titres were higher 28 days after vaccination in those with a longer interval between first and second dose than for those with a short interval (median total IgG titre: 923 EUs [IQR 525–1764] with an 8–12 week interval; 1860 EUs [917–4934] with a 15–25 week interval; and 3738 EUs [1824–6625] with a 44–45 week interval). Among participants who received a third dose of vaccine, antibody titres (measured in 73 [81%] participants for whom samples were available) were significantly higher 28 days after a third dose (median total IgG titre: 3746 EUs [IQR 2047–6420]) than 28 days after a second dose (median 1792 EUs [IQR 899–4634]; Wilcoxon signed rank test p=0·0043). T-cell responses were also boosted after a third dose (median response increased from 200 spot forming units [SFUs] per million peripheral blood mononuclear cells [PBMCs; IQR 127–389] immediately before the third dose to 399 SFUs per milion PBMCs [314–662] by day 28 after the third dose; Wilcoxon signed rank test p=0·012). Reactogenicity after a late second dose or a third dose was lower than reactogenicity after a first dose. Interpretation An extended interval before the second dose of ChAdOx1 nCoV-19 leads to increased antibody titres. A third dose of ChAdOx1 nCoV-19 induces antibodies to a level that correlates with high efficacy after second dose and boosts T-cell responses. Funding UK Research and Innovation, Engineering and Physical Sciences Research Council, National Institute for Health Research, Coalition for Epidemic Preparedness Innovations, National Institute for Health Research Oxford 981 www.thelancet.com Vol 398 September 11, 2021 Articles Articles Procedures Participants who were included as part of this substudy were vaccinated with a standard dose of ChAdOx1 nCoV-19 (5 × 10¹⁰ viral particles). For control participants who had previously received either one or two doses of MenACWY, this vaccination was their first dose of ChAdOx1 nCoV-19. For participants who had previously received ChAdOx1 nCoV-19, this vaccination was either their second dose (44–45 weeks after the first) or their third dose. These late vaccinations occurred 10 months (plus or minus 56 days) from enrolment. 7 days after vaccination participants were unmasked to treatment allocation, so that those who had received only one dose of ChAdOx1 nCoV-19 could subsequently receive a second dose, in line with national vaccination roll-out in the UK. For analysis of immunogenicity after a single dose of ChAdOx1 nCoV-19, we included all participants in COV001 and COV002 who had yet to receive a second dose of vaccine and for whom immunogenicity data were available. A substudy was added to the COV001 trial as a protocol amendment on March 1, 2021, to investigate the immunogenicity and tolerability of a third dose of the vaccine. Participants who had previously received two doses of ChAdOx1 nCoV-19 were recruited for this substudy, along with control participants, who had received two doses of MenACWY previously, to maintain blinding of reactogenicity data. All recruits received ChAdOx1 nCoV-19; for the control participants this was their first dose. For participants from this three-dose cohort to be eligible for inclusion in these analyses, they had to have an 8–16 week interval between first and second doses. Participants enrolled in the substudy had blood samples taken on the day of vaccination, and then at 14 days and 28 days after vaccination to allow immunogenicity assess ments to be made. Binding antibody titres were measured using standardised single dilution total IgG ELISAs as previously described.5 This assay was used to measure antibody responses before and after vaccination to Victoria/01/2020 SARS-CoV-2 spike protein and adapted to measure responses to beta (B.1.351) SARS-CoV-2 protein. Implications of all the available evidence Vaccine shortages have resulted in some people receiving a first dose of ChAdOx1 nCoV-19 without receiving the second dose within the recommended 4–12 week period. We report that increasing the interval up to 45 weeks results in increased antibody titres after the second dose, offering increased flexibility in vaccination schedules. A third dose at an extended interval after the second dose resulted in a further increase in antibody titres, mitigating concerns that antibodies raised against the ChAdOx1 vector would limit repeated use of the vaccine. 982 www.thelancet.com Vol 398 September 11, 2021 Articles April 19, 2020). The protocol for COV001 is provided in appendix 1 and the protocol for COV002 is provided in appendix 2. April 19, 2020). The protocol for COV001 is provided in appendix 1 and the protocol for COV002 is provided in appendix 2. second dose have been previously published.2,4,5 The trials were originally planned as single-dose vaccine studies, but the strong neutralising titres seen in COV001 induced by a second dose of vaccine4 prompted a protocol amendment to allow the addition of booster doses to most study participants across both trials. Most participants in both COV001 and COV002 were invited to receive a second dose from July, 2020, onwards. The timing of the second dose varied and allows for comparisons of immunogenicity between the recom mended vaccination schedule in the UK of 8–12 weeks and a longer interval of 15–25 weeks.3 The initial phase 1 immunogenicity group in COV001 was retained as a single-dose cohort to observe the persistence of immune responses after a single dose. See Online for appendix 1 See Online for appendix 2 www.thelancet.com Vol 398 September 11, 2021 See Online for appendix 1 See Online for appendix 2 Procedures ELISA assays to Victoria/01/2020 were performed on samples Figure 1: Trial profile for three-dose cohort 90 participants were enrolled and received their third dose of ChAdOx1 nCoV-19 80 participants assessable for reactogenicity 10 excluded because included in an open-label subgroup and interval between doses one and two was <28 days 75 assessable for antibody immunogenicity assessment 5 excluded because their prime-boost interval was outside of the deﬁned interval (8–16 weeks) 15 assessable for T-cell response immunogenicity assessments 60 excluded 56 no ELISPot data 4 insuﬃcient ELISpot data Some participants from the COV001 single-dose cohort were also offered a second dose at this time. The single dose cohort originally comprised a 1:1 ratio of ChAdOx1 nCoV-19 recipients to MenACWY controls. These participants were invited back in a 2:1 ratio, so participants who had previously received a single dose of ChAdOx1 nCoV-19 received their second dose, with an interval of 44–45 weeks, and those who had been controls received their first dose of ChAdOx1 nCoV-19. Participants were targeted for inclusion in the substudy if they had not previously been unmasked to treatment allocation or offered a vaccine as part of the UK Government COVID-19 vaccine programme. A subset of two-dose recipients (for whom reactogenicity or immunogenicity data, or both, were available) were selected for inclusion in analyses for comparison with those who received two doses 44–45 weeks apart. Participants who had a positive PCR test for SARS-CoV-2 were removed from the analysis if the infection occurred before the blood draw. Participants for this substudy were only enrolled at the Oxford site. In the UK, the COV001 and COV002 studies were approved by the South Central Berkshire Research Ethics Committee (COV001 reference 20/SC/0145, on March 23, 2020; COV002 reference 20/SC/0179; con ditional approval on April 8, 2020, and full approval on Figure 1: Trial profile for three-dose cohort www.thelancet.com Vol 398 September 11, 2021 983 Articles Articles Articles For the three-dose cohort, Figure 3: Antibody (A) and T-cell (B) persistence after one dose of ChAdOx1 nCoV-19 vaccine Figure 3: Antibody (A) and T-cell (B) persistence after one dose of ChAdOx1 nCoV-19 vaccine Datapoints represent individual participants and the solid line represents estimates from a linear regression model, with shaded areas showing the 95% CI. Antibody levels to SARS-CoV-2 Victoria/01/2020 spike measured by total IgG ELISA over 1 year after a single dose. Data are from 480 participants across COV001 and COV002 who received a standard dose of ChAdOx1 nCoV-19. Vaccine-induced T-cell responses against the SARS-CoV-2 spike insert were monitored up to day 182 in a cohort of 44 participants who received a single dose of ChAdOx1 nCoV-19. For participants who were excluded from these analyses due to positive PCR test result, second dose on trial, or external COVID-19 vaccination, no ELISA results or ELISpot results beyond the date of censoring were used. PBMCs=peripheral blood mononuclear cells. SFUs=spot-forming units Figure 3: Antibody (A) and T-cell (B) persistence after one dose of ChAdOx1 nCoV-19 vaccine Datapoints represent individual participants and the solid line represents estimates from a linear regression model, with shaded areas showing the 95% CI. Antibody levels to SARS-CoV-2 Victoria/01/2020 spike measured by total IgG ELISA over 1 year after a single dose. Data are from 480 participants across COV001 and COV002 who received a standard dose of ChAdOx1 nCoV-19. Vaccine-induced T-cell responses against the SARS-CoV-2 spike insert were monitored up to day 182 in a cohort of 44 participants who received a single dose of ChAdOx1 nCoV-19. For participants who were excluded from these analyses due to positive PCR test result, second dose on trial, or external COVID-19 vaccination, no ELISA results or ELISpot results beyond the date of censoring were used. PBMCs=peripheral blood mononuclear cells. SFUs=spot-forming units data were included only for those who had an interval of 8–16 weeks between first and second doses. See Online for appendix 3 Participants were asked to complete a diary card for 7 days after each vaccination to record solicited local (induration, itch, pain, redness, swelling, tenderness, and warmth at the injection site) and systemic (chills, fatigue, fever of ≥38°C, feverish [self-reported feeling of feverish ness, whereas fever is an objective fever measurement], headache, joint pain, malaise, muscle ache, and nausea) adverse reactions. Articles Mild Moderate Severe Potentially life-threatening or requiring admission to hospital Severity 0 25 50 75 100 Proportion (%) 0 25 50 75 100 Proportion (%) 8–12 15–25 44–45 8–12 15–25 44–45 8–12 15–25 44–45 8–12 15–25 44–45 8–12 15–25 44–45 8–12 15–25 44–45 8–12 15–25 44–45 Interval between ﬁrst and second doses of vaccine (weeks) Interval between ﬁrst and second doses of vaccine (weeks) A Induration Itch Pain Redness Swelling Tenderness Warmth Local adverse reactions 8–12 15–25 44–45 8–12 15–25 44–45 8–12 15–25 44–45 8–12 15–25 44–45 8–12 15–25 44–45 8–12 15–25 44–45 8–12 15–25 44–45 8–12 15–25 44–45 8–12 15–25 44–45 Chills Fever (≥38°C) Feverish Headache Joint pain Malaise Muscle ache Nausea Fatigue Systemic adverse reactions 0 25 50 75 100 Proportion (%) B Induration Itch Pain Redness Swelling Tenderness Warmth Local adverse reactions 0 25 50 75 100 Proportion (%) First dose Second dose Third dose First dose Second dose Third dose First dose Second dose Third dose First dose Second dose Third dose First dose Second dose Third dose First dose Second dose Third dose First dose Second dose Third dose First dose Second dose Third dose First dose Second dose Third dose First dose Second dose Third dose First dose Second dose Third dose First dose Second dose Third dose First dose Second dose Third dose First dose Second dose Third dose First dose Second dose Third dose First dose Second dose Third dose Chills Fever (≥38°C) Feverish Headache Joint pain Malaise Muscle ache Nausea Fatigue Systemic adverse reactions Mild Moderate Severe Potentially life-threatening or requiring admission to hospital Severity 0 25 50 75 100 Proportion (%) 8–12 15–25 44–45 8–12 15–25 44–45 8–12 15–25 44–45 8–12 15–25 44–45 8–12 15–25 44–45 8–12 15–25 44–45 8–12 15–25 44–45 Interval between ﬁrst and second doses of vaccine (weeks) A Induration Itch Pain Redness Swelling Tenderness Warmth Local adverse reactions Proportion (%) Proportion (%) 984 www.thelancet.com Vol 398 September 11, 2021 0 0 25 50 75 100 Proportion (%) 8–12 15–25 44–45 8–12 15–25 44–45 8–12 15–25 44–45 8–12 15–25 44–45 8–12 15–25 44–45 8–12 15–25 44–45 8–12 15–25 44–45 Interval between ﬁrst and second doses of vaccine (weeks) Interval between ﬁrst and second doses of vaccine (weeks) 8–12 15–25 44–45 8–12 15–25 44–45 8–12 15–25 44–45 8–12 15–25 44–45 8–12 15–25 44–45 8–12 15–25 44–45 8–12 15–25 44–45 8–12 15–25 44–45 8–12 15–25 44–45 Chills Fever (≥38°C) Feverish Headache Joint pain Malaise Muscle ache Nausea Fatigue Systemic adverse reactions 0 25 50 75 100 Proportion (%) B Induration Itch Pain Redness Swelling Tenderness Warmth Local adverse reactions 0 25 50 75 100 Proportion (%) First dose Second dose Third dose First dose Second dose Third dose First dose Second dose Third dose First dose Second dose Third dose First dose Second dose Third dose First dose Second dose Third dose First dose Second dose Third dose First dose Second dose Third dose First dose Second dose Third dose First dose Second dose Third dose First dose Second dose Third dose First dose Second dose Third dose First dose Second dose Third dose First dose Second dose Third dose First dose Second dose Third dose First dose Second dose Third dose Chills Fever (≥38°C) Feverish Headache Joint pain Malaise Muscle ache Nausea Fatigue Systemic adverse reactions 0 25 50 75 100 Proportion (%) Interval between ﬁrst and second doses of vaccine (weeks) 8–12 15–25 44–45 8–12 15–25 44–45 8–12 15–25 44–45 8–12 15–25 44–45 8–12 15–25 44–45 8–12 15–25 44–45 8–12 15–25 44–45 8–12 15–25 44–45 8–12 15–25 44–45 Chills Fever (≥38°C) Feverish Headache Joint pain Malaise Muscle ache Nausea Fatigue Systemic adverse reactions B Proportion (%) Proportion (%) B 0 25 50 75 100 Proportion (%) B Induration Itch Pain Redness Swelling Tenderness Warmth Local adverse reactions First dose Second dose Third dose First dose Second dose Third dose First dose Second dose Third dose First dose Second dose Third dose First dose Second dose Third dose First dose Second dose Third dose First dose Second dose Third dose Systemic adverse reactions Proportion (%) Proportion (%) Systemic adverse reactions 0 25 50 75 100 Proportion (%) First dose Second dose Third dose First dose Second dose Third dose First dose Second dose Third dose First dose Second dose Third dose First dose Second dose Third dose First dose Second dose Third dose First dose Second dose Third dose First dose Second dose Third dose First dose Second dose Third dose Chills Fever (≥38°C) Feverish Headache Joint pain Malaise Muscle ache Nausea Fatigue Proportion (%) Proportion (%) www.thelancet.com Vol 398 September 11, 2021 984 Articles Figure 3: Antibody (A) and T-cell (B) persistence after one dose of ChAdOx1 nCoV-19 vaccine Datapoints represent individual participants and the solid line represents estimates from a linear regression model, with shaded areas showing the 95% CI. Articles Neutralisation assays were done in a randomly selected subset of participants (due to laboratory capacity) who received a third dose of vaccine. Timepoints assessed were 28 days after second vaccination and 28 days after third vaccination. from single-dose recipients up to 1 year after vaccination, in two-dose recipients up to 6 months after the second vaccination, and in three-dose recipients up to 28 days after the third vaccination. ELISA assays on the beta SARS-CoV-2 variant were only done on samples from participants recruited to the substudy who received either a late second vaccination or a third vaccination, up to 28 days after the late vaccination. Meso Scale Discovery multiplex immunoassay was used to assess antibody titres against spike proteins from different variants (Victoria/01/2020, D614G, alpha [B.1.17], beta, and gamma [P.1]). V-PLEX SARS-CoV-2 Panel 6 (IgG) kits were used following manufacturer’s instructions (Meso Scale Discovery, K15433U; full details are in appendix 3 [p 1]). Meso Scale Discovery assays were done on samples from participants recruited to the substudy who received a late second dose of vaccine (44–45 weeks after the first dose. Ex-vivo IFN-γ ELISpot assays were done as previously described5 to assess T-cell responses to Victoria/01/2020 SARS-CoV-2 spike overlapping peptide pools before and after vaccination. Isolated peripheral blood mononuclear cells (PBMCs) were stimulated overnight with peptides spanning the SARS-CoV-2 spike insert. ELISpot assays were done on samples from participants in the single dose cohort up to 182 days after vaccination. ELISpot assays were also done in some participants (due to laboratory capacity) recruited to the substudy who received a third dose, up to 28 days after third dose. Focus reduction neutralisation assays were done as described previously6 to measure neutralising antibody titres against alpha, beta, and delta (B.1.617.2) SARS-CoV-2 viral variants. Neutralisation assays were done in a randomly selected subset of participants (due to laboratory capacity) who received a third dose of vaccine. Timepoints assessed were 28 days after second vaccination and 28 days after third vaccination. A 1 10 100 1000 10000 0 50 100 150 200 250 300 350 ELISA units B Time since ﬁrst dose of vaccine (days) 10 100 1000 10000 0 50 100 150 200 SFU per million PBMCs For all immunogenicity assessments, data were excluded upon earliest occurrence of a positive PCR test result or external COVID-19 vaccination. For single dose immunogenicity assessments, data were excluded from after receipt of second dose. Figure 2: Solicited adverse reactions up to 7 days after ChAdOx1 nCoV-19 vaccination by interval between first and second doses (A) and after the first, second, and third dose for participants who received a third dose of vaccine (B) Figure shows maximum severity of respective solicited adverse event recorded for each participant during days 0–7 after vaccination. In panel A, reactogenicity data after the second dose are shown for 263 participants for fever (≥38°C) and 267 participants for all other symptoms for the 8–12 week interval, for 23 participants for fever (≥38°C) and 24 participants for all other symptoms for the 15–25 week interval, and 28 participants for fever (≥38°C) and 30 participants for all other symptoms for the 44–45 week interval. In panel B, reactogenicity data are from after each dose recorded by participants who received a third dose of vaccine, with data available for 80 participants for all symptoms after dose 1; 15 participants for all symptoms after dose 2; and 77 participants for fever and 80 participants for all symptoms after dose 3. Participants included in panel B received their third dose 20–38 weeks after the second dose (median of 30 weeks [IQR 30–30]). www.thelancet.com Vol 398 September 11, 2021 Articles Antibody levels to SARS-CoV-2 Victoria/01/2020 spike measured by total IgG ELISA over 1 year after a single dose. Data are from 480 participants across COV001 and COV002 who received a standard dose of ChAdOx1 nCoV-19. Vaccine-induced T-cell responses against the SARS-CoV-2 spike insert were monitored up to day 182 in a cohort of 44 participants who received a single dose of ChAdOx1 nCoV-19. For participants who were excluded from these analyses due to positive PCR test result, second dose on trial, or external COVID-19 vaccination, no ELISA results or ELISpot results beyond the date of censoring were used. PBMCs=peripheral blood mononuclear cells. SFUs=spot-forming units A B 1 10 100 1000 10000 0 50 100 150 200 250 300 350 ELISA units Time since ﬁrst dose of vaccine (days) 10 100 1000 10000 0 50 100 150 200 SFU per million PBMCs from single-dose recipients up to 1 year after vaccination, in two-dose recipients up to 6 months after the second vaccination, and in three-dose recipients up to 28 days after the third vaccination. ELISA assays on the beta SARS-CoV-2 variant were only done on samples from participants recruited to the substudy who received either a late second vaccination or a third vaccination, up to 28 days after the late vaccination. Meso Scale Discovery multiplex immunoassay was used to assess antibody titres against spike proteins from different variants (Victoria/01/2020, D614G, alpha [B.1.17], beta, and gamma [P.1]). V-PLEX SARS-CoV-2 Panel 6 (IgG) kits were used following manufacturer’s instructions (Meso Scale Discovery, K15433U; full details are in appendix 3 [p 1]). Meso Scale Discovery assays were done on samples from participants recruited to the substudy who received a late second dose of vaccine (44–45 weeks after the first dose. Ex-vivo IFN-γ ELISpot assays were done as previously described5 to assess T-cell responses to Victoria/01/2020 SARS-CoV-2 spike overlapping peptide pools before and after vaccination. Isolated peripheral blood mononuclear cells (PBMCs) were stimulated overnight with peptides spanning the SARS-CoV-2 spike insert. ELISpot assays were done on samples from participants in the single dose cohort up to 182 days after vaccination. ELISpot assays were also done in some participants (due to laboratory capacity) recruited to the substudy who received a third dose, up to 28 days after third dose. Focus reduction neutralisation assays were done as described previously6 to measure neutralising antibody titres against alpha, beta, and delta (B.1.617.2) SARS-CoV-2 viral variants. See Online for appendix 3 Articles between cohorts, participants were excluded from the two-dose reactogenicity cohort if the interval between first and second dose was outside of the intervals defined in the two-dose immunogenicity cohort. All analyses of COV002 participants were restricted to those aged 18–55 years, to align with the inclusion criteria of the COV001 study, which only enrolled participants in this age range. Figure 4: Antibody response by interval between first and second vaccination Datapoints are medians for each group, with error bars showing IQRs. Antibody levels to SARS-CoV-2 Victoria/01/2020 spike measured by total IgG ELISA. Data are shown for 115 participants for the 8–12 week interval; 116 participants for the 15–25 week interval, and 30 participants for the 44–45 week interval. Unadjusted and age-adjusted geometric mean ratios are shown in appendix 3 (p 16). 1 10 100 1000 10 000 −300 −250 −200 −150 −100 −50 0 50 100 150 200 Total IgG ELISA units Time since second dose of vaccine (days) 8–12 weeks 15–25 weeks 44–45 weeks Vaccination interval 1 10 100 1000 10 000 −300 −250 −200 −150 −100 −50 0 50 100 150 200 Total IgG ELISA units Time since second dose of vaccine (days) 8–12 weeks 15–25 weeks 44–45 weeks Vaccination interval Total IgG ELISA units Our analysis of the decay of antibodies and T-cell responses over time after a single dose included all available data from timepoints up until the booster dose was administered (ie, day 28 and the day of the second dose, which varied across all participants). We modelled data using an unadjusted restricted-maximum likelihood-based mixed-effects regression approach (SAS proc mixed) with participant-level random inter cepts fitted to log-transformed antibody values. We used the variance components covariance structure. We estimated GMRs and GMTs from the linear combination of model parameters. We chose the linear models after comparison with quadratic models and generalised additive (smoothed) models (GAM). The quadratic term was non-significant in the linear models and the GAM results were similar and did not substantially improve the model fits (compared using Akaike information criterion [known as AIC] statistics) from the linear models; therefore, the linear models were retained. Figure 4: Antibody response by interval between first and second vaccination Datapoints are medians for each group, with error bars showing IQRs. Antibody levels to SARS-CoV-2 Victoria/01/2020 spike measured by total IgG ELISA. Role of the funding source AstraZeneca reviewed the manuscript before submission, but the academic authors retained editorial control. All other funders of the study had no role in the study design, data collection, data analysis, data interpretation, or writing of the report. We used the Wilcoxon rank sum and Kruskal-Wallis tests for comparisons between independent groups and we used the Wilcoxon sign rank test to compare paired data. Geometric mean ratios (GMRs) with 95% CIs were produced when comparing groups. When appropriate, adjusted GMTs and GMRs were also presented to adjust for the effect of age. We did not do a sample size calculation for the immunogenicity subgroups in this analysis because of logistical considerations, including laboratory capacity. Sample size calculations for COV001 and COV002 were based on the primary efficacy outcome, which have been previously reported. Statistical analysis We present summary statistics for individuals vaccinated with one, two, or three doses of ChAdOx1 nCoV-19 as median (IQR) or geometric mean titre (GMT) with 95% CIs. We do not include data from control participants (who had previously received one or two doses of MenACWY); they received ChAdOx1 nCoV-19 on recruitment to the substudy. For the purposes of ensuring trial personnel were masked to treatment assignment, data for both vaccinnees and controls were collected. Unmasking information was only available to those performing the final data analyses. Upon unmasking of participants and study personnel, control participants were excluded. We did all statistical analyses using R (version 4.0.2 or later) and SAS (version 9.2). p values of less than 0·05 were considered to be significant and we made no adjustments for multiple comparisons. COV001 is registered with ClinicalTrials.gov, NCT04324606, and ISRCTN, 15281137, and COV002 is registered with ClinicalTrials.gov, NCT04400838, and ISRCTN, 15281137, and both are no longer recruiting. Articles Data are shown for 115 participants for the 8–12 week interval; 116 participants for the 15–25 week interval, and 30 participants for the 44–45 week interval. Unadjusted and age-adjusted geometric mean ratios are shown in appendix 3 (p 16). e 4: Antibody response by interval between first and second vaccination www.thelancet.com Vol 398 September 11, 2021 Articles Participants reported the severity of their adverse reactions as mild, moderate, severe, or life threaten ing as per definitions provided (appendix 1 pp 92–94). 985 www.thelancet.com Vol 398 September 11, 2021 Articles Articles Results pp p ) Baseline characteristics for the one-dose, two-dose, and three-dose cohorts are shown in appendix 3 (pp 4–5). More than 90% of participants were White. There were small differences in the median age of reactogenicity cohorts and immunogenicity cohorts. The median age of participants in the two-dose cohort antibody immunogenicity cohort with an 8–12 week interval between the first and second dose was 39 years (IQR 30–49), in the 15–25 weeks interval group was 36 years (30–43), and in the 44–45 weeks interval group was 32 years (25–44). In the three-dose cohort, the median age of participants in the reactogenicity cohort was 37 years (IQR 31–42), in the immunogenicity antibody cohort was 37 years (31–42), and in the immunogenicity T-cell response cohort 40 years (32–44). h f l l d l d d Baseline characteristics for the one-dose, two-dose, and three-dose cohorts are shown in appendix 3 (pp 4–5). More than 90% of participants were White. There were small differences in the median age of reactogenicity cohorts and immunogenicity cohorts. The median age of participants in the two-dose cohort antibody immunogenicity cohort with an 8–12 week interval between the first and second dose was 39 years (IQR 30–49), in the 15–25 weeks interval group was 36 years (30–43), and in the 44–45 weeks interval group was 32 years (25–44). In the three-dose cohort, the median age of participants in the reactogenicity cohort was 37 years (IQR 31–42), in the immunogenicity antibody cohort was 37 years (31–42), and in the immunogenicity T-cell response cohort 40 years (32–44). The severity of local and systemic solicited adverse reactions 7 days after a second dose were mostly mild to moderate irrespective of the interval between doses. Local symptoms occurred after a second dose in 201 (75%) of 267 participants in the 8–12 week interval group, 15 (63%) of 24 participants in the 15–25 week interval group, and 23 (77%) of 30 participants in the 44–45 wee interval Figure 5: Antibody responses in participants who received a third dose of ChAdOx1 nCoV-19 (A) Antibody levels to SARS-CoV-2 Victoria/01/2020 spike protein measured by total IgG ELISA (n=75). Datapoints in lighter colours represent individual participants and darker datapoints show median values with error bars showing the IQRs and with solid lines connecting these median values. Results Between March 11 and 21, 2021, 90 participants were enrolled into the COV001 third-dose boost substudy and vaccinated with ChAdOx1 nCoV-19. Ten participants from this substudy were excluded from analyses because they were enrolled from an open-label subgroup and the interval between their first and second doses was shorter than 28 days. The remaining 80 participants who were assessable for reactogenicity received their third dose of vaccine 20–38 weeks after their second dose (median 30 weeks [IQR 30–30]). A further five participants were excluded from all immunogenicity assessments because their prime-boost interval was outside the defined range of 8–16 weeks. For The reactogenicity cohorts included masked participants who received at least two standard doses of ChAdOx1 nCoV-19 in the two-dose cohort or three standard doses of ChAdOx1 nCoV-19 in the three-dose cohort, and had completed at least one entry in their adverse event diary after each dose. For consistency 986 www.thelancet.com Vol 398 September 11, 2021 Articles Figure 5: Antibody responses in participants who received a third dose of ChAdOx1 nCoV-19 (A) Antibody levels to SARS-CoV-2 Victoria/01/2020 spike protein measured by total IgG ELISA (n=75). Datapoints in lighter colours represent individual participants and darker datapoints show median values with error bars showing the IQRs and with solid lines connecting these median values. (B) Neutralisation titres from a randomly selected subset of participants (45 of 75 participants who received a third dose of vaccine and who had an interval of 8–16 weeks between their first and second dose). Datapoints represent individual participants for the three variants of concern investigated. FRNT50=focus reduction neutralisation titres with 50% neutralisation cutoff. 1 10 100 1000 10000 First dose 28 days after ﬁrst dose Second dose 28 days after second dose Third dose 14 days after third dose 28 days after third dose Timepoint Total IgG ELISA units 28 days after second dose 28 days after third dose 100 101 102 103 28 days after second dose 28 days after third dose 28 days after second dose 28 days after third dose 104 FRNT50 (reciprocal serum dilution) B A Timepoint Timepoint Timepoint Alpha (B.1.1.7) variant Beta (B.1.351) variant Delta (B.1.617.2) variant immunogenicity analysis of T-cell responses, another 60 participants were excluded because they did not have or had insufficient ELISpot data, leaving an analysable cohort of 15 participants (figure 1). www.thelancet.com Vol 398 September 11, 2021 Results To maintain blinding at the time of vaccination, 40 control participants (who had previously received two doses of MenACWY) were also recruited and vaccinated with ChAdOx1 nCoV-19. Data for these participants have not been included in these analyses. 1 10 100 1000 10000 First dose 28 days after ﬁrst dose Second dose 28 days after second dose Third dose 14 days after third dose 28 days after third dose Timepoint Total IgG ELISA units A Of 1110 participants from the COV001 and COV002 studies who had received a single dose of vaccine, 480 were included in the single-dose immunogenicity assessment of antibodies and 44 were included in the immunogenicity assessment of T-cell responses (appendix 3 p 2). 66 participants from the single-dose cohort of COV001 were also offered a second dose, of whom 44 were ChAdOx1 nCoV-19 recipients and 22 were control group participants who had received MenACWY. 32 (73%) of 44 participants who had previously received a single dose of ChAdOx1 nCoV-19 received their second dose, with an interval of 44–45 weeks. Two of these participants were subsequently excluded from reacto genicity and immunogenicity analyses because they had positive PCR tests for SARS-CoV-2 infection within the follow-up period, leaving 30 participants for inclusion in the two-dose cohort analyses. 28 days after second dose 28 days after third dose 100 101 102 103 28 days after second dose 28 days after third dose 28 days after second dose 28 days after third dose 104 FRNT50 (reciprocal serum dilution) B Timepoint Timepoint Timepoint Alpha (B.1.1.7) variant Beta (B.1.351) variant Delta (B.1.617.2) variant B 28 days after second dose 28 days after third dose 100 101 102 103 104 FRNT50 (reciprocal serum dilution) B Timepoint Alpha (B.1.1.7) variant 28 days after second dose 28 days after third dose Timepoint Beta (B.1.351) variant 28 days after second dose 28 days after third dose Timepoint Delta (B.1.617.2) variant Alpha (B.1.1.7) variant FRNT50 (reciprocal serum dilution) y The two-dose cohort comprised 321 participants from COV001 and COV002 with prime-boost intervals of 8–12 weeks (267 [83%] of 321), 15–25 weeks (24 [7%]), or 44–45 weeks (30 [9%]) who had reactogenicity data available and were included in our analyses, and 261 who had immunogenicity data available (115 [44%] of 261 had an 8–12 week interval, 116 [44%] had a 15–25 week interval, and 30 [11%] had a 44–45 week interval; appendix 3 p 3). Articles Figure 6: IFN-γ ELISpot responses in participants who received a third dose of ChAdOx1 nCoV-19 15 participants with an interval of 8 weeks between their first and second doses were assessed for ELISpot responses. These participants received their third dose 37–38 weeks after the second dose (median 38 weeks [IQR 38–38]). Datapoints in lighter colours represent individual participants and darker datapoints show median values with error bars showing the IQRs and with solid lines connecting these median values. The dotted horizontal line represents the lower limit of detection of the assay (48 SFU per million PBMCs). SFU=spot-forming unit. PBMC=peripheral blood mononuclear cells. 100 1000 10000 14 days after second dose 28 days after second dose Third dose 14 days after third dose 28 days after third dose Timepoint SFU per million PBMCs 6 months after the second dose of vaccine, antibody levels remained significantly higher in the group with a 15–25 week interval between doses compared with an 8–12 week interval (median 1240 EUs [IQR 432–2002] vs 278 EUs [166–499]; Wilcoxon rank sum test with con tinuity correction p<0·0001; figure 4; appendix 3 p 16). 100 1000 10000 14 days after second dose 28 days after second dose Third dose 14 days after third dose 28 days after third dose Timepoint SFU per million PBMCs y pi g pp p ) IgG binding titres to all four variants tested (D614G, alpha, beta, and gamma) were significantly greater after second dose than after the first dose (p<0·0001 for all comparisons [pairwise comparisons using Wilcoxon sign rank test]; appendix 3 pp 17, 19). Antibody responses after a third dose of vaccine were assessed in 75 participants who had received their first two doses with an interval of 8–16 weeks, and who subsequently received their third dose 28–38 weeks after the second (median 30 weeks [IQR 30–30]). Administering a third dose of vaccine boosted antibody response to Victoria/01/2020 SARS-CoV-2 spike protein (figure 5A; appendix 3 p 18). Antibody levels after the third dose were significantly higher than after the second dose (median total IgG titre was 1792 EUs [IQR 899–4634] at 28 days after the second dose vs 3746 EUs [2047–6420] 28 days after the third dose; pairwise comparison in 73 participants due to two samples not being available at these timepoints using Wilcoxon signed rank test p=0·0043). Articles Binding antibody titres to the beta variant increased after a third dose (appendix 3 p 19). Neutralising antibody titres after a third dose were higher than those after the second dose against alpha (p=0·0023), beta (p<0·0001), and delta (p<0·0001) variants (Wilcoxon signed rank test; figure 5B; appendix 3 p 20). Figure 6: IFN-γ ELISpot responses in participants who received a third dose of ChAdOx1 nCoV-19 15 participants with an interval of 8 weeks between their first and second doses were assessed for ELISpot responses. These participants received their third dose 37–38 weeks after the second dose (median 38 weeks [IQR 38–38]). Datapoints in lighter colours represent individual participants and darker datapoints show median values with error bars showing the IQRs and with solid lines connecting these median values. The dotted horizontal line represents the lower limit of detection of the assay (48 SFU per million PBMCs). SFU=spot-forming unit. PBMC=peripheral blood mononuclear cells. vaccination compared with 21 (7%) of 321 participants after second vaccinations (appendix 3 pp 6–9). Third dose vaccinations were also less reactogenic than first doses, with four (5%) of 80 participants in the three-dose cohort reporting more than two moderate-to-severe systemic symptoms after a third dose compared with 27 (34%) of 80 participants after the first dose (appendix 3 pp 10–14). Spike-specific cellular immune responses were measured after a third dose of ChAdOx1 nCoV-19 in 15 individuals. These individuals had received their first two doses with an interval of 8 weeks, and subsequently received their third dose 37–38 weeks after the second (median 38 weeks [IQR 38–38]). Median response increased from 200 spot-forming units (SFUs) per million PBMCs (IQR 127–389) immediately before the third dose to 264 SFUs per million PBMCs (131–452) 14 days after the third dose (p=0·57), and to 399 SFUs per million PBMCs (314–662) by 28 days after the third dose (p=0·012; figure 6; appendix 3 p 20). Peak responses at day 28 after the third dose were not significantly different to the responses after the second dose (p=0·060; appendix 3 p 20). Antibody responses after a single dose of vaccine and measured approximately 320 days after vaccination remained higher than responses measured at baseline (GMTs of 66·00 ELISA units [EUs; 95% CI 47·83–91·08 vs 1·75 EUs [1·60–1·93]). Articles At day 180, geometric mean antibody levels were half the levels observed at the day 28 peak (GMR 0·51 [95% CI 0·45–0·57]), and by day 320 were less than a third of the levels at the peak (GMR 0·27 [0·22–0·34; figure 3A; appendix 3 p 15). Vaccine-induced cellular immune responses after a single dose of ChAdOx1 nCoV-19 followed a similar pattern of decay as antibody responses. T-cell responses decreased over the course of 6 months but were maintained above baseline levels, and at day 180 geometric mean T-cell levels were half the levels observed at the day 28 peak (GMR 0·50 [95% CI 0·41–0·60]; figure 3B; appendix 3 p 15). www.thelancet.com Vol 398 September 11, 2021 Results (B) Neutralisation titres from a randomly selected subset of participants (45 of 75 participants who received a third dose of vaccine and who had an interval of 8–16 weeks between their first and second dose). Datapoints represent individual participants for the three variants of concern investigated. FRNT50=focus reduction neutralisation titres with 50% neutralisation cutoff. group (figure 2A; appendix 3 pp 6–9). Systemic reactions occurred in 190 (71%) of 267 participants in the 8–12 week interval group, 18 (75%) of 24 participants in the 15–25 week interval group, and 26 (87%) of 30 participants in the 44–45 week interval group (figure 2A; appendix 3 pp 6–9). 65 (81%) of 80 participants in the three-dose group reported at least one local symptom after a third dose (figure 2B; appendix 3 pp 10–14). Second dose vaccinations in the two-dose cohort were less reactogenic than first dose vaccinations; with 72 (22%) of 321 participants reporting more than two moderate-to-severe systemic symptoms after first 987 Articles Articles Discussion Antibody levels induced by a single dose of ChAdOx1 nCoV-19 decreased gradually but remained above baseline levels after 1 year. We have previously shown that administration of a second dose of vaccine induces higher antibody responses by 1 month after the second dose than before the second dose, with higher responses with a dose interval up to 3 months between the first two doses.3 Here, we found that a long extension of the dose interval (up to 45 weeks) between the first and second dose further Antibody levels 28 days after a second dose of vaccine were higher among those with longer intervals between doses than among those with shorter intervals between doses (median total IgG titre of 923 EUs [IQR 525–1764] for 8–12 week interval; 1860 EUs [917–4934] with 15–25 week interval; and 3738 EUs [1824–6625] with 44–45 week interval; Kruskal-Wallis test p<0·0001; figure 4; appendix 3 p 16). Age was not statistically significant in adjusted models (appendix 3 p 16). 988 Articles enhances the immune response to the second dose when compared with shorter dose intervals. Furthermore, for the first time, we showed that a third dose of ChAdOx1 nCoV-19 can induce a strong boost to immune responses to the transgene product, SARS-CoV-2 spike protein, and that these responses result in increased neutralising antibody titres and enhanced antibody activity against variants. shown to dampen vaccine-induced immune responses,12,13 here we found no evidence that repeated use of a replication-deficient simian adenoviral vector induces antivector immunity at a level sufficient to impair responses to further vaccination. The third dose was well tolerated by participants with lower reactogenicity than after the first dose. i Our study has several limitations, including a paucity of T-cell data after a late second dose, a paucity of tolerability data after the second dose for those who were recruited to receive a third dose, and the small number of participants who were available at 1 year after single dose who still had only received one dose (mainly due to being offered a second dose after unblinding, as per protocol). Thus far, data are only available 28 days after the third dose; however, follow-up at 6 months is planned. Participants were aged 18–55 years and caution should be taken when extrapolating our findings to beyond this age range. Discussion The sample size in this study is not sufficiently large to assess rare vaccine side-effects, but there were no tolerability concerns reported in those receiving a late second dose or a third dose booster. These results are from a mainly White population and cannot necessarily be generalised to other populations. The devastating impact of COVID-19 is most apparent in countries with low vaccine coverage and little health- care infrastructure, including low-income and middle- income countries. Global vaccine shortages and policy decisions implemented at national levels have curtailed vaccine supplies for some countries where substantial numbers of individuals have already received one dose of vaccine. We have previously shown that protection against symptomatic COVID-19 is maintained after a single dose of ChAdOx1 nCoV-19 for at least 3 months, despite some waning of antibody levels3 and we now report that the antibody levels remain above baseline for at least 1 year after single dose immunisation. These data are important for those countries where administration of a second dose is delayed because of a shortage of supply. We also showed here that an extended interval between the first and second dose of ChAdOx1 nCoV-19 results in a significantly higher antibody response 28 days after the second dose than with shorter intervals. This finding is consistent with previous data showing a longer interval between first and second dose of ChAdOx1 nCoV-19 resulted in an increase in antibody titres,3 thus providing further reassurance that a delay in administration of the second dose will not compromise the level of protection attained. Similar findings have been reported with other vaccines;1,7,8 a delayed two-dose regimen against HPV, given at least 6 months apart, results in as good or better antibody response than does three doses. A second dose of ChAdOx1 nCoV-19 is well tolerated in a delayed two-dose schedule and a third dose is also well tolerated. Reports have emerged of thrombosis and thrombocytopenia after the first dose of ChAdOx1 nCoV-19,9 and information from Public Health England indicates that this very rare event might not occur after a second dose.10 Here, we found that immunity induced by the viral vectored vaccine ChAdOx1 nCoV-19 is maintained for long periods after a first dose, with greater boosting of effects after the second dose after a longer interval between doses than shorter intervals. Contributors Contributors AJP, TL, MV, and DJ contributed to the writing and design of the protocol and design of the study. AJP is chief investigator. AF, JA, PKA, BA, SB-R, SB, MB, FC, PC, EAC, SD, WD, CD, KJE, PMF, JF, DJ, AMM, JM, YFM, EP, MNR, HR, HSa, ES, HSm, MDS, RS, and DW contributed to implementation of the study or laboratory experimentation, or both. AF, MV, and NGM accessed and verified the underlying study data and did the statistical analysis. TL, AF, AJP, SCG, FC, NGM, and MV contributed to the preparation of the report. TL, AF, AJP, SCG, and MV were responsible for the decision to submit the manuscript. All authors critically reviewed and approved the final version. All authors had full access to the data in the study and had final responsibility for the decision to submit for publication. g If booster vaccinations against SARS-CoV-2 will be required, perhaps to counter waning immunity or to augment protection against emerging variants, is not yet known. Here, we show that a third dose of ChAdOx1 nCoV-19 is well tolerated and significantly boosts antibody titres above those measured after the second dose to the level associated with 80% vaccine efficacy, or higher, after two vaccine doses (unpublished; preprint data available11). Higher titre neutralising antibodies against alpha, beta, and delta variants of SARS-CoV-2 were induced 28 days after a third dose vaccination than after the second dose. Spike-specific T-cell responses were boosted after a third dose of ChAdOx1 nCoV-19 and were similar in magnitude to the responses measured after two doses. Although pre- existing immunity to human adenoviral vectors has been Discussion Therefore, a single dose of ChAdOx1 nCoV-19 with a second dose given after an extended period might be an effective strategy3,14 in settings where vaccine supplies are scarce in the short term. A third dose resulted in a further increase in immune responses, including increased neutralisation of variant SARS-CoV-2 viruses, and could be used to increase vaccine efficacy against variants in susceptible populations. www.thelancet.com Vol 398 September 11, 2021 Contributors AJP, TL, MV, and DJ contributed to the writing and design of the protocol and design of the study. AJP is chief investigator. AF, JA, PKA, BA, SB-R, SB, MB, FC, PC, EAC, SD, WD, CD, KJE, PMF, JF, DJ, AMM, JM, YFM, EP, MNR, HR, HSa, ES, HSm, MDS, RS, and DW contributed to implementation of the study or laboratory experimentation, or both. AF, MV, and NGM accessed and verified the underlying study data and did the statistical analysis. TL, AF, AJP, SCG, FC, NGM, and MV contributed to the preparation of the report. TL, AF, AJP, SCG, and MV were responsible for the decision to submit the manuscript. All authors critically reviewed and approved the final version. All authors had full access to the data in the study and had final responsibility for the decision to submit for publication. References 1 WHO. Interim recommendations for use of the AZD1222 (ChAdOx1-S [recombinant]) vaccine against COVID19 developed by Oxford University and AstraZeneca. Geneva: World Health Organization, Feb 10, 2021. https://apps.who.int/iris/bitstream/ handle/10665/339477/WHO-2019-nCoV-vaccines-SAGE- recommendation-AZD1222-2021.1-eng.pdf?sequence=5&isAllowed=y (accessed June 23, 2021). Acknowledgments 5 Ramasamy MN, Minassian AM, Ewer KJ, et al. Safety and immunogenicity of ChAdOx1 nCoV-19 vaccine administered in a prime-boost regimen in young and old adults (COV002): a single- blind, randomised, controlled, phase 2/3 trial. Lancet 2021; 396: 1979–93. Acknowledgments Oxford University has entered into a partnership with AstraZeneca for further development of ChAdOx1 nCoV-19. We acknowledge funding for this study from the UK Research and Innovation (MC_PC_19055); Engineering and Physical Sciences Research Council (EP/R013756/1); National Institute for Health Research (COV19 OxfordVacc-01); Coalition for Epidemic Preparedness Innovations (Outbreak Response To Novel Coronavirus [COVID-19]); NIHR Oxford Biomedical Research Centre (BRC4 Vaccines Theme); The Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Science (CIFMS), China (2018-I2M-2-002; to AF and TL); Thames Valley and South Midlands NIHR Clinical Research Network; AstraZeneca; and the Wellcome Trust (220991/Z/20/Z). The views expressed in this publication are those of the authors and not necessarily those of the NIHR or the UK Department of Health and Social Care. Development of SARS-CoV-2 reagents was partially supported by the National Institute of Allergy and Infectious Diseases Centers of Excellence for Influenza Research and Surveillance (CEIRS) contract HHSN272201400008C. We thank all participants in the trial and the international Data and Safety Monitoring Board (DSMB) and the independent members of the Trial Steering Committee for their invaluable advice. We also thank the various teams within the University of Oxford including Medical Sciences Division, Nuffield Department of Medicine and Department of Paediatrics, the Oxford Immunology Network COVID Consortium, Clinical Trials Research Governance, Research Contracts, Public Affairs Directorate, and the Clinical Biomanufacturing Facility, as well as the Oxford University Hospitals NHS Foundation Trust, and Oxford Health NHS Foundation Trust and the trial sites. Named individuals we acknowledge are listed in appendix 3 (p 25). We are grateful for the input of the Protein Production Team at the Jenner Institute and the team at the Pirbright Institute. We acknowledge the philanthropic support of the donors to the University of Oxford’s COVID-19 Research Response Fund. 6 Zhou D, Dejnirattisai W, Supasa P, et al. Evidence of escape of SARS-CoV-2 variant B.1.351 from natural and vaccine-induced sera. Cell 2021; 184: 2348–61. 7 Baden LR, Karita E, Mutua G, et al. Assessment of the safety and immunogenicity of 2 novel vaccine platforms for HIV-1 prevention: a randomized trial. Ann Intern Med 2016; 164: 313–22. 8 Dobson SR, McNeil S, Dionne M, et al. Data sharing The current study protocol for COV001 is provided in appendix 1 and for COV002 in appendix 2. Anonymised participant data will be made available when the trial is complete, upon requests directed to the corresponding author. Proposals will be reviewed and approved by the sponsor, investigator, and collaborators on the basis of scientific merit. After approval of a proposal, data can be shared through a secure online platform after signing a data access agreement. All data will be made available for a minimum of 5 years from the end of the trial. 3 Voysey M, Costa Clemens SA, Madhi SA, et al. Single-dose administration and the influence of the timing of the booster dose on immunogenicity and efficacy of ChAdOx1 nCoV-19 (AZD1222) vaccine: a pooled analysis of four randomised trials. Lancet 2021; 397: 881–91. 4 Folegatti PM, Ewer KJ, Aley PK, et al. Safety and immunogenicity of the ChAdOx1 nCoV-19 vaccine against SARS-CoV-2: a preliminary report of a phase 1/2, single-blind, randomised controlled trial. Lancet 2020; 396: 467–78. Articles 2 Advisory Group of Experts (SAGE). AJP is an NIHR Senior Investigator. All other authors declare no competing interests. Advisory Group of Experts (SAGE). AJP is an NIHR Senior Investigator. All other authors declare no competing interests. 2 Voysey M, Clemens SAC, Madhi SA, et al. Safety and efficacy of the ChAdOx1 nCoV-19 vaccine (AZD1222) against SARS-CoV-2: an interim analysis of four randomised controlled trials in Brazil, South Africa, and the UK. Lancet 2021; 397: 99–111. Acknowledgments Immunogenicity of 2 doses of HPV vaccine in younger adolescents vs 3 doses in young women: a randomized clinical trial. JAMA 2013; 309: 1793–802. 9 Schultz NH, Sørvoll IH, Michelsen AE, et al. Thrombosis and thrombocytopenia after ChAdOx1 nCoV-19 vaccination. N Engl J Med 2021; 384: 2124–30. 10 Public Health England. Vaccine update: issue 322, June 2021, COVID-19 phase 2 special edition. Rare syndrome related to AstraZeneca vaccine. UK Government, June 17, 2021. https://www. gov.uk/government/publications/vaccine-update-issue-322-june- 2021-covid-19-phase-2-special-edition/vaccine-update-issue-322- june-2021-covid-19-phase-2-special-edition (accessed June 24, 2021). 11 Feng S, Phillips DJ, White T, et al Correlates of protection against symptomatic and asymptomatic SARS-CoV-2 infection. medRxiv 2021; published online June 24. https://doi.org/10.1101/ 2021.06.21.21258528 (preprint). 12 Zhu FC, Guan XH, Li YH, et al. Immunogenicity and safety of a recombinant adenovirus type-5-vectored COVID-19 vaccine in healthy adults aged 18 years or older: a randomised, double-blind, placebo-controlled, phase 2 trial. Lancet 2020; 396: 479–88. 13 Roberts DM, Nanda A, Havenga MJ, et al. Hexon-chimaeric adenovirus serotype 5 vectors circumvent pre-existing anti-vector immunity. Nature 2006; 441: 239–43. 14 14 Public Health England. SARS-CoV_2 variants of concern and variants under investigation in England: technical briefing 16. UK Government, June 18, 2021. https://assets.publishing.service. gov.uk/government/uploads/system/uploads/attachment_data/ file/994839/Variants_of_Concern_VOC_Technical_Briefing_16.pdf (accessed June 23, 2021). www.thelancet.com Vol 398 September 11, 2021 Declaration of interests SCG and AVSH are cofounders of and shareholders in Vaccitech (collaborators in the early development of ChAdOx1 nCoV-19) and named as inventors on a patent covering use of ChAdOx1-vectored vaccines (PCT/GB2012/000467) and a patent application covering this SARS-CoV-2 vaccine (SCG only). TL is named as an inventor on a patent covering use of ChAdOx1-vectored vaccines (PCT/GB2012/000467) and was a consultant to Vaccitech. PMF is a consultant to Vaccitech. AJP is chair of the UK Department of Health and Social Care’s Joint Committee on Vaccination and Immunisation, but does not participate in policy advice on coronavirus vaccines, and is a member of the WHO Strategic 989 Articles Articles References 990 www.thelancet.com Vol 398 September 11, 2021
https://openalex.org/W2958497619	https://www.frontiersin.org/articles/10.3389/fmicb.2019.01573/pdf	English	null	Microbial Communities of Polymetallic Deposits’ Acidic Ecosystems of Continental Climatic Zone With High Temperature Contrasts	Frontiers in microbiology	2,019	cc-by	13,203	Microbial Communities of Polymetallic Deposits’ Acidic Ecosystems of Continental Climatic Zone With High Temperature Contrasts Sergey N. Gavrilov1†, Aleksei A. Korzhenkov 2†, Ilya V. Kublanov1, Rafael Bargiela 3, Leonid V. Zamana 4, Alexandra A. Popova1, Stepan V. Toshchakov 1, Peter N. Golyshin 3,5 and Olga V. Golyshina3,5 * 1Laboratory of Metabolism of Extremophiles, Winogradsky Institute of Microbiology, FRC Biotechnology, RAS, Moscow, Russia, 2Laboratory of Bioinformatics, Genomics and Genome Editing, NRC Kurchatov Institute, Moscow, Russia, 3School of Natural Sciences, Bangor University, Bangor, United Kingdom, 4Laboratory of Geoecology and Hydrogeochemistry, Institute of Natural Resources, Ecology and Cryology, SB RAS, Chita, Russia, 5Centre for Environmental Biotechnology, Bangor University, Bangor, United Kingdom Keywords: acid mine drainage (AMD) systems, mine-impacted environments, acidophilic bacteria, unclassified Euryarchaeota/Terrestrial Miscellaneous Euryarchaeotal Group (TMEG), Ignavibacteriae, Transbaikal area Edited by: Golyshina o.golyshina@bangor.ac.uk †These authors have contributed equally to this work Specialty section: This article was submitted to Extreme Microbiology, a section of the journal Frontiers in Microbiology Received: 26 March 2019 Accepted: 24 June 2019 Published: 17 July 2019 Specialty section: This article was submitted to Extreme Microbiology, a section of the journal Frontiers in Microbiology Received: 26 March 2019 Accepted: 24 June 2019 Published: 17 July 2019 Edited by: Edited by: Axel Schippers, Federal Institute for Geosciences and Natural Resources, Germany Edited by: Axel Schippers, Federal Institute for Geosciences and Natural Resources, Germany Acid mine drainage (AMD) systems are globally widespread and are an important source of metal pollution in riverine and coastal systems. Microbial AMD communities have been extensively studied for their ability to thrive under extremely acidic conditions and for their immense contribution to the dissolution of metal ores. However, little is known on microbial inhabitants of AMD systems subjected to extremely contrasting continental seasonal temperature patterns as opposed to maritime climate zones, experiencing much weaker annual temperature variations. Here, we investigated three types of AMD sites in Eastern Transbaikalia (Russia). In this region, all surface water bodies undergo a deep and long (up to 6 months) freezing, with seasonal temperatures varying between −33 and +24°C, which starkly contrasts the common well-studied AMD environments. We sampled acidic pit lake (Sherlovaya Gora site) located in the area of a polymetallic deposit, acidic drainage water from Bugdaya gold-molybdenum-tungsten deposit and Ulan-Bulak natural acidic spring. These systems showed the abundance of bacteria-derived reads mostly affiliated with Actinobacteria, Acidobacteria, Alpha- and Gammaproteobacteria, chloroplasts, Chloroflexi, Bacteroidetes, and Firmicutes. Furthermore, candidate taxa “Ca. Saccharibacteria” (previously known as TM7), “Ca. Parcubacteria” (OD1) and WPS-2 were represented in substantial quantities (10–20%). Heterotrophy and iron redox cycling can be considered as central processes of carbon and energy flow for majority of detected bacterial taxa. Archaea were detected in low numbers, with Terrestrial Miscellaneous Euryarchaeal Group (TMEG), to be most abundant (3%) in acidic spring Ulan-Bulak. Composition of these communities was found to be typical in comparison to other AMD sites; however, certain groups (as Ignavibacteriae) could be specifically associated with this area. This study provides insight into the microbial diversity patterns in acidic ecosystems formed in areas of polymetallic deposits in extreme continental climate zone with contrasting temperature parameters. Reviewed by: Odile Bruneel, Institut de recherche pour le développement (IRD), France Celia Mendez, University of Illinois at Urbana-Champaign, United States *Correspondence: Olga V. ORIGINAL RESEARCH published: 17 July 2019 doi: 10.3389/fmicb.2019.01573 ORIGINAL RESEARCH INTRODUCTION ( , )l y p ( ) Acidic drainage water from Bugdaya gold-copper (tungsten) polymetallic deposit is located in the Eastern Transbaikalia area 310 km south-east from the Chita city on the north-western slope of a mountain ridge, dividing the basins of rivers Argun and Shilka, whose junction is the source of Amur river and at about 1,000 m altitude. The deposit is referred to Climax-type Porphyry Molybdenum deposits, but is characterized by a higher presence of gold. Main minerals are represented by pyrite, scheelite (CaWO4), molybdenite (MoS2), sphalerite, chalcopyrite, galena, fahlore [composed by tennantite (Cu6[Cu4(Fe,Zn)2]As4S13) and tetrahedrite (Cu6[Cu4(Fe,Zn)2]Sb4S13)] and natural gold. The main veined mineral is quartz, some mineral associations contain fluorite (Kovalenker et al., 2011).ht Acidic pit lakes formed as an outcome of opencast mining and groundwater accumulation are widely distributed in mining areas. A significant body of research data has been produced on pit lakes located in the Iberian Pyrite Belt, where typical and atypical acid mine drainage (AMD) organisms were revealed (Falagán et al., 2014). Natural springs that actively discharge groundwater in acidic areas, and hence contribute to the hydro- and geochemistry in these settings may also be considered as AMD ecosystems, microbiology of which has earlier been reviewed (Johnson and Hallberg, 2003; Chen et al., 2016). Numerous natural and mining-impacted acidic sites of Eastern Transbaikalia, l The Ulan-Bulak Urulyunguevsky natural spring (hereinafter, Ulan-Bulak, “Red Spring” from Buryat) is a linearly represented source of surface waters on the bottom of the creek valley TABLE 1 \| Characteristics of samples used for barcoded 16S rRNA gene amplicon sequencing. INTRODUCTION Russia, are located in the area of headwaters of the river Amur, more than 1,000 km eastwards of the lake Baikal, and represent a generous source of distinctive extreme environments. This area remains microbiologically under sampled with only a small number of recent studies available that were mostly dedicated to the diversity of sulfate-reducing bacteria (Karnachuk et al., 2015, 2016; Kadnikov et al., 2016 and others). The present work is focused on the elucidation of diversity of microbial inhabitants in three different types of acidic settings in this area. Sherlovaya Gora opencast pit lake located on the polymetallic deposit, acidic drainage water from pyrite/arsenopyrite Bugdaya deposit (Akatui group) and, finally, Ulan-Bulak natural acidic spring. Mining-impacted, pyrite-containing environments were extensively studied over past decades, and are known to host diverse Bacteria, Archaea, and Eukarya that drive elements transformations, especially those of carbon, iron, and sulfur (Johnson and Hallberg, 2008). Predominant bacterial species typically belong to Proteobacteria, in particular to Classes α and γ, and, more specifically, to the orders Betaproteobacteriales and Acidithiobacillales (early considered to be separate Classes) inside Gammaproteobacteria, according to Silva 132 taxonomy. Furthermore, some members of Nitrospirae, Actinobacteria, Firmicutes, and Acidobacteria were commonly detected in these environments along with Bacteroidetes and some representatives of uncultured candidate divisions (Mendez-Garcia et al., 2015). Archaeal diversity is predominantly represented by mesophilic members of families Ferroplasmaceae, Cuniculiplasmataceae, and some other archaea from the order Thermoplasmatales of the phylum Euryarchaeota without established taxonomic status (Golyshina, 2011; Golyshina et al., 2016, 2019; Korzhenkov et al., 2019). Further archaeal members in mine-impacted environments appearing in lower numbers, are represented by “Candidatus Micrarchaeota” and “Ca. Parvarchaeota”-related organisms (Baker et al., 2010; Golyshina et al., 2017), unclassified Euryarchaeota/Terrestrial Miscellaneous Euryarchaeal Group (TMEG), and some Thaumarchaeota (Falagán et al., 2014; Mendez-García et al., 2014). i Acidic Pit Lake at Sherlovaya Gora (“Schorl Mountain” from Russian) is located in the area of a polymetallic deposit in south-eastern Transbaikalia and composed by cassiterite (SnO2), pyrites (FeS2), arsenopyrite (FeAsS), galena (PbS), sphalerite (ZnS), and chalcopyrite (CuFeS2). The mining in this area was mostly attributed to lead and beryl and was finished in 1995. Our study was particularly focused on opencast pit lake formed as a result of oxidative dissolution of sulfidic minerals (Banks et al., 2015) reflected in a relatively low pH (Table 1). Citation: Gavrilov SN, Korzhenkov AA, Kublanov IV, Bargiela R, Zamana LV, Popova AA, Toshchakov SV, Golyshin PN and Golyshina OV (2019) Microbial Communities of Polymetallic Deposits’ Acidic Ecosystems of Continental Climatic Zone With High Temperature Contrasts. Front. Microbiol. 10:1573. doi: 10.3389/fmicb.2019.01573 July 2019 \| Volume 10 \| Article 1573 Frontiers in Microbiology \| www.frontiersin.org 1 Microbial AMD Communities of Transbaikalia Gavrilov et al. July 2019 \| Volume 10 \| Article 1573 Frontiers in Microbiology \| www.frontiersin.org INTRODUCTION Sample ID Description Coordinates pH Temperature, °C Eh, mV Shg4 Sherlovaya Gora Acidic Pit lake, upper layer/surface water 50°33′00.20″N, 116°16′07.8″E 3.23 17.3 +538 Shg1t Sherlovaya Gora Acidic Pit lake/dense mats growing on surface of plants brunches immersed in water, 1 m depth 50°33′06.70″N, 116°16′05.34″E 2.83 (inside of mat) and 3.20 (on the surface of plant remnants) 18.0 +509 Shg13 Sherlovaya Gora Acidic Pit lake, bottom water, depth 29 m 50°33′00.20″N, 116°16′07.80″E 4.90 7.8 +220 Shg13sed Sherlovaya Gora Acidic Pit lake/bottom sediment, depth 29 m 50°33′00.20″N, 116°16′07.80″E 4.90 7.8 +220 B3 Acidic drainage water in the area Bugdaya Mo-polymetallic deposit 51°09′29.85″N, 117°42′55.08″E 3.48 11.6 +511 UB1 Natural acidic spring Ulan-Bulak/small pond with red-brownish color 50°30′38.00′N, 118°55′04.90′E 2.37 15.0 +485 UB1sed Natural acidic spring Ulan-Bulak/small pond with red-brownish color 50°30′38.00′N, 118°55′04.90′E 2.37 15.0 +485 UB2 Natural acidic spring Ulan-Bulak/small pond with reddish color 50°30′38.00′N, 118°55′04.90′E 2.36 16.8 n.d. Frontiers in Microbiology \| www.frontiersin.org July 2019 \| Volume 10 \| Article 1573 2 Microbial AMD Communities of Transbaikalia Gavrilov et al. immersed in lake water at a depth of about 1 m from the surface, Shg1t was taken as well. intensely colored reddish-orange with iron oxides. The total length of this system is about 600–700 m; it is situated on south-eastern slope of Nerchinsk mountain ridge. Previous geochemical analysis of waters revealed low pH with Ca and Mg being predominant cations (Zamana, 2013). Fe, Al, Mn, Ni, Zn, and Co were found as well. The chemical composition of waters was similar to that of acid mine drainage waters of Ural and Transbaikalia area. Sulfate was detected to be the predominant anion. Furthermore, sulfur compounds were represented by H2S (289.5 mg/L) and elemental S (0.83 mg/L). Weak spontaneous expulsions of CO2 were noted in warm seasons after melting of frozen layers of deposits (Zamana, 2013). Sampling of acidic drainage water seeping through brownish- black ores of Bugdaya Au-Mo(W) polymetallic deposit (B3) and forming a small pond was conducted on 05.09.16. The flow rate of seepage was calculated as ca. 20 ml/s. Water appeared as almost transparent with a low level of mineralization visualized. The sampling spot was also characterized by the presence of plant litter and dried out cyanobacterial mats. On 03.09.16, samples were taken from two small ponds of the system Natural acidic spring Ulan-Bulak (UB) formed by active spring waters. INTRODUCTION UB1 is a small pond with red-brownish color having residues of different types of organic material, and UB2 is another small pond with significant amount of leaves and other plant residues on the bottom and intensively reddish colored water, characteristic for fulvic acids. The sediment with some organic material from UB1 (UB1sed) emplacement was also used for the study. The location of sampling sites is represented on the Figure 1. Main characteristics of these samples are summarized in the Table 1. Sample Collection and DNA Extraction p Sampling of Sherlovaya Gora (Shg) Acidic Pit lake was done on 30.08.16. Samples selected for the assessment of microbial diversity were taken from: (1) upper layer/surface water, Shg4 (2) bottom layer water (30–50 cm above the sediment), Shg13, and (3) bottom sediment, Shg13sed. Bottom layers of the lake were sampled with a bathometer. Additionally, the sample of (4) dense mats growing on surface of herbaceous plants, For DNA extraction from the surface water sample of Sherlovaya Gora pit lake and B3 sample from Bugdaya deposit, 1.2 L of water were filtered through Sterivex 0.2 μm pore size filter (Millipore). For the bottom water sample from Sherlovaya Gora and water samples from Ulan-Bulak, 0.2 L of water were FIGURE 1 \| Localisation of sampling sites in Transbaikalia (the area to the east from the lake Baikal): Acidic pit lake (site Sherlovaya Gora), Acidic drainage water from Bugdaya Au-Mo (W) deposit and Natural acidic spring Ulan-Bulak. FIGURE 1 \| Localisation of sampling sites in Transbaikalia (the area to the east from the lake Baikal): Acidic pit lake (site Sherlovaya Gora), Acidic drainage water from Bugdaya Au-Mo (W) deposit and Natural acidic spring Ulan-Bulak. mpling sites in Transbaikalia (the area to the east from the lake Baikal): Acidic pit lake (site Sherlovaya Gora), Acidic drainage water it and Natural acidic spring Ulan-Bulak. Frontiers in Microbiology \| www.frontiersin.org July 2019 \| Volume 10 \| Article 1573 3 Microbial AMD Communities of Transbaikalia Gavrilov et al. 15 s; primer annealing at 58°C, 15 s; DNA elongation at 72°C, 25 s, followed by final DNA elongation for 5 min at 72°C. Purification of PCR products was done using the Cleanup Mini kit (Evrogen, Russia). The quality of the final libraries was assessed using the electrophoresis in agarose gel. filtered (sample volume in these cases was restricted by water turbidity which caused filter clogging). For each sedimental sample, 0.75 ml of wet sediment (or microbial foulings) were sampled, which roughly corresponds to 1.2 g in weight. The samples were routinely fixed with 50% sterile ethanol on-site for further transportation to the laboratory and DNA isolation. DNA was extracted from sediments using PowerSoil® Isolation Kit (MO BIO Laboratories) according the manufacturer’s instructions. DNA extraction from Sterivex filters was performed using Meta-G-Nome™ DNA Isolation Kit (Epicenter) according the manufacturer’s instructions. All extraction experiments were performed in duplicates. Sample Collection and DNA Extraction Libraries were sequenced with MiSeq™ Personal Sequencing System technology of Illumina Inc. (San Diego, CA, USA) using paired-end 250-bp reads. Demultiplexing was performed as described previously (Fadrosh et al., 2014). After demultiplexing, all reads were subjected to stringent quality filtering, and parts of reads, corresponding to 16S rRNA primers were removed using CLC Genomics Workbench 10.0 (Qiagen, Germany). OTU picking and taxonomy assignment was made using QIIME in open-reference mode (Caporaso et al., 2010) and Silva132 database (Quast et al., 2012), all parameters were set to default values. Data Availabilityh The 100 most abundant OTUs obtained from the analysis of high-throughput 16S rRNA profiling experiments were submitted to GenBank under accession numbers MK521704 – MK521803. Sequencing reads were submitted to MG-RAST server (Keegan et al., 2016) as a project #mgp88315 and are available by the following link: https://www.mg-rast.org/linkin. cgi?project=mgp88315. Chemical Analysis y On-site measurements were performed for oxygen content – with Multi 340i handheld multimeter (WTW, Germany), рН – with portable precision pH meter CG 837 (Schott Instruments, Germany), Eh (redox potential) and temperature – with HI 98120 ORP and temperature meter, electric conductivity – with HI 9033 (Hanna Instruments). For the sediment of Sherlovaya Gora lake, pH, Eh and temperature were measured in a subsample inside bathometer immediately after buoying it up. The measurements were performed by immersing an appropriate sensor (pH, ORP or temperature) into the sediment on a depth of >1 cm, which provided full coverage of ceramic reference junction and glass membrane or platinum sensor of pH or ORP electrodes with wet sediment. For chemical analyzes, all water samples were filtered through ashless paper filters with 2–3 μm pore size. For cations, the samples were acidified with chemically pure nitric acid to pH 2.0. In the filtered samples, Ca, Mg, Al, Fe, Mn, Co, Ni, Cu, Zn, Sr, Cd, and Pb were quantified with flame atomic absorption spectroscopy (FAAS), Na and K – with flame emission spectroscopy, on a SOLAAR M6 spectrometer (Thermo Scientific). Sulfate ions were quantified by titration with BaCl2 in the presence of nitrochromazo dye as the indicator; fluoride – by potentiometry with corresponding ion-selective electrode in acetate-citrate buffer; chloride – by mercurimetry; Si, total phosphorus and nitrogen species (NO3 −, NO2 −, and NH4 +) – by spectrophotometry. For ICP MS analyzes of elemental composition, water samples were sequentially filtered through ashless paper filters and 0.45 μm membrane filters, and final filtrates were commercially analyzed in Vinogradov Institute of Geochemistry SB RAS (Irkutsk, Russia) on an Element-2 mass spectrometer (Finnigan MAT) using the NSAM method No. 480X according the manufacturer’s instructions. Frontiers in Microbiology \| www.frontiersin.org Sherlovaya Gora “Ferrovum,” shown to be widespread in extremely acidic ferruginous mine waters and containing the only cultured species “Ferrovum myxofaciens,” is an extremely acidophilic, psychrotolerant obligate chemolithoautotroph and diazotroph, which utilizes ferrous iron as the only electron donor (Johnson et al., 2014). Heterotrophy together with certain photosynthetic (Kishimoto et al., 1995) and iron(III)-reducing activity with the expenditure of a variety of organic substrates indicate ecological significance of another genus, Acidiphilium, important in the biogeochemical cycling of iron at oxic-anoxic interfaces in acidic environments. A number of potentially hydrolytic enzymes were reported previously for Acidiphilium spp., pointing at their role in carbon (organic polymers) turnover (Ullrich et al., 2015). All other reads from surface water of Sherlovaya Gora pit lake pointed at various taxonomic groups represented in minor quantities (<1%). Among them worth noting are the sequences affiliated with Acidicapsa genus, whose members are capable of Fe(III) reduction and wood polysaccharides degradation, (Kulichevskaya et al., 2012; Falagán et al., 2017). Facultative anaerobic heterotrophic or/and facultative phototrophic iron-reducing species of the genus Acidiphilium and other Acetobacteraceae were previously detected as the most abundant phylotypes in pit lakes of the Iberian Pyrite Belt (Falagán et al., 2014; González-Toril et al., 2014), accounting for almost 80% of total reads in the upper layers (Santofimia et al., 2013). Microbial mats developed on allochthonous organics (plant material remnants), are grown at similar temperature, pH and Eh values as in the surface water (Supplementary Table S1). Bacteria-derived SSU rRNA gene reads predominantly (23.7%) corresponded to various uncultured Acidimicrobiia, of them, 14.2% were related to IMCC26256 deep branch comprised of bacteria isolated from artificial freshwater lake Soyang (South Korea). Other 1.3% of acidimicrobial reads were related to genus Acidithrix and the remaining 8.2% to four distinct uncultured actinobacteria unassigned at the family level. Members of the family Acidimicrobiaceae within the order Acidimicrobiales are recognized heterotrophic iron-metabolizing acidophiles, previously identified in pit lakes (Santofimia et al., 2013). Further major taxa, represented in the microbial mats were Ktedonobacteraceae-related chloroflexi (16.5%), which was a minor component in the surface water community (<0.1%), and chloroplasts from various organisms, which totally account for 16.0% but are distinct from the chloroplasts clade predominant in the surface water. Interestingly, Ktedonobacteria are The deeper samples of Sherlovaya Gora pit lake were characterized by higher pH (4.9) and lower Eh (+220 mV) values in comparison to the surface layers (Supplementary Table S1). Sherlovaya Gora y We have sampled the opencast pit lake of Sherlovaya Gora deposit. According to long-term monitoring of the Institute of Natural Resources, SB RAS, the lake remains presumably holomictic during the last decade with occasional mixing periods in spring or autumn seasons, it is 29 m-deep and is filled by ground- and surface waters. The level of water in the lake was recorded to be almost constant. The lake remains frozen during the winter period for about 6 months, and the ice depth is usually between 1.2 and 1.4 m with a temperature under the ice level being −0.4°C and about +4.1°C at the depth of 14 m. The area around the lake is covered by iron and manganese oxides and deposits of sulfates. Our chemical analysis showed that the concentration of sulfate ions was overwhelmingly high in this system (4,750 mg/L). Cations of the following metals were detected at total concentrations (mg/L): Ca (694), Zn (658), Fe (347), Mg (274), Mn (110), and Al (22.2). Additionally, many minor and trace elements were detected. Interestingly, F was detected at 15.1–23.4 mg/L, which is likely to be attributed to the greisen mineralization. Eh distribution showed the gradient from +567 mV in the upper layer of the lake to +353 in the middle, and +266 on the bottom. Oxygen was found to be present in upper part of the lake only with a measured concentration 0.44 mg/L. Maximum content of ammonium NH4 + (3.59 mg/L) was found in the middle layer of water at ca. 15 m depth (Supplementary Table S1). 16S rRNA V3-V4 Amplicon Metabarcoding Libraries of 16S rRNA amplicons corresponding to V3–V4 variable region were prepared by single PCR with double- indexed fusion primers as described by Fadrosh et al. (2014). 16S rRNA annealing of forward Pro341F (modified) and reverse 806R primers corresponded to 5′CCTAYGGGBGCWSCAG and 5′GGACTACHVGGGTWTCTAAT (Merkel et al., 2015). Single-step PCR amplification was performed by qPCRmix-HS™ SYBR mastermix (Evrogen, Russia) using the following conditions: 30 cycles of denaturation at 95°C for July 2019 \| Volume 10 \| Article 1573 Frontiers in Microbiology \| www.frontiersin.org 4 Microbial AMD Communities of Transbaikalia Gavrilov et al. soil-borne mesophilic and thermophilic heterotrophic hydrolytic filamentous microorganisms with one of their representatives, Ktedonobacter racemifer, possessing a 13 Mbp genome, currently known as one of the largest among prokaryotes (Chang et al., 2011). Recently, Yabe et al. Sherlovaya Gora (2017a) documented a wide distribution of Ktedonobacteraceae related clones in terrestrial environments, including moderately acidic ones. These same authors also described a representative of Ktedonobacteraceae family able to hydrolyse xylan and cellulose, major components of plant biomass (Yabe et al., 2017b). Considering this physiological feature, persistence of Ktedonobacteraceae-related phylotype in surface water and high abundance of this phylotype in the immersed plant-associated microbial mat, we could speculate that ktedonobacteria might be active organic matter decomposers in the pit lake ecosystem. Among other major groups detected in mats were Acidobacteriaceae Subgroup 1-related sequences comprising totally 12.1% of reads, including 2.3% from polysaccharidolytic Acidicapsa representatives and 9.8% related to various isolates unassigned at the genus or family level. Notably, the abundance of Acidicapsa increased threefold in the mats comparing to the surface water. Also, 6.3% reads from mats were affiliated with Granulicella paludicola, aerobic acidophilic heterotrophic bacteria hydrolysing polysaccharides (Pankratov and Dedysh, 2010). In the surface water, Granulicella-related sequences were rare (<0.02%). Uncultured Acetobacteraceae (5.2%) showed similar abundance as in surface water, while representatives of genus Acidiphilium, were twentyfold less represented compared to the surface water (1.2 vs. 22.9%). Also, 3.2% of bacterial WPS-2 clade-related signatures, identified earlier in contaminated soils and acidic environments (García-Moyano et al., 2012) and Beijerinckiaceae- related alphaproteobacteria (3.3%) were found in the mats formed on plant remnants. Worth mentioning are the sequences related to Leptospirillum genus (2.2%) of iron-oxidizing Nitrospirae and 1.6% sequences of CPla-3 termite group belonging to Phycisphaerae (Planctomycetes). Facultative anaerobes of Phycisphaerae class were isolated from freshwater and hypersaline lakes (Fukunaga et al., 2009; Kovaleva et al., 2015; Edwardson and Hollibaugh, 2018). The phylotypes related to Beijerinckiaceae, Leptospirillum, and Phycisphaerae were also detected in surface water, but comprised a negligible part of its microbial diversity (each less than 0.1%).h The 16S rRNA gene amplicon sequencing data showed minor archaeal (less than 0.5%), but mainly bacterial signatures in all studied sampling points of Opencast Pit Lake on the deposit Sherlovaya Gora. Surface water amplicon sequencing dataset was predominantly derived from chloroplasts from several phototrophic eukaryotes, mainly, from mixotrophic freshwater microalgae of Epipyxis genus (39.4% of reads). Small flocks, characteristic to these golden algae, were visible in the surface water. Bacterial sequences were affiliated with “Ferrovum” (24.6%) and Acidiphilium genera (22.9%) and small proportions to uncultured organisms within the family Acetobacteraceae of Alphaproteobacteria (4.7%). Frontiers in Microbiology \| www.frontiersin.org Sherlovaya Gora Microbial composition of the community from the bottom of the lake showed a certain similarity with those from upper water samples and microbial mats. However, a significant downshift in abundance of reads derived from phototrophs, which could only be detected at 0.5% as uncultured Chloroflexi, was observed. Instead, the microbial diversity in bottom water showed predominance of obligate chemolithoautotrophic iron oxidizers of “Ferrovum” genus (12%), and representatives of uncultured Acetobacteraceae (15.5%), Acidimicrobiia (6.3%), Ignavibacteriales (9.7%), and Elusimicrobia (5.2%), potentially harboring organotrophic microorganisms. Ignavibacteriales were represented by one major and two minor phylotypes of uncultured Frontiers in Microbiology \| www.frontiersin.org July 2019 \| Volume 10 \| Article 1573 5 Microbial AMD Communities of Transbaikalia Gavrilov et al. organisms. Surprisingly, such a high abundance of ignavibacteria was detected in a cold (7.6°C) acidic environment far eastwards of the Rift Zone 2,000 km long plate boundary centered beneath Lake Baikal. However, the majority of Ignavibacteriae-related environmental clones, detected previously, came from subterrestrial sources or deep-sea sediments, and the only two cultured species of this phylum were isolated from thermal habitats (Iino et al., 2010; Podosokorskaya et al., 2013). The two known cultured ignavibacteria are facultatively anaerobic organotrophs that use various carbon sources. One of them, Melioribacter roseus, possesses а high hydrolytic potential along with capabilities of arsenate, nitrite and Fe(III) reduction (Podosokorskaya et al., 2013; Gavrilov et al., 2017), suggesting a possible involvement of Ignavibacteriae- related organisms in iron, nitrogen and mixed valence elements cycling in Sherlovaya Gora lake. Clones belonging to the class and phylum of the same name, Elusimicrobia, have been previously detected in various environments with high organic matter content, as well as in the groundwater of a deep subsurface gold mine (Magnabosco et al., 2016). The only cultured representative of this taxon is a strictly anaerobic monosaccharide-fermenting ultramicrobacterium isolated from gut of scarab beetle (Geissinger et al., 2009). Among less abundant phylotypes in the bottom water of the lake(1–5%), were betaproteobacteria of the genus Sulfuricella (1.1%); WPS-2 clade-related clones (4.4% of total reads), which have also been recovered from microbial mats; Sporichthyaceae family (2.6%, two phylotypes); clones related to Fe(III)-reducing acidobacteria of Acidicapsa genus (2.3%), Rhodanobacter genus of order Xanthomonadales (2.4%), as well as representatives of uncultured “Ca. Saccharibacteria” (candidate division TM7, 0.9%). The genus Sulfuricella contains the only species of sulfur-oxidizing, nitrate reducing facultative anaerobes (Kojima and Fukui, 2011). Sherlovaya Gora Among minor (less than 1%) community components of the bottom water of Sherlovaya Gora pit lake representatives of sulfur and As(III) oxidizing betaproteobacteria (Thiomonas genus), iron oxidisers of the genera Acidithiobacillus and Acidithrix (whose maximal abundance was detected in microbial mats on plant remnants), and heterotrophic iron-reducers of the genus Metallibacterium are worth noting. Facultatively anaerobic metallibacteria were found earlier to be the one of predominant groups of organisms in the sediments of acidic pit lake Cueva de la Mora, Iberian Pyrite belt, Spain (Falagán et al., 2014). Major taxa of pit lake sediments were similar, to those in bottom water (Figure 2) except for slightly decreased numbers of sequences related to uncultured Acetobacteraceae (11.8%), and various iron cycling organisms, making up to 9.8% altogether. The latter group was mainly represented by iron-oxidizing acidithiobacilli (3.1%), Gallionella spp. (3.8%) and Leptospirillum spp. (2.6%), as well as by minor numbers of Ferrithrix and Acidithrix (Figure 3). Organisms related to family Acetobacteraceae, including various heterotrophs, were the most abundant in the sediment and mainly represented by uncultured taxa with minor proportions of Acidiphilium and Acidocella. The abundance of Ignavibacteriales-related clones has been very similar, both in the sediment and bottom water (10.7 and 11.2%). The sediments’ third most abundant group (11.1% reads) was attributed to Xanthomonadales with 10.1% of Rhodanobacter-related and 1.0% of Metallibacterium-related Among minor (less than 1%) community components of the bottom water of Sherlovaya Gora pit lake representatives of sulfur and As(III) oxidizing betaproteobacteria (Thiomonas genus), iron oxidisers of the genera Acidithiobacillus and Acidithrix (whose maximal abundance was detected in microbial mats on plant remnants), and heterotrophic iron-reducers of the genus Metallibacterium are worth noting. Facultatively anaerobic metallibacteria were found earlier to be the one of predominant groups of organisms in the sediments of acidic pit lake Cueva de la Mora, Iberian Pyrite belt, Spain (Falagán et al., 2014). Major taxa of pit lake sediments were similar, to those in bottom water (Figure 2) except for slightly decreased numbers of sequences related to uncultured Acetobacteraceae (11.8%), and various iron cycling organisms, making up to 9.8% altogether. The latter group was mainly represented by iron-oxidizing acidithiobacilli (3.1%), Gallionella spp. (3.8%) and Leptospirillum spp. (2.6%), as well as by minor numbers of Ferrithrix and Acidithrix (Figure 3). Sherlovaya Gora Representatives of Sporichthyaceae family are widespread in soils and aquifers, including cold organisms. Surprisingly, such a high abundance of ignavibacteria was detected in a cold (7.6°C) acidic environment far eastwards of the Rift Zone 2,000 km long plate boundary centered beneath Lake Baikal. However, the majority of Ignavibacteriae-related environmental clones, detected previously, came from subterrestrial sources or deep-sea sediments, and the only two cultured species of this phylum were isolated from thermal habitats (Iino et al., 2010; Podosokorskaya et al., 2013). The two known cultured ignavibacteria are facultatively anaerobic organotrophs that use various carbon sources. One of them, Melioribacter roseus, possesses а high hydrolytic potential along with capabilities of arsenate, nitrite and Fe(III) reduction (Podosokorskaya et al., 2013; Gavrilov et al., 2017), suggesting a possible involvement of Ignavibacteriae- related organisms in iron, nitrogen and mixed valence elements cycling in Sherlovaya Gora lake. Clones belonging to the class and phylum of the same name, Elusimicrobia, have been previously detected in various environments with high organic matter content, as well as in the groundwater of a deep subsurface gold mine (Magnabosco et al., 2016). The only cultured representative of this taxon is a strictly anaerobic monosaccharide-fermenting ultramicrobacterium isolated from gut of scarab beetle (Geissinger et al., 2009). Among less abundant phylotypes in the bottom water of the lake(1–5%), were betaproteobacteria of the genus Sulfuricella (1.1%); WPS-2 clade-related clones (4.4% of total reads), which have also been recovered from microbial mats; Sporichthyaceae family (2.6%, two phylotypes); clones related to Fe(III)-reducing acidobacteria of Acidicapsa genus (2.3%), Rhodanobacter genus of order Xanthomonadales (2.4%), as well as representatives of uncultured “Ca. Saccharibacteria” (candidate division TM7, 0.9%). The genus Sulfuricella contains the only species of sulfur-oxidizing, nitrate reducing facultative anaerobes (Kojima and Fukui, 2011). Representatives of Sporichthyaceae family are widespread in soils and aquifers, including cold environments, grow heterotrophilcally using soil humic acids as the only carbon source (Tamura, 2014). Rhodanobacter species are heterotrophic, facultatively anaerobic denitrifiers populating areas with neutral to moderately acidic pH, such as soil, terrestrial subsurface sediments or mine wastewaters, with some of species being able to degrade polysaccharides and oxidize thiosulfate (Green et al., 2012; Koh et al., 2015). (Green et al., 2012; Koh et al., 2015). Sherlovaya Gora Organisms related to family Acetobacteraceae, including various heterotrophs, were the most abundant in the sediment and mainly represented by uncultured taxa with minor proportions of Acidiphilium and Acidocella. The abundance of Ignavibacteriales-related clones has been very similar, both in the sediment and bottom water (10.7 and 11.2%). The sediments’ third most abundant group (11.1% reads) was attributed to Xanthomonadales with 10.1% of Rhodanobacter-related and 1.0% of Metallibacterium-related FIGURE 2 \| Microbial community composition of Sherlovaya Gora pit lake at different microniches according to 16S rRNA gene profiling. Shg4, surface water; shg1t, microbial mat on plant residues at ca. 1 m depth; shg13, bottom water at ca. 29 m depth; shg13.sed, bottom sediment. “Other” group includes: Archaea, other Acidobacteria, other Actinobacteria, other Chloroflexi, “Candidatus Dependentiae” (TM6), Firmicutes, Gemmatimonadetes, Planctomycetes, other Proteobacteria, Candidate Phyla Radiation (CPR) bacteria, Verruccomicrobia without distinct prevalence of any of them. FIGURE 2 \| Microbial community composition of Sherlovaya Gora pit lake at different microniches according to 16S rRNA gene profiling. Shg4, surface water; shg1t, microbial mat on plant residues at ca. 1 m depth; shg13, bottom water at ca. 29 m depth; shg13.sed, bottom sediment. “Other” group includes: Archaea, other Acidobacteria, other Actinobacteria, other Chloroflexi, “Candidatus Dependentiae” (TM6), Firmicutes, Gemmatimonadetes, Planctomycetes, other Proteobacteria, Candidate Phyla Radiation (CPR) bacteria, Verruccomicrobia without distinct prevalence of any of them. July 2019 \| Volume 10 \| Article 1573 Frontiers in Microbiology \| www.frontiersin.org 6 Microbial AMD Communities of Transbaikalia Gavrilov et al. FIGURE 3 \| Relative abundances of 16S rRNA gene V3-V4 amplicon reads in studied sites. organics. Still, considerable proportion of allochtonous organics is likely to be decomposed by oxygen respiring or fermentative organisms in aerobic and anaerobic zones of the lake, respectively, while oxygenic phototrophs could significantly contribute to autochtonous organics production. Our chemical data suggest rather high soluble iron availability in the system (347 mg/L), part of which may also be adsorbed but easily available on the surface of iron minerals from underlying bedrock of Sherlovaya Gora pit lake. Dopson et al. (2008) observed inhibition of microbial Fe oxidation due to fluoride toxicity (F content detected to be in 15.1–23.4 mg/L in Sherlovaya Gora). Falagán et al. (2014) also noted a poor availability of phosphorus in acidic, highly stratified, iron-containing water systems, where ferric phosphate is extremely insoluble and phosphate availability was directly linked with redox variations of iron. Frontiers in Microbiology \| www.frontiersin.org Sherlovaya Gora There, the phosphate was found to be at higher concentrations in the anaerobic monimolimnion zone in comparison to the aerobic mixolimnion. One could also suggest that a part of microbial community might be associated with clay mineral formation, signatures. Representation of Elusimicrobia (3.8%), Acidicapsa (1.6%), Acidimicrobiia (totally 4.9%) and WPS-2 clade (totally 3.8%) decreased in the sediment as compared to bottom water, whereas Holophagaceae representation increased to 2.5%. Within the minor part of the sediment community, of interest are the members of the family Chitinophagaceae (1.3%) with high hydrolytic potential. Several taxa, containing sulfur oxidizers of genera Thiobacillus, Sulfuricella, Sulfuriferula (all, betaproteobacteria) were also present in the sediments, each in minor quantities, but their total share was considerable (1.9%). To sum up, the surface water, which is warmer, more oxygenated and illuminated, was less diverse in comparison to bottom sediment and bottom water samples (Figure 2). Ultimately, metabolic traits in microbial inhabitants of Sherlovaya Gora pit lake might be considered similar to other organisms from acidic pit lakes, where biogeochemical cycles of carbon and iron are linked together via the activity of phototrophic Fe(II) oxidizers and chemoheterotrophic Fe(III) reducers, mineralizing allochtonous, as well as autochtonous July 2019 \| Volume 10 \| Article 1573 July 2019 \| Volume 10 \| Article 1573 7 Microbial AMD Communities of Transbaikalia Gavrilov et al. Frontiers in Microbiology \| www.frontiersin.org Acidic Drainage Water Outlet From Bugdaya Au-Mo(W) Polymetallic Deposit The largest numbers of bacterial reads in B3 sample (28.8%) were affiliated with Acidimicrobiia (mostly with uncultured organisms including 9.6% reads related to IMCC26256, abundant in Shg1t sample, but with a small proportion (0.4%) of signatures related to Ferrithrix genus of iron oxidizers and reducers, Figure 3). The second most abundant group in Bugdaya sample was represented by uncultured phylum-level bacterial lineage “Ca. Patescibacteria,” including 10.4% reads related to “Ca. Parcubacteria” and 9.1% – to “Ca. Saccharibacteria,” which were highly similar to those identified in bottom sediments of Sherlovaya Gora pit lake (Figure 3). Acidobacteriaceae were represented in B3 sample by 13.0% of total reads and included several uncultured phylotypes, similar to those identified in Sherlovaya Gora samples. Mucilaginibacter spp. (Sphingobacteriaceae) were the fourth most-significant group in B3 site (11.9%). Chemoorganotrophic facultative anaerobes of the latter genus were earlier isolated from a range of environments including soils, wetlands, sediments, acidic peat bogs and others (Lee et al., 2015). Actinobacteria and Bacteroidetes may be responsible for organic matter decomposition and, partially, for iron cycling in Bugdaya ecosystem. Among “Ca. Patescibacteria,” formerly known as candidate phylum “Ca. Parcubacteria” (OD1) was previously identified as a minor fraction of reads in various anoxic environments such as sediments and rumen, but in relatively high numbers in freshwater ecosystem of Svalbard (Ntougias et al., 2016). This group is considered to include numerous organisms with diverse metabolisms and phylogenies (León- Zayas et al., 2017). “Ca. Saccharibacteria” was described to be a cosmopolitan and diverse group with sample-specific phylotypes (Ferrari et al 2014) confirmed to be present in The chemical content of water was found to be typical for acid mine drainage waters of Transbaikalia, with high concentrations of sulfates and metals, particularly Al (Zamana, 2011; Zamana and Chechel, 2014). From other anions, high concentration of F− (5.0% of all anions, Supplementary Table S2) was detected, which is originated from formation of complex compounds with Al (Zamana and Bukaty, 2004) and is characteristic for AMD of Transbaikalia, especially if fluorite is presented in ores. Main metal cation was found to be Ca (32.6% of all metals detected), followed by Mn (26.3%), and Al (17.9%, Supplementary Table S2). Low concentration of iron might be explained by precipitation of it as crust-like oxides of Fe, less of Mn, Al and Si on stones surfaces, so-named “sunburn of desert”. Fe was found to be represented rather in ferrous state (Fe2+). Acidic Drainage Water Outlet From Bugdaya Au-Mo(W) Polymetallic Deposit as Sánchez-España et al. (2018) earlier showed in acidic pit lake ecosystem in the Iberyan Pyritic Belt. However, relatively high abundance of iron cycling organisms in Sherlovaya Gora pit lake indicates the possibility of active Fe(II) and Fe(III) compounds turnover in the system. Main physico-chemical characteristics of water from this sample are summarized in Table 1 and Supplementary Table S2. The sampling site was also characterized by the presence of old leafs and dry crusts of cyanobacterial mats contributing to the organic pool in this ecosystem. Despite the high availability of sulfate ions in Shg samples, currently known bacteria responsible for sulfur compounds transformation appeared to be poorly represented in the site. We have only detected sequences of acidophilic sulfate reducing bacteria (SRB) of Desulfosporosinus genus, in minor numbers (0.3%, Figure 3) – from anoxic sediments of Sherlovaya Gora acidic pit lake. Similar situation was observed in Cueva de la Mora and explained by a probable loss in competition by SRB to heterotrophs for electron donors (small molecular weight organic molecules) (Falagán et al., 2014). On the other hand, a large pool of uncultured organisms and newly recognized groups without any background physiological information may also be potentially engaged in sulfur compounds transformations as well as other functions in Sherlovaya Gora pit lake. The chemical content of water was found to be typical for acid mine drainage waters of Transbaikalia, with high concentrations of sulfates and metals, particularly Al (Zamana, 2011; Zamana and Chechel, 2014). From other anions, high concentration of F− (5.0% of all anions, Supplementary Table S2) was detected, which is originated from formation of complex compounds with Al (Zamana and Bukaty, 2004) and is characteristic for AMD of Transbaikalia, especially if fluorite is presented in ores. Main metal cation was found to be Ca (32.6% of all metals detected), followed by Mn (26.3%), and Al (17.9%, Supplementary Table S2). Low concentration of iron might be explained by precipitation of it as crust-like oxides of Fe, less of Mn, Al and Si on stones surfaces, so-named “sunburn of desert”. Fe was found to be represented rather in ferrous state (Fe2+). CO2 was detected either (Supplementary Table S2). Drainage water percolating deposits was found to contain mostly bacterial sequences, however a minor fraction of reads (0.4%) attributed to Thaumarchaeota “Candidatus Nitrosotaleaceae” family were also detected. Acidic Drainage Water Outlet From Bugdaya Au-Mo(W) Polymetallic Deposit CO2 was detected either (Supplementary Table S2). Many similarities between acidic pit lakes of Sherlovaya Gora and Cueva de la Mora became apparent. However, while the seasonal fluctuations in mixolimnion of the Iberian Pyritic Belt were described as rather insignificant (10–25°C) (Falagán et al., 2014), huge differences in seasonal temperature regimes are characteristic for continental climate zone of Transbaikalia. Acidic pit lake remains frozen from mid-November till mid-May, which is the case for all water bodies in this area. Water temperature decreases down to −0.4°C under the ice sheet, which is more than a meter thick. These conditions increase oxygenation of the upper layer of the lake but hinder gas circulation and decrease availability of sun light as the energy source, thus significantly changing the community composition for almost half a year. In this view, the high abundance of phototrophs in surface water of the lake and presence of typical aerobic organisms in the bottom water in summer point at a good fitness and resilience of the microbial community to resist sharp changes in physico- chemical conditions of the environment. In relation to that, the activity of acidophiles at low temperatures was reported for Arctic, Antarctic and northern latitudes’ areas but no detailed information on characteristics of bacterial populations was provided (Dold et al., 2013; Wu et al., 2013). state (Fe2+). CO2 was detected either (Supplementary Table S2). Drainage water percolating deposits was found to contain mostly bacterial sequences, however a minor fraction of reads (0.4%) attributed to Thaumarchaeota “Candidatus Nitrosotaleaceae” family were also detected. The largest numbers of bacterial reads in B3 sample (28.8%) were affiliated with Acidimicrobiia (mostly with uncultured organisms including 9.6% reads related to IMCC26256, abundant in Shg1t sample, but with a small proportion (0.4%) of signatures related to Ferrithrix genus of iron oxidizers and reducers, Figure 3). The second most abundant group in Bugdaya sample was represented by uncultured phylum-level bacterial lineage “Ca. Patescibacteria,” including 10.4% reads related to “Ca. Parcubacteria” and 9.1% – to “Ca. Saccharibacteria,” which were highly similar to those identified in bottom sediments of Sherlovaya Gora pit lake (Figure 3). Acidobacteriaceae were represented in B3 sample by 13.0% of total reads and included several uncultured phylotypes, similar to those identified in Sherlovaya Gora samples. Mucilaginibacter spp. (Sphingobacteriaceae) were the fourth most-significant group in B3 site (11.9%). Acidic Drainage Water Outlet From Bugdaya Au-Mo(W) Polymetallic Deposit Chemoorganotrophic facultative anaerobes of the latter genus were earlier isolated from a range of environments including soils, wetlands, sediments, acidic peat bogs and others (Lee et al., 2015). Actinobacteria and Bacteroidetes may be responsible for organic matter decomposition and, partially, for iron cycling in Bugdaya ecosystem. Among “Ca. Patescibacteria,” formerly known as candidate phylum “Ca. Parcubacteria” (OD1) was previously identified as a minor fraction of reads in various anoxic environments such as sediments and rumen, but in relatively high numbers in freshwater ecosystem of Svalbard (Ntougias et al., 2016). This group is considered to include numerous organisms with diverse metabolisms and phylogenies (León- Zayas et al., 2017). “Ca. Saccharibacteria” was described to be a cosmopolitan and diverse group with sample-specific phylotypes (Ferrari et al., 2014), confirmed to be present in minor quantities in acidic environments including pit lakes According to the present study, moderate acidophiles outnumber extremely acidophilic counterparts in Sherlovaya Gora acidic pit lake. This suggests a discrepancy with previous data of Kadnikov et al. (2016), who studied the composition of microbial communities from another emplacement within Sherlovaya Gora ecosystem, in the wells drilled in the body of ore deposits. We consider that the higher abundance of iron and lower pH values in wells together with their depth and wellheads dimensions (Kadnikov et al., 2016) restrict mass and heat exchange with the surrounding environment. That may smoothen sharp seasonal variations in physico-chemical parameters of the ecosystem and thus shift the abundances of principal microbial taxa in the wells toward more extremely acidophilic iron-transforming organisms. July 2019 \| Volume 10 \| Article 1573 8 Microbial AMD Communities of Transbaikalia Gavrilov et al. organisms constituted the majority of microbial community in comparison to other, mining-impacted, sites in this study. Acidithiobacillus-related sequences were the most significant group in UB1 water sample (23.2%), while in the UB1 sediments, the share of these organisms was smaller (5.0%). Reads related to iron oxidizers of Leptospirillum genus (Nitrospirae), Acidithrix and Ferrithrix actinobacterial genera, as well as betaproteobacteria of the genus Ferritrophicum totally comprised 13.1% of UB1 water community, while the reads affiliated to the genera Metallibacterium (Gammaproteobacteria) and Acidicapsa (Acidobacteriaceae subgroup 1), both of which harbor Fe(III) reducing species, rated at 2.8% in water samples. In the sediments of UB1 site (UB1sed sample), iron oxidizers of the abovementioned groups comprised 5.8% of the community (twice as low as in the water). Acidic Drainage Water Outlet From Bugdaya Au-Mo(W) Polymetallic Deposit At the same time, the share of moderately acidophilic Desulfosporosinus genus in the sediment was much higher than in UB1 water (up to 6.5% vs. 0.5%). Desulfosporosinus (the family Peptococcaceae) representatives are known to populate acidic settings, those include few acidophilic sulfate-reducers isolated so far in pure cultures: D. acidiphilus from a mining site at Chessy-Les-Mines, France (Alazard et al., 2010), D. acididurans from White River sediment in Montserrat (Sánchez-Andrea et al., 2015) and Desulfosporosinus sp. strain BG from oxidized mining wastes in Northern Transbaikalia (Karnachuk et al., 2015, 2016). The latter was reported to tolerate high concentrations of copper and produce crystalline copper sulfides (Karnachuk et al., 2015). In another study, the presence of polyvalent metals in ionic form was proposed to mitigate the toxicity of free sulfide in acidic environments by removing it from the aqueous phase through the formation of insoluble sulfides (Meier et al., 2012). Similarly, higher metal (Falagán et al., 2014; Mendez-Garcia et al., 2015). In the recent SILVA database v.132, this taxon, previously referred to as candidate division TM7, was placed as a family-level subclade within “Saccharimonadia” class of candidate phylum “Ca. Patescibacteria.” The first cultivated organism related to “Ca. Saccharibacteria” group (TM7x) was isolated from the human oral cavity and revealed to possess epibiotic parasitic lifestyle using an actinomycete as a host (He et al., 2015). Considering possible epibiosis between “Ca. Saccharibacteria” and actinomycetes, abundance of TM7-related clones in Bugdaya AMD could be attributed to the prevalence of Actinobacteria (class Acidimicrobiia) in this same ecosystem. However, it is currently hard to consider exact role of “Ca. Patescibacteria” and, in particular, “Saccharibacteria”-related organisms in mine-impacted environments. Remarkably, the candidate divisions found in Bugdaya were previously detected in Svalbard acid mine drainage-affected area (García-Moyano et al., 2015), which is characterized by comparable parameters: moderate pH and transitionally low temperatures below the freezing point for a significant part of the year. However, while low temperatures are constant in high Arctic, Transbaikalia is situated in the continental climatic zone with outstanding temperature contrasts of about 60°C between summer and winter seasons. Chloroplasts of Oxyphotobacteria class (4.5%) from various phototrophic eukaryotes have also been detected in Bugdaya site, being probably washed out from foulings on ore deposits surrounding the sampling spot. Taxa harboring organisms with geochemical activity (generally, sulfur oxidizers, iron oxidizers, and reducers) are only represented as minor components of Bugdaya sample. Acidic Drainage Water Outlet From Bugdaya Au-Mo(W) Polymetallic Deposit These organisms were of different phylogenetic affiliations. With the prevalence of iron-oxidizing Leptospirillum spp. (1%), their total share was of 2.5% of reads derived from the genus Ferrithrix (actinobacteria), genera Rhodanobacter, Acidiphilium, Acidithiobacillus, Metallibacterium, Gallionella, Sulfuricella (all, proteobacteria) and genus Acidicapsa (from subgroup 1 Acidobacteriaceae). FIGURE 4 \| Relative abundance of prokaryotic phyla and candidate divisions in microbial communities of natural acidic spring Ulan Bulak. Low abundance taxa (<1%) are shown together as “Other.” Frontiers in Microbiology \| www.frontiersin.org Natural Acidic Spring Ulan-Bulak This lineage has uncertain taxonomic position within the superphylum, p g Another discrepancy between water and sediment samples of UB1 site is in the composition of putative organotrophic component of the community. In both samples, these organisms were represented by Paludibacteraceae-related (Bacteroidetes) sequences (4.1% from water and 6.1% from sediment) including those belonging to Microbacter and Paludibacter genera. However, in water sample UB1, alphaproteobacteria of Rhizobiales, Sphingomonadales, and Caulobacterales orders, which all harbor aerobic chemoorganotrophic organisms, represent 6.2% of the community, while the sediment sample UB1sed contained much higher percentage of organotrophic Firmicutes. Besides the abovementioned Desulfosporosinus-related clones, the reads related to families Clostridiaceae (group 1), Lachnospiraceae and Ruminococcaceae comprised 17.5% in the sediment sample. Current state of knowledge on metabolic traits of these Firmicutes (Rosenberg et al., 2014) allows predicting fermentative or aerobic organotrophic lifestyle of their representatives in UB1 site. However, the majority of UB1sed Firmicutes-related reads is affiliated with uncultured taxa, which role in the ecosystem is unpredictable to the moment. Altogether, the analysis of Ulan Bulak samples indicates that the main components of microbial population in this natural acidic environment are organisms involved in redox transformations of iron, which soluble forms are highly available in the system, and organotrophic organisms degrading complex organic matter entrapped into iron-rich sediments. Comparison of community composition in two sites of Ulan-Bulak with different content of plant residues revealed influence of these organic sources on the distribution of typical acidophiles and novel uncultured groups in this natural acidic environment. A potential for anaerobic acidophilic Firmicutes and Bacteroidetes as well as for uncultured Acidimicrobiia to play a significant role in the community can also be recognized. Principal components analysis (Figure 5) revealed clustering of samples by pH values and temperatures of the sites. Diversity of surface water samples from Sherlovaya Gora pit lake appeared to be more close to that of surface water bodies of Ulan-Bulak and Bugdaya sites than to the bottom samples of the lake, while pH parameter separates all of the samples into three groups, including surface water samples from Sherlovaya Gora FIGURE 5 \| Principal Components Analysis (PCA) clustering all samples from the three sites studied. Fisrt (PC1) and second (PC2) components explain the 26.07 and 24.46% of the variability. Samples from the same site are surrounded by an ellipse of red (Bugdaya), light green (Ulan-Bulak) or light blue (Sherlovaya Gora) colors. Natural Acidic Spring Ulan-Bulak Community analysis of microbial composition in Ulan-Bulak was performed in two sampling sites (UB1 and UB2), which differed in the thickness of bottom sediment layers (ca. 20 cm thicker in UB1 site, sampled separately) and plant residues amounts correlating with estimated fulvic acids contents (higher in UB2 site as judged by more intense yellow-reddish color of water in this spot). Ulan-Bulak natural acidic spring is characterized as the most acidic environment among all sites studied in this survey (Supplementary Table S3). As a result, a significant proportion of typical acidophilic bacteria known to be involved in iron redox cycling, Acidithobacillus spp., Leptospirillum spp. and Acidimicrobiales have been identified alongside the iron- and sulfur compounds-metabolizing firmicutes (Figures 3, 4). The analysis revealed a high representation in both emplacements of actinobacterial Acidimicrobiia-related sequences (without a clear affiliation to cultured representatives) – 15.0, 14.9, and 10.2% for surface water and bottom sediments of UB1 site and water of UB2 site, respectively. In all Ulan- Bulak samples, the taxa comprising iron-oxidizing and -reducing FIGURE 4 \| Relative abundance of prokaryotic phyla and candidate divisions in microbial communities of natural acidic spring Ulan Bulak. Low abundance taxa (<1%) are shown together as “Other.” Frontiers in Microbiology \| www.frontiersin.org July 2019 \| Volume 10 \| Article 1573 9 Microbial AMD Communities of Transbaikalia Gavrilov et al. ions content together with lower oxygen availability may facilitate proliferation of acidophilic sulfate reducers in UB1 sediments, comparing to the surface water of the site. ions content together with lower oxygen availability may facilitate proliferation of acidophilic sulfate reducers in UB1 sediments, comparing to the surface water of the site. candidate phylum “Ca. Diapherotrites” (Youssef et al., 2015), or to candidate phylum “Ca. Micrarchaeota” with equal probability. “DPANN” superphylum includes Nanoarchaeota and other archaea with reduced genome sizes and was suggested to have nano- sized cells implying symbiotic or parasitic lifestyle. Recently, interaction of a “Ca. Micrarchaeota” Mia14 with its archaeal host of Thermoplasmatales order was confirmed by using CARD-FISH in the co-culture (Golyshina et al., 2017). p g Another discrepancy between water and sediment samples of UB1 site is in the composition of putative organotrophic component of the community. In both samples, these organisms were represented by Paludibacteraceae-related (Bacteroidetes) sequences (4.1% from water and 6.1% from sediment) including those belonging to Microbacter and Paludibacter genera. Natural Acidic Spring Ulan-Bulak However, in water sample UB1, alphaproteobacteria of Rhizobiales, Sphingomonadales, and Caulobacterales orders, which all harbor aerobic chemoorganotrophic organisms, represent 6.2% of the community, while the sediment sample UB1sed contained much higher percentage of organotrophic Firmicutes. Besides the abovementioned Desulfosporosinus-related clones, the reads related to families Clostridiaceae (group 1), Lachnospiraceae and Ruminococcaceae comprised 17.5% in the sediment sample. Current state of knowledge on metabolic traits of these Firmicutes (Rosenberg et al., 2014) allows predicting fermentative or aerobic organotrophic lifestyle of their representatives in UB1 site. However, the majority of UB1sed Firmicutes-related reads is affiliated with uncultured taxa, which role in the ecosystem is unpredictable to the moment. UB2 sample displayed maximum abundance (20.4%) of sequences related to Microbacter genus. The only cultured representative of this genus, anaerobic fermentative propionigenic bacterium Microbacter margulisiae, was isolated from the sediment of an acid rock drainage environment at Rio Tinto (Sánchez- Andrea et al., 2014). Notably lower numbers of Microbacter- related sequences have been retrieved from UB1 site. Considering carbohydrate- and polypeptide-fermenting lifestyle of these bacteria, the difference in their abundancy in UB1 and UB2 samples correlates with higher organics content of the latter site. Another group represented in the UB2 sample in relatively high proportion (10.7%) was affiliated with uncultured Chloroflexi (AD3), previously detected in acidic forest soil, metal-contaminated sites and in an industrial soft coal slags (Selenska-Pobell, 2002; Wegner and Lieasack, 2017), however their metabolic roles remain elusive. Among others, candidate phylum WPS-2 should be named. It contributed 9.2% reads in the sediment sample UB1sed, 2.1% in UB1 water and 5.2% in UB2. Further relatively significant groups in UB2 sample are merged from iron oxidizers of genera Ferrimicrobium (3.4%) and Leptospirillum (3.8%), as well as from heterotrophs of Acidibacillus and Sphingomonas genera (2.8 and 2.2%, respectively). The presence of archaeal phylotypes attributed to uncultured TMEG (Terrestrial Miscellaneous Euryarchaeal group, which is referred to as a group of ambiguous taxa in the class Thermoplasmata in the recent SILVA database v.132) in numbers 3.0% for UB2 and 0.4% for UB1 sediment (i.e., in both sampling spots of Ulan Bulak) seems rather interesting. These archaea have been found in various environments that also include acidic areas (Mendez-García et al., 2014; Söllinger et al., 2016; Korzhenkov et al., 2019). In UB2 site, OTUs, corresponding to uncultured euryarchaea, clustering together within a separate deep phylogenetic lineage of “DPANN” superphylum, are also considerably represented (1.8%). Frontiers in Microbiology \| www.frontiersin.org Natural Acidic Spring Ulan-Bulak Gray ellipses differentiate surface from bottom samples in Sherlovaya Gora. Distances among samples seem to be explained by proximity of the pH. PCA has been developed under R programming environment using the function prcomp from stats package from basic R (R Development Core Team, 2008). FIGURE 5 \| Principal Components Analysis (PCA) clustering all samples from the three sites studied. Fisrt (PC1) and second (PC2) components explain the 26.07 and 24.46% of the variability. Samples from the same site are surrounded by an ellipse of red (Bugdaya), light green (Ulan-Bulak) or light blue (Sherlovaya Gora) colors. Gray ellipses differentiate surface from bottom samples in Sherlovaya Gora. Distances among samples seem to be explained by proximity of the pH. PCA has been developed under R programming environment using the function prcomp from stats package from basic R (R Development Core Team, 2008). FIGURE 5 \| Principal Components Analysis (PCA) clustering all samples from the three sites studied. Fisrt (PC1) and second (PC2) components explain the 26.07 and 24.46% of the variability. Samples from the same site are surrounded by an ellipse of red (Bugdaya), light green (Ulan-Bulak) or light blue (Sherlovaya Gora) colors. Gray ellipses differentiate surface from bottom samples in Sherlovaya Gora. Distances among samples seem to be explained by proximity of the pH. PCA has been developed under R programming environment using the function prcomp from stats package from basic R (R Development Core Team, 2008). FIGURE 5 \| Principal Components Analysis (PCA) clustering all samples from the three sites studied. Fisrt (PC1) and second (PC2) components explain the 26.07 and 24.46% of the variability. Samples from the same site are surrounded by an ellipse of red (Bugdaya), light green (Ulan-Bulak) or light blue (Sherlovaya Gora) colors. Gray ellipses differentiate surface from bottom samples in Sherlovaya Gora. Distances among samples seem to be explained by proximity of the pH. PCA has been developed under R programming environment using the function prcomp from stats package from basic R (R Development Core Team, 2008). July 2019 \| Volume 10 \| Article 1573 Frontiers in Microbiology \| www.frontiersin.org 10 Microbial AMD Communities of Transbaikalia Gavrilov et al. other previously studied AMD sites (Figures 3, 6 for a summary on microbial biodiversity). However, in these emplacements some untypical for AMD taxa, e.g. Ignavibacteriae, were also detected. Natural Acidic Spring Ulan-Bulak Top layers were predominantly populated by photoautotrophic eukaryotes, chemoautotrophic and photoheterotrophic prokaryotes, while the bottom layers were much more diverse with a high proportion of uncultured microorganisms with yet unknown (presumably heterotrophic) type of metabolism. Many of sequences of uncultured taxa originated from candidate phyla and other highly ranked taxa with no previously reported acidophilic members. The question, whether these microorganisms are capable of thriving in a wide range of environmental conditions, or whether this study is a snapshot of a seasonal microbial succession, is widely open and can only be resolved through a continuous monitoring. and Bugdaya sites, water and sedimentary samples from Ulan- Bulak and bottom water and sediment samples from Sherlovaya Gora pit lake with significantly higher pH values of ca. 5 in contrast to 2.4–3.5 in other sites. Rarefaction analysis across all sampled sites (Supplementary Figure S1) showed that B3 and UB1 are likely to host the most diverse communities spots unlike the Shg4 (microbial mat grown on plant remnants) exhibiting the lowest microbial diversity. CONCLUSION Our study has revealed that the composition of microbial communities from these acidic environments with highly contrasting seasonal temperature changes resembles that in Our study has revealed that the composition of microbial communities from these acidic environments with highly contrasting seasonal temperature changes resembles that in FIGURE 6 \| Phylogenetic tree of prokaryotic diversity found in different acidic sites. Sequences belonging to the 16S rRNA gene used in the tree have been obtained from isolated clones (represented in circles) or as reads from barcodes sequencing (squares). Size of circles is related to number of clones of the same taxonomic group found in the site, whereas squares size are related to relative abundance of reads. Labeled branches correspond to reference sequences chosen from each phylum. The tree combines data from different acidic mine drainages (AMDs; Transbailkal area, Parys Mountain and Richmond mine and pit lakes of Transbaikal area, Tinto River and Cueva de la Mora). Sequences have been aligned using Mafft (Katoh and Standley, 2013). Resulting multiple alignment was trimmed using Trimal (Capella-Gutierrez et al., 2009), removing those columns with more than 50% of gaps or similarity score lower than 0.0001. Phylogenetic tree was calculated by maximum likelihood with bootstrapping of 1,000 replicates. Graphical development was performed under R programming environment using basic tools and the package ape (Paradis and Schliep, 2018). FIGURE 6 \| Phylogenetic tree of prokaryotic diversity found in different acidic sites. Sequences belonging to the 16S rRNA gene used in the tree have been obtained from isolated clones (represented in circles) or as reads from barcodes sequencing (squares). Size of circles is related to number of clones of the same taxonomic group found in the site, whereas squares size are related to relative abundance of reads. Labeled branches correspond to reference sequences chosen from each phylum. The tree combines data from different acidic mine drainages (AMDs; Transbailkal area, Parys Mountain and Richmond mine and pit lakes of Transbaikal area, Tinto River and Cueva de la Mora). Sequences have been aligned using Mafft (Katoh and Standley, 2013). Resulting multiple alignment was trimmed using Trimal (Capella-Gutierrez et al., 2009), removing those columns with more than 50% of gaps or similarity score lower than 0.0001. Phylogenetic tree was calculated by maximum likelihood with bootstrapping of 1,000 replicates. Graphical development was performed under R programming environment using basic tools and the package ape (Paradis and Schliep, 2018). Frontiers in Microbiology \| www.frontiersin.org REFERENCES important sources for iron cycling in the Southern Ocean. Environ. Sci. Technol. 47, 6129–6136. doi: 10.1021/es305141b important sources for iron cycling in the Southern Ocean. Environ. Sci. Technol. 47, 6129–6136. doi: 10.1021/es305141b Alazard, D., Joseph, M., Battaglia-Brunet, F., Cayol, J. L., and Ollivier, B. (2010). Desulfosporosinus acidiphilus sp. nov.: a moderately acidophilic sulfate-reducing bacterium isolated from acid mining drainage sediments: new taxa: Firmicutes (class Clostridia, order Clostridiales, family Peptococcaceae). Extremophiles 14, 305–312. doi: 10.1007/s00792-010-0309-4 Dopson, M., Halinen, A. K., Rahunen, N., Boström, D., Sundkvist, J. E., Riekkola-Vanhanen, M., et al. (2008). Silicate mineral dissolution during heap bioleaching. Biotechnol. Bioeng. 99, 811–820. doi: 10.1002/bit.21628 p g g Edwardson, C. F., and Hollibaugh, J. T. (2018). Composition and activity of microbial communities along the redox gradient of an alkaline, hypersaline, Edwardson, C. F., and Hollibaugh, J. T. (2018). Composition and activity of microbial communities along the redox gradient of an alkaline, hypersaline, lake. Front. Microbiol. 9:14. doi: 10.3389/fmicb.2018.00014 Baker, B. J., Comolli, L. R., Dick, G. J., Hauser, L. J., Hyatt, D., Dill, B. D., et al. (2010). Enigmatic, ultrasmall, uncultivated Archaea. Proc. Natl. Acad. Sci. USA 107, 8806–8811. doi: 10.1073/pnas.0914470107 y et al. (2010). Enigmatic, ultrasmall, uncultivated Archaea. Proc. Natl. Acad. Sci. USA 107, 8806–8811. doi: 10.1073/pnas.0914470107 Fadrosh, D. W., Ma, B., Gajer, P., Sengamalay, N., Ott, S., Brotman, R. M., Fadrosh, D. W., Ma, B., Gajer, P., Sengamalay, N., Ott, S., Brotman, R. M., et al. (2014). An improved dual-indexing approach for multiplexed 16S rRNA gene sequencing on the Illumina MiSeq platform. Microbiome. 2:6. doi: 10.1186/2049-2618-2-6 Banks, D., Karnachuk, O. V., Kadnikov, V. V., Watts, M., Boyce, A., Ivasenko, D. A., et al. (2015). Hydrochemical data report from sampling of polymetallic mines in Zaibaikalskii Krai, eastern Siberia, Russian Federation. NGU Report 2014.035. Geological Survey of Norway, Trondheim. Falagán, C., Foesel, B., and Johnson, B. (2017). Acidicapsa ferrireducens sp. nov., Acidicapsa acidiphila sp. nov., and Granulicella acidiphila sp. nov.: novel acidobacteria isolated from metal-rich acidic waters. Extremophiles 21, 459–469. doi: 10.1007/s00792-017-0916-4 Capella-Gutierrez, S., Silla-Martinez, J. M., and Gabaldon, T. (2009). trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics 25, 1972–1973. doi: 10.1093/bioinformatics/btp348 Falagán, C., Sánchez-España, J., and Johnson, D. B. (2014). New insights into the biogeochemistry of extremely acidic environments revealed by a combined cultivation-based and culture-independent study of two stratified pit lakes. FEMS Microbiol. Ecol. 87, 231–243. doi: 10.1111/1574-6941 Caporaso, J. FUNDING The work of OG, PG, AK, and ST was supported by the Royal Society UK – Russia Exchange Grant #IE 160224 (RFBR 16-54-10072 КО project). The work of SG, IK, and AP on the analysis of metabolic potential of detected taxa was supported by the Russian Science Foundation Project #17-74-30025. The work of LZ was supported by the project #0386-2015-0006 (IX.137.1.2.) of SB RAS. OG and PG acknowledge ERA Net IB2 Project MetaCat through UK Biotechnology and Biological Sciences Research Council (BBSRC) Grant BB/M029085/1 and the support of the Centre of Environmental Biotechnology Project funded by the European Regional Development Fund (ERDF) through the Welsh Government. RB and PG acknowledge the support of the Supercomputing Wales project, which is part-funded by the European Regional Development Fund (ERDF) via the Welsh Government. DATA AVAILABILITY The datasets generated for this study can be found in MG-RAST server as a project #mgp88315 and are available by the following link: https://www.mg-rast.org/linkin.cgi?project=mgp88315. MK521704 – MK521803. AUTHOR CONTRIBUTIONS The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2019.01573/ full#supplementary-material ST, SG, PG, and OG designed the study. AK and SG did sampling, AK and ST did DNA isolation and sequencing ST, SG, PG, and OG designed the study. AK and SG did sampling, AK and ST did DNA isolation and sequencing CONCLUSION Frontiers in Microbiology \| www.frontiersin.org July 2019 \| Volume 10 \| Article 1573 11 Microbial AMD Communities of Transbaikalia Gavrilov et al. Nevertheless, the long-term deep freezing of water surface in all studied environments during autumn, winter and spring months, high temperature contrasts between seasons and intensive UV exposure due to very low rainfall and snowfall levels in the area, are the factors shaping microbial communities of investigated extreme environments and making these emplacements unique in comparison to AMD sites studied thus far in temperate climatic zones. Based on their taxonomic affiliation, microorganisms populating these low-pH environments do likely have heterotrophic and iron redox cycling lifestyles. These environments are furthermore of a great potential for exploration of, and prospecting for, biotechnological applications of cold-adapted acidophilic organisms as a source for hydrolases and oxidoreductases, active at low pH and for expanding the toolbox for environmental impact mitigation of AMD. experiments. LZ organized sampling and did chemical analysis. 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This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Copyright © 2019 Gavrilov, Korzhenkov, Kublanov, Bargiela, Zamana, Popova, Toshchakov, Golyshin and Golyshina. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Söllinger, A., Schwab, C., Weinmaier, T., Loy, A., Tveit, A. T., Schleper, C., et al. (2016). Phylogenetic and genomic analysis of Methanomassiliicoccales in wetlands and animal intestinal tracts reveals clade-specific habitat preferences. FEMS Microbiol. Ecol. 92:fiv149. doi: 10.1093/femsec/fiv149 July 2019 \| Volume 10 \| Article 1573 Frontiers in Microbiology \| www.frontiersin.org 14
https://openalex.org/W2433980300	https://orbi.uliege.be/bitstream/2268/198249/1/journal.elementa.000112.pdf	English	null	Air-ice carbon pathways inferred from a sea ice tank experiment	Elementa	2,016	cc-by	13,702	Air-ice carbon pathways inferred from a sea ice tank experimentAir-ice carbon pathways Marie Kotovitch1,2* • Sébastien Moreau3 • Jiayun Zhou1,2,4 • Martin Vancoppenolle5 • Gerhard S. Dieckmann6 • Karl-Ulrich Evers7 • Fanny Van der Linden1,2 • David N. Thomas8,9 • Jean-Louis Tison2 • Bruno Delille1 1Unité d’Océanographie Chimique, MARE, Université de Liège, Liège, Belgium 2Laboratoire de Glaciologie, DSTE, Université Libre de Bruxelles, Brussels, Belgium 3Institute for Marine and Antarctic Studies, University of Tasmania, Hobart, Tas, Australia 3Institute for Marine and Antarctic Studies, University of Tasmania, Hobart, Tas, Australia 4Division of Earth and Ocean Sciences, Nicholas School of the Environment, Duke University, Durham, North Carolina, United States 4Division of Earth and Ocean Sciences, Nicholas School of the Environment, Duke University, Durham, Nor Carolina, United States 5Sorbonne Universités, UPMC Paris 6, LOCEAN-IPSL, CNRS/IRD/MNHN, France 6Alfred Wegener Institute, Helmholtz Center for Polar and Marine Research, Bremerhaven, Germany 7A i T h l D HSVA H b G 5Sorbonne Universités, UPMC Paris 6, LOCEAN-IPSL, CNRS/IRD/MNHN, France 6Alfred Wegener Institute, Helmholtz Center for Polar and Marine Research, Bremerhaven, Germany 7Arctic Technology Department, HSVA, Hamburg, Germany gy p g y 8Marine Research Centre, Finnish Environment Institute (SYKE), Helsinki, Finland 8Marine Research Centre, Finnish Environment Institute (SYKE), Helsinki, Finland 9S h l f O S i B U i i M i B id U i d Ki d 8Marine Research Centre, Finnish Environment Institute (SYKE), Helsinki, Finland 9School of Ocean Sciences, Bangor University, Menai Bridge, United Kingdom 9School of Ocean Sciences, Bangor University, Menai Bridge, United Kingdom Marie.Kotovitch@ulg.ac.be Domain Editor-in-Chief Jody W. Deming, University of Washington Associate Editor Stephen F. Ackley, University of Texas at San Antonio Knowledge Domain Ocean Science Article Type Research Article Part of an Elementa Special Feature Biogeochemical Exchange Processes at Sea-Ice Interfaces (BEPSII) Received: May 29, 2015 Accepted: May 19, 2016 Published: June 28, 2016 Abstract Given rapid sea ice changes in the Arctic Ocean in the context of climate warming, better constraints on the role of sea ice in CO2 cycling are needed to assess the capacity of polar oceans to buffer the rise of atmospheric CO2 concentration. Air-ice CO2 fluxes were measured continuously using automated chambers from the initial freezing of a sea ice cover until its decay during the INTERICE V experiment at the Hamburg Ship Model Basin. Cooling seawater prior to sea ice formation acted as a sink for atmospheric CO2, but as soon as the first ice crystals started to form, sea ice turned to a source of CO2, which lasted throughout the whole ice growth phase. Once ice decay was initiated by warming the atmosphere, the sea ice shifted back again to a sink of CO2. Direct measurements of outward ice-atmosphere CO2 fluxes were consistent with the depletion of dissolved inorganic carbon in the upper half of sea ice. Combining measured air-ice CO2 fluxes with the partial pressure of CO2 in sea ice, we determined strongly different gas transfer coefficients of CO2 at the air-ice interface between the growth and the decay phases (from 2.5 to 0.4 mol m−2 d−1 atm−1). A 1D sea ice carbon cycle model including gas physics and carbon biogeochemistry was used in various configurations in order to interpret the observations. All model simulations correctly predicted the sign of the air-ice flux. By contrast, the amplitude of the flux was much more variable between the different simulations. In none of the simulations was the dissolved gas pathway strong enough to explain the large fluxes during ice growth. This pathway weakness is due to an intrinsic limitation of ice-air fluxes of dissolved CO2 by the slow transport of dissolved inorganic carbon in the ice. The best means we found to explain the high air-ice carbon fluxes during ice growth is an intense yet uncertain gas bubble efflux, requiring sufficient bubble nucleation and upwards rise. We therefore call for further investigation of gas bubble nucleation and transport in sea ice. Associate Editor Stephen F. Ackley, University of Texas at San Antonio Elementa: Science of the Anthropocene • 4: 000112 • doi: 10.12952/journal.elementa.000112 elementascience.org Domain Editor-in-Chief Jody W. Deming, University of Washington 1. Introduction Among the natural systems, the role of sea ice in CO2 cycling is not well constrained. Several studies report air-ice CO2 fluxes that show that sea ice is a permeable medium under certain conditions of temperature and salinity (Semiletov et al., 2004; Nomura et al., 2006, 2010b; Miller et al., 2011b; Delille et al., 2014), thereby refuting the assumption that sea ice impedes the air-ocean gas exchange. However, observational difficulties, in particular the lack of continuous observations covering the entire ice growth and decay cycle, hinder the understanding of carbon exchange processes in ice-covered seas. Some studies suggest that active sea ice processes are significant (Rysgaard et al., 2011; Delille et al., 2014), while others assume them to be Air-ice carbon pathways negligible (Cross et al., 2014). Given the rapid sea ice changes in the Arctic Ocean in the context of global warming (IPCC, 2013), better constraints on the role of sea ice in CO2 cycling are needed to assess the future capacity of polar oceans to buffer the rise of atmospheric CO2 concentration. negligible (Cross et al., 2014). Given the rapid sea ice changes in the Arctic Ocean in the context of global warming (IPCC, 2013), better constraints on the role of sea ice in CO2 cycling are needed to assess the future capacity of polar oceans to buffer the rise of atmospheric CO2 concentration. f Sea ice is a composite of pure ice, brine and gas inclusions. Various biological, chemical and physical parameters may affect the concentration of gases in sea ice (e.g., Tsurikov, 1979; Zhou et al., 2014a). Temperature appears to be one of the main controls of CO2 concentration in sea ice (Geilfus et al., 2012a; Delille et al., 2014). When temperature decreases, brine inclusions shrink, concentrating salts, gases and other impurities in the brine. In contrast, when temperature increases, the melting of ice with the increase in size of brine inclusions dilutes their content (e.g., Vancoppenolle et al., 2013; Delille et al., 2014). The increasing concentration of gases in brine due to the shrinking of brine inclusions triggers the nucleation of bubbles (Tsurikov, 1979; Killawee et al., 1998; Tison et al., 2002; Light et al., 2003). Gases are therefore found in sea ice in both dissolved (i.e., brines) and gaseous forms (i.e., bubbles). Bubbles are indeed observed at the onset of sea ice growth, when air inclusions are trapped within the ice structure. 1. Introduction Crabeck et al. (2014b) and Zhou et al. (2014a) suggested that bubbles further develop during ice growth when the gases concentrates within the brine. Conversely, during ice decay, brine dilution and the related decrease of gas concentration is hypothesized to promote the dissolution of gases in the brines. Sea ice is considered as permeable when the brine fraction is above 5% (Golden et al., 1998). As sea ice becomes permeable, air-ice gas exchange increases (Delille et al., 2014). Brine convection or gravity drainage (Notz and Worster, 2009; Hunke et al., 2011) is the main process responsible for the rejection of dissolved gases to the ocean during ice growth (Moreau et al., 2014). In the absence of convection, the diffusion of dissolved gases becomes the main pathway to transport dissolved gas across the ice (Gosink et al., 1976; Loose et al., 2011; Shaw et al., 2011). This diffusive flux is driven by dissolved gas gradients (Delille et al., 2007; Nomura et al., 2010b; Miller et al., 2011b). Gas bubbles may provide an alternative gas transport pathway (Zhou et al., 2013; Crabeck et al., 2014a). The efficiency of this pathway depends on whether bubbles can only diffuse molecularly (Loose et al., 2011) or rise due to their buoyancy (Moreau et al., 2014). Considering the need to better understand carbon cycling in ice-covered seas, the absence of measurements over the whole ice growth and decay cycle and the difficulty of performing these measurements on natural sea ice, we carried out a controlled ice growth and decay experiment during which the air-ice CO2 fluxes were monitored using the chamber method. The CO2 transfer coefficient during both ice growth and ice decay was computed and compared to a sea ice model integrating carbon dynamics. 2.1 Experimental settingh The experiment was carried out at the Arctic Environmental Test Basin facility of the Hamburg Ship Model Basin (http://www.hsva.de) in the framework of the INTERICE V project. Eleven polyethylene bags of 1.2 m3 were filled with about 1000 L of filtered seawater from the North Sea. The experiment reproducing ice growth and ice decay took place over a period of 19 days. The day “0” of the experiment was 30 May 2012. The air temperature above the mesocosms was set to −14 °C the first 14 days (hereafter denoted as the“growth phase”), then to −1 °C until the end of the experiment (the “melting phase”). More information about the experimental settings are provided by Zhou et al. (2014b).l p g p y We carried out continuous in situ measurements of ice temperature and air-ice CO2 fluxes: A chain of 10 thermistors was placed at 2 cm intervals through the whole ice thickness. Ice cores were also collected regularly (every 1 to 3 days). On a given day, all samples (ice, under-ice water) came from the same mesocosm. Once the ice in a mesocosm was sampled, it was compromised and not used again in the experiment. So each sampling day corresponded to a different mesocosm, except on day 19, when we sampled two mesocosms. Ice cores were wrapped in polyethylene bags for storage below −25 °C in the dark and for subsequent measurements of bulk ice salinity, total alkalinity (TA) and partial pressure of CO2 (pCO2). Elementa: Science of the Anthropocene • 4: 000112 • doi: 10.12952/journal.elementa.000112 2.4 Dissolved inorganic carbon We computed the dissolved inorganic carbon of bulk ice from TAbulk and pCO2 bulk using a 2 step computation. We estimated the salinity of brines according to the ice temperature using the relationship of Cox and Weeks (1983) derived from data compiled by Assur (1960). We then computed TAbrines at the salinity of brines from the linear relationship between TA and salinity. We computed the total dissolved inorganic carbon (DIC) of brines (denoted as DICbrines) from TAbrines and pCO2 bulk using the CO2SYS program for the carbonate system (Lewis and Wallace, 1998). We used the CO2 dissociation constants of Mehrbach (1973) refitted by Dickson and Millero (1987) and the other constants advocated by Dickson and Goyet (1994). We then converted DICbrines to DICbulk assuming a linear relationship between DIC and salinity. Nonetheless, there are some limitations with this approach that should be noted: the dissociation constants have been established for the ranges of temperatures and salinities of open ocean waters (i.e., temperatures above 1 °C and salinities of 35). We assumed that the CO2 dissociation constants were applicable at sub-zero temperatures, as suggested by Marion (2001) and Delille et al. (2007). We refer the reader to Brown et al. (2014) for a discussion on the validity of the constants. 2.2 Ice pCO2 at high vertical resolution We applied the method developed by Geilfus et al. (2012b) and reviewed by Crabeck et al. (2014b) and Geilfus et al. (2015) for the measurement of the bulk pCO2 (denoted as pCO2 bulk) within permeable sea ice. The goal is to equilibrate the sea ice samples with a N2/CO2 gas mixture of known concentration at a temperature as close as possible to the in situ temperature (the temperature of the ice upon sampling). Samples were cut to fit tightly into a square container, 4 x 4 cm, that was 4.4 cm high to minimize the headspace. The container containing the sample was sealed and connected to a vacuum pump for 15 min. A standard gas of known concentration (500 ppm of CO2) was then injected at a pressure of 1013 mbar. Standard gas and ice sample were equilibrated for 20 hours in a constant temperature bath at the in situ temperature. Gas was then recovered and injected in a Varian 3300 gas chromatograph to measure the CO2 concentration. Shortly afterward, the 2 2 Elementa: Science of the Anthropocene • 4: 000112 • doi: 10.12952/journal.elementa.000112 Air-ice carbon pathways sample temperature was measured to check for experimental drift. Measured pCO2 bulk was corrected for the temperature difference between the sample and the in situ temperature according to the corrections proposed by Copin-Montegut (1988). sample temperature was measured to check for experimental drift. Measured pCO2 bulk was corrected for the temperature difference between the sample and the in situ temperature according to the corrections proposed by Copin-Montegut (1988). 2.3 Total alkalinity We measured total alkalinity (TA) on melted sea ice (TAbulk) using 50 g of sea ice melt collected at a 2 cm vertical resolution. TA was measured on all mesocosms except SW 9. We derived TA for all SW 9 ice sections using the strong linear regression between salinity and TA observed for all of the other samples. We also collected seawater for TA measurement. Melted bulk ice and seawater samples were poisoned with a solution of super-saturated HgCl2 and then stored in the dark, until analysis (one year after the sampling). TA was measured by open-cell titration with 0.11 M HCl and the endpoints were determined according to Gran (1952). Routine analyses of Certified Reference Materials (provided by A. G. Dickson, Scripps Institution of Oceanography) ensured that the uncertainty of the TA measurements was less than 4 μmol kg−1. Elementa: Science of the Anthropocene • 4: 000112 • doi: 10.12952/journal.elementa.000112 2.5 Seawater pCO2 To measure the underlying seawater pCO2 a hole was drilled through the sea ice cover. Seawater was pumped from the hole using a peristaltic pump (Masterflex® - Environmental Sampler) and supplied to a sea ice equilibrator system (SIES; Delille et al., 2007) for measurements of the pCO2 and recycled back to the seawater through the same hole. The SIES is based on a membrane contractor equilibrator (Membrana® Liqui-cell) coupled to an infrared gas analyzer (IRGA, Li-Cor® 6262). Seawater flowed into the equilibrator at a maximum rate of 1 L min−1 and a closed air loop ensured circulation through the equilibrator and the IRGA at a rate of 3 L min−1. The IRGA was calibrated before and after the experiment with N2 and CO2:N2 mixtures with mixing ratios of 388 and 813 ppm supplied by Air Liquide Belgium. During the experiment, the drift of the IRGA was corrected with N2. Uncertainty during this experiment was less than 6 µatm. 2.8 Modelling air-ice CO2 fluxes The gas concentration in the bubble reservoir changes due to bubble nucleation/dissolution, upward migration of buoyant gas bubbles, and bubble escape to the atmosphere. Nucleation of gas bubbles transfers dissolved CO2 from brine to the bubble compartment as a function of the CO2 super-saturation. At each time step, a fraction Rbub of the CO2 super-saturation is transferred to bubbles. The bubbles migrate upward when the brine network is connected, which is assumed to happen above a given brine fraction threshold (e.g., eT gas = 0.07, Zhou et al., 2013; Moreau et al., 2014). If the fraction of sea ice with e > eT gas includes the ice surface, all gas bubbles escape to the atmosphere and contribute to the air-ice CO2 flux. For our CTRL simulation, we used Rbub = 10% h−1 and eT gas = 0.07 (Table 1). In order to understand the potential reasons to explain the underestimation of the observed air-ice CO2 fluxes by the model, we performed three series of sensitivity experiments (Table 1). First, we tested the impact of a more intense dissolved CO2 pathway, by changing Ddiff and zBL (runs 2–4). The Ddiff value of Broecker and Peng (1974) is derived from seawater and only includes diffusive effects. We tested the slightly higher Ddiff value of Loose et al. (2011), derived from sea ice experiments, which can potentially include both diffuse and bubble contributions. A lower zBL (0.05 µm) value was also tested. In a second series of experiments The gas bubble pathway was developed in the model to simulate Argon dynamics (Moreau et al., 2014) and implies explicit gas bubble reservoirs in every layer. The gas concentration in the bubble reservoir changes due to bubble nucleation/dissolution, upward migration of buoyant gas bubbles, and bubble escape to the atmosphere. Nucleation of gas bubbles transfers dissolved CO2 from brine to the bubble compartment as a function of the CO2 super-saturation. At each time step, a fraction Rbub of the CO2 super-saturation is transferred to bubbles. The bubbles migrate upward when the brine network is connected, which is assumed to happen above a given brine fraction threshold (e.g., eT gas = 0.07, Zhou et al., 2013; Moreau et al., 2014). If the fraction of sea ice with e > eT gas includes the ice surface, all gas bubbles escape to the atmosphere and contribute to the air-ice CO2 flux. 2.6.2 Computation of a gas transfer coefficient for CO2 p gfi As described above, gases are transported through sea ice to the atmosphere by convection, diffusion and/or the ascent of bubbles to the ice surface. In the present study, we calculated an effective gas transfer coefficient for CO2 (K, in mmol m−2 d−1 atm−1), using the equation developed by Liss and Slater (1974) and Sarmiento and Gruber (2004): F = K ( pCO2 bulk – pCO2 air ) (1) (1) where F is the air-ice CO2 flux in mmol m−2 d−1, pCO2 bulk is the pCO2 in the ice and pCO2 air is the pCO2 in the air, both expressed in atm. In this equation we assume that F and K reflect both diffusive flux and bubble buoyancy (i.e., the rise of bubbles to the ice surface). where F is the air-ice CO2 flux in mmol m−2 d−1, pCO2 bulk is the pCO2 in the ice and pCO2 air is the pCO2 in the air, both expressed in atm. In this equation we assume that F and K reflect both diffusive flux and bubble buoyancy (i.e., the rise of bubbles to the ice surface). Elementa: Science of the Anthropocene • 4: 000112 • doi: 10.12952/journal.elementa.000112 2.6 Air–ice CO2 fluxes l 2.6.1 Measurements at the automated chamberll In this paper, positive CO2 flux refers to CO2 flux from the ice to the atmosphere, while negative CO2 flux refers to a flux from the atmosphere to the ice. We measured air-ice CO2 fluxes using an automated chamber placed above the water surface or on top of the ice. The chamber consisted of a mobile cap and a plastic cylinder, or so-called collar, with a diameter of 20 cm and a height of 9.7 cm. A rubber seal surrounded the cylinder and ensured an airtight connection between the ice and the chamber. Each hour, the cap closed the chamber and the pCO2 was measured over 15 min. At the beginning of the experiment one chamber was set above the surface of the water with the collar lowered a few millimetres below the water surface of a dedicated mesocosm apart from the 21 mesocosms used for ice collection. However, ice freezing and consolidation pushed the collar upward so that the collar was not properly sealed. After the fifth day of the experiment, the chamber was therefore moved to mesocosm 11 and was properly sealed. A pump within the LI-COR Multiplexer (LI-8150) circulated the air in the chamber at a flow rate of 2.1 L min−1. When the pCO2 of ice is higher than atmospheric pCO2, CO2 is transferred from the ice to the atmosphere and the automated chamber records a positive flux. A negative flux is observed in the opposite case. Water-corrected CO2 flux was computed automatically with LI-8100 File Viewer 3.1.0 package provided by LI-COR Biosciences. The flux was either calculated with a linear or an empirical exponential regression depending on which method provided the best fit (assessed from the normalized sums of the squares of the residuals). 3 Elementa: Science of the Anthropocene • 4: 000112 • doi: 10.12952/journal.elementa.000112 Air-ice carbon pathways 2.6.2 Computation of a gas transfer coefficient for CO2f 2.6.2 Computation of a gas transfer coefficient for CO2 2.7 Assessment of the precision of derived variables A Bootstrap resampling statistical analysis procedure, using random values of the measured parameters (temperature, salinity, pCO2, DIC and TA) between the mean ± precision over 1000 iterations, was used to estimate the propagation of errors to the computed parameters (∆DIC and K). This method was used as a way to show the effects of the imprecision of the data set on the calculated parameter. 2.8 Modelling air-ice CO2 fluxes 2.8 Modelling air-ice CO2 fluxes l In order to understand the factors that drive air-ice CO2 fluxes during the experiment, we ran a one-dimensional thermodynamic sea ice model representing sea ice halo-thermodynamics and carbon dynamics, including air-ice CO2 fluxes (Vancoppenolle et al., 2010; Moreau et al., 2015). Vertical carbon transport and air-ice carbon fluxes are explicitly separated between dissolved and gaseous form contributions.h l The dissolved gas pathway combines the vertical transport of dissolved inorganic carbon in brine and the diffusive air-ice CO2 flux, FCO2 (mmol m−2 d−1), which is assumed proportional to the CO2 partial pressure (pCO2) difference between the surface brine (in the top 5 cm of the ice) and the atmosphere, and is a function of the near-surface brine fraction: F CO2 = kCO2 · e 2/3 · (z CO2 – K0 f CO2) (2) (2) where kCO2 (m s−1) is the piston velocity, e is the brine fraction near the ice surface, e 2/3 represents the fractional surface open to brine-air diffusive CO2 fluxes, ζ CO2 is the brine CO2 concentration (mmol m−3), f CO2 is the atmospheric CO2 fugacity (atm), and K0 is the Henry’s constant (mmol m−3 atm−1). We consider that f CO2 = patm r CO2,atm, where patm is the atmospheric pressure (atm) and rCO2,atm is the atmospheric CO2 mixing ratio measured within the chamber. The piston velocity is calculated from the molecular diffusion coefficient of dissolved CO2 (Ddiff in m2 s−1) and the thickness of the diffusive boundary layer (zBL in µm): (3) f kCO2 = Ddiff /ZBL (3) kCO2 = Ddiff /ZBL This piston velocity neglects the effects of snow and wind, absent in these tank experiments, and only includes the contribution of dissolved CO2. In contrast, equation 1 includes both gas bubble and dissolved contributions as F and pCO2 bulk are direct measurements without distinction between dissolved and gaseous forms. The zBL is highly uncertain and therefore has been used as a tuning parameter to adjust the magnitude of air-ice CO2 fluxes, whereas Ddiff is better constrained by observations. For our control simulation (CTRL), we used Ddiff = 0.97 10−9 m2 s−1 (the diffusion coefficient of CO2 in water from Broecker and Peng, 1974) and zBL = 0.5 µm (Moreau et al., 2015; Table 1).h µ The gas bubble pathway was developed in the model to simulate Argon dynamics (Moreau et al., 2014) and implies explicit gas bubble reservoirs in every layer. 2.8 Modelling air-ice CO2 fluxes For our CTRL simulation, we used Rbub = 10% h−1 and eT gas = 0.07 (Table 1). l In order to understand the potential reasons to explain the underestimation of the observed air-ice CO2 fluxes by the model, we performed three series of sensitivity experiments (Table 1). First, we tested the impact of a more intense dissolved CO2 pathway, by changing Ddiff and zBL (runs 2–4). The Ddiff value of Broecker and Peng (1974) is derived from seawater and only includes diffusive effects. We tested the slightly higher Ddiff value of Loose et al. (2011), derived from sea ice experiments, which can potentially include both diffuse and bubble contributions. A lower zBL (0.05 µm) value was also tested. In a second series of experiments Elementa: Science of the Anthropocene • 4: 000112 • doi: 10.12952/journal.elementa.000112 Air-ice carbon pathways Table 1. Description of the sensitivity runs used to test the sensitivity of air-ice CO2 fluxes to model parameterizationa Table 1. Description of the sensitivity runs used to test the sensitivity of air-ice CO2 fluxes to model parameterizationa Run Name Rbub eT gas Ddiff zBL Ikaite precipitation Number of ice layers Misfit 1 CTRL 10 0.07 0.97 0.5 Yes 10 0.1 2 Loose-Ddiff b 0 0.07 24 0.5 Yes 10 0.08 3 No-bub 0 0.07 0.97 0.5 Yes 10 0.08 4 Low-zBL 0 0.07 0.97 0.05 Yes 10 0.08 5 No-ikaite 0 0.07 0.97 0.5 No 10 0.10 6 20-layers 0 0.07 0.97 0.5 Yes 20 0.06 7 Low-bub 1 0.07 0.97 0.5 Yes 10 0.07 8 High-bub 20 0.07 0.97 0.5 Yes 10 −0.05 9 Moreau-bubc 0.1 0.07 0.97 0.5 Yes 10 0.08 bLoose et al. (2011) cMoreau et al. (2015) doi: 10.12952/journal.elementa.000112.t001 (runs 5–6), the impacts of higher resolution (20 layers instead of 10) and ikaite precipitation/dissolution were investigated. Third, we tested the impact of a more intense gas bubble pathway by changing Rbub, the bubble nucleation rate (runs 7–9), which is highly uncertain.li (runs 5–6), the impacts of higher resolution (20 layers instead of 10) and ikaite precipitation/dissolution were investigated. Third, we tested the impact of a more intense gas bubble pathway by changing Rbub, the bubble nucleation rate (runs 7–9), which is highly uncertain.li To evaluate which scenario is closest to the observed fluxes, we computed a model-data misfit for each of the model runs. 2.8 Modelling air-ice CO2 fluxes The misfit were computed from the difference between the flux observed and the flux modelled. We report averages for each of the model runs in Table 1.hh All other parameters are from Moreau et al. (2015). The simulation spans 31 May – 18 June 2012. The time step is 1 h. Based on the present observations, initial seawater TA and DIC concentrations were set to 2300 mmol m−3 and 2090 mmol m−3, and sea ice TA and DIC concentrations were set to 850 mmol m−3 and 750 mmol m−3. Figure 1 Linear regression between TA and salinity within bulk sea ice. The ice growth and melting phases are colored in blue and red, respectively. Figure 1 Linear regression between TA and salinity within bulk sea ice. aDifferent parameterizations include the bubble formation rate (Rbub, % h−1), the molecular diffusion rate (Ddiff , 109 m−2 s−1), the boundary layer thickness (zBL, um), ikaite precipitation and the number of vertical ice layers. Model-data misfit (mmol m−2 d−1) are also presented for each run. doi: 10.12952/journal.elementa.000112.f001 Figure 1 Linear regression between TA and salinity within bulk sea ice. The ice growth and melting phases are colored in blue and red, respectively. doi: 10.12952/journal.elementa.000112.f001 3.1 Total alkalinity TA concentrations in bulk sea ice (i.e., melted sea ice) are consistent with the values reported in the literature (Gleitz et al., 1995; Delille et al., 2007; Nomura et al., 2010b; Fransson et al., 2011; Miller et al., 2011a; Geilfus et al., 2012a; Rysgaard et al., 2013) and were highly correlated with salinity (r2 = 0.95; Figure 1). TA concentrations in bulk sea ice (i.e., melted sea ice) are consistent with the values reported in the literature (Gleitz et al., 1995; Delille et al., 2007; Nomura et al., 2010b; Fransson et al., 2011; Miller et al., 2011a; Geilfus et al., 2012a; Rysgaard et al., 2013) and were highly correlated with salinity (r2 = 0.95; Figure 1).h y g g y y g The repeated inspection of freshly melted ice using a binocular microscope (Leitz Laborlux® with 125 to 500 x magnification) did not reveal the presence of ikaite, a hydrated calcium carbonate polymorph (CaCO3·6H2O). Taking into account the thermodynamic constraints (Papadimitriou et al., 2013) and the kinetics to simulate the ikaite precipitation (Papadimitriou et al., 2014), the maximum value of ikaite simulated by the model in the ice surface layer was 13 µmol kg−1, whereas the error on TA measurement was 4 µmol kg−1.(parameters from the CTRL simulation of Moreau et al., 2015). This concentration falls at the lower end of the range of ikaite concentrations reported in sea ice (7–93 µmol kg−1 in Dieckmann et al., 2008; 15–19 µmol kg−1 in Geilfus et al., 2013; 100–900 µmol kg−1 in Rysgaard et al., 2013). This evidence indicates that if ikaite precipitated during the experiment, it was not significant. 3.2 CO2 exchange at the air-ice interface 3. Results and discussion Detailed information about sea ice physical properties can be found in Zhou et al. (2014b). Briefly, the first phase of the experiment – the growth phase – lasted from day 1 to day 15. Air temperature above the tank was set to −15 °C and sea ice grew continuously, reaching a maximum thickness of 24 cm. A strong temperature gradient was observed between the top and the bottom of the ice. Salinity exhibited a typical C-shape profile with a lower salinity at the ice interior compared to the top and the bottom of the ice. Then from day 16 to 19, the air temperature was set to −1 °C, sea ice thickness slightly decreased, temperature exhibited a more homogeneous profile through the whole thickness, and salinity decreased in the top and bottom parts. The brine volume fraction remained above 5% during the whole experiment. On the whole, the sea ice remained thin, warm and permeable during both the growth and the melting phase. Elementa: Science of the Anthropocene • 4: 000112 • doi: 10.12952/journal.elementa.000112 doi: 10.12952/journal.elementa.000112.f001 Elementa: Science of the Anthropocene • 4: 000112 • doi: 10.12952/journal.elementa.000112 Air-ice carbon pathways Figure 2 Air-ice CO2 fluxes, sea ice surface temperature and pCO2 over time. doi: 10.12952/journal.elementa.000112.f002 3.2.1 Continuous measurements of air-ice CO2 fluxes l During seawater cooling, and before the formation of the ice crystals, CO2 fluxes measured with the automated chamber showed negative values, down to −6 mmol m−2 d−1 (Figure 2). Within hours after the formation of the first ice crystals, CO2 fluxes became mostly positive (ranging between −0.4 mmol m−2 d−1 and 0.75 mmol m−2 d−1 with an average of 0.2 mmol m−2 d−1 for the growth phase), consistent with the observed super-saturation of CO2 in bulk ice (i.e., pCO2 bulk above 400 ppm). During the melting phase, CO2 fluxes turned to negative (ranging between −2.1 mmol m−2 d−1 and 0 mmol m−2 d−1 with an average of −0.24 mmol m−2 d−1) in parallel with the decrease of pCO2 bulk that passed below saturation. The pCO2 of the underlying seawater remained under-saturated during the whole experiment, while the surface (first 5 cm) pCO2 bulk showed values above or below the atmospheric pCO2 during the growth and the melting phase, respectively.l p p g g g p p y Although the chamber was not properly sealed between day 0 and day 5, air-ice CO2 fluxes from the whole measurement period were consistent with previous measurements carried out with chambers over artificial sea ice (between 0 and 0.27 mmol m−2 d−1; Nomura et al., 2006) and slightly lower than measurements over natural sea ice. Delille et al. (2014) measured CO2 fluxes ranging from −5.2 mmol m−2 d−1 to 1.9 mmol m−2 d−1 on Antarctic pack ice in spring, and ascribed these fluxes to seasonal pCO2 gradients between the brine and the atmosphere. Geilfus et al. (2012a) measured CO2 fluxes at the sea ice interface ranging from −2.63 mmol m−2 d−1 up to 0.84 mmol m−2 d−1 in the Arctic coastal zone, while Nomura et al. (2010a) measured CO2 fluxes ranging from -1 mmol m−2 d−1 to 0.7 mmol m−2 d−1 over land fast ice in Barrow at the end of spring. Elementa: Science of the Anthropocene • 4: 000112 • doi: 10.12952/journal.elementa.000112 s s Figure 2 Air-ice CO2 fluxes, sea ice surface temperature and pCO2 over time. Figure 2 Nevertheless, the results can still be interpreted in terms of the main factors driving the CO2 fluxes, and the overall pattern of observed air-ice CO2 fluxes is consistent with in situ observation of a seasonal cycle, with upward CO2 fluxes during ice growth (Nomura et al., 2010a; Geilfus et al., 2012a; Delille et al., 2014) and downward CO2 fluxes during ice melt (Delille et al., 2014; Geilfus et al., 2015). The temperature and the pCO2 air measured within the automated chamber exhibited daily variations, rising during “working hours” due to the presence of the researchers in the experimental room and decreasing outside “working hours”. The pattern of air-ice CO2 fluxes is clearly opposite and strongly affected by the rise of atmospheric pCO2 during working hours. These imperfections are inherent to the environmental constraints of this experimental study and could hardly be avoided. Nevertheless, the results can still be interpreted in terms of the main factors driving the CO2 fluxes, and the overall pattern of observed air-ice CO2 fluxes is consistent with in situ observation of a seasonal cycle, with upward CO2 fluxes during ice growth (Nomura et al., 2010a; Geilfus et al., 2012a; Delille et al., 2014) and downward CO2 fluxes during ice melt (Delille et al., 2014; Geilfus et al., 2015). 3.2.2 Integrated estimates of air-ice CO2 fluxes l In parallel with direct measurements of air-ice CO2 fluxes, we derived air-ice CO2 fluxes from DIC depletion in the top layers of the ice (Geilfus et al., 2013). DIC was normalized to a salinity of 7 (7 corresponds to the mean bulk ice salinity during the whole experiment, noted as DIC7) to remove the salinity-related changes (brine rejection, concentration and dilution; Figure 3). If no biogeochemical processes occurred (biological activity, CaCO3 precipitation, CO2 transfer to the gas phase and CO2 exchange with the atmosphere), DIC7 profiles should be homogeneous over the ice column. During the growth phase, a clear decrease in the top 12.5 cm of the young ice was measured compared to the bottom ice horizons. Because no significant primary production was possible due to absence of primary producers (Zhou et al., 2014b) and ikaite precipitation was insignificant (section 3.1), we assumed that DIC7 depletion in the top 12.5 cm of the growing ice was due to CO2 release from the super-saturated ice to the atmosphere above. Whereas, during the melting phase we observed a DIC7 increase in the top 12.5 cm. This increase can be linked to the downward air-ice CO2 flux measured during the melting phase.l l Besides the measurements of air-ice CO2 fluxes, the amount of CO2 released to the atmosphere during the sea ice growth was assessed using a method proposed by Geilfus et al. (2013). First, a “theoretical DIC”(DICth) was calculated from the raw DIC concentration at 12.5 cm (DIC12.5 cm), assuming that, if biogeochemical processes are null, DIC and salinity (S) should follow a linear relationship: DICth, i = DIC12.5 cm · (Si / S12.5 cm) (4) (4) DICth, i = DIC12.5 cm · (Si / S12.5 cm) where DICth, i and DIC12.5 cm are computed for each sampling event and expressed in µmol kg−1. The index, i, refers to the upper two sampling depths, 2.5 cm and 7.5 cm. From the theoretical DIC, we derive the “DIC anomaly” for each sampling day (ΔDICt in mmol m−2): where DICth, i and DIC12.5 cm are computed for each sampling event and expressed in µmol kg−1. The index, i, refers to the upper two sampling depths, 2.5 cm and 7.5 cm. Figure 2 Figure 2 Air-ice CO2 fluxes, sea ice surface temperature and pCO2 over time. a) Evolution of the air-ice CO2 flux (black corresponds to a period with the chamber not properly sealed, blue to a period with a correct sealing of the chamber) and temperature 2 cm above the air-ice interface (in red). The green horizontal step line corresponds to the flux calculated from the DIC anomaly. The shaded areas refer to working hours and the related increase of atmospheric pCO2. b) Atmospheric (black line), ice surface (green triangles down) and seawater (blue triangles up) pCO2. The ice surface pCO2 corresponds to the first 5 cm of the bulk sea ice measured with the method of high vertical resolution. doi: 10.12952/journal.elementa.000112.f002 Elementa: Science of the Anthropocene • 4: 000112 • doi: 10.12952/journal.elementa.000112 6 Air-ice carbon pathways The temperature and the pCO2 air measured within the automated chamber exhibited daily variations, rising during “working hours” due to the presence of the researchers in the experimental room and decreasing outside “working hours”. The pattern of air-ice CO2 fluxes is clearly opposite and strongly affected by the rise of atmospheric pCO2 during working hours. These imperfections are inherent to the environmental constraints of this experimental study and could hardly be avoided. Nevertheless, the results can still be interpreted in terms of the main factors driving the CO2 fluxes, and the overall pattern of observed air-ice CO2 fluxes is consistent with in situ observation of a seasonal cycle, with upward CO2 fluxes during ice growth (Nomura et al., 2010a; Geilfus et al., 2012a; Delille et al., 2014) and downward CO2 fluxes during ice melt (Delille et al., 2014; Geilfus et al., 2015). Figure 3 Bulk sea ic DIC depleti a) Bulk DIC profiles nor of 7 (DIC7) refers to the doi: 10.12952/jo ir-ice carbon pathways a) Bulk DIC profiles and b) DIC profiles normalized to a salinity of 7 (DIC7), where the grey area refers to the DIC depletion zone. doi: 10.12952/journal.elementa.000112.f003 The temperature and the pCO2 air measured within the automated chamber exhibited daily variations, rising during “working hours” due to the presence of the researchers in the experimental room and decreasing outside “working hours”. The pattern of air-ice CO2 fluxes is clearly opposite and strongly affected by the rise of atmospheric pCO2 during working hours. These imperfections are inherent to the environmental constraints of this experimental study and could hardly be avoided. Figure 3 Bulk sea ice DIC and surface DIC depletion during ice growth. Figure 3 Bulk sea ice DIC and surface DIC depletion during ice growth. a) Bulk DIC profiles and b) DIC profiles normalized to a salinity of 7 (DIC7), where the grey area refers to the DIC depletion zone. doi: 10.12952/journal.elementa.000112.f003 3.2.2 Integrated estimates of air-ice CO2 fluxes From the theoretical DIC, we derive the “DIC anomaly” for each sampling day (ΔDICt in mmol m−2): (5) DDICt = S (i = 2.5 to 12.5 cm) (DICth, i – DICi ) (5) As the DIC anomaly was mainly due to the release of CO2 from the ice to the atmosphere during the present experiment (see above), integrating DIC anomalies over time gives the theoretical air-ice CO2 flux between two sampling events: As the DIC anomaly was mainly due to the release of CO2 from the ice to the atmosphere during the present experiment (see above), integrating DIC anomalies over time gives the theoretical air-ice CO2 flux between two sampling events: F = ((DDICt + Dt – DDICt ) / Dt ) · dx · r (6) F = ((DDICt + Dt – DDICt ) / Dt ) · dx · r (6) Elementa: Science of the Anthropocene • 4: 000112 • doi: 10.12952/journal.elementa.000112 Elementa: Science of the Anthropocene • 4: 000112 • doi: 10.12952/journal.elementa.000112 Air-ice carbon pathways Table 2. Gas transfer coefficient calculated for each day of the experimenta Table 2. Gas transfer coefficient calculated for each day of the experimenta Table 2. Gas transfer coefficient calculated for each day of the experimenta Day of the experiment T Bulk S K 2 −3.0 11.5 2.43 (± 0.9) 5 −5.1 8.8 2.59 (± 0.9) 8 −5.0 9.5 2.66 (± 0.9) 14 −5.8 8.0 2.22 (± 0.9) 15b −3.3 3.0 2.1 16b −2.5 6.1 2.9 19 −1.8 6.9 0.5 (± 0.08) 19 −1.8 6.9 0.3 (± 0.08) aMean ice surface temperature (T, °C), bulk salinity (Bulk S) and gas transfer coefficient for CO2 at the air-ice interface (K, mol m−2 d−1 atm−1) for each day of the experiment bRefers to the transition period doi: 10 12952/journal elementa 000112 t002 aMean ice surface temperature (T, °C), bulk salinity (Bulk S) and gas transfer coefficient for CO2 at the air-ice interface (K, mol m−2 d−1 atm−1) for each day of the experiment bRefers to the transition period doi: 10 12952/journal elementa 000112 t002 where ∆t is the time frame between two sampling events in days, dx is the distance in meters between two sampling depths, and ρ is the sea ice density for each layer defined from temperature and salinity and using relationships given by Cox and Weeks (1982), in kg m−3. 3.2.2 Integrated estimates of air-ice CO2 fluxes Based on the precision of the measured variables (salinity, temperature, TA, pCO2; see Methods section), the precision on F is assessed at ± 0.03 mmol m−2 d−1 (which represents an error of 15% for the averaged CO2 flux of 0.2 mmol m−2 d−1).l l From these DIC anomalies, the calculated air-ice CO2 fluxes (black triangles in Figure 2a) are in good agreement with the observed air-ice CO2 fluxes, except on day 16 where the difference represents more than 1 mmol m−2 d−1; integrated measurements cannot accurately capture the rapid transition from the freezing to the melting decay phase. With the exception of this transition phase, the consistency between the continuous and integrated fluxes suggest that in an indoor experiment, the automated chamber provides accurate measurements of air-ice CO2 fluxes when snow cover is absent, despite the large variation of the atmospheric pCO2. 3.3 Determination of a gas transfer coefficient for CO2 in artificial sea icefi fii Using equation 1 for gas exchange, we calculated a gas transfer coefficient for CO2 at the air-ice interface for each sampling day of the experiment (see K; Table 2), where F corresponds to the air-ice CO2 fluxes measured with the automated chamber, pCO2 air to the pCO2 at the air interface and pCO2 bulk to the pCO2 at the ice interface (0 cm to 5 cm). The numbers in light grey in Table 2 refer to the transition phase which was not taken into account in the final value computed for K. The values for K and ancillary data detailed in Table 2 are close during the whole growth period. The mean K for the sea ice growth period, from day 2 to day 14 inclusive, is K = 2.5 mol m−2 d−1 atm−1. A Bootstrap resampling statistical analysis of the propagation of the uncertainties of the measured variables (salinity, temperature, measured CO2 fluxes, pCO2 bulk and pCO2 air) gives an uncertainty for K of 0.9 mol m−2 d−1 atm−1 where standard error deviation is only 0.25 mol m−2 d−1 atm−1. For the melting phase (which corresponds to two measurements on day 19) the mean K value was 0.4 mol m−2 d−1 atm−1 (the uncertainty derived from a Bootstrap resampling statistical analysis was 0.08 mol m−2 d−1 atm−1). K was therefore 6 times higher during the growth phase than during the melting phase. In order to get a measure of gas exchange that does not depend on the gas solubility, Wanninkhof (1992) proposed to use k, the gas transfer velocity, defined from: (7) K = k. Sol where Sol (mmol m−3 atm−1) is the solubility of CO2 in salt water (here brine), a function of temperature (T) and salinity (S), following Weiss (1974): ln Sol = A1 + A2 (100/T ) + A3 ln (T/100) + S [B1 + B2 (T/100) + B3 (T/100)2] (8) ln Sol = A1 + A2 (100/T ) + A3 ln (T/100) + S [B1 + B2 (T/100) + B3 (T/100)2] 3.4 Model sensitivity experiments on the CO2 transport pathways through sea ice 3.4 Model sensitivity experiments on the CO2 transport pathways through sea ice Here we use the model to frame the CO2 flux into further theoretical considerations. The various model simulations described in Table 1 are compared to the observations, in order to understand which parameters are most influential in shaping the model response (Figure 5).l l First, the sign of the CO2 flux is consistent between observations and all model simulations. All model runs show an efflux of CO2 during the growth period and an influx during the melt period. The direction of the flux depends mostly on the dissolved gas pathway, itself driven by the air-brine pCO2 difference, ultimately set by the near-surface ice temperature evolution, which is quite well understood. In addition, some of the short-term variations of the observed CO2 fluxes are reproduced. The model gives a good estimation of downward CO2 fluxes during ice melting although the time of the onset of downward CO2 fluxes occurs earlier than observed in the model.lf By contrast, the magnitude of the air-ice CO2 flux is highly variable among the different model runs, in particular during the growth phase. The most influential but uncertain factors clearly lie within the gas bubble pathway (Figure 5c). By contrast, the dissolved pathway is found rather insensitive to parameterization choices (e.g., gas diffusivity Ddiff, boundary layer thickness zBL, vertical resolution, ikaite, etc.). In turn, the total (dissolved + bubble) air-ice CO2 flux is essentially determined by the gas bubble nucleation rate Rbub, which acts as a bottleneck parameter for the efflux of CO2 to the atmosphere via the gas bubble pathway. This bottleneck effect explains why Rbub was used as a tuning parameter, set to 10% h−1 in the CTRL run, the value that gave the best agreement with observations. g g Why is the dissolved pathway rather insensitive between the different runs? As in Moreau et al. (2015), we investigated whether the dissolved gas pathway could have been more intense than simulated. To answer this question, we tested to what extent modifying the uncertain parameters zBL and Ddiff could increase the upward CO2 fluxes during ice growth. None of our trials was successful. For example, increasing Ddiff up to 2.4 x 10−8 m2 s−1 (run 2: Loose-Ddiff) barely affects the upward CO2 fluxes during ice growth. Decreasing zBL down to 0.05 µm did not significantly increase the simulated air-ice CO2 fluxes either. ln Sol = A1 + A2 (100/T ) + A3 ln (T/100) + S [B1 + B2 (T/100) + B3 (T/100)2] (8) We used the T and S of sea ice brine. The resulting value, k = 0.164 (± 10%) cm h−1, falls within the range of values given by Liss and Merlivat (1986) for a smooth surface regime and the values given by Crusius and Wanninkhof (2003) for gas exchange over a lake at low wind speed. However, solubility as described by eq. 8 is most likely not adequate for the range of temperature and salinity encountered within sea ice, as suggested by Zhou et al. (2014a). K, the gas transfer coefficient, is solely deduced from measurements and does not depend on an uncertain solubility value.h Thin sections of the ice cores were processed following Tison et al. (2002). Bubbles were observed during the growth phase, particularly near the ice surface (Figure 4). We suggest that these bubbles (trapped at the very beginning of ice growth or newly formed) were likely moving upward due to their own buoyancy. Once Elementa: Science of the Anthropocene • 4: 000112 • doi: 10.12952/journal.elementa.000112 Air-ice carbon pathways Air-ice carbon pathways Figure 4 Thin sections o Thin sections o 2, 5, 14 and 15 The contrasted are bubbles whi features are brin doi: 10.12952/journ Air-ice carbon pathways bubbles reached the ice surface, they collapsed and CO2 was released to the atmosphere, corresponding to an ebullition flux. K-values calculated during the growth and melting phases indicate that gas transport was about 6 times faster during ice growth than during ice decay. We hypothesize that air-ice CO2 fluxes during the melting phase are driven mainly by molecular diffusion, while during the growth phase, ebullition fluxes add to molecular diffusion This hypothesis suggests that the ebullition fluxes are a dominant pathway for Figure 4 Thin sections of surface ice cores. Thin sections of ice cores for day 2, 5, 14 and 15 of the experiment. The contrasted circles and tubes are bubbles while the gray-shaded features are brine inclusions. doi: 10.12952/journal.elementa.000112.f004 Air-ice carbon pathways Figure 4 Thin sections of surface ice cores. Thin sections of ice cores for day 2, 5, 14 and 15 of the experiment. The contrasted circles and tubes are bubbles while the gray-shaded features are brine inclusions. doi: 10.12952/journal.elementa.000112.f004 bubbles reached the ice surface, they collapsed and CO2 was released to the atmosphere, corresponding to an ebullition flux. ln Sol = A1 + A2 (100/T ) + A3 ln (T/100) + S [B1 + B2 (T/100) + B3 (T/100)2] K-values calculated during the growth and melting phases indicate that gas transport was about 6 times faster during ice growth than during ice decay. We hypothesize that air-ice CO2 fluxes during the melting phase are driven mainly by molecular diffusion, while during the growth phase, ebullition fluxes add to molecular diffusion. This hypothesis suggests that the ebullition fluxes are a dominant pathway for gas transport and exchange during the growth phase. Elementa: Science of the Anthropocene • 4: 000112 • doi: 10.12952/journal.elementa.000112 Figure 4 Thin sections of surface ice cores. doi: 10.12952/journal.elementa.000112.f004 Thin sections of ice cores for day 2, 5, 14 and 15 of the experiment. The contrasted circles and tubes are bubbles while the gray-shaded features are brine inclusions. 3.4 Model sensitivity experiments on the CO2 transport pathways through sea ice This model behavior arises because the ultimate limitation for the air-ice CO2 diffusive flux is the near surface stock of DIC: supply to the near-surface DIC is not rapid enough to sustain CO2 fluxes to the observed magnitude. This result was already obtained by Moreau et al. (2015; see their Figure 9b where the simulated outward CO2 fluxes during winter at Barrow reach asymptotically 1.3 mmol m−2 d−1 when zBL is decreased down to 10−3 µm). By contrast, the gas bubble pathway is much more variable between the different runs and characterized by much larger uncertainties, because gas bubble flux can originate further down in the ice and is not limited by near-surface DIC stocks. The model-data misfit presented in Table 1 shows that the best model-observation agreement was achieved for a strong gas bubble pathway, which corresponds to high values of Rbub (10-20% h−1). Yet, the bubble nucleation rate Rbub is far from being well constrained from observations. In Moreau et al. (2014), this value was tuned to 0.1% h−1, corresponding to a characteristic time scale of 40 days, in order to match observed gas bubble concentrations over simulations spanning several months. In the present context (curve Moreau-bub in Figure 5c), such value gives a weak gas bubble pathway and an underestimation of CO2 fluxes by the model. Forty days is rather long for bubble nucleation, much larger than expected, typically less than one hour (see, e.g., Brennen, 1995). The most obvious reason why such an unrealistically large 40 day Elementa: Science of the Anthropocene • 4: 000112 • doi: 10.12952/journal.elementa.000112 Air-ice carbon pathways Figure 5 Observed and simulated air-ice CO2 fluxes. Figure 5 Observed and simulated air-ice CO2 fluxes. Observed and simulated air-ice CO2 fluxes. a) Simulated air-ice CO2 fluxes (mmol m−2 d−1) for runs Loose- Ddiff (grey, where Ddiff = 24 10−9 m2 s−1), No-bub (green, where Rbub = 0% h−1 and Ddiff = 0.97 10−9 m2 s−1) and Low-zBL (dark blue in dash, where zBL = 0.05 µm). The three curves for runs Loose-Ddiff, No- bub and Low-zBL are confounded on the plot. Observed CO2 fluxes are given in blue. b) Simulated air-ice CO2 fluxes (mmol m−2 d−1) for runs No-ikaite (dark red, where there is no ikaite precipitation) and 20-layers (dark blue, where the number layers of ice was set to 20 instead of 10). Observed CO2 fluxes are given in blue. 3.4 Model sensitivity experiments on the CO2 transport pathways through sea ice c) Simulated air-ice CO2 fluxes (mmol m−2 d−1) for runs CTRL (in black), Low-bub (grey, where Rbub = 1% h−1), High-bub (cyan, where Rbub = 20% h−1) and Moreau-bub (dark grey, Rbub = 0.1% h−1). Observed CO2 fluxes (mmol m−2 d−1) are given in blue. value had to be used by Moreau et al. (2014) is that pressure effects are missing in the model, which should quickly inhibit gas bubble nucleation once a large enough stock of gas bubbles is built up, which would typically occur for impervious sea ice over a few days. Larger Rbub values of 10–20% h−1 strongly intensify the gas bubble pathway. They also correspond to more realistic time scales for gas bubble nucleation (4–8 hours).hl g p yh y p g The influence of several other processes, known to occur in natural conditions and contribute to the ice- air CO2 fluxes, was also considered. As far as frost flowers are concerned, they were not observed in the tank experiment, and hence are not relevant to the present discussion of air-ice CO2 fluxes. The precipitation of ikaite could have influenced ice-atmosphere CO2 fluxes (e.g., Geilfus et al., 2013; Rysgaard et al., 2014); however, as discussed above, ikaite precipitation was not observed under the microscope and the model CTRL simulation predicts a rather low ikaite concentration of 13 µmol kg−1. The lack of ikaite influence is further corroborated by the model run with no ikaite precipitation (and no bubbles; Run 6, Table 1) which barely differs from the no-BUB run in terms of air-ice CO2 flux (Figure 5b).l fl We also tested the role of vertical resolution of the simulated CO2 fluxes by running the model with 20 vertical ice layers instead of 10, and with no bubbles (Figure 5b). More layers better resolve and increase the near-surface pCO2, which slightly intensifies the dissolved gas pathway during the growth phase. Elementa: Science of the Anthropocene • 4: 000112 • doi: 10.12952/journal.elementa.000112 doi: 10.12952/journal.elementa.000112.f005 3.5 Synthesis As detailed in Zhou et al. (2014b), the artificial sea ice was likely permeable to gas exchange during the whole experiment. Convection was likely only present during the growth period and limited to near the ice- water interface (see Rayleigh numbers in Figure 3 of Zhou et al., 2014b) and was nearly negligible during 10 Elementa: Science of the Anthropocene • 4: 000112 • doi: 10.12952/journal.elementa.000112 Air-ice carbon pathways the whole period of decay. We therefore consider that only diffusion and/or buoyancy processes occurred in the upper parts of the ice. Both the computation of the gas transfer coefficient and the model sensitivity analysis support this assumption. As described previously, bubbles were observed during the growth phase, particularly near the ice surface (Figure 4).h the whole period of decay. We therefore consider that only diffusion and/or buoyancy processes occurred in the upper parts of the ice. Both the computation of the gas transfer coefficient and the model sensitivity analysis support this assumption. As described previously, bubbles were observed during the growth phase, particularly near the ice surface (Figure 4).h The values of K were about 6 times lower during the melting phase as compared with the growth phase (Table 2). At this stage, based on model simulations, we cannot explain this difference without assuming a significant contribution of the gas bubble pathway during the growth of sea ice. However, this explanation is not based on direct observation of gas bubble processes but rather on inference from model simulations characterized by large uncertainties, in particular in terms of the gas bubble nucleation rate. The latter is the key tuning bottleneck parameter sourcing gas bubbles to the model gas bubble ice-atmosphere pathway. All the other surface processes that we could envision were, to the best of our present investigation capability, not able to supply enough carbon to the atmosphere. During ice melt, the model is in agreement with the much lower K values, suggesting that air-ice CO2 fluxes are dominated by the dissolved pathway, apart from the short under-sampled transition period when other processes such as episodic convection events might have taken place. Using our best model run (CTRL), we compared the contribution of all processes based on the computation of the model DIC budget, following Moreau et al. (2015; see Figure 6). Sea ice is gaining DIC throughout its growth (Figure 6a). doi: 10.12952/journal.elementa.000112.f006 3.5 Synthesis Large quantities of DIC are incorporated into sea ice by growth, although 80% of this DIC is rejected through brine drainage (Figure 6b). Ikaite precipitation does not significantly contribute to the budget of DIC. Ice-atmosphere CO2 fluxes (Figure 6c) are dominated by the escape of gas bubbles during ice growth. However, only the dissolved pathway affects CO2 fluxes during ice melt. As suggested by Moreau et al. (2015) the budget of DIC in sea ice is driven mainly by physics while biogeochemical processes (only chemical here), although significant, are secondary. y g gi y Based on these considerations, we expand to ice-atmosphere exchanges of CO2 the suggestion that gas bubbles provide important contributions to the stocks of gaseous compounds in sea ice (Mock, 2002; Tison et al., 2002; Rysgaard and Glud, 2004; Zhou et al., 2013; Moreau et al., 2014). The conceptual view that we propose is based on three key arguments. 1. Gas bubbles easily form in sea ice because brine shrinking with temperature induces drastic increases in gas concentrations, and because there is a net decrease in gas solubility with decreasing temperature in brine inclusions. 2. Gas bubbles in the liquid brine rise upward if the connectivity of the brine network is sufficient. 3. The dilution of brines during warming brings CO2 concentration in brine below saturation, which brings trapped gas bubbles back into dissolved form. Because there is no analytical method to assess the partitioning of gases between dissolved and gaseous forms in sea ice brine, it is difficult for the time being to confirm this conceptual view directly. In natural sea ice, other surface processes likely affect air-ice CO2 fluxes as well (e.g., the formation of frost flowers, brine skim; Geilfus et al., 2013; Barber et al., 2014). In the present experiments, however, they were not observed. 4. Conclusions The first aim of this study was to determine experimentally the air-ice CO2 transfer coefficient from continuous CO2 flux measurements during an ice growth and decay cycle in an ice-tank experiment. Discrete measurements of air-ice pCO2 gradients and DIC anomalies reflected well the amplitude and the patterns of the air-ice CO2 fluxes, supporting the reliability of our methods and results, including the calculation of a bulk gas transfer coefficient. The second aim was to discriminate the different drivers of air-ice CO2 fluxes using a 1D sea ice carbon cycle model (Vancoppenolle et al., 2010; Moreau et al., 2015), including explicit empirical representations of dissolved gas and gas bubble ice-atmosphere pathways and testing for several gases (Ar, O2, CO2) at a few locations.hihfi There are three key findings in the paper. 1. The observation-based gas transfer coefficient, retrieved by dividing the observed CO2 flux by the air-brine pCO2 difference, was ~6 times higher during growth (K = 2.5 mol m−2 d−1 atm−1) than during melt (K = 0.4 mol m−2 d−1 atm−1). 2. The time evolution of the sign of the air-ice CO2 flux, characterized by an efflux from the ice during growth and an influx during melting, was consistent between observations and the nine model simulations, as well as with previous literature (Nomura et al., 2010a; Geilfus et al., 2012a; Delille et al., 2014; Geilfus et al., 2015). Such evolution must therefore be seen as typical and robust. 3. The magnitude of the observed CO2 flux is consistent with previous literature but not between the different model simulations. The simulated dissolved CO2 flux clearly underestimates the observed value because of an intrinsic limitation by DIC stocks in the model already identified by Moreau et al. (2015). The observed magnitude of the ice-air CO2 flux can only be reached in the model by invoking an intense escape of gas bubbles through an ice-atmosphere pathway. Such an intense pathway was achieved by tuning the gas bubble nucleation time scale down to a few hours, which contrasts with the value of a few weeks used in a previous 6-month simulation of Argon dynamics in natural sea ice (Moreau et al., 2014).i Based on these concordant findings, we infer that the gas bubble ice-atmosphere pathway is likely a significant contributor to air-ice flux in young growing sea ice, next to diffusion. Air-ice carbon pathways Finally let us stress that there are only a few processes able to sustain enhanced CO2 fluxes during a long period. The presence of frost flowers would be transient and have only short-term effects on CO2 fluxes. Other processes (e.g., melting/freezing cycle of the ice surface) modulate air-ice CO2 fluxes, but few of them can actually sustain CO2 fluxes for extended periods of time as we observed during the experiment. Processes that sustain CO2 for extended periods of time must involve either the production of CO2, like bacterial respiration, or the transfer of CO2 from a large reservoir (like the ice interior or the ocean) to the surface. 4. Conclusions This intense gas bubble pathway in growing sea ice proposed here is plausible at this stage but subject to caution, because it does not rely on direct process observations, but rather on inference from air-ice CO2 flux and a single sea ice model, characterized by a number of assumptions. Further evaluation of the proposed scenario and reduction of uncertainties rely on improvements of both observation and modelling techniques. Observations of gas bubbles from X-ray tomography (Crabeck et al., 2016) provide new insights on gas bubble size and number distribution, and might be used to document the upward migration of gas bubbles. Future models should include at least pressure effects on gas bubble nucleation and perhaps tortuosity effects on gas bubble rise, as well as improved representations of brine dynamics (e.g., Griewank and Notz, 2013; Turner et al., 2013; Rees Jones and Worster, 2014).h The multiphase nature of CO2 in sea ice is well established, with contributions of dissolved species, gas bubbles (Tison et al., 2002; Zhou et al., 2013; Crabeck et al., 2014a; Moreau et al., 2014, 2015) and ikaite crystals (Papadimitriou et al., 2004; Dieckmann et al., 2008), a solid inorganic compound that contains inorganic carbon. This multiphase nature of CO2 is, to our best knowledge, a unique feature in marine environments. Clarifying the partitioning of CO2 between the gaseous and dissolved phases and the gas transport pathways, as well as the contribution of surface ice processes, is a critical challenge for future research related to gas dynamics and the carbon cycle in ice-covered seas. Elementa: Science of the Anthropocene • 4: 000112 • doi: 10.12952/journal.elementa.000112 Figure 6 CO2 budget. 11 Elementa: Science of the Anthropocene • 4: 000112 • doi: 10.12952/journal.elementa.000112 Figure 6 CO2 budget. a) Daily budget of vertically integrated DIC (TDIC, mmol m−2) dissolved into CO2 (black), CO3 2- (light grey), and HCO3- (grey) for the CTRL run. b) Corresponding daily mean changes (mmol m−2 day−1) in TDIC due to total sea ice growth and melt (dark blue), brine drainage (light blue), and ikaite precipitation/dissolution (red). c) Corresponding daily mean changes (mmol m−2 day−1) in TDIC due to diffusive CO2 fluxes (light blue) and gas bubble fluxes (dark blue). doi: 10.12952/journal.elementa.000112.f006 a) Daily budget of vertically integrated DIC (TDIC, mmol m−2) dissolved into CO2 (black), CO3 2- (light grey), and HCO3- (grey) for the CTRL run. b) Corresponding daily mean changes (mmol m−2 day−1) in TDIC due to total sea ice growth and melt (dark blue), brine drainage (light blue), and ikaite precipitation/dissolution (red). c) Corresponding daily mean changes (mmol m−2 day−1) in TDIC due to diffusive CO2 fluxes (light blue) and gas bubble fluxes (dark blue). a) Daily budget of vertically integrated DIC (TDIC, mmol m−2) dissolved into CO2 (black), CO3 2- (light grey), and HCO3- (grey) for the CTRL run. b) Corresponding daily mean changes (mmol m−2 day−1) in TDIC due to total sea ice growth and melt (dark blue), brine drainage (light blue), and ikaite precipitation/dissolution (red). c) Corresponding daily mean changes (mmol m−2 day−1) in TDIC due to diffusive CO2 fluxes (light blue) and gas bubble fluxes (dark blue). doi: 10.12952/journal.elementa.000112.f006 Elementa: Science of the Anthropocene • 4: 000112 • doi: 10.12952/journal.elementa.000112 11 11 Air-ice carbon pathways References Mar Chem 51(2): 81–91.h Golden KM, Ackley SF, Lytle VI. 1998. 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Contributions • Lead of the INTERICE V experiment: DT • Lead of the INTERICE V experiment: DT p • Contributed to planning and experimental design: DT, GD, K-UE, J-LT, BD • Contributed to planning and experimental design: DT, GD, K-UE, J-LT, BD C b d h d f d Z G p p • Contributed to carry out the experiment and acquisition of data: JZ, GD, K-UE, DT, J-LT, BD • Contributed to carry out the experiment and acquisition of data: JZ, GD, K-UE, DT, J-LT, BD y p q J J • Contributed to analysis and interpretation of data: MK, SM, JZ, FVdL, J-LT, BD, MV Contributed to analysis and interpretation of data: MK, SM, J • Contributed to analysis and interpretation of data: MK, SM, JZ, FVdL, J-LT, BD, M C b d h d l l d SM MV • Contributed to the model simulations and interpretation: SM, MV p MK wrote the paper, with the help of SM, JZ, BD, MV and comments and inputs from all coauthors. • MK wrote the paper, with the help of SM, JZ, BD, MV and comments and inputs from all coauthors. Acknowledgmentsh The authors would like to thank the Hamburg Ship Model Basin (HSVA), and the ice tank crew, for the hospitality, technical and scientific support and the professional execution of the test program in the Research Infrastructure ARC- TICLAB. We are grateful to Michael Fischer for his help with the deployment of the automated chambers and treatment of flux data, to Nicolas-Xavier Geilfus, one anonymous reviewer, the Editor Jody Deming and the associate editor for their comments that improve the overall quality of the manuscript. References Carbonate system evolution at the Arctic Ocean surface during autumn freeze-up J Geophys Res Oceans 116(C9) LA, Carnat G, Else BGT, Sutherland N, Papakyriakou TN. 2011a. Carbonate system evolution at the Arctic Ocean rface during autumn freeze-up. J Geophys Res-Oceans 116(C9).l Miller LA, Carnat G, Else BGT, Sutherland N, Papakyriakou TN. 2011 surface during autumn freeze-up. J Geophys Res-Oceans 116(C9). iller LA, Carnat G, Else BGT, Sutherland N, Papakyriakou TN. 2011a. Carbonate system evolution at the Arctic Ocean surface during autumn freeze-up. J Geophys Res-Oceans 116(C9).l p y y surface during autumn freeze-up. J Geophys Res-Oceans 116(C9).l p y surface during autumn freeze-up. J Geophys Res-Oceans 116(C9). g p p y Miller LA, Papakyriakou TN, Collins RE, Deming JW, Ehn JK, et al. 2011b. Carbon dynamics in sea ice: A winter flux time series. J Geophys Res-Oceans 116(C2). p y Mock T. 2002. In situ primary production in young Antarctic sea ice. Hydrobiologia 470(1–3): 127–132. Moreau S, Vancoppenolle M, Delille B, Tison J-L, Zhou J, et al. 2015. Drivers of inorganic carbon dynamics in first-ye Moreau S, Vancoppenolle M, Delille B, Tison J-L, Zhou J, et al. 2015. Drivers of inorganic carbon dynam sea ice : A model study J Geophys Res-Oceans 120(1): 471–495 doi: 10 1002/2014JC010388 Moreau S, Vancoppenolle M, Delille B, Tison J-L, Zhou J, et al. 2015. Drivers of inorganic carbon dynamics in first-year sea ice : A model study. J Geophys Res-Oceans 120(1): 471–495. doi: 10.1002/2014JC010388. Moreau S, Vancoppenolle M, Delille B, Tison J L, Zhou J, et al. 2015. Drivers of inorganic carbon dynamics in first year sea ice : A model study. J Geophys Res-Oceans 120(1): 471–495. doi: 10.1002/2014JC010388.i y p y Moreau S, Vancoppenolle M, Zhou J, Tison J-L, Delille B, et al. 2014. Modelling argon dynamics in first-year sea ice. Ocean Modelling 73: 1–18. 13 Elementa: Science of the Anthropocene • 4: 000112 • doi: 10.12952/journal.elementa.000112 13 Air-ice carbon pathways Nomura D, Eicken H, Gradinger R Shirasawa K. 2010a. Rapid physically driven inversion of the air–sea ice CO2 flux in the seasonal landfast ice off Barrow, Alaska after onset of surface melt. Cont Shelf Res 30(19): 1998–2004.hfl f f Nomura D, Inoue HY, Toyota T. 2006. The effect of sea ice growth on air–sea CO2 flux in a tank experiment. Tellus B 58(5): 418–426. Nomura D, Inoue HY, Toyota T Shirasawa K. 2010b. References Zhou J, Delille B, Kaartokallio H, Kattner G, Kuosa H, et al. 2014b. Physical and bacterial controls on inorganic nutrients and dissolved organic carbon during a sea ice growth and decay experiment. Mar Chem 166: 59–69. Zhou J, Delille B, Eicken H, Vancoppenolle M, Brabant F, et al. 2013. Physical and biogeochemical properties in landfast sea ice (Barrow, Alaska): Insights on brine and gas dynamics across seasons. J Geophys Res-Oceans 118(6): 3172–3189. Zhou J, Delille B, Kaartokallio H, Kattner G, Kuosa H, et al. 2014b. Physical and bacterial controls on inorganic nutrients and dissolved organic carbon during a sea ice growth and decay experiment. Mar Chem 166: 59–69. Zhou J, Delille B, Eicken H, Vancoppenolle M, Brabant F, et al. 2013. Physical and biogeochemical properties in landfast sea ice (Barrow, Alaska): Insights on brine and gas dynamics across seasons. J Geophys Res-Oceans 118(6): 3172–3189. Zhou J Delille B Kaartokallio H Kattner G Kuosa H et al 2014b Physical and bacterial controls on inorganic nutrients References Cryosphere 7(2): 707–718.h p y y y p Rysgaard S, Wang F, Galley RJ, Grimm R, Notz D, et al. 2014. Temporal dynamics of ikaite in experimental sea ice. The Cryosphere 8(4): 1469–1478. doi: 10.5194/tc-8-1469-2014. y p Sarmiento JL, Gruber N. 2004. Ocean Biogeochemical Dynamics. Princeton University Press.h Semiletov I, Makshtas A, Akasofu S, Andreas EL. 2004. Atmospheric CO2 balance: The role of Arctic sea ice. Geophys Res Lett 31(5): doi: 10.1029/2003GL017996.hf ( ) Shaw MD, Carpenter LJ, Baeza-Romero MT, Jackson AV. 2011. Thermal evolution of diffusive transport of atmospher halocarbons through artificial sea–ice. Atmos Environ 45(35): 6393–6402. doi: 10.1016/j.atmosenv.2011.08.023. haw MD, Carpenter LJ, Baeza-Romero MT, Jackson AV. 2011. Thermal evolution of diffusive transport of atmospheric i , p J, , Jhf p p halocarbons through artificial sea–ice. 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Technical Note: On the use of the mushy-layer Rayleigh number for the interpretation of sea-ice-core data. The Cryosphere Discussions 7(4): 3209–3230. h Wanninkhof R. 1992. Relationship between wind speed and gas exchange over the ocean. J Geophys Res-Oceans 97(C5): 7373–7382. s RF. 1974. Carbon dioxide in water and seawater: The solubili h Zhou J, Delille B, Brabant F, Tison J-L. 2014a. Insights into oxygen transport and net community production in sea ice from oxygen, nitrogen and argon concentrations. Biogeosciences Discussions 11(2): 2045–2081. yg g g g Zhou J, Delille B, Eicken H, Vancoppenolle M, Brabant F, et al. 2013. Physical and biogeochemical properties in landfast sea ice (Barrow, Alaska): Insights on brine and gas dynamics across seasons. J Geophys Res-Oceans 118(6): 3172–3189. Data accessibility statementh The observation data and the model code will be made available to any scientist upon request and which is encouraged in order to reproduce the computations and test the model results in other locations (contacts: marie.kotovitch@ulg.ac.be, s.moreau@uclouvain.be and bruno.delille@ulg.ac.be). Funding informationh This study was supported by the European Community's 7th Framework Programme through the grant to the budget of the Integrated Infrastructure Initiative HYDRALAB-IV, Contract no. 261520. The work was supported by a FiDiPro award by the Academy of Finland, the Walter and Andree Nottbeck Foundation, and the BIGSOUTH project funded by the Belgian Science Federal Policy Office. MK and JZ, SM and BD are respectively research fellows, postdoctoral researcher and research associate of the Fonds de la Recherche Scientique – FNRS. MV acknowledges support from the EU, through BISICLO (FP7 CIG grant no 321938). This is a MARE contribution. 14 Elementa: Science of the Anthropocene • 4: 000112 • doi: 10.12952/journal.elementa.000112 Air-ice carbon pathways Competing interests The authors have declared that no competing interests exist. Elementa: Science of the Anthropocene • 4: 000112 • doi: 10.12952/journal.elementa.000112 Copyright © 2016 Kotovitch et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 15 Elementa: Science of the Anthropocene • 4: 000112 • doi: 10.12952/journal.elementa.000112 15
https://openalex.org/W2050874614	https://www.pure.ed.ac.uk/ws/files/21171975/07029702.pdf	English	null	Spectral and Energy Efficiency Analysis for Cognitive Radio Networks	IEEE transactions on wireless communications	2,015	cc-by	14,026	Edinburgh Research Explorer General rights C i h f h General rights Copyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights. Spectral and Energy Efficiency Analysis for Cognitive Radio Networks Citation for published version: Haider, F, Wang, C-X, Haas, H, Hepsaydir, E, Ge, X & Yuan, D 2015, 'Spectral and Energy Efficiency Analysis for Cognitive Radio Networks', IEEE Transactions on Wireless Communications, vol. 14, no. 6, pp. 2969-2980. https://doi.org/10.1109/TWC.2015.2398864 Citation for published version: Haider, F, Wang, C-X, Haas, H, Hepsaydir, E, Ge, X & Yuan, D 2015, 'Spectral and Energy Efficiency Analysis for Cognitive Radio Networks', IEEE Transactions on Wireless Communications, vol. 14, no. 6, pp. 2969-2980. https://doi.org/10.1109/TWC.2015.2398864 Digital Object Identifier (DOI): 10.1109/TWC.2015.2398864 Link: Link to publication record in Edinburgh Research Explorer Document Version: Publisher's PDF, also known as Version of record Published In: IEEE Transactions on Wireless Communications I. INTRODUCTION T T HE continuous evolution of wireless communication with more sophisticated technologies has had a massive impact on changing how people on the globe can communicate with each other in all aspects of life including business operations, individuals, and society. There is an increasing number of smart phones and laptops every year. All of them are demanding advanced multimedia and high data rate services. More and more people crave better Internet access on the move resulting in a boundary-less global information world. One way to meet the continuously increasing demand for high-speed data is to secure new spectrum bands. However, achieving this is a very difﬁcult task as the spectrum is a rare resource. Hence, the radio spectrums are congested and there are limited new spectrum bands available for wireless uses. Despite this fact, the federal communications commission (FCC) has reported that a signiﬁcant amount of the radio spectrum is underutilized during the day [1]. This ignited the research activities to improve the usage of the highly sought-after radio spectrum and as a result, the cognitive radio (CR) concept has been proposed [2], [3]. Manuscript received March 2, 2014; revised July 16, 2014 and November 12, 2014; accepted January 8, 2015. Date of publication February 3, 2015; date of current version June 6, 2015. The authors gratefully acknowledge the support of this work from the International Science & Technology Coopera- tion Program of China (No. 2014DF11640), the 863 project in 5G wireless networking (No. 2014AA01A701), Ministry of Science and Technology of China, the Opening Project of the Key Laboratory of Cognitive Radio and Information Processing (Guilin University of Electronic Technology), Ministry of Education (No. 2013KF01), EU FP7 QUICK project (No. PIRSES-GA- 2013-612652), Hutchison 3G U.K., EPSRC under Established Career Fellow- ship Grant EP/K008757/1, Major International Joint Research Project of the National Natural Science Foundation of China (NSFC) (No. 61210002), EU FP7-PEOPLE-IRSES, project acronym WiNDOW (No. 318992) and project acronym CROWN (No. 610524). The associate editor coordinating the review of this paper and approving it for publication was M. Bennis. CR is an innovative radio technique that aims to utilize the radio spectrum more efﬁciently by intelligently exploiting the licensed spectrum. Hence, a CR network, i.e., secondary network, shares radio spectrum owned by a licensed network, i.e., primary network. Spectral and Energy Efﬁciency Analysis for Cognitive Radio Networks Fourat Haider, Cheng-Xiang Wang, Senior Member, IEEE, Harald Haas, Member, IEEE, Erol Hepsaydir, Xiaohu Ge, Senior Member, IEEE, and Dongfeng Yuan, Senior Member, IEEE Fourat Haider, Cheng-Xiang Wang, Senior Member, IEEE, Harald Haas, Member, IEEE, Erol Hepsaydir Xiaohu Ge, Senior Member, IEEE, and Dongfeng Yuan, Senior Member, IEEE between the secondary transmitter and PRs) have no impact on the minimum energy efﬁciency. Abstract—Cognitive radio (CR) is considered one of the promi- nent techniques for improving the utilization of the radio spec- trum. A CR network (i.e., secondary network) opportunistically shares the radio resources with a licensed network (i.e., primary network). In this work, the spectral-energy efﬁciency trade-off for CR networks is analyzed at both link and system levels against varying signal-to-noise ratio (SNR) values. At the link level, we analyze the required energy to achieve a speciﬁc spectral efﬁciency for a CR channel under two different types of power constraint in different fading environments. In this aspect, besides the transmit power constraint, interference constraint at the primary receiver (PR) is also considered to protect the PR from a harmful inter- ference. Whereas at the system level, we study the spectral and energy efﬁciency for a CR network that shares the spectrum with an indoor network. Adopting the extreme-value theory, we are able to derive the average spectral and energy efﬁciency of the CR network. It is shown that the spectral efﬁciency depends upon the number of the PRs, the interference threshold, and how far the secondary receivers (SRs) are located. We characterize the impact of the multi-user diversity gain of both kinds of users on the spectral and energy efﬁciency of the CR network. Our analysis also proves that the interference channels (i.e., channels Index Terms—Cognitive radio networks, spectral efﬁciency, en- ergy efﬁciency, extreme-value theory, multi-user diversity gain. Manuscript received March 2, 2014; revised July 16, 2014 and November 12, 2014; accepted January 8, 2015. Date of publication February 3, 2015; date of current version June 6, 2015. The authors gratefully acknowledge the support of this work from the International Science & Technology Coopera- tion Program of China (No. 2014DF11640), the 863 project in 5G wireless networking (No. 2014AA01A701), Ministry of Science and Technology of China, the Opening Project of the Key Laboratory of Cognitive Radio and Information Processing (Guilin University of Electronic Technology), Ministry of Education (No. 2013KF01), EU FP7 QUICK project (No. PIRSES-GA- 2013-612652), Hutchison 3G U.K., EPSRC under Established Career Fellow- ship Grant EP/K008757/1, Major International Joint Research Project of the National Natural Science Foundation of China (NSFC) (No. 61210002), EU FP7-PEOPLE-IRSES, project acronym WiNDOW (No. 318992) and project acronym CROWN (No. 610524). The associate editor coordinating the review of this paper and approving it for publication was M. Bennis. F. Haider is with the Joint Research Institute for Signal and Image Pro- cessing, School of Engineering and Physical Sciences, Heriot-Watt University, Edinburgh EH14 4AS, U.K. (e-mail: fsh12@hw.ac.uk). This work is licensed under a Creative Commons Attribution 3.0 License. For more information, see http://creativecommons. er a Creative Commons Attribution 3.0 License. For more information, see http://creativecommons.org/licenses/by/3.0/ Take down policy Take down policy The University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorer content complies with UK legislation. If you believe that the public display of this file breaches copyright please contact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately and investigate your claim. Download date: 24. Oct. 2024 2969 IEEE TRANSACTIONS ON WIRELESS COMMUNICATIONS, VOL. 14, NO. 6, JUNE 2015 I. INTRODUCTION The secondary network is authorized of dynamically and autonomously adapting its radio operating parameters to coexist with the primary network, providing that the performance primary network is protected or above a certain level of quality. CR networks can be classiﬁed under two cat- egories, namely interference-free and interference-tolerant CR networks [4]. In the former CR networks, secondary transmit- ters (STs) can only use those spectrums which are not occupied by primary receivers (PRs) [5], [6]. Whereas in the latter CR networks, the STs can share the spectrum as long as they do not cause any outage to the primary network operation and the interference to PRs is kept below a threshold [7]. Therefore, it is essential that interference-tolerant CR systems acquire the interference level, in real-time feedback, from the PRs. To this end, some modiﬁcation on the primary system is unavoidable. In this paper, we focus on the spectral and energy efﬁciency for the interference-tolerant CR networks. The spectral efﬁciency is deﬁned as the number of bits per second transmitted over F. Haider is with the Joint Research Institute for Signal and Image Pro- cessing, School of Engineering and Physical Sciences, Heriot-Watt University, Edinburgh EH14 4AS, U.K. (e-mail: fsh12@hw.ac.uk). C.-X. Wang is with the Joint Research Institute for Signal and Image Pro- cessing, School of Engineering and Physical Sciences, Heriot-Watt University, Edinburgh EH14 4AS, U.K., and also with the School of Information Science and Engineering, Shandong University, Jinan 250100, China (e-mail: cheng- xiang.wang@hw.ac.uk). H. Haas is with the Joint Research Institute for Signal and Image Processing, Institute for Digital Communications, University of Edinburgh, Edinburgh EH9 3JL, U.K. (e-mail: h.haas@ed.ac.uk). E. Hepsaydir is with Network Technology, Hutchison 3G, U.K., Maidenhead SL6 1EH, U.K. (e-mail: erol.hepsaydir@three.co.uk). E. Hepsaydir is with Network Technology, Hutchison 3G, U.K., Maidenhead SL6 1EH, U.K. (e-mail: erol.hepsaydir@three.co.uk). X. Ge is with the School of Electronic Information and Communications, Huazhong University of Science and Technology, Wuhan 430074, China (e-mail: xhge@mail.hust.edu.cn). D. Yuan is with the School of Information Science and Engineering, Shandong University, Jinan 250100, China (e-mail: dfyuan@sdu.edu.cn). D. Yuan is with the School of Information Science and Engineering, Shandong University, Jinan 250100, China (e-mail: dfyuan@sdu.edu.cn). Color versions of one or more of the ﬁgures in this paper are available online at http://ieeexplore.ieee.org. Color versions of one or more of the ﬁgures in this paper are available online at http://ieeexplore.ieee.org. I. INTRODUCTION 1) We extend the work of [30] to compare the spectral- energy efﬁciency trade-off in the low and high SNR regimes when transmitting a signal under average power constraint with transmitting a signal under peak power constraint while keeping the interference on primary re- ceiver below an acceptance level for both. 2) We propose a CR-based cellular network where a sec- ondary network shares a spectrum that belongs to an indoor system. The spectral efﬁciency for the proposed network with multiple primary and secondary users is analyzed using extreme value theory. The analysis will highlight the impact of the multi-user diversity gain of both the primary and secondary users on the achievable spectral efﬁciency. 3) A general analytical framework to evaluate the energy- spectral efﬁciency trade-off of CR-based cellular network is established for all SNR values using peak-power in- terference constraint. The framework takes into account the numbers of primary and secondary receivers, transmit power, and interference threshold. The remainder of this paper is organized as follows. Section II introduces two analytical tools to study the spectral- energy efﬁciency trade-off in low and high SNR regimes. Section III describes the link-level model of the proposed study and analyzes the resulting link-level spectral-energy efﬁciency trade-off, along with numerical results and discussions. The system-level spectral efﬁciency and energy efﬁciency of mul- tiple cognitive links are subsequently derived in Section IV. Finally, conclusions are drawn in Section V. g y p y Energy-efﬁcient communication, or as it is globally well- known as Green Radio [15], has been attracting more and more attention in various societies. Studying the energy con- sumption is crucial to ﬁnd efﬁcient strategies that minimize the carbon footprint from wireless networks and its impact on the environment [16]–[18]. Jointly attaining both enhanced energy efﬁciency and spectral efﬁciency is unfortunately a challenging problem to solve. Often, achieving enhancement of one of them means sacriﬁcing the other. Therefore, it is important to study different trade-offs between the two perfor- mance indicators to decide the minimum energy consumption that is required to achieve the target spectral efﬁciency, or vice versa [20]–[22]. Two analytical tools used to analyze the spectral-energy efﬁciency trade-off for any given wireless networks were proposed in [19] and [23] in low and high signal- to-noise ratio (SNR) regimes, respectively. These tools were used to analyze the energy consumption in different network scenarios [24]–[30]. I. INTRODUCTION p p g Digital Object Identiﬁer 10.1109/TWC.2015.2398864 This work is licensed under a Creative Commons Attribution 3.0 License. For more information, see http://creativeco IEEE TRANSACTIONS ON WIRELESS COMMUNICATIONS, VOL. 14, NO. 6, JUNE 2015 2970 a given bandwidth (in bps/Hz) while the energy efﬁciency is deﬁned as the required energy per bit (in joules/bit) for reliable communication, normalized to the background noise level. tolerant link-level CR network under an assumption of average transmit power constraint. To extend this work further, we will compare the outcomes of [30] with a different CR power policy. Furthermore, to the best of our knowledge, no other research has considered similar analysis in a system-level CR network with multiple primary and secondary receivers. Therefore, our novel contributions are summarized as follows: There are various studies that analyzed the spectral efﬁciency of CR networks at the link level [8]–[11] as well as at the system level [12]–[14]. For link-level CR networks, the spectral efﬁ- ciency for additive white Gaussian noise (AWGN) channels was derived in [8] under an assumption of average power constraint. In [9], the spectral efﬁciency of a CR channel was analyzed against various fading channel distributions. The authors of [10] analyzed the spectral efﬁciency of a CR channel under different power allocation policies. In [11], ergodic and outage capacities of CR channels were evaluated under both peak and average interference power constraints. The spectral efﬁciency for both the link level and system level cooperative CR networks was studied in [12]. However, the power control of STs did not consider the interference threshold that the PRs can tolerate. In [13], the authors derived the average throughput of a system level CR network and studied its asymptotic behavior. However, the analysis was limited to a single PR. The spectral efﬁciency for hybrid CR networks was studied in [14] under average interference power constraint. Hybrid CR allows a network to be simultaneously both primary and secondary networks, thus gaining the advantages of both networks. Hybrid CR networks can be adopted in cellular networks to explore additional bands and enhance the spectral efﬁciency. It is noticeable that all the aforementioned studies focused on analyzing the spectral efﬁciency but neglected to study the spectral-energy efﬁciency trade-off which is an increasingly important area nowadays. I. INTRODUCTION Using the low-SNR tool, the interplay of the spectral and energy efﬁciency was studied for single-user multiple-input multiple-output (MIMO) channels [24], single- user relay channels [25], [26], and multi-user relay channels [27]. The authors of [28], [29] used the high-SNR tool to analyze the energy efﬁciency of MIMO channels. The works of [24]–[28], [30] can only be applied to primary networks where the transmit power and spectral efﬁciency depend on the characteristics of the PRs’ channels only. In CR networks, however, the network performance is characterized by both the primary and secondary receivers’ channels. In [30], we investi- gated the spectral-energy efﬁciency trade-off in an interference- II. SPECTRAL-ENERGY EFFICIENCY TRADE-OFF UNDER LOW AND HIGH SNR REGIMES In this section, we summarize two tools that analyze the con- sumed energy per transmitted bit for a given spectral efﬁciency in low and high SNR regimes [23]. The spectral efﬁciency, C, here refers to the number of bits per second transmitted over a given bandwidth (in bps/Hz). The energy efﬁciency, Eb N0 , is deﬁned as the required energy per bit (in joules/bit) normalized to the background noise power N0 for reliable communications. B. High SNR Regime penalty the power offset in dB with respect to a reference channel having the same high SNR regime slope but with an unfaded channel (i.e., AWGN) and it is calculated by [23] Eb N0 penalty = lim SNR→∞ log2(SNR) − ¯C(SNR) S∞ . (6) (6) A. Fading Channels With Average Transmit Power Constraint where ˙¯C(0) and ¨¯C(0) are the ﬁrst and second derivative, respectively, when SNR = 0. If we consider a CR channel under the average transmit power and peak interference power constraints, the spectral efﬁciency in this case can be calculated by [10] A. Low SNR Regime In the low SNR regime, Eb N0 can be approximated as an afﬁne function with respect to the spectral efﬁciency and can be expressed by [19] Eb N0 dB = Eb N0 min + 3 S0 C (1) (1) HAIDER et al.: SPECTRAL AND ENERGY EFFICIENCY ANALYSIS FOR COGNITIVE RADIO NETWORKS 2971 where Eb N0 min is the minimum energy efﬁciency required for transmitting information reliably over a channel and it is given by knowledge of the instantaneous channel status information (CSI) for the cognitive and interference channels. It is further assumed that the interference from the primary transmitter (PT) to the SR is considered as background noise [9], [10]. There are two constraints that the ST has to take into the account before transmitting a signal to the SR. The ﬁrst constraint is the allowable received peak interference power at the PR. This constraint is essential in CR networks to avoid harmful interference at the PR. The second constraint is the available transmit power that the ST has. In this paper, we consider two types of power constraint which are the average and peak transmit power constraints. Eb N0 min = lim SNR→0 SNR ¯C(SNR). (2) (2) min Here, ¯C(SNR) is the spectral efﬁciency as a function of SNR. In (1), S0 is the wideband slope of the spectral efﬁciency, deﬁned as the increase of bits per second per hertz per 3 dB (bps/Hz/ (3 dB)), and can be expressed by [19] Here, ¯C(SNR) is the spectral efﬁciency as a function of SNR. In (1), S0 is the wideband slope of the spectral efﬁciency, deﬁned as the increase of bits per second per hertz per 3 dB (bps/Hz/ (3 dB)), and can be expressed by [19] S0 = 2 ˙¯C(0) −¨¯C(0) (3) (3) B. High SNR Regime C = max γs(gc,gi)≥0 E log2 1 + gcγs(gc, gi) N0 (7) s.t. E [γs(gc, gi)] ≤γavg (8) giγs(gc, gi) (9) (7) In the high SNR regime, the required energy efﬁciency to obtain a speciﬁc spectral efﬁciency can be expressed by [23] (8) (9) (8) (8) (9) Eb N0 dB ≈C S∞ 10 log10 2 −10 log(C) Eb N0 dB ≈C S∞ 10 log10 2 −10 log(C) + Eb N0 penalty 10 log10 2 (4) (9) where γs(gc, gi), γavg, and Q are the instantaneous transmit power, allowed average transmit power, and peak received interference power that the PR can tolerate, respectively, and E[.] denotes the statistical expectation. The optimum power allocation can then be expressed by + Eb N0 penalty 10 log10 2 (4) where S∞is the slope of the spectral efﬁciency in the high SNR regime in bps/Hz/(3 dB) and is given by [23] γ∗ s(gc, gi) = min 1 γ0 −N0 gc + , Q gi (10) S∞= lim SNR→∞SNR ˙¯C(SNR). (5) (5) (10) In (4), Eb N0 p In (4), Eb N0 penalty is the horizontal penalty which represents the power offset in dB with respect to a reference channel having the same high SNR regime slope but with an unfaded channel (i.e., AWGN) and it is calculated by [23] ty is the horizontal penalty which represents where γ0 is the water-ﬁlling cutoff value that can be calculated from the constraint in (8) and (x)+ = max{0, x}. Numerical optimization is required to get the optimum value of γ0 [30]. Unlike in the primary network, where only the CSI of the PR is required at the PT, the CSIs of both the PR and SR are needed at the ST as inputs for the power allocation algorithm. Depending upon the gains of the two types of channel, the CR transmission resides in different modes. No communication is allowed as long as the CR channel gain is below the cutoff value, i.e., gc ≤γ0. The classical water ﬁlling algorithm can be adopted if 1 γ0 −N0 gc ≤Q gi . If 1 γ0 −N0 gc > Q gi , the transmit power is equal to Q gi . B. Fading Channels With Peak Transmit Power Constraint The optimum power allocation in this case is equal to γ∗ s(gc, gi) = min γpk, Q gi (14) (14) where γpk is the peak transmit power of the ST. Unlike (10), only gi is required as input for power policy. That makes it more straight forward as it only requires the CSI of the interference channel as an input. The minimum energy efﬁciency can be calculated by where γpk is the peak transmit power of the ST. Unlike (10), only gi is required as input for power policy. That makes it more straight forward as it only requires the CSI of the interference channel as an input. The minimum energy efﬁciency can be calculated by Eb N0 min = lim γpk→0 E [γ∗ s(gc, gi)] N0E log2 1 + γ∗ s (gc,gi)gc N0 . (15) (15) In the high SNR regime, Fig. 1(b) shows the spectral-energy efﬁciency trade-off when gc and gi are both changing according to Rayleigh fading distributions. It is worth noting that the SNR regime in which the cognitive channel resides is not only decided by the transmit power but also by the interference threshold Q. Regardless Q and the fading distribution of the cognitive and interference channels, the slope of the spectral efﬁciency goes to 0 as Eb N0 grows. The reason is that for CR channel the spectral efﬁciency is limited by interference thresh- old of the primary channel, i.e., even without Gaussian noise it achieves a bounded spectral efﬁciency Cmax. If the cognitive and interference channels follow Rayleigh distributions, then Cmax is equal to In the low SNR regime, E[γ∗ s(gc, gi)] = γpk and when we take this into consideration, Eb N0 min is always equal −1.59 dB for all types of cognitive channel fading distribution. Fig. 2 compares spectral-energy efﬁciency trade-off when the ST transmits a signal under the average and peak power constraints. In the low SNR regime, transmitting a signal with average power constraint provides better energy efﬁciency than transmitting a signal with peak power constraint. Moreover, reliable communication is no possible for Eb N0 < −1.59 dB when transmitting a signal under peak power constraint. This is due to that fact that the power policy with peak power constraint does not beneﬁt from the available energy at those moment in which the cognitive channel fading is exceedingly high. III. LINK-LEVEL SPECTRAL-ENERGY EFFICIENCY TRADE-OFF In this section, we consider a link-level CR channel. It con- sists of an interference-tolerant secondary transmitter-receiver pair that shares a spectrum with a primary transmitter-receiver pair [9]–[11]. We assume a point-to-point ﬂat fading channel that is corrupted by AWGN. All nodes in this model are assumed to be equipped with a single antenna. The channel between the ST and secondary receiver (SR) is deﬁned as the cognitive channel, while the channel between the ST and the PR is deﬁned as the interference channel. The cognitive and interference channel gains are denoted by gc and gi, re- spectively. They are random variables drawn from an arbitrary continuous distribution with an expected value of unity and they are mutually independent. The ST is assumed to have perfect gi Theorem 1: Under average transmit and peak interference power constraints, the minimum energy efﬁciency required for reliable information over the cognitive channel is given by Eb N0 min = ln 2 gc(max) (11) (11) ■ Proof: See Appendix A. ■ Proof: See Appendix A. ■ where gc(max) is the supremum of a random variable gc. Unsurprisingly, the minimum energy is only affected by the cognitive channel while the interference channel has no inﬂu- ence on it. If the cognitive channel, for instance, is an AWGN IEEE TRANSACTIONS ON WIRELESS COMMUNICATIONS, VOL. 14, NO. 6, JUNE 2015 2972 Fig. 1. (a) Per-link spectral-energy efﬁciency trade-off with different fading distributions of the interference channel in the low SNR regime (Q = −5, Fig. 2. Per-link spectral-energy efﬁciency trade-off for CR channel with average and peak power constraints (Rician factor K = 5). Fig. 2. Per-link spectral-energy efﬁciency trade-off for CR channel with average and peak power constraints (Rician factor K = 5). Fig. 1. (a) Per-link spectral-energy efﬁciency trade-off with different fading distributions of the interference channel in the low SNR regime (Q = −5, Rician factor K = 5). (b) Per-link spectral-energy efﬁciency trade-off for different interference threshold values in the high SNR regime (gc and gi ≡ exponential distribution). Fig. 2. Per-link spectral-energy efﬁciency trade-off for CR channel with average and peak power constraints (Rician factor K = 5). Fig. 1. (a) Per-link spectral-energy efﬁciency trade-off with different fading distributions of the interference channel in the low SNR regime (Q = −5, Rician factor K = 5). B. Fading Channels With Peak Transmit Power Constraint In the high SNR regime, both power policies behave similarly and approach the same maximum spectral efﬁciency Cmax since the transmit power for both policies is controlled by Q gi . Therefore, (13) can yet be applied in the high SNR regime if Q is high enough. Cmax = log2 Q N0 1 −N0 Q . (12) (12) The detailed derivation for (12) is given in Appendix B. Hence, Cmax is characterized by Q and it is independent of the fading distribution of the cognitive and interference channels. How- ever, we can notice that If Q is high enough, the spectral-energy efﬁciency trade-off can be approximated by III. LINK-LEVEL SPECTRAL-ENERGY EFFICIENCY TRADE-OFF (b) Per-link spectral-energy efﬁciency trade-off for different interference threshold values in the high SNR regime (gc and gi ≡ exponential distribution). [23]. The only difference is that Cmax has no limited value for the primary channel while it is limited for the cognitive channel. channel, Eb N0 min will be equal to −1.59 dB. For the Rayleigh fading channel, Eb N0 min = 0 (−∞dB) as the fading channel gain is unbounded, i.e., gc(max) = ∞. Fig. 1(a) presents the spectral-energy efﬁciency trade-off when the cognitive chan- nel is under Rayleigh and AWGN channels against different interference channel fading distributions. Here, Q is assumed to be −5 dB. We can see that Eb N0 min depends only on the fading statistics of the cognitive channel regardless of the dis- tribution of the interference channel, which veriﬁes Theorem 1. Better energy efﬁciency can be obtained when the cognitive channel follows a Rayleigh distribution due to additional gain in the fading distribution. channel, Eb N0 min will be equal to −1.59 dB. For the Rayleigh fading channel, Eb N0 min = 0 (−∞dB) as the fading channel gain is unbounded, i.e., gc(max) = ∞. Fig. 1(a) presents the spectral-energy efﬁciency trade-off when the cognitive chan- nel is under Rayleigh and AWGN channels against different interference channel fading distributions. Here, Q is assumed to be −5 dB. We can see that Eb N0 min depends only on the fading statistics of the cognitive channel regardless of the dis- tribution of the interference channel, which veriﬁes Theorem 1. Better energy efﬁciency can be obtained when the cognitive channel follows a Rayleigh distribution due to additional gain in the fading distribution. IV. SYSTEM-LEVEL SPECTRAL-ENERGY EFFICIENCY TRADE-OFF First, since the primary network is assumed to be an indoor one, the mutual interference between the primary and secondary networks will be scaled down because of the penetration losses. Secondly, as the PRs, are all ﬁxed in position, this offers an opportunity to easily detect them by the ST. Hence, the STs can detect the pilot channel broadcast from indoor PRs and decide how many PRs with which they are surrounded [31]. The ST can then rely on channel reciprocity and estimate the channel coefﬁcient of the interference channel using injected pilots in the uplink channel of the PRs. Finally, it is also possible that the interference channel status information (ICSI) is sent from all PRs along with their identities and collected by a certain central unit. In fact, using a separate wireline control channel that broadcasts the interference measured over a broadband connection is a very practical solution. Before the secondary network can utilize the spectrum, it must register itself with the central unit ﬁrst to be updated regarding the ICSI. However, the PRs do not necessarily need to identify each registered ST. The ICSI can inform the STs regarding the status of the worst aggregate interference that a PR suffers. The STs can also use ICSI as an alternative way to estimate the channel status to that PR and regulate their transmit power accordantly. In this work, we assume that there is only one registered CR network with a single secondary cell. (17) where gs(n) and gm(n) represent the power gain of the shad- owing and multi-path fading of the nth SR, respectively. We use the composite channel model for both shadowing and fast fading [32]. This model is the result of the multiplication of the log-normal distribution with the Nakagami distribution. Thus, the composite channel gain can be approximated by log-normal distribution [32, pp. 132], i.e., where gs(n) and gm(n) represent the power gain of the shad- owing and multi-path fading of the nth SR, respectively. We use the composite channel model for both shadowing and fast fading [32]. This model is the result of the multiplication of the log-normal distribution with the Nakagami distribution. Thus, the composite channel gain can be approximated by log-normal distribution [32, pp. 132], i.e., fh(x) = ξ √ 2πσx exp −(10 log10(x) −μ)2 2σ2 (18) (18) where ξ = 10 log(10). IV. SYSTEM-LEVEL SPECTRAL-ENERGY EFFICIENCY TRADE-OFF The mean μ and variance σ2 can be given by μ = m−1 k=1 1 k −ln(m) + μΩ (19) σ2 = ∞ k=0 1 (m + k)2 + σ2 Ω (20) (19) (20) respectively, where μΩ and σ2 Ω are the mean and variance of the shadowing, respectively, while m represents the Nakagami fading factor. If we recall (17) and (18), the distribution of gc(n) can be expressed by fgc(g)= Be( 2 a (μ−ξ log g)) g erfc a(Lmax+ξ log g−μ) −2σ2 a √ 2σ2 −Be( 2 a (μ−ξ log g)) g erfc a(Lmin+ξ log g −μ)−2σ2 a √ 2σ2 (21) (21) where Lmax and Lmin are the maximum and minimum path- loss values in dB, respectively, and they depend on the cell radius d and minimum distance to the ST, i.e., dmin. In (21), a = ξβ and B is a constant given by B = ξe 2σ2 a2 aA2(d2 −d2 min). (22) IV. SYSTEM-LEVEL SPECTRAL-ENERGY EFFICIENCY TRADE-OFF First, since the primary network is assumed to be an indoor one, the mutual interference between the primary and secondary networks will be scaled down because of the penetration losses. Secondly, as the PRs, are all ﬁxed in position, this offers an opportunity to easily detect them by the ST. Hence, the STs can detect the pilot channel broadcast from indoor PRs and decide how many PRs with which they are surrounded [31]. The ST can then rely on channel reciprocity and estimate the channel coefﬁcient of the interference channel using injected pilots in the uplink channel of the PRs. Finally, it is also possible that the interference channel status information (ICSI) is sent from all PRs along with their identities and collected by a certain central unit. In fact, using a separate wireline control channel that broadcasts the interference measured over a broadband connection is a very practical solution. Before the secondary network can utilize the spectrum, it must register itself with the central unit ﬁrst to be updated regarding the ICSI. However, the PRs do not necessarily need to identify each registered ST. The ICSI can inform the STs regarding the status of the worst aggregate interference that a PR suffers. The STs can also use ICSI as an alternative way to estimate the channel status to that PR and regulate their transmit power accordantly. In this work, we assume that there is only one registered CR network with a single secondary cell. belongs to another licensed network. We assume that a CR network consists of a single ST, i.e., macro BS, which transmits signals to multiple SRs. The CR network shares a spectrum owned by an indoor primary network. The primary network also consists of multiple PRs, i.e., primary indoor access points (APs). The SRs and PRs are indexed by n ∈N = {1, . . . , N} and k ∈K = {1, . . . , K}, respectively. The SRs and PRs are uniformly distributed in a cell of radius d and a cell of radius D(d ≤D), respectively, as shown in Fig. 3. The downlink transmissions of the CR network are considered and assumed to occur in the uplink transmission of the primary network. There are many advantages for sharing the spectrum of the uplink transmissions of an indoor network. IV. SYSTEM-LEVEL SPECTRAL-ENERGY EFFICIENCY TRADE-OFF Eb N0 dB ≈C×10 log10 2−10 log(C)+2.5067, ∀C <Cmax. (13) In this section, we will study the spectral and energy efﬁ- ciency for a CR-based cellular network. The intention here is not to build a complete cellular network using the concept of CR, but rather to enhance the spectral efﬁciency of the cellular networks for a short period of time by sharing a spectrum that (13) Expression (13) is similar to spectral-energy efﬁciency trade-off approximation that can be applied to the single primary channel HAIDER et al.: SPECTRAL AND ENERGY EFFICIENCY ANALYSIS FOR COGNITIVE RADIO NETWORKS 2973 Fig. 3. System model of CR-based cellular network with secondary BS, multiple PRs, and multiple SRs. The cognitive and interference channels experience pathloss, shadowing, and multi-path fading. The focus in this section will be on the cognitive channel. However, the same analysis can be applied to the interference channel. The pathloss is a function of the distance r between the ST and the nth SR, and can be expressed by gp(n) = Arβ(n) (16) (16) with β representing the pathloss exponent. The propagation coefﬁcient A includes parameters related to antenna height, antenna gain, path-loss frequency dependence, and, in the case of the interference channel, the indoor loss. The combined channel gain gc(n) is given by Fig. 3. System model of CR-based cellular network with secondary BS, multiple PRs, and multiple SRs. gc(n) = gm(n)gs(n) gp(n) (17) belongs to another licensed network. We assume that a CR network consists of a single ST, i.e., macro BS, which transmits signals to multiple SRs. The CR network shares a spectrum owned by an indoor primary network. The primary network also consists of multiple PRs, i.e., primary indoor access points (APs). The SRs and PRs are indexed by n ∈N = {1, . . . , N} and k ∈K = {1, . . . , K}, respectively. The SRs and PRs are uniformly distributed in a cell of radius d and a cell of radius D(d ≤D), respectively, as shown in Fig. 3. The downlink transmissions of the CR network are considered and assumed to occur in the uplink transmission of the primary network. There are many advantages for sharing the spectrum of the uplink transmissions of an indoor network. B. Interference Constraint and CR Power Control To keep a certain level of performance for the primary network, the aggregate interference Ik at any PR must always be below a predeﬁned threshold, i.e., Q. The interference Ik that a PR experiences consists of the aggregate interference Ip from all transmit nodes in the primary network as well as the interference Ic = γsgi(k) from the CR network, where γs is the ST transmit power. In other words, the aggregate interference at a PR can be calculated by ´γ = γsgc(n∗) I = Q I X Y , Y > Q γpk γpk I X, Y ≤ Q γpk (25) (25) where X and Y are random variables that represent max n∈N {gc(n)} and max k∈K {gi(k)}, respectively, and n∗is the index for an SR who has the maximum value of the channel gain. In (25), I is interference plus noise power. The spectral efﬁciency can then be evaluated by where X and Y are random variables that represent max n∈N {gc(n)} and max k∈K {gi(k)}, respectively, and n∗is the index Ik = Ip + Ic ≤Q. (23) for an SR who has the maximum value of the channel gain. In (25), I is interference plus noise power. The spectral efﬁciency can then be evaluated by (23) It is assumed that the adjacent indoor APs are using orthogo- nal radio recourses to avoid strong interference among them. It is assumed that the adjacent indoor APs are using orthogo- nal radio recourses to avoid strong interference among them. Moreover, the interference between non-adjacent indoor APs, i.e., Ip, can be negligible or considered as background noise. This is because signals that come from a primary user should travel through at least two walls to reach the other primary APs [33]. However, Ic = γsgi(k) is dominate compared with Ip because typically macro BS transmits a signal with high power and then its signal could be high enough to propagate through the walls of the building where the PR is deployed and generate interference. Given there are many PRs, it is important to ensure that ST transmit power γs should always be tightly controlled to avoid harmful interference on a PR which has the maximum channel gain toward ST. This will inevitably protect the other PRs and (23) remains true for all PRs. See Appendix D for the detailed derivation. In (27), This means that any gain of power control under average power constraint is minor. Finally, the power control with peak power constraint is more straight forward as it requires gi only as an input rather than gi and gc. To this end, the ST can request the worst ICSI, which belong to the surrounding PRs, from the central unit. The ST then uses this ICSI to estimate the channel status and regulate the transmit power. Alternatively, or concurrently, the ST can use injected uplink pilots to estimate the channel status assuming that the channel is reciprocal. (30) where δc and δp are the scale parameters for the cognitive and interference channels, respectively. A numerical integration of (26) provides convenient spectral efﬁciency evaluation. D. Spectral-Energy Efﬁciency Trade-Off The average energy efﬁciency is given by Eb N0 sys = ¯γavg N0Csys , where ¯γavg is the average transmit power. From (24), we have ¯γavg = E[γs] = γs(y)fY (y)dy = ∞ Q γpk Q 2 δ0.5 p y−2.5 exp − δp y 0.5 dy + γpk exp − δpγpk Q 0.5 = P1 + P2 (31) (31) A. The Distribution of the Channel Gain (22) The cognitive channel power gain between the ST and the nth SR is denoted by gc(n), while the interference channel gain between the ST and the kth PR is denoted by gi(k). The detailed derivation of (21) is given in Appendix C. The detailed derivation of (21) is given in Appendix C. IEEE TRANSACTIONS ON WIRELESS COMMUNICATIONS, VOL. 14, NO. 6, JUNE 2015 2974 B. Interference Constraint and CR Power Control (TDMA) is assumed by which the ST chooses an SR whose CSI implies the largest channel gain among all other SRs. In this case, the received signal to interference plus noise ratio, ´γ, for the scheduled SR is equal to See Appendix D for the detailed derivation. In (27), K = Iδp Qδc 0.5 (28) K1 = γpkδc I 0.5 (29) K2 = γpkδp Q 0.5 (30) γs = ⎧ ⎨ ⎩ Q max k∈K gi(k), max k∈K gi(k) > Q γpk γpk, max k∈K gi(k) ≤ Q γpk (24) γs = ⎧ ⎨ ⎩ Q max k∈K gi(k), max k∈K gi(k) > Q γpk γpk, max k∈K gi(k) ≤ Q γpk (24) (28) (24) (29) where γpk is the peak transmit power. The above power control is similar to (14) which demonstrates the power control under peak power constraint. It is more suitable for the proposed system to use the power policy with peak power constraint rather than power policy with average power constraint for many reasons. Firstly, in typical cellular networks, the BS has a limited maximum power that it can transmit with. The power control with average power constraint does not take into account this limitation. Secondly, to get as much beneﬁt as possible of CR network, the SRs would usually be close to the ST, and so they would be within high SNR regime. This means that any gain of power control under average power constraint is minor. Finally, the power control with peak power constraint is more straight forward as it requires gi only as an input rather than gi and gc. To this end, the ST can request the worst ICSI, which belong to the surrounding PRs, from the central unit. The ST then uses this ICSI to estimate the channel status and regulate the transmit power. Alternatively, or concurrently, the ST can use injected uplink pilots to estimate the channel status assuming that the channel is reciprocal. where γpk is the peak transmit power. The above power control is similar to (14) which demonstrates the power control under peak power constraint. It is more suitable for the proposed system to use the power policy with peak power constraint rather than power policy with average power constraint for many reasons. Firstly, in typical cellular networks, the BS has a limited maximum power that it can transmit with. The power control with average power constraint does not take into account this limitation. Secondly, to get as much beneﬁt as possible of CR network, the SRs would usually be close to the ST, and so they would be within high SNR regime. B. Interference Constraint and CR Power Control Therefore, the transmit power of ST is controlled according to Csys = log2(1 + ´γ)f(´γ)d´γ (26) (26) where f(´γ) is the probability density function (PDF) of ´γ. By adopting the extreme value theory [34], f(´γ) converges to f(´γ) →K1´γ−1.5 2 exp(−K1´γ−0.5 −K2) − K1K´γ−1 2(K´γ0.5 + 1) exp(−K1´γ−0.5 −KK1) + K 2(K + ´γ−0.5)2´γ 3 2 1 −exp(−K1´γ−0.5 −KK1) . (27) (27) See Appendix D for the detailed derivation. In (27), See Appendix D for the detailed derivation. In (27), C. Spectral Efﬁciency Analysis They are given by where Cmax and Eb N0 max are the maximum spectral efﬁciency and energy efﬁciency that the CR network can reach, respec- tively. They are given by with k(X) is the kurtosis of X. with k(X) is the kurtosis of X. 2) High SNR Regime: In the high SNR regime, the avail- able transmit power and Q are important. If Q is set to be high, the spectral and energy efﬁciency converges to Cmax and Eb N0 max, respectively. Using (5), the slope of spectral efﬁciency for the cognitive network in this case is equal to 1. The horizontal penalty with respect to the AWGN channel, Cmax = log(1 + ´γ) K 2(K + ´γ−0.5)2´γ 3 2 d´γ (34) Eb N0 max = P1 N0Cmax = ∞ Q γpk Q 2 δ0.5 p x−2.5 exp− δp x 0.5 dx N0Cmax . (35) (34) p y Eb N0 penalty, is given by (35) y Eb N0 penalty = E log2 1 X . (39) (39) See Appendix E for the detailed derivation of (34). We can notice that Cmax and Eb N0 max can be considered as new parameters that characterize the CR network. In (33), γpk can be numerically calculated as a function of Csys from (26) and (27). See Appendix F for the detailed derivation. See Appendix F for the detailed derivation. C. Spectral Efﬁciency Analysis where fY (y) is the PDF of Fréchet distribution (refer to (55) in Appendix D). The transmit power can be modeled as a The ST schedules SRs in orthogonal mode to avoid the intra- cell interference. In this work, time division multiple access HAIDER et al.: SPECTRAL AND ENERGY EFFICIENCY ANALYSIS FOR COGNITIVE RADIO NETWORKS 2975 Fig. 4. The average transmit power as a function of γpk Q (N = 20, Q = 0 dB, D = 1000 m, I N0 = 1, AdB = 128 dB). summation of two power components, i.e., P1 and P2. Fig. 4 shows how P1, P2, and ¯γavg change as a function of γpk Q . We can notice that P1 is a monotonously increasing function while P2 is a waterfall curve with respect to γpk Q . Hence, ¯γavg is dominated by P2 until a point, after which ¯γavg is relatively constant and dominated by P1. This gives the conclusion that Fig. 5. The average network spectral efﬁciency of the CR network as a function of the number of the secondary users (γpk = 43 dB, K = 20, D = 1000 m, I N0 = 1, AdB = 128 dB). where the expectation is with respect to the random variable X. Applying L’Hopital’s Rule into (36), Eb N0 min can then be calculated by Eb = I ln 2 (37) Fig. 4. The average transmit power as a function of γpk Q (N = 20, Q = 0 dB, D = 1000 m, I N0 = 1, AdB = 128 dB). Fig. 5. The average network spectral efﬁciency of the CR network as a function of the number of the secondary users (γpk = 43 dB, K = 20, D = 1000 m I 1 A 128 dB) Fig. 4. The average transmit power as a function of γpk Q (N = 20, Q = 0 dB, D = 1000 m, I N0 = 1, AdB = 128 dB). Fig. 5. The average network spectral efﬁciency of the CR network as a function of the number of the secondary users (γpk = 43 dB, K = 20, D = 1000 m, I N0 = 1, AdB = 128 dB). Fig. 4. C. Spectral Efﬁciency Analysis The average transmit power as a function of γpk Q (N = 20, Q = 0 dB, D = 1000 m, I N0 = 1, AdB = 128 dB). Fig. 5. The average network spectral efﬁciency of the CR network as a function of the number of the secondary users (γpk = 43 dB, K = 20, D = 1000 m, I N0 = 1, AdB = 128 dB). summation of two power components, i.e., P1 and P2. Fig. 4 shows how P1, P2, and ¯γavg change as a function of γpk Q . We can notice that P1 is a monotonously increasing function while P2 is a waterfall curve with respect to γpk Q . Hence, ¯γavg is dominated by P2 until a point, after which ¯γavg is relatively constant and dominated by P1. This gives the conclusion that ¯γavg can be approximated by where the expectation is with respect to the random variable X. Applying L’Hopital’s Rule into (36), Eb N0 min can then be calculated by Eb N0 min = I ln 2 N0E max n∈N {gc(n)} . (37) (37) ¯γavg ≈max(P1, P2). (32) (32) We can conclude that in a very noisy region, the minimum energy efﬁciency in the CR network is characterized by the cognitive channels of the best SR. Hence, the interference channels have no impact on Eb N0 min. The slope of the spectral efﬁciency versus Eb N0 is given by We can conclude that in a very noisy region, the minimum energy efﬁciency in the CR network is characterized by the cognitive channels of the best SR. Hence, the interference channels have no impact on Eb N0 min. The slope of the spectral efﬁciency versus Eb N0 is given by If we allow the ST to transmit to the best user who has the maximum channel gain, then the average energy efﬁciency is given by Eb N0 sys = ⎧ ⎪ ⎪ ⎨ ⎪ ⎪ ⎩ γpk exp − δpγpk Q 0.5 N0Csys , Csys < Cmax Eb N0 max , otherwise (33) 0 S0 = 2E2 max n∈N {gc(n) E max n∈N {gc(n)}2 = 2 k(X) (38) (33) (38) where Cmax and Eb N0 max are the maximum spectral efﬁciency and energy efﬁciency that the CR network can reach, respec- tively. E. Numerical Results and Discussions Per-network spectral-energy efﬁciency trade-off as a function of (a) the number of secondary receivers (K = 50, D = 1000 m, I N0 = 1, AdB = 128 dB) and (b) the number of primary receivers (N = 50, D = 1000 m, I N0 = 1, AdB = 128 dB). Fig. 7. Per-network spectral-energy efﬁciency trade-off for CR network with different values of Q (N = 30, K = 20, D = 1000 m, I N0 = 1, AdB = 128 dB). Fig. 7 shows the spectral-energy efﬁciency trade-off for the CR network. The numbers of SRs and the PRs are assumed to be 30 and 20, respectively. As we can see, for a given Q, the spectral-energy efﬁciency trade-off curve tends to a point that corresponds to the maximum spectral efﬁciency, i.e., Cmax. It also indicates that the high SNR asymptotic tool is valid only if the CR network is working below its maximum spectral efﬁciency. The minimum energy efﬁciency, deﬁned in (37), is the same for all curves. The impact of the multiuser diversity gain on the spectral- energy trade-off is pointed out in Fig. 8(a) and (b). Thus, Fig. 8(a) illustrates the spectral-energy efﬁciency trade-off as a function of the number of the SRs. We can notice that, for a given spectral efﬁciency, increasing the SRs improves the energy efﬁciency. This improvement is because the horizontal penalty spectral-energy efﬁciency trade-off and Eb N0 min are characterized by the cognitive channels only. The impact of the number of the PRs on the spectral-energy efﬁciency trade- off, however, has a different aspect. Thus, from Fig. 8(b), the number of PRs has impact only on the maximum spectral efﬁciency but not on the horizontal penalty nor Eb N0 min. Fig. 7. Per-network spectral-energy efﬁciency trade-off for CR network with different values of Q (N = 30, K = 20, D = 1000 m, I N0 = 1, AdB = 128 dB). transmit power that can be used in a typical cellular network. The number of PRs is assumed to be 20. We can notice from this ﬁgure that the maximum spectral efﬁciency is still achievable even with a reasonable transmit power and does not need to be unlimited. E. Numerical Results and Discussions So far, the theoretical framework, which numerically evalu- ates the spectral-energy efﬁciency trade-off, is established for system-level CR networks. In the following two sub-sections, we will focus on low and high SNR regimes for the proposed network. This section presents the simulation results of the spectral and energy efﬁciency for a multi-user CR network. The sim- ulation is based on the Monte Carlo method, which consists of 106 channel realizations. The analysis is carried out with the following parameters: I N0 = 1, β = 3.7, the indoor loss is 8 dB, and A = 120 dB (128 dB in the case of the interference channel). network. 1) Low SNR Regime: If we recall (2), Eb N0 min is giving by 1) Low SNR Regime: If we recall (2), Eb N0 min is giving by Eb N0 min = lim ¯γavg→0 ¯γavg N0E log2 1 + ¯γavgX I (36) (36) Fig. 5 shows the spectral efﬁciency of the CR network as a function of the number of SRs. We assume a reasonable IEEE TRANSACTIONS ON WIRELESS COMMUNICATIONS, VOL. 14, NO. 6, JUNE 2015 2976 Fig. 8. Per-network spectral-energy efﬁciency trade-off as a function of (a) the number of secondary receivers (K = 50, D = 1000 m, I N0 = 1, AdB = 128 dB) and (b) the number of primary receivers (N = 50, D = 1000 m, I N0 = 1, AdB = 128 dB). Fig. 6. The average network spectral efﬁciency of the secondary network as a function of the primary receivers (a) with different values of Q (b) with different values of d D , (γpk = 43 dB, N = 20, D = 1000 m, I N0 = 1, AdB = 128 dB). Fig. 6. The average network spectral efﬁciency of the secondary network as a function of the primary receivers (a) with different values of Q (b) with different values of d D , (γpk = 43 dB, N = 20, D = 1000 m, I N0 = 1, AdB = 128 dB). Fig. 8. E. Numerical Results and Discussions The spectral efﬁciency of the CR network improves with the increase of the number of the SRs due to the additional gain that comes from the multi-user diversity. The theoretical results obtained from the numerical integration of (34) agree with the simulation results. V. CONCLUSION The increase in the spectral efﬁciency is, however, sensi- tive to the interference threshold and the number of the PRs. Fig. 6(a) shows the impact of the number of the PRs on the spectral efﬁciency of the CR network. The number of the SRs is assumed to be 20 in this example. As shown, the spectral efﬁciency decreases quickly with the increase of the number of PRs. It indicates that adopting the spectrum sharing with another licensed network is unsuitable if the density of the PRs is relatively very high. However, the spectral efﬁciency can be improved by relaxing the interference threshold of the primary network, as it is shown in Fig. 6(a). The spectral efﬁciency can also be improved if the CR network considers only the SRs that are within a short distance to the ST. Hence, Fig. 6(b) shows the spectral efﬁciency of the CR network as a function of the number of the PRs with different values of d for a given D, assuming Q = 0 dB. Clearly, for the given number of the PRs, the spectral efﬁciency increases dramatically with the decreasing of d. This paper has investigated the spectral and energy efﬁciency in interference-tolerant CR networks. The initial analysis has studied the spectral-energy efﬁciency trade-off for a link-level CR network under transmit power and interference constraints. In the low SNR regime, transmitting a signal with average power constraint provides better energy efﬁciency than trans- mitting a signal with peak power constraint. In addition to that, the interference channel has no impact on Eb N0 min required for reliable communications. In the high SNR regime, however, transmitting signals with either power constraint gives the same energy efﬁciency. We have also proposed a CR-based cellular network in which a secondary network shares a spectrum belonging to an indoor system. This paper has also demonstrated that with CR technology, cellular operators can share their spectrum opportunistically with each other to increase the performance HAIDER et al.: SPECTRAL AND ENERGY EFFICIENCY ANALYSIS FOR COGNITIVE RADIO NETWORKS 2977 Expression (11) can then be obtained after applying γ0 → gc(max). Expression (11) can then be obtained after applying γ0 → gc(max). of their network. One way to do so is to share a spectrum in the uplink phase of an indoor system. APPENDIX A PROOF OF THEOREM 1 By applying integration by parts method to (46), (12) can be obtained. The minimum energy efﬁciency Eb No min occurs when γavg approaches zero, i.e., occurs when γavg V. CONCLUSION This is indeed an opportunity to make the implementation of the CR-based cel- lular network more feasible in the near future without the necessity of modiﬁcation at the end user’s handset. An example of a practical application of CR and its integration with existing technology, is the use of carrier aggregation applied with CR to allow greater spectrum accessibility whilst remaining efﬁcient by using the underutilized spectrum. The challenge is how to practically estimate the interference channels by the STs. Relying on channel reciprocity or broadcasting ICSIs can give some insight to solving this issue. gc(max). APPENDIX B DERIVATION OF (12) Let us ﬁrst rewrite (7) as C = ∞ 0 ∞ 0 log2 1 + gcγ∗ s(gc, gi) N0 f(gc)f(gi)dgcdgi. (43 APPENDIX B DERIVATION OF (12) Let us ﬁrst rewrite (7) as C = ∞ 0 ∞ 0 log2 1 + gcγ∗ s(gc, gi) N0 f(gc)f(gi)dgcdgi. (43 C = ∞ 0 ∞ 0 log2 1 + gcγ∗ s(gc, gi) N0 f(gc)f(gi)dgcdgi. (43 (43) In the high SNR regime, for sufﬁciently large transmit power, γs(gc, gi))∗will be equal to Q gi . Let x = gc gi and y = gi. In this case, The spectral and energy efﬁciency of the proposed network have been analyzed. By adopting the extreme value theory, we have derived the spectral efﬁciency of the system-level CR network under optimal power allocation. We have studied the impact of multi-user diversity gain in both the primary and secondary receivers on the spectral and energy efﬁciency. The spectral efﬁciency of the CR network is relatively large when the number of primary receivers is small. The spectral efﬁciency, however, diminishes rapidly with the increase in the number of primary receivers. This degradation can be compen- sated by relaxing the interference threshold or by increasing the number of SRs that are within a short distance from the ST. f(x) = ∞ 0 yfgc(xy)fgi(y)dy. (44) (44) If both the cognitive and interference channels follow Rayleigh distribution (i.e., both gc and gi would be exponentially dis- tributed with unit-mean), then f(x) = 1 (1 + x)2 . (45) (45) Therefore, C = ∞ 0 log2 1 + xQ No 1 (1 + x)2 dx. (46) (46) APPENDIX C DERIVATION OF (21) It has been found that the following condition is sufﬁcient to determine if FX(x) and FY (y) belong to type II or Fréchet distribution domain of attraction [34, Theorem 1.6.1] to Weilbull. If ¯ζ < 0, it converges to Fréchet distribution. If ¯ζ = 0, then it converges to Gumbel distribution. It has been found that the following condition is sufﬁcient to determine if FX(x) and FY (y) belong to type II or Fréchet distribution domain of attraction [34, Theorem 1.6.1] where a = ξβ. The channel gain gc(n) = S(n) gp(n), where S(n) = gm(n)gs(n) is a random variable derived from (18) to represent the combined shadowing and fading channel status. The CDF of gc(n) can be expressed by (50), shown at the bottom of the page. By differentiating (50) with respect to the random variable g and by applying Leibniz’s rule, the PDF of gc(n) can be calculated by (51), shown at the bottom of the page, where we have used d dxerfc(x) = 2 √πe−x2 and w = (ξ log(g) + y − μ). By some manipulations, (21) can be obtained. lim t→∞ tf(t) 1 −F(t) = α > 0. (53) (53) Therefore, FX(x) →exp − x −λc δc β (54) FY (y) →exp − y −λp δp β (55) Therefore, (54) APPENDIX D DERIVATION OF (27) Here, λc and λp both approach 0, and β always equals −0.5 for all cases. Due to the complicated distribution of channel gain, ﬁnding closed from expressions for δc and δp is difﬁcult. In this paper, however, we adopt the maxi- mum likelihood method to estimate the parameters of (52), and consequently δc and δp [35]. Other methods to estimate these parameters can be found in [36]. Now, the conditional distribution of ´γ, i.e., Pr{´γ ≤´γ0}, can be expressed by (56), shown at the top of the next page. Expression (27) is obtained by differentiating (56) with respect to ´γ. δp = δp ¯ζp . By applying Theorem 9.5 of [35], δc and δp can , , gi(1), gi(2), . . . , gi(k) are independent random variables that are drawn from a common distribution, i.e., (21), then the CDF of the maximum for any size of the samples is equal to [Fgc(g)]N and [Fgi(g)]K, respectively [35]. Using these two new distributions may not provide understandable results. However, the distribution of the maximum function can be tracked using the extreme-value theory, as we will see in the following Lemma. Lemma 1: Let z1, z2, . . . , zn be independent and identically distributed (i.i.d.) random variables drawn from a common CDF FZ(z). By setting Z = max n=1,...,N{zn}, there exist a se- quence of constants ¯λ, ¯λ, ¯δ and some non-degenerate distri- bution function ¯HZ(¯λ, ¯δ, ¯ζ) such that f(z) converges to the distribution ¯HZ(¯λ, ¯δ, ¯ζ). The distribution ¯HZ(¯λ, ¯δ, ¯ζ) is called generalized extreme value distribution (GEVD) [35] and it is equal to APPENDIX C DERIVATION OF (21) pp Eb N0 min = lim γavg→0 E [γ∗ s(gc, gi)] N0E log2 1 + γ∗ s (gc,gi)gc N0 (40) (40) The distance from the ST to a given SR is an independent and identical random variable following a uniform distribution. Thus, the probability that R ≤r holds is given by where the expectation in the dominator and nominator is with respect to two random variables, i.e. gi and gc. We can notice from (10) that γavg vanishes when γ0 approaches gc(max). Then, (40) can be re-written as (41), shown at the bottom of the page. Applying L’Hôpital’s Rule into (41) and following Leibniz integral rule, Eb No min is then calculated by Fd(r) = 2r d2−d2 min dmin ≤r ≤d 0 otherwise (47) (47) where dmin(0 ≤dmin ≤d) is the minimum distance between the ST and a SR. The cumulative distribution function (CDF) of pathloss in the dB scale can be calculated by Eb No min = lim γ0→gc(max) × 1 γ2 0 gc(max) γ0 Q 1 γ0 −N0 gc 0 f(gc)f(gi)dgcdgi 1 γ0 ln 2 gc(max) γ0 Q 1 γ0 −N0 gc 0 f(gc)f(gi)dgcdgi . (42) FL(y) = Pr {a log(Ar) ≤y} = e( y a) A dmin 2r d2 −d2 min dr. (48) (48) min The PDF, i.e., dFL(r) dr , is then given by The PDF, i.e., dFL(r) dr , is then given by The PDF, i.e., dFL(r) dr , is then given by fL(y) = 2e( 2y a ) aA2 (d2 −d2 min) (49) (49) (42) (42) Eb N0 min = lim γ0→gc(max) gc(max) γ0 ⎛ ⎝ Q 1 γ0 −N0 gc 0 1 γ0 −N0 gc + ∞ Q 1 γ0 −N0 gc Q gi ⎞ ⎠f(gc)f(gi)dgcdgi N0 gc(max) 0 ⎛ ⎝ Q 1 γ0 −N0 gc 0 log2 gc γ0N0 + ∞ Q 1 γ0 −N0 gc log2 1 + gcQ giN0 ⎞ ⎠f(gc)f(gi)dgcdgi (41) (41) IEEE TRANSACTIONS ON WIRELESS COMMUNICATIONS, VOL. 14, NO. 6, JUNE 2015 2978 to Weilbull. If ¯ζ < 0, it converges to Fréchet distribution. If ¯ζ = 0, then it converges to Gumbel distribution. APPENDIX D DERIVATION OF (27) (55) To derive an explicit expression for the PDF of the ´γ, we have to ﬁrst ﬁnd the distribution of X = max n=1,...,N{gc(N)} and Y = max k=1,...,K{gi(K)}. Since gc(1), gc(2), . . . , gc(n) and gi(1), gi(2), . . . , gi(k) are independent random variables that are drawn from a common distribution, i.e., (21), then the CDF of the maximum for any size of the samples is equal to [Fgc(g)]N and [Fgi(g)]K, respectively [35]. Using these two new distributions may not provide understandable results. However, the distribution of the maximum function can be tracked using the extreme-value theory, as we will see in the following Lemma. To derive an explicit expression for the PDF of the ´γ, we have to ﬁrst ﬁnd the distribution of X = max n=1,...,N{gc(N)} and Y = max k=1,...,K{gi(K)}. Since gc(1), gc(2), . . . , gc(n) and (1) (2) (k) i d d d i bl h where λc = ¯λc − ¯δc ¯ζc , δc = ¯δc ¯ζc , λp = ¯λp − ¯δp ¯ζp , β = −1 ¯ζ , and δp = ¯δp ¯ζp . By applying Theorem 9.5 of [35], δc and δp can be calculated by δc = F −1 gc 1 −1 N and δp = F −1 gi 1 −1 K , respectively. Here, λc and λp both approach 0, and β always equals −0.5 for all cases. Due to the complicated distribution of channel gain, ﬁnding closed from expressions for δc and δp is difﬁcult. In this paper, however, we adopt the maxi- mum likelihood method to estimate the parameters of (52), and consequently δc and δp [35]. Other methods to estimate these parameters can be found in [36]. Now, the conditional distribution of ´γ, i.e., Pr{´γ ≤´γ0}, can be expressed by (56), shown at the top of the next page. Expression (27) is obtained by differentiating (56) with respect to ´γ. where λc = ¯λc − ¯δc ¯ζc , δc = ¯δc ¯ζc , λp = ¯λp − ¯δp ¯ζp , β = −1 ¯ζ , and ¯ ζc ζc ζp ζ δp = ¯δp ¯ζp . By applying Theorem 9.5 of [35], δc and δp can be calculated by δc = F −1 gc 1 −1 N and δp = F −1 gi 1 −1 K , respectively. APPENDIX E DERIVATION OF (34) In (56), if we assume ¯γ →∞, then the term exp{−(K1´γ−β)} vanishes. Therefore, the PDF of the random variable ´γ is given by ¯HZ(¯λ, ¯δ, ¯ζ) = exp − 1 + ¯ζ z −¯λ ¯δ −1 ζ (52) (52) f(´γ) ≈ K 2(K + ´γ−0.5)2´γ 3 2 (57) where ¯λ, ¯δ, and ¯ζ are the location, scale, and shape parameters, respectively. The GEVD inherently contains the three well- known extreme value distributions, i.e., Gumbel, Weilbull, and Fréchet distributions. If ¯ζ > 0, then the distribution converges (57) which is then used to calculate the Cmax, as expressed by (34). Fgc(g) = Lmax Lmin 2e( 2y a ) aA2 (d2 −d2 min) 1 2 + 1 2erfc ξ log(geξ−1y) −μ √ 2σ2 dy (50) (50) fgc(g) = 2ξ g√πaA2 (d2 −d2 min) √ 2σ2 e( 2 a (μ−ξ log g)) Lmax+ξ log g−μ Lmin+ξ log g−μ e( 2w a )e− w2 2σ2 dw = Be( 2 a (μ−ξ log g)) g erfc aw −2σ2 a √ 2σ2 Lmax+ξ log g−μ Lmin+ξ log g−μ (51) (51) HAIDER et al.: SPECTRAL AND ENERGY EFFICIENCY ANALYSIS FOR COGNITIVE RADIO NETWORKS 2979 HAIDER et al.: SPECTRAL AND ENERGY EFFICIENCY ANALYSIS FOR COGNITIVE RADIO NETWORKS 2979 F(´γ) = exp − γpkδc I´γ β − δpγpk Q β + βδβ p ∞ Q γpk y−(β+1) exp − Qδc I´γy β − δp y β dy = exp −(K1´γ−β) −K2 ! + K´γβ (K´γβ + 1) 1 −exp " − K1´γ−β + KK1 # (56) APPENDIX F DERIVATION OF (39) [14] X. Hong, C.-X. Wang, M. Uysal, X. Ge, and S. Ouyang, “Ca- pacity analysis of hybrid cognitive radio networks with distributed VAAs,” IEEE Trans. Veh. Tech., vol. 59, no. 7, pp. 3510–3523, Sep. 2010. APPENDIX F DERIVATION OF (39) [14] X. Hong, C.-X. Wang, M. Uysal, X. Ge, and S. Ouyang, “Ca- pacity analysis of hybrid cognitive radio networks with distributed VAAs,” IEEE Trans. Veh. Tech., vol. 59, no. 7, pp. 3510–3523, Sep. 2010. [14] X. Hong, C.-X. Wang, M. Uysal, X. Ge, and S. Ouyang, “Ca- pacity analysis of hybrid cognitive radio networks with distributed VAAs,” IEEE Trans. Veh. Tech., vol. 59, no. 7, pp. 3510–3523, Sep. 2010. From (6), we have pp p [15] C. Han et al., “Green radio: Radio techniques to enable energy efﬁcient wireless networks,” IEEE Commun. Mag., vol. 49, no. REFERENCES [1] Federal Communications Commission, “Spectrum policy task force,” Washington, DC, USA,Tech. Rep., ET Docket No. 02-155, Nov. 2002. [24] A. Lozano, A. Tulino, and S. Verdu, “Multiple-antenna capacity in the low-power regime,” IEEE Trans. Inf. Theory, vol. 49, no. 10, pp. 2527–2544, Oct. 2003. g p [2] J. Mitola and G. Maguire, “Cognitive radio: Making software radios more personal,” IEEE Pers. Commun. Mag., vol. 6, no. 6, pp. 13–18, Aug. 1999. pp [25] Y. Yao, X. Cai, and G. Giannakis, “On energy efﬁciency and optimum resource allocation of relay transmissions in the low-power regime,” IEEE Trans. Wireless Commun., vol. 4, no. 6, pp. 2917–2927, Nov. 2005. [3] C.-X. Wang, H.-H. Chen, X. Hong, and M. Guizani, “Cognitive radio network management,” IEEE Veh. Technol. Mag., vol. 3, no. 1, pp. 28–35, Mar. 2008. pp [4] X. Hong, C.-X. Wang, H.-H. Chen, and Y. Zhang, “Secondary spectrum access networks: Recent developments on the spatial models,” IEEE Veh. Technol. Mag., vol. 4, no. 2, pp. 36–43, Jun. 2009. [26] J. Gomez-Vilardebo, A. Perez-neira, and M. Najar, “Energy efﬁcient communications over the AWGN relay channel,” IEEE Trans. Wireless Commun., vol. 9, no. 1, pp. 32–37, Jan. 2010. pp [27] O. Oyman and M. Win, “Power-bandwidth trade-off in multiuser relay channels with opportunistic scheduling,” in Proc. IEEE ALLERTON, Champaign, IL, USA, Sep. 2008, pp. 72–78. [5] S. A. Jafar and S. Srinivasa, “Capacity limits of cognitive radio with distributed and dynamic spectral activity,” in Proc. IEEE ICC, Istanbul, Turkey, Jun. 2006, pp. 5742–5747. y pp [6] D. Ugarte and A. B. McDonald, “On the capacity of dynamic spectrum access enable networks,” in Proc. IEEE DySPAN, Baltimore, MD, USA, Nov. 2005, pp. 630–633. [28] N. Jindal, “High SNR analysis of MIMO broadcast channels,” in Proc. ICSIT, Adelaide, SA, Australia, Sep. 2005, pp. 2310–2314. [29] A. Lozano, A. Tulino, and S. Verdu, “High-SNR power offset in multi- antenna communication,” IEEE Trans. Inf. Theory, vol. 51, no. 12, pp. 4134–4151, Dec. 2005. [7] S. Haykin, “Cognitive radio: Brain-empowered wireless communica- tions,” IEEE J. Select. Areas Commun., vol. 23, no. 2, pp. 201–220, Feb. 2005. [30] F. Haider et al., “Spectral-energy efﬁciency trade-off in cognitive radio networks with peak interference power constraints,” in Proc. IEEE ICCT, Jinan, China, Sep. 2011, pp. 368–372, invited paper. [8] M. Gastpar, “On capacity under receive and spatial spectrum-sharing constraints,” IEEE Trans. Inf. Theory, vol. 53, no. 2, pp. 471–487, Feb. 2007. APPENDIX E DERIVATION OF (34) [19] S V d “S l fﬁi i h id b d i ” IEEE T I f , , , pp , [18] X. Ge et al., “Spectrum and energy efﬁciency evaluation of two-tier fem- tocell networks with partially open channels,” IEEE Trans. Veh. Technol., vol. 63, no. 3, pp. 1306–1319, Mar. 2014. pp [19] S. Verdu, “Spectral efﬁciency in the wideband regime,” IEEE Trans. Inf. Theory, vol. 48, no. 6, pp. 1319–1343, Jun. 2002. Applying L’Hôpital’s rule into (59) leads to Applying L’Hôpital’s rule into (59) leads to y pp [20] I. Ku, C.-X. Wang, and J. S. Thompson, “Spectral-energy efﬁciency trade- off in relay-aided cellular networks,” IEEE Trans. Wireless Commun., vol. 12, no. 10, pp. 4970–4982, Oct. 2013. Eb N0 penalty = ∞ 0 log2 1 X f(X)dX (60) (60) [21] X. Hong, Y. Jie, C.-X. Wang, J. Shi, and X. Ge, “Energy-spectral efﬁ- ciency trade-off in virtual MIMO cellular systems,” IEEE J. Sel. Areas Commun., vol. 31, no. 10, pp. 2128–2140, Oct. 2013. which is the expected value of log2 1 X and (39) is then obtained. [22] X. Hong, J. Wang, C.-X. Wang, and J. Shi, “Cognitive radio in 5G: A perspective on energy-spectral efﬁciency trade-off,” IEEE Commun. Mag., vol. 52, no. 7, pp. 46–53, Jul. 2014. [23] S. Shamai and S. Verdu, “The impact of frequency-ﬂat fading on the spectral efﬁciency of CDMA,” IEEE Trans. Inf. Theory, vol. 47, no. 4, pp. 1302–1327, May 2001. APPENDIX E DERIVATION OF (34) 6, pp. 46–54, Jun. 2011. Eb N0 penalty = lim SNR→∞ log2(SNR)−E[log2(1+SNRX)] S∞ (58) [16] L. Xiang, X. Ge, C.-X. Wang, F. Y. Li, and F. Reichert, “Energy efﬁciency evaluation of cellular networks based on spatial distributions of trafﬁc load and power consumption,” IEEE Trans. Wireless Commun., vol. 12, no. 3, pp. 961–973, Mar. 2013. where the expectation is with respect to the random variable X. Knowing that ∞ 0 f(X)dX = 1, (58) can be re-written as where the expectation is with respect to the random variable X. Knowing that ∞ 0 f(X)dX = 1, (58) can be re-written as where the expectation is with respect to the random variable X. Knowing that ∞ 0 f(X)dX = 1, (58) can be re-written as E ∞ SNR pp. 961 973, Mar. 2013. [17] X. Ge et al., “Energy efﬁciency optimization for MIMO-OFDM mobile multimedia communication systems with QoS constraints,” IEEE Trans. Veh. Technol., vol. 63, no. 5, pp. 2127–2138, Jun. 2014. here the expectation is with respect to the random variable X. nowing that ∞ 0 f(X)dX = 1, (58) can be re-written as E ∞ SNR [17] X. Ge et al., “Energy efﬁciency optimization for MIMO-OFDM mobile multimedia communication systems with QoS constraints,” IEEE Trans. Veh. Technol., vol. 63, no. 5, pp. 2127–2138, Jun. 2014. [17] X. Ge et al., “Energy efﬁciency optimization for MIMO-OFDM mobile multimedia communication systems with QoS constraints,” IEEE Trans. Veh. Technol., vol. 63, no. 5, pp. 2127–2138, Jun. 2014. 0 Eb N0 penalty = ∞ 0 log2 lim SNR→∞ SNR (1 + SNRX) f(X)dX. (59) Eb N0 penalty = ∞ 0 log2 lim SNR→∞ SNR (1 + SNRX) f(X)dX. (59) Veh. Technol., vol. 63, no. 5, pp. 2127 2138, Jun. 2014. [18] X. Ge et al., “Spectrum and energy efﬁciency evaluation of two-tier fem- tocell networks with partially open channels,” IEEE Trans. Veh. Technol., vol. 63, no. 3, pp. 1306–1319, Mar. 2014. Eb N0 penalty = ∞ 0 log2 lim SNR→∞ SNR (1 + SNRX) f(X)dX. (59) , , , pp , [18] X. Ge et al., “Spectrum and energy efﬁciency evaluation of two-tier fem- tocell networks with partially open channels,” IEEE Trans. Veh. Technol., vol. 63, no. 3, pp. 1306–1319, Mar. 2014. IEEE TRANSACTIONS ON WIRELESS COMMUNICATIONS, VOL. 14, NO. 6, JUNE 2015 He received the Best Paper Awards from IEEE Globecom 2010. He is leading several projects funded by NSFC, China MOST, and industries. He is taking part in several international joint projects, such as the EU FP7-PEOPLE-IRSES: project acronym S2EuNet (grant no. 247083), project acronym WiNDOW (grant no. 318992) and project acronym CROWN (grant no. 610524). Cheng-Xiang Wang (S’01–M’05–SM’08) received the B.Sc. and M.Eng. degrees in communication and information systems from Shandong University, Jinan, China, in 1997 and 2000, respectively, and the Ph.D. degree in wireless communications from Aalborg University, Aalborg, Denmark, in 2004. Since 2005, he has been with Heriot-Watt Uni- versity, Edinburgh, U.K., where he was promoted to Professor in 2011. He is also an Honorary Fel- low of the University of Edinburgh, Edinburgh, and a Chair/Guest Professor with Shandong University, and with Southeast University, Nanjing, China. From 2001 to 2005, he was a Research Fellow with the University of Agder, Grimstad, Norway. In 2004, he was a Visiting Researcher with Siemens AG-Mobile Phones, Munich, Germany. From 2000 to 2001, he was a Research Assistant with the Hamburg University of Technology, Hamburg, Germany. He is the Editor of one book. He has published one book chapter and over 210 papers in refereed journals and conference proceedings. His research interests include wireless channel modeling and simulation, green communications, cognitive radio networks, vehicular communication networks, massive multiple-input multiple-output systems, and ﬁfth-generation wireless communications. g Dr. Ge is a Senior Member of the China Institute of Communications and a member of the National Natural Science Foundation of China and the Chinese Ministry of Science and Technology Peer Review College. He has been actively involved in organizing more the ten international conferences since 2005. He served as the Executive Chair for the 2013 IEEE International Conference on Green Computing and Communications (IEEE GreenCom) and as the Cochair of the Workshop on Green Communication of Cellular Networks at the 2010 IEEE GreenCom. He serves as an Associate Editor for the IEEE ACCESS, Wireless Communications and Mobile Computing Journal (Wiley) and the International Journal of Communication Systems (Wiley). Moreover, he served as the guest editor for IEEE Communications Magazine Special Issue on 5G Wireless Communication Systems and ACM/SpringMobile Communications and Application Special Issue on Networking in 5G Mobile Communication Systems. Dr. IEEE TRANSACTIONS ON WIRELESS COMMUNICATIONS, VOL. 14, NO. 6, JUNE 2015 2980 Fourat Haider received the B.Sc. degree in elec- trical and electronic engineering/communication en- gineering from the University of Technology, Iraq, in 2004, and the M.Sc. degree (with distinction) from Brunel University, U.K., in 2009. He has been pursuing the Ph.D. degree at Heriot-Watt University and the University of Edinburgh, Edinburgh, U.K. since December 2010 From 2005 to 2007, he worked as a Senior Network Engineer at Asia cell company (a mobile operator in Iraq). In 2009, he began work- ing on a European project (pre-drive project) to develop a HLA-based simulation integration at the University of Surrey. He has also been working as a RAN Strategy and Architecture Engineer at Hutchison 3G, U.K. His main research interests include spectral-energy efﬁciency trade- off, wireless channel capacity analysis, small cells, femtocell and mobile femtocell, and conventional and massive MIMO systems. He received Best Paper Awards from IEEE ICCT 2011. Erol Hepsaydir received the electronics engineering degree in 1985 and the Ph.D. degree in telecommu- nications in 2001 from the University of Technology, Sydney. He is currently the Head of Radio and Devices Strategy at Hutchison 3G UK. He has been working in the cellular mobile industry for 22 years. He has worked for various mobile operators as Net- work Technologist. He has recently been appointed as RAEng Visiting Professor at the University of Kent. His earlier research work is in the design, de- velopment of communications systems. His current work is to contribute to next generation mobile network developments as well as analysis of new mobile technologies. He is the IF&E Chair of IEEE ICC 2015 conference. Xiaohu Ge (M’09–SM’11) received the Ph.D. de- gree in communication and information engineering from Huazhong University of Science and Technol- ogy (HUST), Wuhan, China, in 2003. He is currently a Professor with the School of Electronic Informa- tion and Communications, HUST. He has worked at HUST since November 2005. Prior to that, he worked as a Researcher at Ajou University, Suwon, Korea, and Politecnico Di Torino, Italy, from January 2004 to October 2005. He was a Visiting Researcher at Heriot-Watt University, Edinburgh, U.K., from June to August 2010. His research interests are in the area of mobile commu- nications, trafﬁc modeling in wireless networks, green communications, and interference modeling in wireless communications. He has published about 90 papers in refereed journals and conference proceedings and has been granted about 15 patents in China. IEEE TRANSACTIONS ON WIRELESS COMMUNICATIONS, VOL. 14, NO. 6, JUNE 2015 Wang is a Fellow of the Institution of Engineering and Technology and the HEA and a member of the Engineering and Physical Research Council Peer Review College. He has served as an Editor for eight international journals, including the IEEE TRANSACTIONS ON VEHICULAR TECHNOLOGY (since 2011) and the IEEE TRANSACTIONS ON WIRELESS COMMUNICA- TIONS (2007–2009). He was the lead Guest Editor for the IEEE JOURNAL ON SELECTED AREAS IN COMMUNICATIONS, Special Issue on Vehicular Com- munications and Networks. He has served as a Technical Program Committee (TPC) Member, TPC Chair, and General Chair for more than 70 international conferences. He received the Best Paper Awards from IEEE Globecom 2010, IEEE ICCT 2011, ITST 2012, and IEEE VTC 2013-Spring. Harald Haas (SM’98–AM’00–M’03) received the Ph.D. degree from the University of Edinburgh in 2001. He currently holds the Chair of Mobile Com- munications at the University of Edinburgh. His main research interests are in optical wireless com- munications, hybrid optical wireless and RF com- munications, spatial modulation, and interference coordination in wireless networks. He ﬁrst intro- duced and coined spatial modulation and “Li-Fi.” Li-Fi was listed among the 50 best inventions in TIME Magazine 2011. He was an invited speaker at TED Global 2011, and his talk has been watched online more than 1.5 million times. He is Co-Founder and Chief Scientiﬁc Ofﬁcer (CSO) of pureLiFi Ltd. He holds 31 patents and has more than 30 pending patent applications. He has published 300 conference and journal papers including a paper in Science. He was co-recipient of a Best Paper Award at the IEEE Vehicular Technology Conference in Las Vegas, NV, USA, in 2013. In 2012, he was the only recipient of the prestigious Established Career Fellowship from the Engineering and Physical Sciences Research Council (EPSRC) within Information and Communications Technology in the U.K. He is a recipient of the Tam Dalyell Prize 2013 awarded by the University of Edinburgh for excellence in engaging the public with science. In 2014, he was selected by EPSRC as one of ten Recognizing Inspirational Scientists and Engineers (RISE) Leaders. Dongfeng Yuan (SM’01) received the M.Sc. de- gree from the Department of Electrical Engineering, Shandong University, Shandong, China, in 1988, and the Ph.D. degree from the Department of Electrical Engineering, Tsinghua University, Beijing, China, in January 2000. Currently, he is a Full Professor with the School of Information Science and Engineering, Shandong University, China. REFERENCES Jinan, China, Sep. 2011, pp. 368–372, invited paper. [31] B. Wild and K. Ramchandran, “Detecting primary receivers for cogni- tive radio applications,” in Proc. IEEE DySPAN, Baltimore, MD, USA, Nov. 2005, pp. 124–130. [9] A. Ghasemi and E. S. Sousa, “Fundamental limits of spectrum-sharing in fading environments,” IEEE Trans. Wireless Commun., vol. 6, no. 2, pp. 649–658, Feb. 2007. [32] G. L. Stuber, Principles of Mobile Communication, 3rd ed. New York, NY, USA: Springer-Verlag, 2011. pp [10] X. Kang, Y.-C. Liang, A. Nallanathan, H. Garg, and R. Zhang, “Optimal power allocation for fading channels in cognitive radio networks: Ergodic capacity and outage capacity,” IEEE Trans. Wireless Commun., vol. 8, no. 2, pp. 940–950, Feb. 2009. [33] Y. Sun, R. P. Jover, and X. Wang, “Uplink interference mitigation for OFDMA femtocell networks,” IEEE Trans. Wireless Commun., vol. 11, no. 2, pp. 614–625, Feb. 2012. [34] M. R. Leadbetter, G. Lindgren, and H. Rootzen, Extremes and Related Properties of Random Sequences and Processes. New York, NY, USA: Springer-Verlag, 1983. [11] L. Musavian and S. Aissa, “Capacity and power allocation for spec- trum sharing communications in fading channels,” IEEE Trans. Wireless Commun., vol. 8, no. 1, pp. 148–156, Jan. 2009. pp [12] C.-X. Wang, X. Hong, H.-H. Chen, and J. Thompson, “On capacity of cognitive radio networks with average interference power constraints,” IEEE Trans. Wireless Commun., vol. 8, no. 4, pp. 1620–1625, Apr. 2009. [35] E. Castillo, A. S. Hadi, N. Balakrishnan, and J. Sarabia, Extreme Value and Related Models With Applications in Engineering and Science. New York, NY, USA: Wiley, 2001. [13] J. Hong and W. Choi, “Throughput characteristics by multiuser diversity in a cognitive radio network,” IEEE Trans. Signal Process., vol. 59, no. 8, pp. 3749–3763, Aug. 2011. [36] J. Maritz and A. Munro, “On the use of the generalized extreme-value distribution in estimating extreme percentiles,” Biometrics, vol. 23, no. 1, pp. 79–103, Mar. 1969. IEEE TRANSACTIONS ON WIRELESS COMMUNICATIONS, VOL. 14, NO. 6, JUNE 2015 IEEE TRANSACTIONS ON WIRELESS COMMUNICATIONS, VOL. 14, NO. 6, JUNE 2015 From 1993 to 1994, he was with the Electrical and Computer Department, University of Calgary, Calgary, AB, Canada. He was with the Department of Electrical Engineering, University of Erlangen, Germany, from 1998 to 1999, with the Department of Electrical Engineering and Computer Science, University of Michigan, Ann Arbor, MI, USA, from 2001 to 2002, with the Department of Electrical Engineering, Munich University of Technology, Germany, in 2005, and with the Department of Electrical Engineering, Heriot-Watt University, U.K., in 2006. His current research interests include cognitive radio systems, cooperative (relay) communications, and 4G wireless communications.
https://openalex.org/W4241947618	https://www.qeios.com/read/ZN3XA1/pdf	English	null	Low Interval Point in Time Date Data Type	Definitions	2,020	cc-by	65	Qeios · Definition, February 2, 2020 Open Peer Review on Qeios Low Interval Point in Time Date Data Type National Cancer Institute Qeios ID: ZN3XA1 · https://doi.org/10.32388/ZN3XA1 Source National Cancer Institute. Low Interval Point in Time Date Data Type. NCI Thesaurus. Code C102556. Code C102556. A data type comprised of a set of consecutive low values of an ordered point in time date datatype. 1/1
https://openalex.org/W3126128582	https://europepmc.org/articles/pmc7850389?pdf=render	English	null	Inhaled nitric oxide and acute kidney injury risk: a meta-analysis of randomized controlled trials	Renal failure	2,021	cc-by	6,010	ABSTRACT Second, most included studies in these meta-analyses focused on ARDS and there was less evidence on other neglected. Two previous meta-analyses raised concerns about the relationship between the use of iNO and the risk for acute kidney injury (AKI) [3,4]. In 2007, Adhikari and colleagues included four randomized control trials (RCTs) involving 895 participants in a pooled analysis and suggested that iNO increased the risk for renal dys- function (risk ratio 1.50, 95% confidence interval 1.11–2.02) [3]. Another meta-analysis published in 2015 by Ruan et al. [4] found that the use of iNO was associ- ated with higher renal dysfunction risk especially with prolonged use in ARDS patients. Despite these interest- ing findings, there were some limitations to the meta- analyses. First, a sensitivity analysis was not performed and the stability of the results were not evaluated. Second, most included studies in these meta-analyses focused on ARDS and there was less evidence on other CONTACT Jiaqiang Zhang jqzhang@henu.edu.cn Department of Anesthesiology and Perioperative Medicine, Henan Provincial People’s Hospital, People’s Hospital of Zhengzhou University, No. 7, Weiwu Road, Zhengzhou 450003, China Supplemental data for this article can be accessed here. 2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Junqiu Wanga, Xuhui Congb, Mengrong Miaob, Yitian Yangb and Jiaqiang Zhangb Junqiu Wanga, Xuhui Congb, Mengrong Miaob, Yitian Yangb and Jiaqiang Zhang aJournal Editorial Department, Henan Provincial People’s Hospital, People’s Hospital of Zhengzhou University, Zhengzhou, China; bDepartment of Anesthesiology and Perioperative Medicine, Henan Provincial People’s Hospital, People’s Hospital of Zhengzhou University, Zhengzhou, China ARTICLE HISTORY Received 8 October 2020 Revised 2 January 2021 Accepted 2 January 2021 ABSTRACT ABSTRACT Purpose: There are conflicting results as to the effect of inhaled nitric oxide (iNO) therapy on the risk of acute kidney injury (AKI). The aim of this study was to perform a meta-analysis to assess the updated data. Methods: We systematically searched Web of Science, the Cochrane Library, Wanfang, and PubMed for relevant randomized control trials between database inception and 9/07/2020. Relative risks (RRs) with 95% confidence intervals (CIs) predicting the risk of AKI were extracted to obtain summary estimates using fixed-effects models. The Trim and Fill method was used to evaluate the sensitivity of the results and adjust for publication bias in meta-analysis. KEYWORDS Inhaled nitric oxide; acute kidney injury; randomized controlled trial; acute respiratory distress syndrome; cardiac surgery; organ transplantation y j y Results: 15 randomized controlled studies from 14 articles involving 1853 patients were included in the study. Analyzing the eligible studies we found: (1) iNO therapy significantly increased the risk of AKI in acute respiratory distress syndrome patients (RR 1.55, 95% CI 1.15–2.10, p ¼ 0.004; I2 for heterogeneity 0%; Phet ¼ 0.649). (2) The use of iNO was associated with reduced AKI risk in patients undergoing cardiac surgery (RR 0.80, 95% CI 0.64–0.99, p ¼ 0.037; I2 for heterogeneity 0%; Phet ¼ 0.528). (3) For organ transplantation recipients, there was no effect of iNO administra- tion on the risk of AKI (RR 0.50, 95% CI 0.16–1.56, p ¼ 0.233; I2 for heterogeneity 0%; Phet ¼ 0.842). The Trim and Fill analysis showed that the overall effect of this meta-analysis was stable. Conclusions: The effect of iNO on AKI risk might be disease-specific. Future RCTs with larger patient populations should aim to validate our findings. neglected. Two previous meta-analyses raised concerns about the relationship between the use of iNO and the risk for acute kidney injury (AKI) [3,4]. In 2007, Adhikari and colleagues included four randomized control trials (RCTs) involving 895 participants in a pooled analysis and suggested that iNO increased the risk for renal dys- function (risk ratio 1.50, 95% confidence interval 1.11–2.02) [3]. Another meta-analysis published in 2015 by Ruan et al. [4] found that the use of iNO was associ- ated with higher renal dysfunction risk especially with prolonged use in ARDS patients. Despite these interest- ing findings, there were some limitations to the meta- analyses. First, a sensitivity analysis was not performed and the stability of the results were not evaluated. RENAL FAILURE 2021, VOL. 43, NO. 1, 281–290 https://doi.org/10.1080/0886022X.2021.1873805 RENAL FAILURE 2021, VOL. 43, NO. 1, 281–290 https://doi.org/10.1080/0886022X.2021.1873805 CLINICAL STUDY jqzhang@henu.edu.cn Department of Anesthesiology and Perioperative Medicine, Henan Provincial People’s Hospital ou University, No. 7, Weiwu Road, Zhengzhou 450003, China Meta-analyses For dichotomous data, we used risk ratios (RRs) as the effect measure with 95% confidence intervals (CIs) cal- culated using the fixed-effects model, where a RR > 1 indicates an increased likelihood of renal dysfunction when treated with iNO compared with placebo. We generated summary forest plots to show the RRs and 95% CIs. Statistical heterogeneity between trial results was assessed using the I2 statistic and the Chi-square test. It was classified as large (75%), moderate (50%), and low (25%) [7]. We considered it to be substantial heterogeneity when the I2 was greater than 50% and was accompanied by a statistically significant Chi2 stat- istic. The cause of heterogeneity was explored using subgroup analyses. Finally, publication bias was eval- uated by graphical analysis of the funnel plot. Egger’s regression test was used to test the funnel plot [8]. All analyses were performed in Stata software, version 12.0 (College Station, TX). The significance level for all statis- tical tests was p < 0.05 (two-tailed). Search methods Applying a predetermined search strategy, two inde- pendent investigators (JW and XC) searched Web of Science, the Cochrane Library, Wanfang, and PubMed in order to identify potentially relevant articles between database inception and 9 July 2020. The following search terms were used: ‘inhaled nitric oxide’ and ‘randomized controlled trial’. Secondary searching included a manual search of reference lists in previous meta-analyses, reviews, and all included studies. There were no language restrictions. The search details are shown in Supplementary Table S1. Results The literature search yielded 741 articles. After the removal of duplicates, they were reduced to 596. At the screening of study titles and abstracts, an additional 572 articles were excluded as they did not meet our inclusion criteria. Figure 1 shows how we picked the studies. Ultimately, a total of 15 studies from 14 articles were analyzed in our final meta-analysis [9–22]. All stud- ies were performed in North America, Europe, China, Brazil, and Uganda. Among the included studies, 4 stud- ies reported the results for ARDS [9,11–13], 4 studies reporting cardiac surgery [15,16,21,22], 3 studies report- ing organ transplantation [14,17], and 4 studies report- ing other diseases [10,18–20]. The number of participants in each trial varied with 29 subjects in the smallest study and 385 subjects in the largest study. The majority were men, except in one study. All the studies provided data for AKI. Table 1 shows these Inclusion and exclusion criteria Studies were included in this meta-analysis if they met the following inclusion criteria: (1) the eligible studies were RCTs; (2) they compared iNO with placebo or usual treatment; and (3) the number of patients with renal dysfunction was reported in iNO and control groups. The exclusion criteria were: (1) retrospective studies, cohort studies, and non-randomized controlled studies; (2) conference abstracts; and (3) data for renal dysfunction were not reported. Assessment for risk of bias diseases. Third, the number of relevant researches has increased rapidly since these meta-analyses were pub- lished. The accumulating evidence needs to be reeval- uated. Therefore, we aimed to perform an up-dated quantitative assessment of the relationship between iNO and AKI. diseases. Third, the number of relevant researches has increased rapidly since these meta-analyses were pub- lished. The accumulating evidence needs to be reeval- uated. Therefore, we aimed to perform an up-dated quantitative assessment of the relationship between iNO and AKI. Two investigators (JW and XC) assessed the risk of bias for each trial by using criteria according to the Cochrane Risk of Bias Tool, which allows for evaluating six domains: method of random sequencing, allocation concealment, blinding of participants and personnel, blinding of outcome assessors, incomplete outcome data, selective reporting, and other factors that may affect bias [6]. Discrepancies were resolved by discus- sion or by consultation with the third investigator, JZ. Methods This meta-analysis was conducted and reported accord- ing to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses statement [5]. There was no registered protocol for this meta-analysis. Introduction Nitric oxide (NO) is an important signaling substance and vasodilator. It can relax vascular smooth muscle cells as well as pericytes by binding to the heme moiety of cytosolic guanylate cyclase and ultimately causing a fall in intracellular Ca2þ [1]. Inhalation of NO (iNO) leads to selective pulmonary vasodilatation and reduces pul- monary vascular resistance, increases arterial oxygen- ation, and improves pulmonary angiogenesis and lung alveolarization [2]. Given these beneficial effects, iNO is used widely as therapy in the field of critical care and medicine in general, including acute respiratory distress syndrome (ARDS), neonatal pulmonary hypertension, and cardiac surgery. Although iNO has excellent effectiveness and safety profile, its potential adverse effects should not be J. WANG ET AL. 282 Data extraction From all RCTs found eligible, the following information was extracted: the family name of the first author, country/region of research, year of publication, study designs and methods, participant number and details, intervention details, and the number of patients with renal dysfunction in iNO and control groups. Two inves- tigators (JW and XC) performed data extraction. Any disagreements were resolved by discussion between the two investigators and JZ where necessary. RENAL FAILURE 283 Figure 1. Flow chart for identification of studies in the meta-analysis. Figure 1. Flow chart for identification of studies in the meta-analysis. Figure 4(C)). The robustness of the results was eval- uated by a sensitivity analysis using the trim and fill method [23]. As shown in Table 4, the results of the trim and fill test showed that sensitivity analyses for ARDS and cardiac surgery remained significant. studies selected for the final meta-analysis. Detailed risk of bias assessments are presented in Table 2. Table 1 presents the RRs for AKI from each study. The pooled meta-analysis showed that iNO treatment was not associated with AKI risk, with a pooled RR of 1.00 (95% CI 0.84–1.18; p ¼ 0.977; I2 for heterogeneity 31.6%; Phet ¼ 0.116) (Table 3 and Figure 2). Egger’s test (p ¼ 0.461), Begg’s test (p ¼ 0.198), and visual evaluation of the funnel plot (Figure 3) indicated no publication bias. In subgroup analysis based on the fixed-effects model, the use of iNO was associated with increased risk of AKI in ARDS patients (RR 1.55, 95% CI 1.15–2.10, p ¼ 0.004; I2 for heterogeneity 0%; Phet ¼ 0.649) (Table 3 and Figure 4(A)), but iNO treatment decreased AKI risk for individuals who received cardiac surgery (RR 0.80, 95% CI 0.64–0.99, p ¼ 0.037; I2 for heterogeneity 0%; Phet ¼ 0.528) (Table 3 and Figure 4(B)). In addition, there was no effect of iNO therapy on the risk of AKI in patients undergoing organ transplantation (Table 3 and Heterogeneity To quantify the between-study heterogeneity, we uti- lized the I2 statistic and the Chi-square test. There was no significant heterogeneity across the studies in both the overall meta-analysis and subgroup analyses. This enabled us to pool evidence from the included studies using the fixed-effects model. Discussion We carried out a meta-analysis by combining evidence from 15 randomized clinical studies to evaluate the 284 J. WANG ET AL. J. WANG ET AL. 284 Table 1. Characteristics of included studies. Trial Location n Men (%) Age Intervention description AKI definition Number of AKI patients RR with 95% CI p-Value Disease iNO Control Dellinger et al. 1998 [9] USA iNO: 120 Control: 57 iNO: 64 Control: 70 iNO: 49 ± 18 years Control: 47 ± 18 years iNO at concentrations of 1.25, 5, 20, 40, or 80 ppm for up to 28 days or till FiO2 < 0.5 Creatinine above 2 mg/dL 20 7 1.36 (0.61–3.02) 0.448 ARDS Lundin et al. 1999 [11] 11 European countries iNO: 80 Control: 74 iNO: 75 Control: 62 iNO: 58 ± 15 years Control: 56 ± 17 years 1–40 ppm of NO at the lowest effective dose for up to 30 days or until an end point was reached Creatinine above 300 mmol/L and/or institution of RRT 28 12 2.16 (1.19–3.92) 0.008 ARDS Kinsella et al. 1999 [10] USA iNO: 48 Control: 32 iNO: 58 Control: 63 iNO: Gestational 27.1 weeks Control: Gestational 26.8 weeks 5 ppm of NO for up to 7 days Acute renal failure 2 2 0.67 (0.10–4.49) 0.675 Severe hypoxaemic respiratory failure Payen et al. 1999 [12] European countries iNO: 98 Control: 105 Unknown Adults 10 ppm of NO for 5 days Requiring RRT 33 26 1.36 (0.88–2.10) 0.162 ARDS Taylor et al. 2004 [13] USA iNO: 192 Control: 193 iNO: 52 Control: 54 iNO: 50 ± 17 years Control: 50 ± 17 years 5 ppm of NO until 28 days Creatinine above 265.2 mmol/L 12 8 1.51 (0.63–3.61) 0.352 ARDS Perrin et al. 2006 [14] France iNO: 15 Control: 15 iNO: 60 Control: 53 iNO: 37 ± 12 years Control: 33 ± 11 years 20 ppm of NO during a 12-h period Requiring RRT 1 1 1.00 (0.07–14.55) 1.000 Lung transplantation Fernandes et al. 2011 [15] Brazil iNO: 14 Control: 15 iNO: 7 Control: 20 iNO: 48 ± 11 years Control: 44 ± 13 years 10 ppm of NO for 48 h Acute renal failure 0 1 0.36 (0.02–8.07) 0.517 Cardiac surgery Potapov et al. Discussion 2018 [21] China iNO: 117 Control: 127 iNO: 44 Control: 41 iNO: 49 ± 10 years Control: 48 ± 9 years 80 ppm of NO for 24 h or less if patients were ready to be extubated early. An increase in serum creatinine by 50% within 7 days of surgery, or an increase in serum creatinine by 0.3 mg/dL within 2 days of surgery from preoperative baseline levels of serum creatinine 58 81 0.78 (0.62–0.97) 0.025 Cardiac surgery Kamenshchikov et al. 2019 [22] Russia iNO: 30 Control: 30 iNO: 83 Control: 70 iNO: 62 years Control: 58 years 40 ppm of NO during CPB Unknown 1 3 0.33 (0.04–3.03) 0.301 Cardiac surgery AKI: acute kidney injury; ARDS: acute respiratory distress syndrome; CPB: cardiopulmonary bypass; NO: nitric oxide; RRT: renal replacement therapy; ULN: upper limit of normal. It is widely accepted that the effects of NO are lim- ited to the lungs when delivered by inhalation. Many published RCTs suggest that iNO is associated with a very low incidence of adverse effects in the usual range of dosage. However, because iNO forms many metabo- lites that can act as endocrine carriers of NO in circula- tion, the safety aspects of iNO remain the focus of research. Lundin and coworkers focused on the adverse effects of iNO on ARDS in a European multicentre study [11]. They found that although iNO administration was not associated with bleeding complications, marked methemoglobinemia, or increased frequency of pneumothorax, there was an association between acute renal failure and iNO in ARDS (RR 2.16, 95% CI 1.19–3.92) [11]. This raised a concern about iNO-related renal dysfunction in ARDS patients. Several other stud- ies from the USA and Europe also evaluated this pos- sible relationship, including Dellinger et al. [9], Payen et al. [12], and Taylor et al. [13]. Based on the findings, two meta-analyses reviewed the data and concluded that the use of iNO increased AKI risk on ARDS [3,4]. Our meta-analysis confirmed the association between iNO administration and renal dysfunction in ARDS patients (RR 1.55). In addition, using the trim and fill method (Table 4), the meta-analysis results were shown to be stable, and no evidence of publication bias was detected. Previous meta-analyses and ours highlighted the potential AKI risk associated with iNO therapy in ARDS. It remains unknown why iNO administration in ARDS is linked to increased AKI risk. Discussion 2011 [16] Germany and USA iNO: 73 Control: 77 iNO: 88 Control: 84 iNO: 58 ± 10 years Control: 54 ± 12 years 40 ppm of NO for 48 h Requiring RRT 10 8 1.32 (0.55–3.16) 0.533 Cardiac surgery Lang et al. 2014 (A) [17] USA iNO: 20 Control: 20 iNO: 80 Control: 80 iNO: 54 (36–72) years Control: 55 (45–68) years 80 ppm of NO during the operative phase of liver transplantation Renal dysfunction 2 5 0.40 (0.09–1.83) 0.212 Liver transplantation Lang et al. 2014 (B) [17] USA iNO: 20 Control: 20 iNO: 75 Control: 85 iNO: 59 (30–67) years Control: 57 (24–70) years 80 ppm of NO during the operative phase of liver transplantation Renal dysfunction 1 2 0.50 (0.05–5.08) 0.548 Liver transplantation Trzeciak et al. 2014 [18] USA iNO: 26 Control: 23 iNO: 42 Control: 57 iNO: 59 ± 15 years Control: 58 ± 20 years 40 ppm of NO for 6 h Requiring RRT 2 1 1.77 (0.17–18.26) 0.626 Severe sepsis or septic shock Hawkes et al. 2015 [19] Uganda iNO: 88 Control: 92 iNO: 60 Control: 53 iNO: 2 (1–3) years Control: 2 (1–3) years 80 ppm of NO for up to 72 h Serum creatinine >1.5 ULN (children age 1–2) or >2.0 ULN (age 2–10) AND an abrupt (within 48 h) reduction in kidney function 7 3 2.44 (0.65–9.14) 0.169 Severe malaria (continued) RENAL FAILURE 285 effect of iNO therapy on the risk of AKI. The main find- ings of the meta-analysis are the following: (i) iNO ther- apy significantly increased the risk of AKI in ARDS patients; (ii) the use of iNO was associated with reduced AKI risk in patients undergoing cardiac surgery; (iii) for organ transplantation recipients, there was no effect of iNO administration on the risk of AKI. Trial Location n Men (%) Age Intervention description AKI definition Number of AKI patients RR with 95% CI p-Value Disease iNO Control Liu et al. 2016 [20] China iNO: 19 Control: 13 iNO: 68 Control: 62 iNO: Gestational 37.4 weeks Control: Gestational 36.1 weeks 15–20 ppm of NO for 96 h Acute renal failure 1 0 2.10 (0.09–47.89) 0.642 Hypoxic respiratory failure Lei et al. Discussion However, it is com- monly believed that prolonged treatment with iNO might cause detrimental cell damage by reacting with reactive oxygen species and the resulting formation of reactive nitrogen species [24]. NO oxidative products can oxidize DNA bases and create DNA strand damage [25]. They also disrupt the maintenance of oxidation- sensitive enzymes, inhibit mitochondrial respiration, induce nuclear factor-kappaB-mediated protein degrad- ation, and enhance caspase activation [26,27]. Dellinger and coworkers found a positive link between iNO doses and circulating levels of NO oxidative products (such as NO2) in patients with ARDS [9]. There is evidence that the generation of NO oxidative products contributed to glomerular cell apoptosis [28]. In addition, animal stud- ies showed that the prolonged administration of iNO 286 J. WANG ET AL. J. WANG ET AL. 286 Table 2. Assessment of risk of bias in individual studies. Trial Sequence generation Allocation concealment Blinding Incomplete outcome data Selective outcome reporting Other sources of bias Dellinger et al. [9] Low risk Low risk Low risk Low risk Low risk Low risk Lundin et al. [11] Low risk Low risk High risk Low risk Low risk High risk Kinsella et al. [10] Low risk Low risk Low risk Low risk Low risk Low risk Payen et al. [12] Low risk Low risk Low risk Low risk Low risk High risk Taylor et al. [13] Low risk Low risk Low risk Low risk Low risk Unclear risk Perrin et al. [14] High risk Unclear risk High risk Low risk Low risk Unclear risk Fernandes et al. [15] Low risk Low risk Low risk Low risk Low risk Low risk Potapov et al. [16] Low risk Low risk Low risk Low risk Low risk Low risk Lang et al. (A) [17] Low risk Low risk Low risk Low risk Low risk Low risk Lang et al. (B) [17] Low risk Low risk Low risk Low risk Low risk Low risk Trzeciak et al. [18] Low risk Low risk Low risk Low risk Low risk Low risk Hawkes et al. [19] Low risk Low risk Low risk Low risk Low risk Low risk Liu et al. [20] High risk Unclear risk Low risk Low risk Low risk Low risk Lei et al. [21] High risk Low risk Low risk Low risk Low risk Unclear risk Kamenshchikov et al. [22] Low risk Low risk Low risk Low risk Low risk Low risk Table 2. Discussion Assessment of risk of bias in individual studies. Trial Sequence generation Allocation concealment Blinding Incomplete outcome data Selective outcome reporting Other sources of bias Dellinger et al. [9] Low risk Low risk Low risk Low risk Low risk Low risk Lundin et al. [11] Low risk Low risk High risk Low risk Low risk High risk Kinsella et al. [10] Low risk Low risk Low risk Low risk Low risk Low risk Payen et al. [12] Low risk Low risk Low risk Low risk Low risk High risk Taylor et al. [13] Low risk Low risk Low risk Low risk Low risk Unclear risk Perrin et al. [14] High risk Unclear risk High risk Low risk Low risk Unclear risk Fernandes et al. [15] Low risk Low risk Low risk Low risk Low risk Low risk Potapov et al. [16] Low risk Low risk Low risk Low risk Low risk Low risk Lang et al. (A) [17] Low risk Low risk Low risk Low risk Low risk Low risk Lang et al. (B) [17] Low risk Low risk Low risk Low risk Low risk Low risk Trzeciak et al. [18] Low risk Low risk Low risk Low risk Low risk Low risk Hawkes et al. [19] Low risk Low risk Low risk Low risk Low risk Low risk Liu et al. [20] High risk Unclear risk Low risk Low risk Low risk Low risk Lei et al. [21] High risk Low risk Low risk Low risk Low risk Unclear risk Kamenshchikov et al. [22] Low risk Low risk Low risk Low risk Low risk Low risk Table 3. Meta-analysis evaluation of the relationship between inhaled nitric oxide and acute kidney injury risk. Subgroup Number of studies RR (95% CI) p Heterogeneity I2 Phet All 15 1.00 (0.84–1.18) 0.977 31.6% 0.116 ARDS 4 1.55 (1.15–2.10) 0.004 0 0.649 Cardiac surgery 4 0.80 (0.64–0.99) 0.037 0 0.528 Organ transplantation 3 0 50 (0 16–1 56) 0 233 0 0 842 Table 2. Assessment of risk of bias in individual studies. Table 3. Meta-analysis evaluation of the relationship between inhaled nitric oxide and acute kidney injury risk. Discussion It is worth mentioning that several RCTs (such as NCT02836899 and NCT03527381) are underway to fur- ther assess the nephroprotective effect of NO in cardiac surgery. These trials may help to elucidate the underly- ing mechanisms of NO therapy for protecting AKI post- cardiac surgery. Figure 3. Begg’s funnel plot for evaluating publication bias. could induce apoptosis of the cells of the collecting ducts and the distal convoluted tubular cells, leading to renal injury [29]. In a rat model of diabetic kidney dis- ease, endothelial nitric oxide synthase (eNOS) and indu- cible nitric oxide synthase (iNOS)-derived high NO production and oxidative stress contributed to apop- tosis in the kidney [30]. However, the precise mecha- nisms of iNO-induced AKI in ARDS patients remain largely unknown, deserving further research in the future. In addition to ARDS and cardiac surgery, our meta-analysis also evaluated the effect of iNO on AKI incidence in other conditions, including organ trans- plantation. Some animal studies and RCTs reported that administrating iNO during the intraoperative period had protective effects on ischemia-reperfusion induced injury post-transplantation [14,17,35,36]. However, the results of our meta-analysis did not show any difference in the incidence of AKI post-transplantation between the iNO group and the control group. Cardiac surgery is a procedure that is commonly car- ried out in the world. Despite promising technical advances, it is still a high-risk surgery. AKI is a common and serious complication post cardiac surgery. AKI has been found in approximately 40–70% of patients undergoing cardiac surgery and is associated with an unfavorable prognosis [31]. The primary mechanisms of cardiac surgery-associated AKI include hemolysis, renal ischemia, and inflammation [31]. Unlike patients with ARDS, clinical studies showed that patients undergoing cardiac surgery developed a NO deficient state due to hemolysis. NO depletion induces a proinflammatory cascade, causes oxidative stress, produces vasoconstric- tion, and impairs endothelial function and tissue perfu- sion [32]. Preclinical and clinical data demonstrated that NO was a renal-protective agent during hemolysis and might thus prevent cardiac surgery-associated AKI. In a canine model of water-induced hemolysis, the use of iNO significantly reversed the vasoconstrictor effect of hemolysis, reduced serum creatinine, and attenuated renal impairment [33]. Several RCTs evaluated the effect of iNO therapy on renal dysfunction in patients under- going cardiac surgery. In a Chinese RCT of 244 patients undergoing multiple valve replacement, Lei and coworkers found that NO administration during Our meta-analysis has several limitations. Discussion Subgroup Number of studies RR (95% CI) p Heterogeneity I2 Phet All 15 1.00 (0.84–1.18) 0.977 31.6% 0.116 ARDS 4 1.55 (1.15–2.10) 0.004 0 0.649 Cardiac surgery 4 0.80 (0.64–0.99) 0.037 0 0.528 Organ transplantation 3 0.50 (0.16–1.56) 0.233 0 0.842 Others 4 1.67 (0.65–4.27) 0.286 0 0.747 ARDS: acute respiratory distress syndrome; CI: confidence interval; RR: relative risk. 2. Meta-analysis of the effect of inhaled nitric oxide on acute kidney injury risk by pooling the 15 randomized trials. Table 3. Meta-analysis evaluation of the relationship between inhaled nitric oxide and acute kidney injury risk. Heterogeneity Meta-analysis evaluation of the relationship between inhaled nitric oxide and acute kidney k Table 3. Meta-analysis evaluation of the relationship between inhaled nitric oxide and acute kidne injury risk. Table 3. Meta-analysis evaluation of the relationship between inhaled nitric oxide and acute kidney injury risk. Subgroup Number of studies RR (95% CI) p Heterogeneity I2 Phet All 15 1.00 (0.84–1.18) 0.977 31.6% 0.116 ARDS 4 1.55 (1.15–2.10) 0.004 0 0.649 Cardiac surgery 4 0.80 (0.64–0.99) 0.037 0 0.528 Organ transplantation 3 0.50 (0.16–1.56) 0.233 0 0.842 Others 4 1.67 (0.65–4.27) 0.286 0 0.747 ARDS: acute respiratory distress syndrome; CI: confidence interval; RR: relative risk. ure 2. Meta-analysis of the effect of inhaled nitric oxide on acute kidney injury risk by pooling the 15 randomized ed trials Figure 2. Meta-analysis of the effect of inhaled nitric oxide on acute kidney injury risk by pooling the 15 randomized con- trolled trials. Figure 2. Meta-analysis of the effect of inhaled nitric oxide on acute kidney injury risk by pooling the 15 randomized con- trolled trials. RENAL FAILURE 287 Figure 3. Begg’s funnel plot for evaluating publication bias. cardiopulmonary bypass and for the first 24 h postoper- atively was associated with a decreased incidence of postoperative AKI and major adverse kidney events [21]. The trial by Lei et al. also indicated that the neph- roprotective effect of iNO might be attributed to iNO’s impact on hemodynamics and right ventricular after- load. Their findings were supported by a recent European study in which Lomivorotov et al. demon- strated cardioprotective and nephroprotective effects of NO administration in cardiac surgery [22]. Combining results from different RCTs, this meta-analysis indicated a 20% decreased risk of postoperative AKI in cardiac surgery patients who received iNO therapy. Our results supported a previous meta-analysis by Hu et al. [34]. Discussion Firstly, it remains unclear if the effect of iNO therapy on AKI risk in different diseases is dose-related. Although there was no significant heterogeneity in our meta-analysis, the included studies did vary in study design and iNO administration, making it difficult to assess dose-related effects. Secondly, most of the included studies were performed in adults and the number of pediatric stud- ies was small. There were only three pediatric studies on the topic. It requires more research to investigate the effect of iNO therapy on renal function and AKI in pediatric patients. Thirdly, the incidence of AKI was not constantly reported by RCTs evaluating the effect of iNO therapy. Although Egger’s test did not suggest the presence of publication bias, we further performed the Trim and Fill test to assess the sensitivity of the results and adjust for publication bias in meta-analysis. As shown in Table 4, the results indicated that the overall effect of this meta-analysis was stable. In conclusion, this meta-analysis suggested that iNO therapy increased the risk of AKI in ARDS patients, but the use of iNO was associated with a decreased risk of 288 J. WANG ET AL. J. WANG ET AL. 288 e 4. Meta-analysis of the effect of inhaled nitric oxide on acute kidney injury risk. (A) Acute respiratory distress syndro rdiac surgery. (C) Organ transplantation. Figure 4. Meta-analysis of the effect of inhaled nitric oxide on acute kidney injury risk. (A) Acute respiratory distress syndrome. (B) Cardiac surgery. (C) Organ transplantation. RENAL FAILURE RENAL FAILURE 289 Table 4. Trim and Fill test results. [6] Higgins JP, Altman DG, Gøtzsche PC, et al. The Cochrane collaboration’s tool for assessing risk of bias in randomised trials. Version 2. BMJ. 2011;343:d5928. Table 4. Trim and Fill test results. Subgroup Model RR (95% CI) p Heterogeneity I2 Phet ARDS Fixed-effect 1.55 (1.15–2.10) 0.005 0 0.651 Random-effect 1.55 (1.15–2.10) 0.005 0 0.651 Cardiac surgery Fixed-effect 0.80 (0.64–0.99) 0.037 0 0.528 Random-effect 0.80 (0.64–0.99) 0.037 0 0.528 Organ transplantation Fixed-effect 0.40 (0.15–1.05) 0.061 0 0.914 Random-effect 0.40 (0.15–1.05) 0.061 0 0.914 ARDS: acute respiratory distress syndrome; CI: confidence interval; RR: relative risk. [7] Higgins JPT, Thompson SG, Deeks JJ, et al. Measuring inconsistency in meta-analyses. BMJ. 2003;327(7414): 557–560. [8] Egger M, Davey Smith G, Schneider M, et al. Bias in meta-analysis detected by a simple, graphical test. BMJ. 1997;315(7109):629–634. [9] Dellinger RP, Zimmerman JL, Taylor RW, et al. Discussion Effects of inhaled nitric oxide in patients with acute respira- tory distress syndrome: results of a randomized phase II trial. Inhaled Nitric Oxide in ARDS Study Group. Crit Care Med. 1998;26(1):15–23. AKI in patients undergoing cardiac surgery. For organ transplantation recipients, iNO therapy had no effects on AKI incidence post-transplantation. Future RCTs are necessary to evaluate if the effects of iNO on AKI inci- dence is disease-specific. In addition, future RCTs should focus on the relation of iNO duration and dose with renal function. [10] Kinsella JP, Walsh WF, Bose CL, et al. Inhaled nitric oxide in premature neonates with severe hypoxaemic respiratory failure: a randomised controlled trial. Lancet. 1999;354(9184):1061–1065. [11] Lundin S, Mang H, Smithies M, et al. Inhalation of nitric oxide in acute lung injury: results of a European multicentre study. Intensive Care Med. 1999;25(9): 911–919. [12] Payen D, Vallet B. Results of the French prospective multicentric randomized double-blind placebo-con- trolled trial on inhaled nitric oxide in ARDS. Intensive Care Medicine. 1999;25S:166. Author contributions JZ contributed to the conception of research. JW and XC performed the search, extracted data, and conducted statis- tical analyses. All authors contributed to the writing and revi- sion of the manuscript. [13] [13] Taylor RW, Zimmerman JL, Dellinger RP, et al. Inhaled Nitric Oxide in ARDS Study Group. Low-dose inhaled nitric oxide in patients with acute lung injury: a randomized controlled trial. JAMA. 2004;291(13): 1603–1609. Funding [15] Fernandes JL, Sampaio RO, Brand~ao CM, et al. Comparison of inhaled nitric oxide versus oxygen on hemodynamics in patients with mitral stenosis and severe pulmonary hypertension after mitral valve sur- gery. Am J Cardiol. 2011;107(7):1040–1045. This work was supported by Natural Science Foundation of Henan Province (202300410353). [16] Potapov E, Meyer D, Swaminathan M, et al. Inhaled nitric oxide after left ventricular assist device implant- ation: a prospective, randomized, double-blind, multi- center, placebo-controlled trial. J Heart Lung Transplant. 2011;30(8):870–878. 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Prolonged expos- ure to inhaled nitric oxide transiently modifies tubular
https://openalex.org/W3097945111	https://www.granthaalayahpublication.org/journals/index.php/granthaalayah/article/download/IJRG17_A07_472/2008	English	null	THE RELATIONSHIP BETWEEN WATER, POVERTY AND HEALTH EXPENDITURE: AN ANALYSIS	International journal of research - granthaalayah	2,017	cc-by	2,818	Social Social Abstract Even though the human being cannot live without water it has the negative effect on human health too. Its effect depends on the quality of water which human beings drink. The contaminated water used by human beings may degrade human health with the help of some toxic substances, like, fluoride, arsenic, mercury, lead etc. The poor people are prone to suffer from these toxic substances although they, generally, cannot afford for the water of good quality for drinking and washing. Moreover, there is a lot of water-washed diseases, water based diseases, water born diseases etc. which affect human health. These diseases and toxic substances increase health expenditure of the people. Therefore, contaminated water forces the poor people to stay in the vicious circle of poverty if they use it. This paper will analyze water, poverty and health expenditure nexus. Keywords: Disease; Health Expenditure; Poverty; Water. Keywords: Disease; Health Expenditure; Poverty; Water. Cite This Article: Mangal Sing Kro. (2017). “THE RELATIONSHIP BETWEEN WATER, POVERTY AND HEALTH EXPENDITURE: AN ANALYSIS.” International Journal of Research - Granthaalayah, 5(7), 214-218. 10.29121/granthaalayah.v5.i7.2017.2125. Cite This Article: Mangal Sing Kro. (2017). “THE RELATIONSHIP BETWEEN WATER, POVERTY AND HEALTH EXPENDITURE: AN ANALYSIS.” International Journal of Research - Granthaalayah, 5(7), 214-218. 10.29121/granthaalayah.v5.i7.2017.2125. THE RELATIONSHIP BETWEEN WATER, POVERTY AND HEALTH EXPENDITURE: AN ANALYSIS Mangal Sing Kro 1 1 Gauhati University, India ISSN- 2350-0530(O), ISSN- 2394-3629(P) DOI: https://doi.org/10.29121/granthaalayah.v5.i7.2017.2125 ISSN- 2350-0530(O), ISSN- 2394-3629(P) DOI: https://doi.org/10.29121/granthaalayah.v5.i7.2017.2125 [Kro , Vol.5 (Iss.7): July, 2017] Social ISSN- 2350-0530(O), ISSN- 2394-3629(P) DOI: 10.5281/zenodo.836441 ISSN- 2350-0530(O), ISSN- 2394-3629(P) DOI: 10.5281/zenodo.836441 [Kro , Vol.5 (Iss.7): July, 2017] cleaning are dug well, river, spring water, ponds etc. But due to the dearth of safe drinking water the water-borne diseases like cholera, dysentery, diarrheal disease, guinea worm disease etc., infects the poor people which ultimately increases their household expenditure for the disease treatment. Moreover, due to the lack of safe drinking water also increases financial burden which eventually leads to increase the dropout rate in schools, colleges among the poor. The contaminated water used by the poor people enhance the expenditure on health and it again pushes back to the cycle of poverty. In India, around 37.7 million populations are affected by waterborne diseases annually and 1.5 million children are estimated to die only of diarrhea (Khurana & Sen). 3. The Selected Existing Literature The human beings use water for multipurpose, like for drinking, washing, bathing, for agriculture, industries etc. But the use of contaminated water effect negatively on human beings. The contaminated water creates diseases that ultimately effect on human health as well as on its households expenditure. There is a number of water-washed diseases, water based diseases, water born diseases and water-related diseases. According to the World Health Organization (WHO) 2000, about 202 million people deaths per year for diarrheal disease only. Around 10,000 people die every day from water and sanitation related diseases and illnesses in the world and majority of them belong to developing countries (Abayawardana, S. & Hussain, I., 2002). The payments on healthcare push a large number of families into poverty (Van Doorslaer et al, 2006). In the absence of insurance, all illness not only reduce welfare directly, it also increases the risk of impoverishment due to high treatment expenditures (Gupta, I.,2009). 2. Objective of the Study 1) To look into the water, poverty and health expenditure nexus. 1) To look into the water, poverty and health expenditure nexus. 2) To investigate the impact of contaminated water on human heal 2) To investigate the impact of contaminated water on human health. 4. Methodology Good health is one of the components of human capital and the human capital is one of the factors for development. Therefore, weak health reduces not welfare only, rather it increases the financial burden of the households too. The safe drinking water and sanitation is one of the important factors to keep good health of the people. The dearth of safe drinking water may create a number of diseases which may drain the limited income of the poor people. This paper will analyze the interlinkage among water, poverty and health expenditure. The data used for analyzing are taken from secondary sources collecting from national and international reports, books, and journals as well as through my own observation. 1. Introduction Poverty is generally defined as the people those who are unable to afford for their basic needs. The basic needs are like food, shelter, clothing, water, and sanitation, health and education. Various methods have been given by the economists to measure the poverty, like Head Count Ratio, Poverty Gap, Foster-Greer-Thorbecke Index, Human Poverty Index etc. The World Bank has given the minimum norms for poverty based on the 2011 purchasing power parity (PPP) of the currency. Previously, it was pegged at $1.25 a day but, now it has been revised to $1.90 a day based on the average of the national poverty line of 15 poorest economies of the world. According to the World Bank report, 12.8 % global populations are extremely poor. In 2012, more than 78.8% of extremely poor people lived in South Asia and Sub-Saharan Africa (The Hindu). Most of the poor people are living in rural area in all developing countries. These poor people are generally landless and small landholders and agriculture is the main source of their livelihood. Some of the poor people work as the carpenter, blacksmith, pottery, grocery, herders, milkman, and daily wage workers etc. Their main sources of water for drinking, washing, and [214] Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH 5. Water and Human Beings Out of Pocket Expenditure on Health The contaminated water used by the people increases the financial burden of the household because the contaminated water or lack of safe drinking water creates a number of diseases. In the developing countries, like India out of pocket expenditure (OOP) on health is more than public expenditure. The expenditure on health out of their pocket by the poor people affects on Table 1: Water-related Diseases Sl.No. Water-Related Diseases Name of Diseases 1 Water Borne Diseases Cholera, typhoid, amoebic and bacillary dysentery and other diarrheal diseases. 2 Water Washed Diseases Scabies, trachoma and flea, lice and tick-borne diseases. 3 Water based Diseases Dracunculiasis, schistosomiasis, and other helminths. 4 Water-Related Diseases Dengue, malaria, filariasis, onchocerciasis, trypanosomiasis and yellow fever. Table 1: Water-related Diseases Sl.No. Water-Related Diseases Name of Diseases 1 Water Borne Diseases Cholera, typhoid, amoebic and bacillary dysentery and other diarrheal diseases. 2 Water Washed Diseases Scabies, trachoma and flea, lice and tick-borne diseases. 3 Water based Diseases Dracunculiasis, schistosomiasis, and other helminths. 4 Water-Related Diseases Dengue, malaria, filariasis, onchocerciasis, trypanosomiasis and yellow fever. Table 1: Water-related Diseases 6. Out of Pocket Expenditure on Health 5. Water and Human Beings Life without water is impossible. It is used for drinking, washing, bathing, agriculture, industries etc. The food grains for human consumption come from agriculture and the agriculture is not possible without water. The agriculture is the main income source of the rural poor. In developing countries, due to lack of adequate irrigation facilities, natural rainwater is the main [215] Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH ISSN- 2350-0530(O), ISSN- 2394-3629(P) DOI: 10.5281/zenodo.836441 ISSN- 2350-0530(O), ISSN- 2394-3629(P) DOI: 10.5281/zenodo.836441 [Kro , Vol.5 (Iss.7): July, 2017] runner for harvesting. However in some area, basically, nearby forest area people manage water supply in their agriculture field by indigenous water management technique like making canal by the villagers on the small river. But, natural rain water is still playing a pivotal role in agriculture of developing countries. The natural rainwater used by poor farmers is shown in following figure 1 Figure 1: Use of run-off water in Agriculture field Figure 1: Use of run-off water in Agriculture field In the fig.1, The poor farmers used runoff water in agriculture is shown by the upward extreme left side arrow in the figure, but the run-off water used in agriculture also provides other services such as, for cleaning, washing, etc., i.e., shown on left side downward arrow. The upper arrow indicates the run-off water used in agriculture and the lower arrow indicates stabilizing of the water table in the concerned area; the right side upward arrow indicates agriculture practiced by poor farmers and the extreme right side downward arrow depicts how agriculture supports poor farmers. Even though human life is impossible without water, the contaminated water affects on human health. There are a lot of diseases that related to water. A few examples of water-related diseases are given in the table1. Even though human life is impossible without water, the contaminated water affects on human health. There are a lot of diseases that related to water. A few examples of water-related diseases are given in the table1. Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH [216] Table 1: Water-related Diseases Sl.No. Water-Related Diseases Name of Diseases 1 Water Borne Diseases Cholera, typhoid, amoebic and bacillary dysentery and other diarrheal diseases. 2 Water Washed Diseases Scabies, trachoma and flea, lice and tick-borne diseases. 3 Water based Diseases Dracunculiasis, schistosomiasis, and other helminths. 4 Water-Related Diseases Dengue, malaria, filariasis, onchocerciasis, trypanosomiasis and yellow fever. 6. 6. Out of Pocket Expenditure on Health In other words, out-of-pocket health expenditure is lowest in developed countries and it is very high in developing countries. The table 2 shows that the out-of-pocket health expenditure is 47.9%, 53.4%, 32.2% and 17.8% in low income, lower middle income, upper middle income and high-income countries respectively. The out-of-pocket expenditure on health is lowest in high-income countries and it is highest in lower middle-income countries. In other words, out-of-pocket health expenditure is lowest in developed countries and it is very high in developing countries. 6. Out of Pocket Expenditure on Health The contaminated water used by the people increases the financial burden of the household because the contaminated water or lack of safe drinking water creates a number of diseases. In the developing countries, like India out of pocket expenditure (OOP) on health is more than public expenditure. The expenditure on health out of their pocket by the poor people affects on Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH [216] [Kro , Vol.5 (Iss.7): July, 2017] ISSN- 2350-0530(O), ISSN- 2394-3629(P) DOI: 10.5281/zenodo.836441 their children education, food requirements etc. The World Bank has divided the economies into four groups according to 2009 GNI per capita using Atlas Method, such as Low Income ($995 or less), Lower Middle Income ($996-$3945), Upper Middle Income ($3946-$12195) and High Income ($12196 or more) and accordingly WHO has shown the Out-of-Pocket health expenditure with respect to this different groups. Table 2: Out-of-Pocket Health Expenditure, 2012 Sl.No. Country Group Out-of-Pocket Health Expenditure as a % of Total Health Expenditure 1 Low Income 47.9 2 Lower Middle Income 53.4 3 Upper Middle Income 32.2 4 High Income 17.8 Source: WHO, 2014 (Global Health Expenditure Database). The table 2 shows that the out-of-pocket health expenditure is 47.9%, 53.4%, 32.2% and 17.8% in low income, lower middle income, upper middle income and high-income countries respectively. The out-of-pocket expenditure on health is lowest in high-income countries and it is highest in lower middle-income countries. In other words, out-of-pocket health expenditure is lowest in developed countries and it is very high in developing countries. Table 2: Out-of-Pocket Health Expenditure, 2012 Sl.No. Country Group Out-of-Pocket Health Expenditure as a % of Total Health Expenditure 1 Low Income 47.9 2 Lower Middle Income 53.4 3 Upper Middle Income 32.2 4 High Income 17.8 Source: WHO, 2014 (Global Health Expenditure Database). Table 2: Out-of-Pocket Health Expenditure, 2012 Sl.No. Country Group Out-of-Pocket Health Expenditure as a % of Total Health Expenditure 1 Low Income 47.9 2 Lower Middle Income 53.4 3 Upper Middle Income 32.2 4 High Income 17.8 Source: WHO, 2014 (Global Health Expenditure Database). Table 2: Out-of-Pocket Health Expenditure, 2012 The table 2 shows that the out-of-pocket health expenditure is 47.9%, 53.4%, 32.2% and 17.8% in low income, lower middle income, upper middle income and high-income countries respectively. The out-of-pocket expenditure on health is lowest in high-income countries and it is highest in lower middle-income countries. 7. Contaminated water, Diseases and Poverty’s relationship Contaminated water is polluted water having foreign toxic substances, like arsenic, fluoride, mercury, lead etc. which is not fit for human consumption. Consumption such water affects on human health which ultimately reduces human welfare. But, unfortunately, millions of the people are still drinking this contaminated water which increases mortality and morbidity and reduces the longevity of the people. The consumption of such water increases households' expenditure too because of consumption of such water harm human health through different types of diseases, like cholera, malaria, diarrheal, polio, dengue etc. The relationship among contaminated water, diseases, and poverty is explained through the following figure 2. Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH [217] The lack of safe drinking water or contaminated water used by poor people forces them to stay in a poverty trap. The contaminated water creates diseases; as a result, a part of their income is expended for disease treatment. Although, the poor people mean low-income group, therefore, The lack of safe drinking water or contaminated water used by poor people forces them to stay in a poverty trap. The contaminated water creates diseases; as a result, a part of their income is expended for disease treatment. Although, the poor people mean low-income group, therefore, [217] Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH [Kro , Vol.5 (Iss.7): July, 2017] ISSN- 2350-0530(O), ISSN- 2394-3629(P) DOI: 10.5281/zenodo.836441 the income of the poor are expended on disease treatment and on other household expenditures. Generally, they have either zero or low saving or negative saving. The zero or low saving or negative saving means low capital formation like they cannot be able to afford for their children education and for physical capital. The low capital formation ultimately pulls back the poor people into the poverty trap. 8. Conclusion and Policy Implication The diseases are one of the main factors for creating vicious circle poverty. These diseases are mainly come out from contaminated water. They add extra expenditure to the households’ budget that leads to deficit budget of the poor families and deprives the children’ education as well. This low capital formation (both human capital and physical capital) pulls back into the earlier position Therefore different steps can be taken to reduce diseases as well as the poverty p p p y p p earlier position. Therefore, different steps can be taken to reduce diseases as well as i. The government should increase health expenditure out of its GDP so that out-of-pocket expenditure is reduced. ii. The safe drinking water and sanitation facilities should be provided so that diseases and expenditure on diseases are reduced. ii. The safe drinking water and sanitation facilities should be provided so that diseases and expenditure on diseases are reduced. iii. Awareness, as well as health education, should be effectively provided through health workers. iii. Awareness, as well as health education, should be effectively provided through health workers. iv. Nutritional education is also needed to mitigate diseases. The out-of-pocket health expenditure of the poor people means non-fulfilment of other needs. Therefore, high out-of-pocket health expenditure, especially in developing countries, will impact on human capital formation. The low human capital formation will impact negatively on the economy. The public health expenditure only on diseases treatment is not the end of the government; it should give importance on other sides too, like health education, effective health awareness programme, nutritional education etc. Corresponding author. E-mail address: mangalkro@ gmail.com Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH E-mail address: mangalkro@ gmail.com References [1] Gupta, I. (2009). Out-of-pocket expenditure and poverty: Estimates from NSS 61st round. Paper presented for consideration of the expert group on poverty, planning commission. Retrieved from http://planning commission.nic.in/reports/genrep/indrani.pdf. [2] Gleick, P.H. (2002). Dirty water: Estimated deaths from water-related diseases 200 [3] Von Doorslaer, E. et al (2006). Effect of payment for health care on poverty estimates in 11 countries in Asia: An analysis of household survey data. [3] Von Doorslaer, E. et al (2006). Effect of payment for health care on poverty estimates in 11 countries in Asia: An analysis of household survey data. [4] World Bank estimates show fall in India’s Poverty Rate. Retrieved from http://www.thehindu.com/news/national/world-bank-estimates-show-fall-in-indias-poverty- rate/article7727591.ece. [5] Water aid. Drinking water quality in rural India: Issues and approaches. Retrieved from http://www.wateraid.org/≈/media/Publications/drinking-water-quality-rural-india.pdf. [5] Water aid. Drinking water quality in rural India: Issues and approaches. Retrieved from http://www.wateraid.org/≈/media/Publications/drinking-water-quality-rural-india.pdf. p g g q y [6] Country Classification. Retrieved from http://go.worldbank.org/L547EEP5C0. y p g g [7] Abayawardana, S. & Hussain, I. (2002). Water, health and poverty linkages: A case study from Sri Lanka. Paper prepared for the Asian Development Bank Regional Consultation Workshop on Water and Poverty, Dhaka, Bangladesh. [7] Abayawardana, S. & Hussain, I. (2002). Water, health and poverty linkages: A case study from Sri Lanka. Paper prepared for the Asian Development Bank Regional Consultation Workshop on Water and Poverty, Dhaka, Bangladesh. [218] Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH
https://openalex.org/W3119941335	https://zenodo.org/records/2011911/files/article.pdf	English	null	Flowers of Prey	Scientific American	1,903	public-domain	2,275	FLOWERS OF PREY. BY J CARTER BltAlm. B r a z i l ian birds, fly catcherrs, dis play a brh Upper Part of American iIIan tis with an Insect Which It is About to Devour. Upper Part of American iIIan tis with an Insect Which It is About to Devour. Immature Form ot Rose-Leaf Mantis of the Genus Gongylus, trom India. Deroplatys Truucata (IrUntated M.aut!:;), from Singapore. col'lected them from the twigs of a bush where they were hanging and brought them alive to a Mr. Lary more, who forwarded them to Mr. Buckland, who in turn gave them to the doctor. They are said to particularly frequent rose bushes, and at Mindipur are known as rose·leaf insects. Their wings, when the mantis is mature, are furnished with foliaceous expansions that perfectly counterfeit rose leaves. They were fed upon flies and grasshoppers, preferring the flies because the grasshoppers were evidently too vigorous for them to easily manage. The insects were immature; they did not exhibit, look iag at their upper surfaces, any particularly striking peculiarities, except the leaf· like di'lation of the pro thorax, and the foliaceous appendages to the legs, all of which, together with the rest of the upper parts of of which, together with the rest of the upper parts of some particular plant. The Thomi Deroplatys Sarwace, trom Borneo. Curious Orcbid Lately Dlscovered by Wood Mason. FLOWER TBAPS. Curious Orcbid Lately Dlscovered by Wood Mason. FLOWER TBAPS. B r a z i l ian birds, fly catcherrs, dis play a brh liantly colored c r e s t easily mistaken for a flower cup. In sects, attract- ed by what ap pears to be a scovered by Wood Mason. freshly opened b}{)ssom, f 11 r nish the birds with food. An Asiatic lizard is entirely colored like the surface of the desert plains where it lives, except that at each angle of the mouth blooms a brilliant red folding of the flesh exactly resembling a little flower that grows in the sa.nd. Insects lured- by the seeming flower are incontinently disillusioned when they settle upon it. Deroplatys Sarwace, trom Borneo. Curious Orcbid Lately Dlscovered by Wood Mason. FLOWER TBAPS. the insect, was green. On the other hand, the mantis presents an entirely different appearance when its under surface is exposed to view. FLOWERS OF PREY. BY J CARTER BltAlm. Probably in some respects the most surprising result of late entomologica'l exploration is the discovery of semblances of orchidaceous flowers endowed with ani mal life and voracious carnivorous appetites, that seize and incontinently devour insect vegetarians which, al lured by their form and color, incautiously alight upon them. These flower insects belong to the curious fam ily Mantidre, of which we have a well-known member in our southern States, Phasmomantis· Oarolina, com monly cailled "praying mantis," though if the flrst part of the name was spelled with an "e" instead of an "a," it would be far more appropriate, since no known insect is more bloodthirsty and destructive of smaller and weaker individuals belonging to its class. Its form is characteristic of its predatory habits. The mantis is really a four-legged insect, for the fore limbs are so modifled that they cannot under any circumstanoes be u s e d in walking an d are no more properly term ed legs than would be the arms of men or the winge of birds. They are, in fact, t h e natural weapons of the insect and are used for noth ing else than flghting a n d for capturing \. \ \ \ " Probably in some respects the most surprising result of late entomologica'l exploration is the discovery of semblances of orchidaceous flowers endowed with ani mal life and voracious carnivorous appetites, that seize and incontinently devour insect vegetarians which, al lured by their form and color, incautiously alight upon them. These flower insects belong to the curious fam ily Mantidre, of which we have a well-known member in our southern States, Phasmomantis· Oarolina, com monly cailled "praying mantis," though if the flrst part of the name was spelled with an "e" instead of an "a," it would be far more appropriate, since no known insect is more bloodthirsty and destructive of smaller and weaker individuals belonging to its class. Its form is characteristic of its predatory habits. The mantis is really a four-legged insect, for the fore limbs are so modifled that they cannot under any circumstanoes The curious forms shown in our illustratlone belong to the same genus, Deroplatys, ailthough the flrst is a native of Singapore, south of the Malay Peninsula, while the Sarawaca is from Borneo. In these species the outspread wings simvlate the petals of a flower. .. . • In the city of New York there are only 737,477 white persons bOrD ot Dative parenti. FLOWERS OF PREY. As the mantis rests, head downward, amid the stems and leaves of a plant, the fore legs drawn in so that they c a n n o t b e s e e n, t h e thighs of the two hind ones radiating out on each side, and the thor ax and the ab domen raised at right an gles to each other, the in s e c t might easily at flrst sight deceive more discrim inating ento mologists than the h 0 n e y seekers t h a t settle upon it. An allied spe c i e s, exactly resembling a pink orchid, is mentioned by D r. Wallace, on the author ity 0 f S i r Charles Dilke, as inhabiting Java. Its spe cialty is all ur ing and cap turing butter flies. The ex pected guest Deroplatys Sarwace, trom B having arriv- ed, the seeming feast spread out for his delectation s 139 139 AUGUST 22, 1903. mouth of the tube of a corolla. In addition to this, the long, slender upper part of the prothorax reo sembles a flower, while the fore limbs, when restiƓg drawn up in the center of the oorolla, add to and heighten the imposture. touched by gentle zephyrs. A bright violet·blue dUa tion of the thorax, in front of which its fore legs, band ed violet and black, extended like petals, simulated the corolla of a papilionaceous flower so perfectly as to deceive the eyes of a practised botanist. An ac count is given in the proceedings of the Asiatic So ciety, Bengal, of a number of specimens of this mantis in the possession of Dr. J. Anderson. These imects came from Mindipur. Santal women and children liad FLOWERS OF PREY. BY J CARTER BltAlm. The leaf-like expan sion of the prothorax, instead of green, presents a pale, clear lavender· violet hue with a faint pink bloom along the margin. The resemblance to the coro'lla of an orchidaceous flower thus presented is perfected by the presence of a dark brown spot in the middle over the prothoru or breast, which looks precisely like the FLOWERS OF PREY. FLOWERS OF PREY. BY J CARTER BltAlm. Probably in some respects the most surprising result of late entomologica'l exploration is the discovery of semblances of orchidaceous flowers endowed with ani mal life and voracious carnivorous appetites, that seize and incontinently devour insect vegetarians which, al lured by their form and color, incautiously alight upon them. These flower insects belong to the curious fam ily Mantidre, of which we have a well-known member in our southern States, Phasmomantis· Oarolina, com monly cailled "praying mantis," though if the flrst part of the name was spelled with an "e" instead of an "a," it would be far more appropriate, since no known insect is more bloodthirsty and destructive of smaller and weaker individuals belonging to its class. Its form is characteristic of its predatory habits. The mantis is really a four-legged insect, for the fore limbs are so modifled that they cannot under any circumstanoes be u s e d in walking an d are no more properly term ed legs than would be the arms of men or the winge of birds. They are, in fact, t h e natural weapons of the insect and are used for noth ing else than flghting a n d for capturing prey. T h e insect shown at Fig. 2, discovered by Wood Ma- \. \ \ \ " Upper Part of American iIIan tis with an Insect Which It is About to Devour. son, masquerades sometimes as a pink and at others as a white orchid. The whole flower insect is either conspicuously white or of a resplendent pink color, and both in color and form perfectly imi tates a flower. The lower or apparently anterior petal of an orchidaceolls blossom, the labellum, often of a very curious shape, is represented by the abdomen of the insect, while the parts which might be taken, re garding it as an insect, for its wings, are actua'lly the femurs of the two pairs of posterior limbs, so greatly expanded, flattened, and shaped in such manner as to represent the remaining petals of the flower. FLOWERS OF PREY. BY J CARTER BltAlm. Honey bees as well as other i n - s e c t s have been found in their murder Blossoms of the Vibernum lantana, a Eliropean shrub having large ovate leaves and dense cymes of small w h i t e flowers, and it may be added, our American species (Viber num lentago ) , or sheep berry, a 1 s 0 bea.ring Immature Form ot Rose-Leaf Mantis of the Genus Gongylus, trom India. \. \ \ \ " Upper Part of American iIIan tis with an Insect Which It is About to Devour. hue as that of the blossoms, and their glo b u 1 a r abdo mens m i m i c the unopened buds (of which Immature Form ot Rose-Leaf Mantis of there are many the Genus Gongylus, trom India. in each clus- ter) Dot only in color but in shape and size. These l!illiders spin no web, depending upon strategy to secure their ppef, and live upon their insect visitors. Later in the sea son spiders apparently identical with these, except in color, are found in the blossoms of the Orchis macu lata. The spiders found here have dark reddish brown spots on the abdomen; and in the position usu ally assumed by it, the Aranima, standing with de pressed head, closely mimics in shape and size, in re'lative position, and a little way off in color, the dark purple pollinia of the flowers. Recent investiga tions render it extremely probable that, as flrst sug gested by Prof. N ottridge, as the season advances, these spiders change color, and that each successive change adapts them for concealment in the flowers of some particular plant. The ThomiBu8 citrens poses it- self in t h e midd'le of a c ompo s i t e flower w i t h legs expanded like ita exte rior ray. They have been ob se r v ed i n o r c h id a c e o u s flowers with t h e i r legs expanded h o r i z on tally. Honey bees as well as other i n - s e c t s have been found in their murder ous clutches. FLOWERS OF PREY. BY J CARTER BltAlm. A whole tribe of spiders, members of the Thom isadre family, livmƔ in flower cups, assume the colors and markings of the flowers in which they lie in wait for victims. Deroplatys Truucata (IrUntated M.aut!:;), from Singapore for victims. Blossoms of the Vibernum lantana, a Eliropean shrub having large ovate leaves and dense cymes of small w h i t e flowers, and it may be added, our American species (Viber num lentago ) , or sheep berry, a 1 s 0 bea.ring broad, flat clus ters of white flowers, are at times occupied by spiders of ·t h e s a m e creamy - white hue as that of the blossoms, and their glo b u 1 a r abdo mens m i m i c the unopened buds (of which Immature Form ot Rose-Leaf Mantis of there are many the Genus Gongylus, trom India. in each clus- ter) Dot only in color but in shape and size. These l!illiders spin no web, depending upon strategy to secure their ppef, and live upon their insect visitors. Later in the sea son spiders apparently identical with these, except in color, are found in the blossoms of the Orchis macu lata. The spiders found here have dark reddish brown spots on the abdomen; and in the position usu ally assumed by it, the Aranima, standing with de pressed head, closely mimics in shape and size, in re'lative position, and a little way off in color, the dark purple pollinia of the flowers. Recent investiga tions render it extremely probable that, as flrst sug gested by Prof. N ottridge, as the season advances, these spiders change color, and that each successive change adapts them for concealment in the flowers of some particular plant. The ThomiBu8 citrens poses it- self in t h e midd'le of a c ompo s i t e flower w i t h legs expanded like ita exte rior ray. They have been ob se r v ed i n o r c h id a c e o u s flowers with t h e i r legs expanded h o r i z on tally.
W2564705626.txt	https://fermet.misis.ru/jour/article/download/961/876	en		OPTIMAL PARAMETERS OF THE STEAM COAL GASIFICATION IN THE GASIFIER WITH DAMPED CIRCULATING FLUIDIZED BED	Izvestiâ vysših učebnyh zavedenij. Černaâ metallurgiâ	2,016	cc-by	4,081	М ISSN: 0368-0797 . Известия высших учебных заведений. Черная металлургия. 2016. Том 59. № 11. С. 773 – 780. © 2016. Дубинин А.М., Маврин С.П. УДК 621.1:662 ОПТИМАЛЬНЫЕ ПАРАМЕТРЫ ПАРОВОЙ ГАЗИФИКАЦИИ УГЛЕЙ В ГАЗОГЕНЕРАТОРЕ С ЗАТОРМОЖЕННЫМ ЦИРКУЛЯЦИОННЫМ ПСЕВДООЖИЖЕННЫМ СЛОЕМ Дубинин А.М., д.т.н., профессор кафедры теплоэнергетики и теплотехники Маврин С.П., заведующий лабораторий ( mspfr@mail.ru ) Уральский федеральный университет имени первого Президента России Б.Н. Ельцина (620002, Россия, Екатеринбург, ул. Мира, 19) Аннотация. Представлены эксперименты по паровой газификации бородинского угля в двухкамерном газогенераторе с заторможенным насадкой циркуляционным псевдоожиженным слоем. В одной из двух камер сжигали уголь с воздухом, в другой газифицировали уголь водяным паром. Средний размер частиц угля 0,2 мм. Теплота из камеры сгорания в камеру газификации поступала благодаря циркуляции дисперсного теплоносителя (электрокорунда) между камерами. Камеры заполнены сферической насадкой диаметром 50 мм и порозностью 0,5, размер частиц электрокорунда 0,3 мм. Теплота сгорания продуктов газификации 8195 кДж/м3. Химический недожог 5 %, механический недожог 2 %. Составлена модель газогенератора, состоящая из систем двух уравнений теплового баланса камер сгорания и газификации, удовлетворительно описывающая эксперимент. На основании модели найдены оптимальные параметры газогенератора: температура в камере газификации 900 °С, в камере сгорания 1020 °С; доля угля, поступающего в камеру газификации 0,36; химический КПД 40 %. Ключевые слова: уголь, газогенератор, воздух, водяной пар, дисперсный теплоноситель, температура, химический состав, теплота сгорания, моделирование, система уравнений, оптимальные параметры, химический КПД. DOI: 10.17073/0368-0797-2016-11-773-780 Современные технологии использования твердых топлив базируются на их термохимической переработке в синтез газ, который затем сжигается в технологических и энергетических установках или преобразуется в жидкое топливо [1, 2]. Одним из направлений развития таких технологий является снижение температуры в активной зоне термохимического реактора, поскольку расширяется возможность использования низкосортных топлив [3]. Продолжает исследоваться низкотемпературная технология с циркуляционным псевдоожиженным слоем большой единичной производительностью для энергоблоков угольных ТЭС [4]. В США исследуется цикл парогазовой установки (ПГУ) с частичной газификацией угля в реакторе с пузырьковым псевдоожиженным слоем [5]. Основным элементом цикла является разработанная система высокотемпературной газоочистки при температуре 500 °С и более. В целях снижения капиталоемкости оборудования и упрощения условий эксплуатации использован метод автотермической паровой газификации с комбинированной схемой движения дисперсных и газовых потоков. Уголь поступает на газификацию в одном направлении с воздухом и водяным паром, поэтому реализуются преимущества обращенного газогенератора. В данной работе рассматривается случай, когда требуется получение чистого газа без очистки, поскольку для удаления смоляных включений необходимо создание дорогостоящих очистных блоков [6]. В работе [7] показано, что в газогенераторе с псевдоожиженным слоем при паровой газификации углей существуют параметры, при которых химический КПД достигает оптимальной величины. Эксперименты по паровой газификации бородинского угля следующего состава: W r = 33; A r = 7,4; S r = 0,2; C r = 42,6; H r = 3; N r = 0,6; O r = 13,2; V daf = 47 % (по = 15 280 кДж/кг; = 4,5; = 4,89 м3/кг и массе); фракционным составом: 0 – 40 мкм (6,9 %); 40 – 80 мкм (10,9 %); 80 – 150 мкм (18,1 %); 150 – 200 (13,3 %); 200 – 300 мкм (19,3 %); 300 – 400 мкм (15,8 %); 400 – 500 мкм (15,7 %) проведены в газогенераторе с циркуляционным псевдоожиженным слоем (рис. 1), смонтированном на базе эндотермического генератора ЭН-60-ИЗ. В цилиндрической реторте диаметром 280 мм имеются две камеры, заполненные инертной сферической насадкой диаметром 50 мм и порозностью εн = 0,5. Высота камер 1 м. Во внутренней реторте 18 диаметром 120 мм угольную пыль газифицировали водяным паром. В кольцевой камере между внутренней 18 и наружной 19 ретортами осуществляли сжигание угля для получения необходимой теплоты. Между камерами располагали опускные стояки 8 и 15, по которым осуществлялась направленная циркуляция электрокорунда размером 300 мкм. Нагретый в камере сгорания 11 электрокорунд поступал по стоякам 8 в псевдоожиженный слой 5 и через подъемные стояки 20 под камеру газификации 16, где отдавал теплоту на эндотермическую реакцию и подогрев продуктов паровой газификации 773 И . Ч . 2016. Т 5 9 . № 11 Рис. 1. Экспериментальная установка для паровой газификации углей в заторможенном циркуляционном псевдоожиженном слое: 1 – штуцер для ввода водяного пара; 2 – беспровальная парораспределительная решетка; 3 – штуцер для удаления золы и шлака; 4 – устройство (шибер) для регулирования расхода электрокорунда по опускным стоякам; 5, 21 – псевдоожиженный слой; 6 – воздухораспределительная беспровальная решетка; 7 – штуцер для ввода угля в камеру сгорания; 8 – опускной стояк; 9 – трубка для замера температуры по высоте камеры сгорания; 10 – тепловая изоляция; 11 – сферическая насадка в камере сгорания; 12 – штуцер для выхода продуктов сгорания; 13 – трубка для замера температуры по высоте камеры газификации; 14 – штуцер для выхода продуктов газификации; 15 – опускной стояк в камере газификации; 16 – сферическая насадка в камере газификации; 17 – устройство для забора проб продуктов газификации по высоте; 18 – внутренняя реторта; 19 – наружная реторта; 20 – подъемные стояки; 22 – штуцер для ввода воздуха на горение; 23 – перегородка; 24 – штуцер для вывода золы; 25 – штуцер для ввода угля на газификацию Fig.1. Experimental unit for coal steam gasiﬁcation in a damped circulating ﬂuidized bed: 1 – ﬁtting for steam input; 2 – steam distribution unfailing grid; 3 – ﬁtting for removing of ash and slag; 4 – device (gate) to control electrocorundum loss at downcomers; 5, 21 – ﬂuidized bed; 6 – air distribution unfailing grid; 7 – ﬁtting for coal input info the combustion chamber; 8 – downcomer; 9 – tube for measuring the temperature at the height of combustion chamber; 10 – thermal insulation; 11 – spherical nozzle in the combustion chamber; 12 – ﬁtting for the exit of combustion products; 13 – tube for measuring the gasiﬁcation chamber temperature heightwise; 14 – ﬁtting for the exit of gasiﬁcation products; 15 – downcomer in the gasiﬁcation chamber; 16 – spherical head in the gasiﬁcation chamber; 17 – device for sampling of gasiﬁcation products heightwise; 18 – inside retort; 19 – outside retort; 20 – lifting risers; 22 – ﬁtting for entering the combustion air; 23 – partition; 24 – ﬁtting for the withdrawal of ashes; 25 – coal input ﬁtting for gasiﬁcation угля. Охлажденный электрокорунд по стояку 15 возвращался в псевдоожиженный слой 21 для нагрева. Уголь в камеру сгорания поступал через штуцер 7, а воздух через штуцер 22. Уголь в камеру газификации посту774 пал через штуцер 25, а перегретый водяной пар через штуцер 1. Продукты сгорания удалялись через штуцера 12, а продукты газификации – через штуцер 14. Расход электрокорунда по стоякам 8 изменяли устройством 4. М Благодаря разности гидравлических сопротивлений заторможенного насадкой циркуляционного слоя и столба электрокорунда, опускающегося по стоякам 8 и 15, создавалась устойчивая циркуляция частиц между камерами. Расход циркулирующего теплоносителя (электрокорунда) определяли по скорости опускного движения щупа, помещаемого в опускные стояки 8 и 15, площади поперечного сечения стояков и насыпной плотности электрокорунда. Расходы угля и воздуха в камеру сгорания составляли 22,4 и 126 м3/ч при коэффициенте избытка воздуха 1,25. Расходы угля и водяного пара в камеру газификации равнялись 12,5 и 8 кг/ч соответственно. Температуру по высоте камер газификации и сгорания измеряли хромель-алюмелевой (ХА) термопарой, перемещаемой в тонкостенных трубках 9 и 13, заваренных с нижнего торца. Забор продуктов газификации по высоте камеры газификации производили устройством 17, анализ осуществляли на хроматографе. Доля угля, подаваемого в камеру газификации, x = 12,5 / (12,5 + 22,4) = 0,358. Расход воздуха измеряли ротаметром, водяного пара, перегретого до 450 °С, дроссельной шайбой, угля – калиброванными шайбами, заранее протарированными. Удельную внешнюю площадь поверхности угольных частиц в слое определяли по рекомендациям, приведенным в работах [8, 9]. Поля температур в камерах газификации t1 и сгорания t2 представлены на рис. 2. Температура по высоте камер распределяется практически равномерно. С увеличением расхода электрокорунда температуры в камерах газификации и сгорания сближаются и находятся на достаточно высоком уровне, необходимом для паровой газификации угля. Состав продуктов по высоте камеры газификации при разных температурах представлен в табл. 1. Видно, что процесс взаимодействия углерода топлива с водяным паром проходит через образование, накапливание и расходование диоксида углерода. Мольное отношение расходов водяного пара и углерода топлива равнялось 1:1. При температуре 860 °C и высоте камеры газификации 1 м степень расходования водяного пара составляла 83,9 %. Теплота сгорания продуктов газификации = 8195 кДж/м3. С повышением температуры от 720 до 860 °С теплота сгорания продуктов возрастала с 5847 до 8195 кДж/м3, т.е. на 40 %. Моделирование газогенератора с циркуляционным заторможенным псевдоожиженным слоем Синтез газ при паровой газификации углерода топлива образуется в соответствии с [10] (1) прямой (2) Рис. 2. Экспериментальная зависимость температуры в камере газификации t1 и в камере сгорания t2 от относительной циркуляции электрокорунда μ между камерами; Z – расстояние от нижнего торца камеры. Доля угля, подаваемого в камеру газификации x = 0,358 Fig.2. Experimental temperature dependence in the gasiﬁcation chamber t1 and in the combustion chamber t2 on the relative circulation of electrocorundum μ between the chambers; Z – distance from the lower end of the chamber. The share of coal supplied to the gasiﬁcation chamber x = 0.358 и обратной реакцией водяного газа (3) с эндотермическим эффектом qx = 10 955 кДж/кг угле1 рода [11]. Для обеспечения теплотой эндотермической реакции паровой газификации углерода топлива и подогрева продуктов этой реакции до оптимальной температуры организована камера сгорания 11 (см. рис. 1), в которой протекает экзотермическая реакция горения углерода топлива (4) с тепловым эффектом qx = 32 833 кДж/кг углерода 0 (примерно равна теплоте сгорания угля 35 868 кДж/кг углерода). Система из двух уравнений теплового баланса камер: – камера газификации 775 И . Ч . 2016. Т 5 9 . № 11 Таблица 1 Зависимость состава продуктов паровой газификации от высоты камеры и температуры t1 (x = 0,358) Table 1. Dependence of composition of steam gasiﬁcation products on the chamber height and temperature t1 (x = 0.358) Температура, °С Высота, z, м 720 790 860 Состав влажного газа, % об. СО2 N2 СH4 СО H2 Н2О 0,2 17,4 0,4 0,4 21,6 0,6 5,6 8,3 12,7 55,6 5,1 11,3 15,9 45,5 0,6 21,6 0,5 5,1 13,2 18,4 41,1 0,8 22,6 0,4 4,1 14,5 21,3 37,1 1,0 22,1 0,3 4,1 15,9 22,3 35,5 0,2 18,7 0,3 3,0 12,1 16,1 49,8 0,4 20,9 0,3 2,6 16,7 21,2 38,3 0,6 18,9 0,2 2,0 20,6 25,1 33,2 0,8 27,6 0,1 2,0 23,7 28,6 28,0 1,0 16,9 0,2 1,9 25,4 29,9 25,7 0,2 18,3 0,3 2,9 14,9 18,9 44,7 0,4 20,2 0,4 2,4 20,2 24,6 32.2 0,6 18,4 0,3 1,8 25,2 29,3 25,0 0,8 16,1 0,2 1,8 29,1 33,5 19,3 1,0 15,8 0,2 1,4 30,8 35,7 16,1 (5) – камера сгорания (6) В левой части уравнения (5) приход теплоты с электрокорундом из камеры сгорания с температурой t2 ; сухим углем qy ; влагой угля qв(у) ; водяным паром qп и приход теплоты теплопередачей через стенку внутренней реторты qт из камеры сгорания. В правой части уравнения (5) расход теплоты на эндотермическую реакцию газификации, на нагрев продуктов газификации; с электрокорундом, поступающим в камеру сгорания при температуре t1 ; на нагрев, испарение и перегрев пара от влаги угля qнив ; с золой q6 . Все члены в уравнении измеряются в кДж/кг углерода топлива. В левой части уравнения (6) приход теплоты от сгорания углерода топлива в камере сгорания; с сухим углем qy ; влагой угля qв(у) ; воздухом qв ; с электрокорундом, поступающим из камеры газификации с температурой t1 . В правой части уравнения (6) расход теплоты на нагрев продуктов сгорания и электрокорунда до температуры t2 ; нагрев, испарение и перегрев пара от влаги 776 Теплота сгорания на выходе QH1 кДж/м3 5 847 7 075 8 195 угля qнив , теплопередачей через стенку реторты qт в камеру газификации и с золой q6 . Все члены в уравнении (6) измеряются в кДж/кг углерода. Удельные массовые теплоемкости: продуктов газификации Cпг = 5, сгорания Cпс = 13,6, электрокорунда Ck = 1,17 кДж/кг на 1 кг углерода; μ – относительный расход электрокорунда, кг на 1 кг углерода, подаваемого в газогенератор; x – доля угля, подаваемого в камеру газификации (0,358), а (1 – x) = 0,642 – в камеру сгорания; теплота, подведенная с углем qy = 60; с влагой угля qв(у) = 66; с воздухом qв = 390; с теплопередачей qт = 2800; нагрев, испарение и перегрев пара от влаги угля qнив = 3000; qп = 1330; уходящей золой q6 = 170 кДж на 1 кг углерода. Удельные тепловые потери с химическим недожогом q3 = 0,05; механическим недожогом q4 = 0,02; в окружающую среду q5 = 0,23. Результат решения системы уравнений (5) и (6) представлен на рис. 3. Там же нанесены экспериментально полученные значения температуры при μ = 18 и x = 0,358. Материальные балансы газогенератора [10]: – концентрация диоксида углерода: (7) – концентрация оксида углерода: М (10) (8) При Z = h (высота камеры) имеем состав на выходе из газогенератора. Насадка разрушает пузыри в псевдоожиженном слое, поэтому используем эффективные константы скорости для плотного слоя [12]. Константа скорости взаимодействия водяного пара с углеродом бородинского угля м3/(м2с) – концентрация водяного пара: Константа скорости взаимодействия оксида углерода с водяным паром, м3/(м2с) (9) – концентрация водорода: Константа скорости взаимодействия диоксида углерода с водородом, м3/(м2с) где Kр – константа равновесия реакции водяного газа [13]. Удельная внешняя площадь поверхности угольных частиц в потоке определялась по выражению [8, 9], м2/м3 где ρ = ρн (1 – Z * ) + ρk Z * – средняя плотность потока, кг/м3; ρн и ρk – истинные плотности инертного материала (электрокорунда, золы) и кокса, равные соответственно 3760 и 1650 кг/м3; Z * – концентрация кокса в потоке, кг/кг смеси; ε – порозность потока (~0,25 – 0,3), м3/м3; dk – размер частиц кокса, м. Скорость продуктов газификации, м/с, возрастает по высоте камеры в 2 раза (см. уравнение (1)) и описывается выражением а средняя равна Рис. 3. Результаты моделирования зависимости температуры в камере газификации t1 и в камере сгорания t2 от относительной циркуляции дисперсного теплоносителя μ между камерами: – эксперимент при μ = 18; x = 0,358 в камере газификации, – в камере сгорания; Z – расстояние от нижнего торца камеры, м Fig. 3. The modeling results of temperature dependence in the gasiﬁcation chamber t1 and the combustion chamber t2 on a relative particulate heat carrier circulation μ between the chambers. Points – experiment with μ = 18; x = 0.358 in the gasiﬁcation chamber, – in the combustion chamber. Z – distance from the lower end of the chamber, m где wo – скорость продуктов на выходе из камеры газификации при реальных параметрах в расчете на свободное сечение, w* – скорость на выхо- де из камеры при нормальных условиях в расчете на свободное сечение камеры. В табл. 2 показано сравнение экспериментально полученных концентраций на выходе из камеры газификации с рассчитанным составом по представленной модели. 777 И . Ч Таблица 2 Сравнение расчетного и экспериментально полученного состава продуктов паровой газификации бородинского угля на выходе из газогенератора: μ = 18; x = 0,358; w = 3,16 м/с; h = 1 м; t1 = 860 °С, S = 1200 м2/м3 Table 2. Comparison of the calculated and experimental composition of Borodino coal products of steam gasiﬁcation at the exit of the gasiﬁer: μ = 18; x = 0,358; w = 3,16 m/s; h = 1 m; t1 = 860 °С, S = 1200 m2/m3 Состав продукта, % об. СО2 СО N2 Н2О СH4 H2 Эксперимент 15,8 30,8 0,2 16,1 1,4 35,7 Моделирование 12,4 45,5 – 4,2 – 37,9 Моделирование камеры сгорания . 2016. Т 5 9 . № 11 Для дожигания CO над слоем подается воздух. Концентрация CO2 на выходе Концентрация азота Химический недожог q3 = 0,05 (5 %); механический недожог q4 = 0,02 (2 %) найден по методике, представленной в работе [9]. Формулы для определения константы скорости взаимодействия углерода бородинского угля с кислородом воздуха и с диоксидом углерода Реакция горения углерода топлива с кислородом воздуха (4) заканчивается практически на входе в камеру сгорания, а удельная площадь поверхности коксовых частиц в потоке, проходящем через насадку, высока, S = 20 м2/м3. Образовавшийся при горении CO2 реагирует с коксовыми частицами по реакции С + CO2 + 3,76 N2 = 2 CO + 3,76 N2 . Поэтому в составе продуктов полного сгорания на выходе из камеры присутствует оксид углерода (2,63 %), концентрация которого находится из решения уравнения материального баланса (11) с граничным условием rCO Z = 0 = 0, где w2 – скорость, м/с, продуктов сгорания в насадке при рабочих параметрах t и Р, w2 = 6,3 м/с. Решение уравнения (11) совместно с (7) при указанном граничном условии дает выражение для расчета концентрации rCO, м3/м3, на выходе из камеры сгорания. взяты из работы [14]. Моделирование оптимальных параметров процесса паровой газификации угля в газогенераторе с циркуляционным псевдоожиженным слоем Поскольку модель удовлетворительно отражает эксперимент, то она применима для оптимизации параметров паровой газификации угля. Для оптимизации задается ряд значений доли x от 0,25 до 0,4 с интервалом 0,05 и из системы уравнений тепловых балансов (5) и (6) находим значения температуры t1 и t2 при этих же заданных значениях доли x. При этих же температурах из уравнений (7) – (10) рассчитывается состав продуктов газификации. Далее определяется теплота сгорания продуктов газификации по уравнению Д.И. Менделеева при этих же температурах [15] При тех же температурах рассчитывается химический КПД, %, без учета возврата физической теплоты продуктов сгорания и газификации в газогенератор Поскольку щается: 778 то последнее выражение упро- где v = 3,73 м3 продуктов газификации, образующихся из 1 кг углерода топлива по реакции (1); qxo – теплота сгорания топлива, кДж/кг углерода. Строится зависимость (рис. 4) t1 , t2 и ηx от доли x. Графическим методом находится максимальное зна- М чение и соответствующие этому значению , , x опт. Они равны: = 900 °С; = 1020 °С; x опт = 0,36; = 40 %. Там же нанесены экспериментально полученные значения t1 , t2 и ηx . Выводы. Удовлетворительное качественное и количественное совпадение моделирования и эксперимента дает основание утверждать о правильном подходе к процессу моделирования и оптимизации паровой газификации бородинского угля в газогенераторе с заторможенным циркуляционным псевдоожиженным слоем. БИБЛИОГРАФИЧЕСКИЙ СПИСОК 1. Хоффман Е. Энерготехнологическое использование угля: Пер. с англ. – М.: Энергоатомиздат, 1983. – 328 с. 2. Корчевой Ю.П., Майстренко А.Б., Топал А.И. Экологически чистые угольные энерготехнологии. – Киев: Научная мысль, 2004. – 187 с. 3. Табакаев Р.Б., Казаков А.В., Заворин А.С. Перспективность низкосортных топлив Томской области для теплотехнического использования // Известия Томского политехнического университета. 2013. Т. 323. № 4. С. 41 – 46. 4. Combined Cycle Systems for Near-Zero Emission Power Generation. Ashok Rao ed.Woodhead Publishing. 2012. – 360 p. 5. Khan J., Wang T. Implementation of demoisturiration and devolatization model in multi – phase simulation of hubrid entrained and ﬂuidized bed mild qasiﬁer // International Jornal of Clean Coal and Energy. 2013. No. 2. P. 35 – 53. 6. Загрутдинов Р.Ш., Нагорнов А.Н., Сенчин П.К. Наладочные испытания газогенераторов Lurgi и перспективы газогенераторных технологий // Ползуновский вестник. 2007. № 3. С. 40 – 47. 7. Дубинин А.М., Тупоногов В.Г., Филиппов Д.В. Оптимизация процесса паровой газификации угля в кипящем слое // Энергосбережение и водоподготовка. 2008. № 4 (54). С. 60 – 62. 8. Мунц В.А., Баскаков А.П., Ашихмин А.А. Расчет газообразования при горении твердого топлива в кипящем слое // Инженерно-физический журнал.1988.Т. 54. № 3. С. 432 – 438. 9. Баскаков А.П., Мацнев В.В., Распопов И.В. Котлы и топки с кипящим слоем. – М.: Энергоиздат, 1996. – 352 с. 10. Дубинин А.М., Кагарманов Г.Р., Обожин О.А. Моделирование газификации углей водяным паром // Химия твердого топлива. 2012. № 3. С. 30 – 33. 11. Жуховицкий А.А., Шварцман Л.А. Физическая химия: Учеб. для вуз. – М.: Металлургия, 2001. – 688 с. 12. Путрова Е.Е., Свирида Л.В., Федосеев С.Д. О распределении концентрации CO2 и H2O по высоте реакционной зоны газогенератора в изотермических условиях // Химия твердого топлива. 1982. № 2. С. 94 – 96. I VUZ . C M = I Рис. 4. Результаты моделирования зависимости температуры в камере газификации t1 , в камере сгорания t2 и химического КПД от доли углерода (угля), подаваемого в камеру газификации. Относительная циркуляция электрокорунда μ = 18 кг/кг. Точки экспериментальных данных: – t1 ; – t2 ; – ηx Fig. 4. The modeling results of temperature dependence in the gasiﬁcation chamber t1 , the combustion t2 and chemical eﬃciency on the proportion of carbon (coal) supplying into the gasiﬁcation chamber. Relative circulation of electrocorundum μ = 18 kg/kg. Points of experimental data: – t1 ; – t2 ; – ηx 13. Жоров Ю.М. Термодинамика химических процессов. Нефтехимический синтез, переработка нефти, угля и природного газа. – М.: Химия, 1985. – 464 с. 14. Виленский Т.В., Хзмалян Д.М. Динамика горения пылевидного топлива. – М.: Энергия, 1978. – 248 с. 15. Теплотехника: Учеб. для вуз. / А.П.Баскаков, Б.В.Берг, О.К.Витт и др.; Под ред. А.П. Баскакова. –3-е изд., перераб. и доп. – М.: ООО «ИД Бастет», 2010. – 328 с. Поступила 15 сентября 2015 г. . F M . 2016. V . 5 9 . N . 11 , pp. 773–780. OPTIMAL PARAMETERS OF THE STEAM COAL GASIFICATION IN THE GASIFIER WITH DAMPED CIRCULATING FLUIDIZED BED A.M. Dubinin, S.P. Mavrin Ural Federal University named after the ﬁrst President of Russia B.N. Yeltsin, Ekaterinburg, Russia Abstract. The authors have made several experiments of Borodino coal steam gasiﬁcation in a two-chamber gasiﬁer with a circulating ﬂuidized bed damped by the checker. In one of the two chambers the coal was burnt with excess air coeﬃcient of 1.25, in another the coal was gasiﬁed by steam. Average size of coal pieces was 0.2 mm. The heat acted from the combustion chamber into the gasiﬁcation chamber through the particulate coolant circulation (fused 0.3 mm) between the chambers. The chambers were ﬁlled with spherical jets with diameter of 50 mm and porosity of 0.5; electrocorundum particle size was 0.3 mm. Caloriﬁc value was 8195 kJ/m3. Mechanical underburning was 2 %. Chemical underburning was 5 %. The model of the gasiﬁer consisting of a system of two equations of thermal balance of the combustion chambers and gasiﬁcation satisfactorily describes the experiment. The optimum parameters of the gasiﬁer were found based on the model: the temperature in the gasiﬁcation chamber – 779 И . Ч 900 °C, the temperature in the combustion chamber – 1020 °C, the share of coal supplied to the gasiﬁcation chamber – 0.36, chemical eﬃciency – 40 %. Keywords: coal, gasiﬁer, air, steam, particulate heat carrier, temperature, chemical composition, heat of combustion, modeling, system of equations, optimal parameters, chemical eﬃciency. DOI: 10.17073/0368-0797-2016-11-773-780 REFERENCES 1. 2. 3. 4. 5. 6. 7. 780 Hoﬀman E.J. Coal conversion. Laramie, Wyoming, 1980. (Russ. ed.: Hoﬀman E.J. Energotekhnologicheskoe ispol’zovanie uglya. Moscow: Energoatomizdat, 1983, 328 p.). Korchevoi Yu.P., Maistrenko A.B., Topal A.I. Ekologicheski chistye ugol’nye energotekhnologii [Clean coal energy technologies]. Kiev: Nauchnaya mysl’, 2004, 187 p. (In Russ.). Tabakaev R.B., Kazakov A.V., Zavorin A.S. Prospect of lowgrade fuels of the Tomsk region for heat engineering use. Izvestiya Tomskogo politekhnicheskogo universiteta. 2013, vol. 323, no. 4, pp. 41–46. (In Russ.). Combined Cycle Systems for Near-Zero Emission Power Generation. Ashok Rao ed. Woodhead Publishing. 2012, 360 p. Khan J., Wang T. 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https://openalex.org/W4389430002	https://publishing.mpda.ru/index.php/theological-herald/article/download/1479/1302	Russian	null	A. V. Kartashev’s Journalistic and Scientific Publications in Church Journals	Bogoslovskij vestnik	2,023	cc-by-sa	3,358	Иеромонах Серафим (Тищенко) Иеромонах Серафим (Тищенко) магистр богословия аспирант Санкт-Петербургской духовной академии проректор по воспитательной работе Воронежской духовной семинарии 393033, Воронеж, Ленинский проспект, 91 s.tishenko2009@ya.ru магистр богословия аспирант Санкт-Петербургской духовной академии проректор по воспитательной работе Воронежской духовной семинарии магистр богословия аспирант Санкт-Петербургской духовной академии проректор по воспитательной работе Воронежской духовной семинарии магистр богословия аспирант Санкт-Петербургской духовной академии проректор по воспитательной работе Воронежской духовной семинарии 393033, Воронеж, Ленинский проспект, 91 s.tishenko2009@ya.ru Для цитирования: Серафим (Тищенко), иером. Публицистические и научные публикации А. В. Карташёва в церковных журналах // Богословский вестник. 2023. № 3 (50). С. 314–323. DOI: 10.31802/GB.2023.50.3.015 Аннотация Статья посвящена дебюту будущего обер-прокурора А. В. Карташёва как учёного и публи- циста на страницах церковных журналов Санкт-Петербургской духовной академии. В ходе исследования осуществляется обзор публикаций А. В. Карташёва в журналах «Христианское чтение» и «Церковный вестник», анализируется тематика и проблематика материалов. Особое внимание уделяется качествам автора статей как учёного. В статье выделяется те- матика материалов, проводится сравнительный анализ с более поздним творчеством мыс- лителя и историка на схожую тематику, определяется место этих первых опытов в раннем творчестве А. В. Карташёва. В этих материалах А. В. Карташёв показывает себя исследова- телем с большим кругозором и задатками публициста-аналитика. Первые материалы, опу- бликованные им в печатных изданиях, составляют неотъемлемый контекст становления его как публициста, служат фундаментом некоторых его творческих подходов в будущем. Ключевые слова: Антон Владимирович Карташёв, церковная периодика, «Христианское чтение», «Церковный вестник», духовное образование в начале XX в., научные рецензии, церковная публицистика, редакция дореволюционного журнала. 315 ПУБЛИК АЦИИ А. В. К АРТАШЁВА В ЦЕРКОВНЫХ Ж УРНА ЛА Х A. V. Kartashev’s Journalistic and Scientific Publications in Church Journals Hieromonk Serafim (Tischenko) Hieromonk Serafim (Tischenko) Hieromonk Serafim (Tischenko) MA in Theology PhD student at the St. Petersburg Theological Academy vice-rector for educational work of the Voronezh Theological Seminary 91, Leninsky av., Voronezh, 393033, Russia s.tishenko2009@ya.ru Hieromonk Serafim (Tischenko) MA in Theology PhD student at the St. Petersburg Theological Academy vice-rector for educational work of the Voronezh Theological Seminary 91, Leninsky av., Voronezh, 393033, Russia s.tishenko2009@ya.ru For citation: Seraﬁm (Tischenko), hieromonk. “A. V. Kartashev’s Journalistic and Scientiﬁc Publications in Church Journals”. Theological Herald, no. 3 (50), 2023, pp. 314–323 (in Russian). DOI: 10.31802/GB.2023.50.3.015 Abstract. The article is devoted to the ﬁrst publications of A.V. Kartashev in the periodical press. The debut of the future chief prosecutor as a scientist and publicist on the pages of church magazines of the St. Petersburg Theological Academy. In these materials, A.V. Kartashev shows himself as a researcher with a broad outlook and the makings of a publicist-analyst. The ﬁrst materials published by A.V. Kartashev in print media constitute an integral context of his formation as a publicist in the future, serve as the foundation for some of his creative approaches in the future. In the course of the research, the review of A.V. Kartashev's publications in the journals «Christian Reading» and «Church Bulletin» is carried out, the subject matter and problems of the materials are analyzed. Special attention is paid to the manifestation of the author of the articles as a scientist. Keywords: Anton Vladimirovich Kartashev, church periodicals, Christian reading, Church Bulletin, spiritual education at the beginning of the XX century, scientific reviews, church journalism, editorial board of the pre-revolutionary journal. ИЕРОМОНА Х СЕРАФИМ (ТИЩЕНКО) 316 Ш Ш ирокому кругу читателей Антон Владимирович Карташёв известен, в первую очередь, как учёный-историк, богослов и преподаватель. Без сомнения, такие его письменные ра- боты, как «История Русской Церкви» или «Вселенские Со- боры» стали классикой отечественной историографии. Однако эти вер- шины творчества Антона Владимировича едва ли могли бы состояться без раннего этапа его творчества, одним из проявлений которого явля- ются первые публицистические и научные опыты будущего историка. В бытность преподавателем Санкт-Петербургской духовной академии (СПбДА) А. В. Карташёв подаёт несколько статей в академические жур- налы «Христианское чтение» и «Церковный вестник». Представляется, что изучение этих материалов весьма важно, поскольку в них Антон Владимирович проявляет себя и историком, и общественным деятелем. 1 Христианское чтение // ЭС. 1903. Т. 37. С. 635. 3 Христианское чтение // ЭС. 1903. Т. 37. С. 635. 2 Барсов Н. И. Академии духовные православные // ЭС. 1890. Т. 1. С. 256–257. р с с ое е е // ЭС. 903. . 3 . С. 635. 2 Барсов Н. И. Академии духовные православные // ЭС. 1890. Т. 1. С. 256–257. 3 Христианское чтение // ЭС. 1903. Т. 37. С. 635. р у р 3 Христианское чтение // ЭС. 1903. Т. 37. С. 635. «Христианское чтение» и «Церковный вестник» Ежемесячный журнал «Христианское чтение» был основан в 1821 г. по инициативе ректора СПбДА архимандрита Григория (Постникова)1 и по праву считается первым научно-богословским журналом в Русской Православной Церкви. В нём публиковались рецензии, статьи, перево- ды и научные исследования, как правило, членов профессорско-препо- давательской корпорации СПбДА, которые имели весьма существен- ное значение для развития и церковных, и светских гуманитарных наук. Например, журнал печатал синодальный перевод Библии (с на- чала 1860-х гг.), переводы древних и византийских историков (Евсевия Памфила, Сократа Схоластика, Никифора Вриенния, Никиты Хониата и т.д.), святоотеческие творения свт. Иоанна Златоуста, прп. Феодора Студита и т. д.2. С 1875 г. журнал «Христианское чтение» стал официальным при- ложением к еженедельному журналу «Церковный вестник» с общей редакцией. Тем не менее, он по-прежнему оставался наиболее важ- ным печатным органом академии, поскольку имел возможность до- нести до широкого круга общественности свои научные изыскания3. Чаще всего в научно-академическом журнале публиковались работы наиболее опытных учёных с солидным исследовательским материалом. ПУБЛИК АЦИИ А. В. К АРТАШЁВА В ЦЕРКОВНЫХ Ж УРНА ЛА Х 317 С 1899 по 1905 г. наибольшее число работ опубликовали было у трёх профессоров: у Н. Н. Глубоковского — пятьдесят три публикации, А. А. Бронзова — сорок одна и у А. П. Лопухина — сорок. Для сравнения можно представить количество публикаций у других академических пре- подавателей в указанный период: П. Н. Жукович — три, А. Л. Катанский — десять, И. Е. Евсеев — шесть, И. Г. Троицкий — пятнадцать. У молодых сотрудников профессорско-преподавательского коллек- тива (профессорских стипендиатов, доцентов и исполняющих должность доцента) также была возможность публиковать свои труды, которые были связаны, как правило, со стипендиатской работой или магистер- ской диссертацией. Так, С. М. Зарин в 1899 г. был назначен профессор- ским стипендиатом и до защиты магистерской диссертации (в 1907 г.) на тему «Аскетизм по православно-христианскому учению. Том пер- вый. Этико-богословское исследование. Книги I и II»4 опубликовал де- вять статей. Д. П. Миртов был назначен профессорским стипендиатом в 1891 г. Спустя девять лет он защитил магистерскую диссертацию на тему: «Нравственное учение Климента Александрийского как моралиста»5. С 1900 по 1905 г. он опубликовал восемнадцать статей, из которых лишь три были посвящены его магистерской работе. Остальная часть публикаций освещала философские воззрения Эммануила Канта, Куно Фишера, В. С. Соловьёва и т. д. 5 Шевцов А. В. Философское учение профессора Санкт-Петербургской духовной академии Д. П. Миртова (к 150-летию со дня рождения) // ХЧ. 2017. № 4. С. 262, 267. 6 Там же. 4 Богданова Т. А. Зарин С. М. // ПЭ. 2013. Т. 19. С. 650. 11 Карташёв А. В. Краткий историко-критический очерк систематической обработки рус- ской церковной истории // ХЧ. 1903. № 7. С. 77–93. 7 Карташёв А. В. Краткий историко-критический очерк систематической обработки рус- ской церковной истории // ХЧ. 1903. № 6. С. 909–922; № 7. С. 77–93. 8 Карпук Д. А. Кафедра русской церковной истории в Санкт-Петербургской духовной ака- демии на рубеже XIX–XX вв. // ХЧ. 2015. С. 185. 12 Карташёв А. В. Свящ. Н. Стеллецкий. Князь А. Н. Голицын и его церковно-государствен- ная деятельность. Киев, 1901 год // ХЧ. 1904. № 12. С. 888–889. 1 С 8 9 Карташёв А. В. Краткий историко-критический очерк систематической обработки рус- ской церковной истории // ХЧ. 1903. № 6. С. 909–912. 7 Карташёв А. В. Краткий историко-критический очерк систематической обработки рус- ской церковной истории // ХЧ. 1903. № 6. С. 909–922; № 7. С. 77–93. 8 Карпук Д. А. Кафедра русской церковной истории в Санкт-Петербургской духовной ака- демии на рубеже XIX–XX вв. // ХЧ. 2015. С. 185. 9 Карташёв А. В. Краткий историко-критический очерк систематической обработки рус- ской церковной истории // ХЧ. 1903. № 6. С. 909–912. 10 Там же. С. 912–922. 11 Карташёв А. В. Краткий историко-критический очерк систематической обработки рус- ской церковной истории // ХЧ. 1903. № 7. С. 77–93. 12 Карташёв А. В. Свящ. Н. Стеллецкий. Князь А. Н. Голицын и его церковно-государствен- ная деятельность. Киев, 1901 год // ХЧ. 1904. № 12. С. 888–889. 13 Там же. С. 877. 7 Карташёв А. В. Краткий историко критический очерк систематической обработки рус ской церковной истории // ХЧ. 1903. № 6. С. 909–922; № 7. С. 77–93. 8 Карпук Д. А. Кафедра русской церковной истории в Санкт-Петербургской духовной ака- демии на рубеже XIX–XX вв. // ХЧ. 2015. С. 185. Публикации А. В. Карташёва 1903–1905 гг. В свете приведённой статистики степень участия А. В. Карташёва в из- дании «Христианское чтение» является незначительным. За годы науч- ной и преподавательской деятельности (с 1899 по 1905 гг.) А. В. Карташёв опубликовал две работы в журнале «Христианское чтение»: в 1903 г. была напечатана стипендиатская работа «Краткий историко-критиче- ский очерк систематической обработки русской церковной истории», а в 1904 г. — рецензия на монографию священника Николая Стеллецкого «Князь А. Н. Голицын и его церковно-государственная деятельность»6. 318 ИЕРОМОНА Х СЕРАФИМ (ТИЩЕНКО) Первая из этих работ вышла в двух частях7. Она являлась вводной частью лекционного материала А. В. Карташёва, который был им создан на основе упомянутой ранее стипендиатской работы8. Как уже было отмечено, данное исследование А. В. Карташёва являлось во многом не самостоятельным и по большей части было разработано Н. К. Никольским в 1888 г. Вначале автор написал о том, что в XVIII в. подготавливалась почва для формирования истории Русской Православной Церкви как самостоятельной дисциплины: разными исследователями нарабатывался и аккумулировался фактический материал (иером. Никодимом (Селлием), прот. Петром Алексеевым и др.), а также излагались различные суждения о тех или иных церковно-исторических событиях (В. Н. Татищевым, М. М. Щербатовым и И. Н. Болтиным)9. Далее были проанализированы труды церковных историков XIX — начала XX столетия: митр. Платона (Левшина)10, архиеп. Филарета (Гумилевского), митр. Макария (Булгакова), П. В. Знаменского, Е. Е. Голубинского и А. П. Доброклонского11. Вторая публикация А. В. Карташёва представляет собой анализ изданной в 1901 г. монографии выпускника Киевской духовной академии прот. Алексея Стеллецкого «Князь А. Н. Голицын и его церковно- государственная деятельность». В целом Антон Владимирович дал положительную оценку работе автора, считая его более или менее объективным и обстоятельным в исследовании поставленной темы12. Одним из немногих существенных недостатков монографии рецензент счёл то, что автор пользовался только печатными источниками, не привлекая архивные материалы13. Примечательно, что в данной рецензии можно обнаружить те же черты А. В. Карташёва как основательного и скрупулёзного ПУБЛИК АЦИИ А. В. К АРТАШЁВА В ЦЕРКОВНЫХ Ж УРНА ЛА Х 319 рецензента. Среди недостатков работы Антон Владимирович выявил несколько десятков заимствований из разных источников, которые автор использовал без указания на оригинал, а также ряд неточностей в приводимых цитатах. При этом на каждую погрешность А. В. Карташёв приводил краткие библиографические ссылки. Тем не менее, эти «положительные прегрешения» рецензент посчитал несущественными, поскольку автор в исследовании проявил самостоятельность в обработке и изложении достаточно большого фактического материала14. Еженедельный журнал «Церковный вестник» был основан в 1875 г. по инициативе ректора СПбДА прот. Иоанна Янышева15. 14 Карташёв А. В. Свящ. Н. Стеллецкий. Князь А. Н. Голицын и его церковно-государствен- ная деятельность. Киев, 1901 год // ХЧ. 1904. № 12. С. 876–877. 15 Церковный вестник // ЭС. 1903. Т. 38. С. 93. 16 Там же. 17 Карпук Д. А. Академическое издание «Церковный вестник» // ХЧ. 2012. № 4. С. 41–43. 18 Там же. С. 65. 14 Карташёв А. В. Свящ. Н. Стеллецкий. Князь А. Н. Голицын и его церковно-государствен- ная деятельность. Киев, 1901 год // ХЧ. 1904. № 12. С. 876–877. 17 Карпук Д. А. Академическое издание «Церковный вестник» // ХЧ. 2012. № 4. С. 41–43. 18 Там же. С. 65. Публикации А. В. Карташёва 1903–1905 гг. В журнале освещались церковные, общественные и государственные события, а также производился обзор наиболее актуальных публикаций как в духовных, так и в светских журналах. За несколько десятков лет существования статус и структура редакции неоднократно менялись. Так, с 1875 по 1888 г. он был официальным органом Святейшего Синода (до организации журнала «Церковные ведомости»)16. Точнее, его содержание делилось на официальную часть, в которой печатались важные синодальные объявления, распоряжения и т.п., и неофициальную, всецело принадлежащую академической редакции. В финансовом плане это имело несомненное преимущество, ибо Святейший Синод обязывал приходы и монастыри выписывать это издание. Тираж журнала составлял несколько десятков тысяч экземпляров, что, в свою очередь, было выгодно не только с экономической точки зрения, но и давало возможность освещать актуальные темы для широкого круга подписчиков. После того как «Церковный вестник» потерял статус официального органа Святейшего Синода, академической редакции приходилось неоднократно сталкиваться с финансовыми затруднениями17. Тем не менее, журнал оставался актуальным и востребованным: более чем за десять лет он приобрёл постоянный круг читателей и продолжал освещать наиболее острые вопросы церковной и общественной жизни18. 320 ИЕРОМОНА Х СЕРАФИМ (ТИЩЕНКО) Периодически редакции обоих академических журналов то объединялись (с 1875 по 1881 гг. и с 1893 по 1902 г.), то снова разделялись. Редакторов избирала профессорско-преподавательская корпорация тайным голосованием на определённый срок (от 3-х до 5-и лет)19. Каждый раз новый редактор формировал редакционную коллегию, определял, кто будет вести отделы журнала, и в случае необходимости привлекал внешних сотрудников. Сформированной редакции приходилось просматривать и отбирать огромное количество статей, очерков и эссе, поэтому от каждого редактора «Церковного вестника» существенно зависела «повестка дня», а также общая тональность публикаций20. Когда журналом руководили прот. Александр Рождественский (с 1903 по 1905 гг.) и профессор Д. П. Миртов (с 1906 по 1908 г.), издание имело либеральный характер. В этот период «Церковный вестник» выступал за церковные реформы21. Например, ряд статей «Церковного вестника» за 1905–1906 гг. были посвящены участию мирян в церковном управлении22, свободе Церкви при синодальной системе23, единству Церкви, сословному разделению в обществе и т. п.24. Вероятно, по поручению прот. Александра Рождественского А. В. Карташёв написал статью «Русская Церковь в 1904 году», которая вышла в январском номере 1905 г. и по традиции журнала подводила итоги ушедшего года. В целом статью можно условно разделить на две тематические части. В первой Антон Владимирович писал о наиболее тревожных событиях 1904 г. Во второй части, большей по объёму, автор описал текущее состояние Церкви и её актуальные проблемы на различных уровнях, которые либо постепенно решаются, либо ещё ждут решений. В числе тревожных событий А. 19 Карпук Д. А. Академическое издание «Церковный вестник» // ХЧ. 2012. № 4. С. 35, 57. 20 Там же. С. 64. 21 Там же. С. 61. 22 К вопросу об участии мирян в церковном Соборе: догматическое обоснование всецер- ковности // ЦВ. 1905. № 34. С. 1063–1066; Участие мирян в церковном управлении // ЦВ. 1906. № 49. С. 1606–1607. 23 К вопросу об участии мирян в церковном Соборе: догматическое обоснование всецер- ковности // ЦВ. 1905. № 34. С. 1063–1066. 24 Необходимость объединения разрозненных сил Церкви // ЦВ. 1906. № 8. С. 234; Церков- ный Собор и сословная рознь в Церкви // ЦВ. 1905. № 26. С. 803–806. 22 К вопросу об участии мирян в церковном Соборе: догматическое обоснование всецер- ковности // ЦВ. 1905. № 34. С. 1063–1066; Участие мирян в церковном управлении // ЦВ. 1906. № 49. С. 1606–1607. 19 Карпук Д. А. Академическое издание «Церковный вестник» // ХЧ. 2012. № 4. С. 35, 57. 20 Там же. С. 64. 21 Там же. С. 61. 23 К вопросу об участии мирян в церковном Соборе: догматическое обоснование всецер- ковности // ЦВ. 1905. № 34. С. 1063–1066. 24 Необходимость объединения разрозненных сил Церкви // ЦВ. 1906. № 8. С. 234; Церков- ный Собор и сословная рознь в Церкви // ЦВ. 1905. № 26. С. 803–806. 25 Карташёв А. В. Русская Церковь в 1904 году // ЦВ. 1905. № 1. С. 2–5. 26 Там же. С. 3. 27 Там же. С. 8. 28 Та же. С. 5–10. 29 Там же. С. 10. Выводы Подводя итоги публикационной активности А. В. Карташёва в пери- од преподавания в Санкт-Петербургской духовной академии, следует констатировать, что небольшое количество публикаций всё же дают известную почву для размышления. Во-первых, как исследователь А. В. Карташёв проявляет большой кругозор и способность к анализу материалов. Прекрасно исполненный отзыв на работу свящ. Николая Стеллецкого продемонстрировал, что А. В. Карташёв владеет акаде- мическим стилем, а также способен работать и критически оценивать даже обширные исследовательские работы. Во-вторых, статья о библи- ографии по русской церковной истории, без сомнения, представляет собой первый шаг к созданию знаменитого курса лекций, который осу- ществится в Париже. Разумеется, исследователь на много лет прервёт свои работы, но даже известные нам наработки позволяют судить вы- соко о том фундаменте, который был заложен во время краткого пери- ода преподавания. Наконец, аналитическая статья о состоянии Русской Церкви в 1904 г., по сути, являлась переходным моментом к следую- щему этапу интеллектуальной деятельности А. В. Карташёва — публи- цистике в светских изданиях. Недаром одна из его статей, написанная в дальнейшем, будет носит название «Русская Церковь в 1905 году». Публикации А. В. Карташёва 1903–1905 гг. В. Карташёв упомянул о тяжёлых поражениях во время Русско-японской войны: уничтожении Тихоокеанской эскадры и падении многострадального Порт-Артура. ПУБЛИК АЦИИ А. В. К АРТАШЁВА В ЦЕРКОВНЫХ Ж УРНА ЛА Х 321 При этом автор подчеркнул, что Церковь приняла активное участие в помощи российской армии и флоту: от пастырского окормления войсковых частей военно-морским духовенством до общецерковных сборов пожертвований в помощь русским воинам25. Кроме этого, 1904 г. был омрачён возмутительной кражей Казанской иконы Божией Матери из Богородицкого женского монастыря26, а также смертью архиепископа Владимирского и Суздальского Сергия (Спасского), доктора богословия и автора ряда агиологических исследований27. В отличие от первой части статьи, вторая имеет более отрадное содержание. Здесь Антон Владимирович описал состояние Церкви в области управления и законодательства, положения приходского духовенства, духовного просвещения и миссионерства. Автор отметил некоторые позитивные изменения в Церкви: увеличение материального содержания для некоторых категорий духовенства, небольшие изменения в либерализации брачного законодательства, обсуждение в Москве, Киеве и Санкт-Петербурге о степени участия духовенства в общественной жизни, культурной и научной деятельности. Автор также отметил некоторые, хотя и скромные, успехи на миссионерском поприще как в России (преимущественно в среде старообрядцев), так и за рубежом (в Азии и Северной Америке)28. В целом в данной статье А. В. Карташёв выразил осторожный оптимизм относительно возможных грядущих преобразований в Церкви, основывая его на Высочайшем указе от 12 декабря 1904 г., в котором давались обещания большой свободы самоуправления в Церкви, свободы печати и религиозной совести29. Таким образом, деятельность А. В. Карташёва в академических изданиях была также незначительной — всего три публикации. Тем не менее, они характеризуют Антона Владимировича как основательного и педантичного учёного и рецензента, переживающего за судьбу Отечества в годы Русско-японской войны, а также имеющего в то время некий осторожный оптимизм касательно грядущих реформ Русской Православной Церкви. ИЕРОМОНА Х СЕРАФИМ (ТИЩЕНКО) 322 Источники Карташёв А. В. Краткий историко-критический очерк систематической обработки рус- ской церковной истории // ХЧ. 1903. № 6. С. 909–922; № 7. С. 77–93. Карташёв А. В. Краткий историко-критический очерк систематической обработки рус- ской церковной истории // ХЧ. 1903. № 6. С. 909–922; № 7. С. 77–93. Карташёв А. В. Свящ. Н. Стеллецкий. Князь А.Н. Голицын и его церковно-государствен- ная деятельность. Киев, 1901 год // ХЧ. 1904. № 12. С. 888–889. Карташёв А. В. Русская церковь в 1904 году // ЦВ. 1905. № 1. С. 2–5. Литература Барсов Н. И. Академии духовные православные // ЭС. 1890. Т. 1. С. 256–257. Богданова Т. А. За рин С. М. // ПЭ. 2013. Т. 19. С. 650–657. Богданова Т. А. За рин С. М. // ПЭ. 2013. Т. 19. С. 650–657. К вопросу об участи и мирян в церковном соборе: догматическое обоснование всецер- ковности // ЦВ. 1905. № 34. С. 1063–1066. Карпук Д. А. Академическое издание Церковный вестник // ХЧ. 2012. №4. С. 41–67. ПУБЛИК АЦИИ А. В. К АРТАШЁВА В ЦЕРКОВНЫХ Ж УРНА ЛА Х 323 Карпук Д. А. Кафедра Русской церковной истории в Санкт-Петербургской духовной ака- демии на рубеже XIX – XX вв. // ХЧ. 2015. С. 175–218. Необходимость объединен ия разрозненных сил Церкви // ЦВ. 1906. № 8. С. 234. Участие мирян в церковном управлении // ЦВ. 1906. № 49. С. 1606–1607. Участие мирян в церковном управлении // ЦВ. 1906. № 49. С. 1606–1607. Христианское чтение // ЭС. 1903. Т. 37. С. 635. Христианское чтение // ЭС. 1903. Т. 37. С. 635. Христианское чтение // ЭС. 1903. Т. 37. С. 635. Церковный вестник // ЭС. 1903. Т. 38. С. 93. Церковный вестник // ЭС. 1903. Т. 38. С. 93. Церковный собор и сослов ная рознь в Церкви // ЦВ. 1905. № 26. С. 803–806. Шевцов А. В. Философское учение профессора Санкт-Петербургской Духовной Академии Д. П. Миртова (к 150-летию со дня рождения) // ХЧ. 2017. № 4. С. 262–274.
https://openalex.org/W4388338190	https://www.nature.com/articles/s42003-023-05503-9.pdf	English	null	Absence of the primary cilia formation gene Talpid3 impairs muscle stem cell function	Communications biology	2,023	cc-by	12,001	1 School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich, UK. 2 The Earlham Institute, Norwich Research Park, Norwich, UK. 3Present address: Barcelona Institute for Science & Technology, Center for Genome Regulation CRG, Dr Aiguader 88, 08003 Barcelona, Spain. 4Present address: Alberta Children’s Hospital Research Institute (ACHRI), University of Calgary, Calgary, AB, Canada. 5Present address: Clinical Biotechnology Center, NHSBS, Bath, UK. 6Present address: Wellcome Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Saffron Walden CB10 1RQ, UK. ✉email: u.mayer@uea.ac.uk; a.munsterberg@uea.ac.uk COMMUNICATIONS BIOLOGY \| (2023) 6:1121 \| https://doi.org/10.1038/s42003-023-05503-9 \| www.nature.com/commsbio Absence of the primary cilia formation gene Talpid3 impairs muscle stem cell function Victor Martinez-Heredia 1,3, Danielle Blackwell 1,4, Sujith Sebastian1,5, Timothy Pearson1, Gi Fay Mok1, Laura Mincarelli 2,6, Charlotte Utting2, Leighton Folkes 1, Ernst Poeschl1, Iain Macaulay 2, Ulrike Mayer 1✉& Andrea Münsterberg 1✉ https://doi.org/10.1038/s42003-023-05503-9 OPEN Smoothened (Smo), a seven-pass transmembrane protein and Hh co-receptor, moves into the ciliary membrane depending on pathway activity. A conserved ciliary localisation motif has been identiﬁed6,7. Here we examine the role of primary cilia as a nexus for sig- nalling pathways to regulate MuSC behaviour during regenera- tion in response to injury. We use the ﬂoxed allele of TA3,11,15, to generate an inducible conditional knock-out in MuSC, termed TA3iSC-KO, using PAX7-CreERT2. In adult mice Pax7, and thus CreERT2, is speciﬁcally expressed in MuSC. Recombination of the ﬂoxed TA3 locus is triggered in these cells after tamoxifen administration40,41. Using cardiotoxin (ctx)-induced injury, we show that TA3 function is required for efﬁcient muscle repair. In TA3iSC-KO mutants, ﬁbre diameters were smaller and the number of Pax7-positive MuSC was reduced after injury-induced regen- eration. Repeat injury enhanced the phenotype, together sug- gesting that MuSC self-renewal is affected. Many ciliary proteins have now been characterised and mutations in some of these are associated with diseases in human, collectively termed ciliopathies8. Thus, cilia are important for human health, however, their role in skeletal muscle remains enigmatic9,10. Talpid3 (TA3) is a critical component of primary cilia. The protein is located to the distal end of centrioles and necessary for docking of the basal body with the apical cell membrane and thus, for primary cilia formation11,12. The TA3 gene was ﬁrst identiﬁed in a spontaneous chicken mutant and shown to be essential for embryonic patterning and myogenesis13. The defects seen in the original TA3 mutant include features that can be attributed to abnormal Hh signalling, such as limb and neural tube defects14. These were phenocopied in a mouse knock- out where exons essential for TA3 protein function were deleted11,15. g g We used two reporter alleles, Pax7-ZsGreen42 or Rosa-ﬂ-stop- ﬂ-Td-Tomato to FACS isolate MuSC for molecular analysis. Pax7-ZsGreen-positive quiescent MuSC were isolated at day 0. In addition, Td-Tomato-positive MuSC and their progeny were isolated from cultured myoﬁbers at 72 h. Transcriptomics revealed effects on ECM-related genes and de-regulated signalling pathways in absence of TA3; in particular, many Wnt ligands were downregulated. Consistent with the latter, we show that muscle regeneration was rescued by co-injection of 6-bro- moindirubin-3′-oxime (BIO), an activator of β-catenin- dependent Wnt signalling43, but not by purmorphamine (Pur), an activator of the Hh co-receptor, Smo44. Myoﬁber diameter and the number of Pax7-positive (Pax7+ve) MuSC were restored by BIO co-injection. Results TA3 is necessary for primary cilia formation in MuSCs. To examine the requirement of Talpid3 (TA3) for primary cilia formation in MuSCs, we used a conditional allele of the TA3 gene15 with the Pax7CreERT2 driver40,41,45. After only three con- secutive tamoxifen injections, we observed a recombination efﬁ- ciency of 92-94% in myoﬁbers isolated from TA3iSC-KO mutants or controls. MuSC were identiﬁed using Pax7-ZsGreen ﬂuores- cence and recombination was conﬁrmed using the Td-Tomato reporter (Supplementary Fig. 1). In all experiments we used a well-established regime of tamoxifen injections40,45 prior to myoﬁber isolation. Thus, homozygous TA3ﬂox/ﬂox/Pax7CreERT2/+ animals (TA3iSC-KO) lack expression of the gene in >92% of MuSC. Heterozygous TA3ﬂox/+ litter mates or animals lacking Pax7CreERT2 were used as controls. In addition, Wnt and Shh signalling pathways are linked to primary ciliogenesis and depending on the context, they act cooperatively or antagonistically31–34. Quiescent muscle stem cells (MuSC) have a primary cilium, which is disassembled upon activation and cell cycle entry and reassembled in self-renewing MuSC35. Knock-down of IFT88 in C2C12 myoblasts leads to ablation of ciliogenesis and reduced expression of p27, a marker for quiescence36. In mice, conditional deletion of IFT88 in MuSC, impairs recovery of overall muscle strength and alters the expression of cell-cycle-related genes. This group also found that primary cilia are lost during aging, suggesting a possible role of cilia in age-related muscle loss37. Repression of Hh signalling seems to be important in maintaining MuSC quiescence in G037,38. This requires the repressor form of GLI3, since condi- tional deletion of GLI3, or inhibition of its processing into GLI3R, leads to cell cycle entry39. Together this suggests that Hh sig- nalling regulates MuSC activation and proliferation. However, the To detect primary cilia in MuSC, myoﬁbers were isolated, cultured for 0, 48 and 96 h and immunostained using Arl13b and Pax7 antibodies (Fig. 1a). At 0 and 48 h, the percentage of Pax7+ve cells with cilia was similar in TA3iSC-KO and control ﬁbres. This was expected, as primary cilia are disassembled only upon entry into the cell cycle35, which occurs >40 h after activation. After 96 h in culture, MuSC have undergone multiple rounds of cell division, a proportion of cells re-express Pax7, re- enter the stem cell niche and re-assemble a cilium. At this point 60% of Pax7 expressing cells had a primary cilium in control myoﬁbers, similar to previous reports35,39. Absence of the primary cilia formation gene Talpid3 impairs muscle stem cell function Victor Martinez-Heredia 1,3, Danielle Blackwell 1,4, Sujith Sebastian1,5, Timothy Pearson1, Gi Fay Mok1, Laura Mincarelli 2,6, Charlotte Utting2, Leighton Folkes 1, Ernst Poeschl1, Iain Macaulay 2, Ulrike Mayer 1✉& Andrea Münsterberg 1✉ https://doi.org/10.1038/s42003-023-05503-9 OPEN Absence of the primary cilia formation gene Talpid3 impairs muscle stem cell function Victor Martinez-Heredia 1,3, Danielle Blackwell 1,4, Sujith Sebastian1,5, Timothy Pearson1, Gi Fay Mok1, Laura Mincarelli 2,6, Charlotte Utting2, Leighton Folkes 1, Ernst Poeschl1, Iain Macaulay 2, Ulrike Mayer 1✉& Andrea Münsterberg 1✉ https://doi.org/10.1038/s42003 023 05503 9 OPEN Skeletal muscle stem cells (MuSC) are crucial for tissue homoeostasis and repair after injury. Following activation, they proliferate to generate differentiating myoblasts. A proportion of cells self-renew, re-enter the MuSC niche under the basal lamina outside the myoﬁber and become quiescent. Quiescent MuSC have a primary cilium, which is disassembled upon cell cycle entry. Ex vivo experiments suggest cilia are important for MuSC self-renewal, however, their requirement for muscle regeneration in vivo remains poorly understood. Talpid3 (TA3) is essential for primary cilia formation and Hedgehog (Hh) signalling. Here we use tamoxifen- inducible conditional deletion of TA3 in MuSC (iSC-KO) and show that regeneration is impaired in response to cytotoxic injury. Depletion of MuSC after regeneration suggests impaired self-renewal, also consistent with an exacerbated phenotype in TA3iSC-KO mice after repeat injury. Single cell transcriptomics of MuSC progeny isolated from myoﬁbers identiﬁes components of several signalling pathways, which are deregulated in absence of TA3, including Hh and Wnt. Pharmacological activation of Wnt restores muscle regeneration, while purmorphamine, an activator of the Smoothened (Smo) co-receptor in the Hh pathway, has no effect. Together, our data show that TA3 and primary cilia are important for MuSC self-renewal and pharmacological treatment can efﬁciently restore muscle regeneration. COMMUNICATIONS BIOLOGY \| (2023) 6:1121 \| https://doi.org/10.1038/s42003-023-05503-9 \| www.nature.com/commsbio 1 ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-023-05503-9 P role of primary cilia as a nexus for signalling pathways to facilitate MuSC self-renewal after skeletal muscle regeneration in response to injury has not yet been investigated in vivo. P rimary cilia are non-motile microtubule-based structures found on many cells. They serve as a cellular antenna for multiple signalling pathways and are implicated in numerous developmental disorders. The importance of primary cilia for Sonic hedgehog (Hh) signalling in vertebrates was uncovered in a mouse mutagenesis screen, which identiﬁed genes encoding intraﬂagellar transport (IFT) proteins1 required to build and maintain primary cilia. The sequestration of Hh pathway components into the cilium regulates the down-stream processing of GLI effector proteins, with GLI1/2 acting as transcriptional activators and GLI3 acting predominantly as a transcriptional repressor2–5. Absence of the primary cilia formation gene Talpid3 impairs muscle stem cell function Victor Martinez-Heredia 1,3, Danielle Blackwell 1,4, Sujith Sebastian1,5, Timothy Pearson1, Gi Fay Mok1, Laura Mincarelli 2,6, Charlotte Utting2, Leighton Folkes 1, Ernst Poeschl1, Iain Macaulay 2, Ulrike Mayer 1✉& Andrea Münsterberg 1✉ https://doi.org/10.1038/s42003-023-05503-9 OPEN Therefore, we propose that during the regen- erative cycle modulation of signalling via the primary cilium promotes MuSC efﬁcient self-renewal. This might be important in muscle disease or in ageing when MuSC function declines. The Hh pathway is the most investigated cilia-related signalling pathway, however, there is extensive crosstalk between Wnt and Hh signalling pathways, which share common regulators— including GSK3β, CK1α and Smoothened (Smo). Both pathways are important in embryonic myogenesis16–18. GLI proteins have positive and negative regulatory functions controlling expression of the myogenic regulatory factors (MRF), Myf5 and MyoD during development19–23. Shh also promotes the proliferation and differentiation of cultured myogenic cells24,25. Wnt signalling is active during embryonic myogenesis and it has multiple functions during the muscle regenerative cycle (reviewed in ref. 26). Its timely activation is important to control muscle progenitor cell proliferation and differentiation27–30. A transient activation of Wnt/β-catenin (canonical) signalling in myoblasts is critical, while constitutive β-catenin activation leads to a pro- longed regenerative response28. Interestingly, both MuSC-speciﬁc β-catenin gain- or loss-of-function mutations perturb muscle regeneration, suggesting appropriate levels of signalling are essential for MuSC function30. Results b The percentage of Pax7+ve cells positive for Arl13b in ﬁbres from TA3iSC-KO and control mice, n = 3 mice, >10 ﬁbres per mouse, p < 0.05 two-tail t test, mean and SD are shown. c The average number of Pax7+ve cells per ﬁbre after 0, 48 and 96 h in culture, control (green) and TA3iSC-KO (red), n = 3 mice, >10 ﬁbres per mouse, p < 0.05 unpaired, two-tailed t test, mean and SD are shown. After 96 h, Pax7+ve cells are reduced on mutant ﬁbres. myogenic differentiation was not impaired, although there were fewer cells in total (Fig. 2e, f). MuSCs lacking TA3 have a proliferation defect. To determine cellular defects arising from lack of TA3, control and TA3iSC-KO myoﬁbers were cultured ex vivo to characterise activation, pro- liferation and differentiation of MuSCs in their physiological niche (Fig. 2a). Myoﬁbers were analysed at different time points by immunostaining for Pax7, Myf5, MyoD and MyoG, and nuclei were counted using DAPI. Pax7 expression marks quiescent MuSCs. Once cells are activated, they express Myf5, followed by MyoD and MyoG, which indicates proliferation and differentia- tion. A proportion of cells maintains or re-expresses Pax7 and downregulates MyoD to self-renew the stem cell pool46. At 0, 24 and 48 h, we detected no differences in either the total number of cells or the proportion of cells expressing different markers (Supplementary Figs. 2 and 3 and Supplementary Data 2). However, from 72 h TA3iSC-KO mutant myoﬁbers had fewer MuSC-derived cells compared to controls (Fig. 2b–f and Sup- plementary Data 1). The number of cell clusters per myoﬁber was not affected, but there were fewer cells per cluster (Fig. 2b), suggesting a defect in MuSC activation or proliferation. Caspase 3 staining showed no difference between TA3iSC-KO and control myoﬁbers suggesting the reduced cell number was not due to an increase in apoptosis (Supplementary Fig. 4 and Supplementary Data 2). In TA3iSC-KO mutants, the proportion of cells positive for Pax7, or MyoD, or both was not affected, although there were fewer double positive cells (Fig. 2c, d). This was also the case at 96 h (Supplementary Figs. 2 and 3 and Supplementary Data 2). Immunostaining for MyoD and MyoG showed no difference in the proportion of single and double positive cells, indicating that Loss of TA3 in MuSC impairs skeletal muscle repair after injury. COMMUNICATIONS BIOLOGY \| (2023) 6:1121 \| https://doi.org/10.1038/s42003-023-05503-9 \| www.nature.com/commsbio Results In TA3iSC-KO mutants, there were fewer Pax7+ve cells with a primary cilium, and the number of Pax7+ve cells was reduced in TA3iSC-KO mutant ﬁbres (Fig. 1b, c and Supplementary Data 1). Remaining Pax7+ve cells with a primary cilium are most likely the progeny of MuSC that have escaped recombination. COMMUNICATIONS BIOLOGY \| (2023) 6:1121 \| https://doi.org/10.1038/s42003-023-05503-9 \| www.nature.com/commsbio 2 ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-023-05503-9 Fig. 1 Cilia assembly in MuSCs requires TA3. a Representative images of EDL muscle ﬁbres from TA3iSC-KO and control mice at 0, 48 and 96 h. Immunoﬂuorescence detects Pax7 expressing MuSCs (green) and primary cilia using Arl13b (red), DAPI (blue) stains nuclei (scale bars = 10 µm). b The percentage of Pax7+ve cells positive for Arl13b in ﬁbres from TA3iSC-KO and control mice, n = 3 mice, >10 ﬁbres per mouse, p < 0.05 two-tail t test, mean and SD are shown. c The average number of Pax7+ve cells per ﬁbre after 0, 48 and 96 h in culture, control (green) and TA3iSC-KO (red), n = 3 mice, >10 ﬁbres per mouse, p < 0.05 unpaired, two-tailed t test, mean and SD are shown. After 96 h, Pax7+ve cells are reduced on mutant ﬁbres. Fig. 1 Cilia assembly in MuSCs requires TA3. a Representative images of EDL muscle ﬁbres from TA3iSC-KO and control mice at 0, 48 and 96 h. Immunoﬂuorescence detects Pax7 expressing MuSCs (green) and primary cilia using Arl13b (red), DAPI (blue) stains nuclei (scale bars = 10 µm). b The percentage of Pax7+ve cells positive for Arl13b in ﬁbres from TA3iSC-KO and control mice, n = 3 mice, >10 ﬁbres per mouse, p < 0.05 two-tail t test, mean and SD are shown. c The average number of Pax7+ve cells per ﬁbre after 0, 48 and 96 h in culture, control (green) and TA3iSC-KO (red), n = 3 mice, >10 ﬁbres per mouse, p < 0.05 unpaired, two-tailed t test, mean and SD are shown. After 96 h, Pax7+ve cells are reduced on mutant ﬁbres. Fig. 1 Cilia assembly in MuSCs requires TA . a Representative images of EDL muscle ﬁbres from TA and control mice at 0, 48 and 96 h. Immunoﬂuorescence detects Pax7 expressing MuSCs (green) and primary cilia using Arl13b (red), DAPI (blue) stains nuclei (scale bars = 10 µm). Results Next, we determined the effect of primary cilia loss on the ability of skeletal muscle to repair in vivo, following injury caused by cardiotoxin (ctx) injection. Deletion of TA3 was induced in MuSC prior to injury and hindlimb muscles were dissected on day 5, 10 or 15 post-injury. Histology of transverse cryosections and nidogen staining of basement membranes showed disorganised myoﬁbers at day 10 post-injury (Fig. 3a, b). DAPI staining conﬁrmed centrally located myonuclei in regen- erated myoﬁbers compared to non-injured control muscles, where myonuclei are found in the periphery (Fig. 3b). In unin- jured muscles, the distribution of Feret diameters was identical in control and TA3iSC-KO myoﬁbers (Fig. 3c). However, following injury regenerating TA3iSC-KO myoﬁbers had smaller average Feret diameters compared to controls (Fig. 3c, d and Supple- mentary Data 1). 3iSC KO To determine whether TA3iSC-KO mice would eventually regenerate muscle more completely, we also examined Feret diameters at 25 days post-injury. The average Feret diameters were similar when control muscles had reached normal ﬁbre size (Fig. 3e, g and Supplementary Data 1). This shows muscle regeneration is delayed in absence of TA3. Furthermore, the number of MuSC positive for Pax7 and Td-Tomato was reduced 25 days post-injury (Fig. 3e, f and Supplementary Data 1). This MMUNICATIONS BIOLOGY \| (2023) 6:1121 \| https://doi.org/10.1038/s42003-023-05503-9 \| www.nature.com/commsbio 3 ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-023-05503-9 ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-023-05503-9 Fig. 2 Efﬁcient proliferation of MuSC requires TA3. a Schematic illustration of tamoxifen injection regime followed by ex vivo culture of myoﬁbers for th times indicated, 72 h shown here. Genotypes of mutant and control mice are shown. b Clusters of MuSC were counted and plotted as the total number nuclei per ﬁbre or the number of clusters per ﬁbre. Fewer MuSC-derived cells were present in TA3iSC-KO compared to controls. c Pax7 and MyoD or e MyoD and MyoG immunostaining of MuSC progeny on myoﬁbers isolated from control or TA3iSC-KO mice (scale bar = 50 μm). d Graphs show the tot number of nuclei per myoﬁber in control and TA3iSC-KO ﬁbres, or the percentage of Pax7+ (red), MyoD+ (green) or Pax7+/MyoD+ (blue) double positiv cells p < 0.05, n = 9, unpaired, two-tailed t test. Results d Distribution of minimal Feret diameters of myoﬁbers in control (green) and TA3iSC-KO (red) tibialis anterior muscles after 10dpi. e Histology of control and TA3iSC-KO transverse muscle sections at 25dpi, red shows Td-Tomato, green shows the basement membrane (BM) stained with wheat germ agglutinin (WGA), DAPI nuclei in blue. White arrowheads show MuSC identiﬁed based on Td-Tomato and their location beneath the BM. f At 25dpi fewer MuSC were counted in the mutant. g Distribution of minimal Feret diameters of myoﬁbers in control (green) and TA3iSC-KO (red) tibialis anterior muscles after 25dpi. Scale bars = 50 µm, all conditions n = 3 mice per genotype, p < 0.05, unpaired, two-tailed t test, mean and SD are shown. suggests an effect on MuSC self-renewal in TA3iSC-KO mice compared to controls. loss of MuSC after 72 h in ex vivo cultures and the impaired muscle regeneration after injury, we established molecular pro- ﬁles of MuSC and their progeny. We ﬁrst examined whether there are immediate effects on gene expression after TA3 deletion and performed differential transcriptomics of quiescent MuSC iso- lated from control and TA3iSC-KO mice. Pax7-Zs-green ﬂuorescence42 was used to FACS-isolate between 30,000 and 50,000 MuSCs from dissected hindlimb muscles, immediately following tamoxifen-induced TA3 deletion, for RNAseq. Principle component analysis did not clearly separate the control and TA3iSC-KO samples and only few differentially expressed genes were identiﬁed, with 35 genes up-regulated and 27 genes down- regulated. GO-term analysis did not highlight speciﬁc pathways or processes that were enriched. Deletion of TA3 did not lead to signiﬁcant molecular differences in quiescent MuSCs, however, we noticed that several genes associated with ECM were differ- entially expressed, such as TIMP1, LAMB1 and ITGA8 (Sup- plementary Fig. 5). Regeneration is more severely compromised in TA3iSC-KO after repeat injury. We used repeat injury to conﬁrm that self-renewal of MuSCs was affected. A second injury was applied after a 25- day recovery period (Fig. 4a) and muscle was examined on day 10 after the second injury. In control mice, histology was similar 10 days after repeat injury compared to single injury. However, TA3iSC-KO mutants had more severe ﬁbrosis after repeat injury compared to single injury (Fig. 4b). Furthermore, myoﬁber dia- meters were smaller in TA3iSC-KO mutant compared to control muscles 10 days post repeat injury (Fig. Results f Graphs show the total number of nuclei per myoﬁber in control and TA3iSC-KO ﬁbres, or the percentag of MyoG+ (red), MyoD+ (green) or MyoG+/MyoD+ (blue) double positive cells p < 0.05; p < 0.01, n = 9, unpaired, two-tailed t test, mean and SD are shown. Fig. 2 Efﬁcient proliferation of MuSC requires TA3. a Schematic illustration of tamoxifen injection regime followed by ex vivo culture of myoﬁbers for the times indicated, 72 h shown here. Genotypes of mutant and control mice are shown. b Clusters of MuSC were counted and plotted as the total number of nuclei per ﬁbre or the number of clusters per ﬁbre. Fewer MuSC-derived cells were present in TA3iSC-KO compared to controls. c Pax7 and MyoD or e MyoD and MyoG immunostaining of MuSC progeny on myoﬁbers isolated from control or TA3iSC-KO mice (scale bar = 50 μm). d Graphs show the total number of nuclei per myoﬁber in control and TA3iSC-KO ﬁbres, or the percentage of Pax7+ (red), MyoD+ (green) or Pax7+/MyoD+ (blue) double positive cells p < 0.05, n = 9, unpaired, two-tailed t test. f Graphs show the total number of nuclei per myoﬁber in control and TA3iSC-KO ﬁbres, or the percentage of MyoG+ (red), MyoD+ (green) or MyoG+/MyoD+ (blue) double positive cells p < 0.05; p < 0.01, n = 9, unpaired, two-tailed t test, mean and SD are shown COMMUNICATIONS BIOLOGY \| (2023) 6:1121 \| https://doi.org/10.1038/s42003-023-05503-9 \| www.nature.com/commsbio 4 4 ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-023-05503-9 COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-023-05503-9 Fig. 3 Regeneration of myoﬁbers is impaired after deletion of TA3 in MuSCs. a Histology images of tibialis anterior (TA) muscle transverse sections stained with haematoxylin and eosin. b Representative images of TA transverse sections, nidogen staining visualises basement membrane (green), DAPI stains nuclei (blue). Sections from non-injured (intact), and injured control and TA3iSC-KO muscles are shown as indicated, 10dpi = 10 days post-injury. c Raincloud plots show size distribution of myoﬁber minimal Feret diameters in control (green) and TA3iSC-KO (red) mice, non-injured or after cardiotoxin (ctx) injury, p < 0.05, 95% CI (conﬁdence interval) of the mean is shown. d Distribution of minimal Feret diameters of myoﬁbers in control (green) and TA3iSC-KO (red) tibialis anterior muscles after 10dpi. e Histology of control and TA3iSC-KO transverse muscle sections at 25dpi, red shows Td-Tomato, green shows the basement membrane (BM) stained with wheat germ agglutinin (WGA), DAPI nuclei in blue. Results White arrowheads show MuSC identiﬁed based on Td-Tomato and their location beneath the BM. f At 25dpi fewer MuSC were counted in the mutant. g Distribution of minimal Feret diameters of myoﬁbers in control (green) and TA3iSC-KO (red) tibialis anterior muscles after 25dpi. Scale bars = 50 µm, all conditions n = 3 mice per genotype, p < 0.05, unpaired, two-tailed t test, mean and SD are shown. f TA3 in MuSCs. a Histology images of tibialis anterior (TA) muscle transverse sections Fig. 3 Regeneration of myoﬁbers is impaired after deletion of TA3 in MuSCs. a Histology images of tibialis anterior (TA) muscle transverse sections stained with haematoxylin and eosin. b Representative images of TA transverse sections, nidogen staining visualises basement membrane (green), DAPI stains nuclei (blue). Sections from non-injured (intact), and injured control and TA3iSC-KO muscles are shown as indicated, 10dpi = 10 days post-injury. c Raincloud plots show size distribution of myoﬁber minimal Feret diameters in control (green) and TA3iSC-KO (red) mice, non-injured or after cardiotoxin (ctx) injury, p < 0.05, 95% CI (conﬁdence interval) of the mean is shown. d Distribution of minimal Feret diameters of myoﬁbers in control (green) and TA3iSC-KO (red) tibialis anterior muscles after 10dpi. e Histology of control and TA3iSC-KO transverse muscle sections at 25dpi, red shows Td-Tomato, green shows the basement membrane (BM) stained with wheat germ agglutinin (WGA), DAPI nuclei in blue. White arrowheads show MuSC identiﬁed based on Td-Tomato and their location beneath the BM. f At 25dpi fewer MuSC were counted in the mutant. g Distribution of minimal Feret diameters of myoﬁbers in control (green) and TA3iSC-KO (red) tibialis anterior muscles after 25dpi. Scale bars = 50 µm, all conditions n = 3 mice per genotype, p < 0.05, unpaired, two-tailed t test, mean and SD are shown. Fig. 3 Regeneration of myoﬁbers is impaired after deletion of TA3 in MuSCs. a Histology images of tibialis anterior (TA) muscle transverse sections stained with haematoxylin and eosin. b Representative images of TA transverse sections, nidogen staining visualises basement membrane (green), DAPI stains nuclei (blue). Sections from non-injured (intact), and injured control and TA3iSC-KO muscles are shown as indicated, 10dpi = 10 days post-injury. c Raincloud plots show size distribution of myoﬁber minimal Feret diameters in control (green) and TA3iSC-KO (red) mice, non-injured or after cardiotoxin (ctx) injury, p < 0.05, 95% CI (conﬁdence interval) of the mean is shown. Results 4d, e and Supplementary Data 1), and myoﬁber diameters were more reduced after repeat injury compared to single injury (~10 µm compared to ~18 µm) (Figs. 3c and 4d). This conﬁrms that MuSC are depleted after a ﬁrst injury and is consistent with the notion that self-renewal is affected. In non-injured control and mutant muscle the ﬁbre diameters were similar. To determine the effects of TA3-loss on MuSC and their progeny as they go through the regenerative cycle, we performed single cell transcriptomics. Myoﬁbers were cultured ex vivo as before. After 72 h, Td-Tomato ﬂuorescent MuSCs were isolated COMMUNICATIONS BIOLOGY \| (2023) 6:1121 \| https://doi.org/10.1038/s42003-023-05503-9 \| www.nature.com/commsbio Single cell analysis reveals deregulated signalling pathways in TA3iSC-KO MuSCs. To determine the mechanisms underlying the Single cell analysis reveals deregulated signalling pathways in TA3iSC-KO MuSCs. To determine the mechanisms underlying the 5 COMMUNICATIONS BIOLOGY \| (2023) 6:1121 \| https://doi.org/10.1038/s42003-023-05503-9 \| www.nature.com/commsbio COMMUNICATIONS BIOLOGY \| (2023) 6:1121 \| https://doi.org/10.1038/s42003-023-05503-9 \| www.nature.com/commsbio ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-023-05503-9 Fig. 4 A second injury enhances regeneration defects in TA3iSC-KO mice. a Schematic of the tamoxifen (T) and ctx injection regime. The ﬁrst injury on day eight was followed by a second cdx injection 25 days later. Analysis was performed 10 days post-second injury (10dp2ndi). b Histology shows good recovery in controls but poor recovery in TA3iSC-KO mice. c Transverse sections stained with nidogen (green) and DAPI (blue) at 10dp2ndi. Sections from non-injured (intact) and injured muscles from control and TA3iSC-KO mice are shown as indicated. d Raincloud plots show the size distribution of myoﬁber minimal Feret diameters in control (green) and TA3iSC-KO (red) mice, either non-injured or 10 days after the second injury (10dp2ndi) (ctx). e Distribution of minimal Feret diameters in control (green) and TA3iSC-KO (red) tibialis anterior muscles after repeat injury. Scale bars = 50 µm, n ≥3 mice per genotype, p < 0.05, p < 0.01, unpaired, two-tailed t test, mean and SD, or 95% CI (conﬁdence interval) of the mean are shown. Fig 4 A second injury enhances regeneration defects in TA3iSC-KO mice a Schematic of the tamoxifen (T) and ctx injection regime The ﬁrst injury on day Fig. 4 A second injury enhances regeneration defects in TA3iSC-KO mice. a Schematic of the tamoxifen (T) and ctx injection regime. The ﬁrst injury on day eight was followed by a second cdx injection 25 days later. Analysis was performed 10 days post-second injury (10dp2ndi). b Histology shows good recovery in controls but poor recovery in TA3iSC-KO mice. c Transverse sections stained with nidogen (green) and DAPI (blue) at 10dp2ndi. Sections from non-injured (intact) and injured muscles from control and TA3iSC-KO mice are shown as indicated. d Raincloud plots show the size distribution of myoﬁber minimal Feret diameters in control (green) and TA3iSC-KO (red) mice, either non-injured or 10 days after the second injury (10dp2ndi) (ctx). e Distribution of minimal Feret diameters in control (green) and TA3iSC-KO (red) tibialis anterior muscles after repeat injury. Scale bars = 50 µm, n ≥3 mice per genotype, p < 0.05, *p < 0.01, unpaired, two-tailed t test, mean and SD, or 95% CI (conﬁdence interval) of the mean are shown. Single cell analysis reveals deregulated signalling pathways in TA3iSC-KO MuSCs. To determine the mechanisms underlying the The top 40 genes identiﬁed as predominantly expressed in these three clusters are shown (Fig. 5b). A population of cells characterised by Pax7 was separated further into two distinct clusters (Fig. 5a, d). GO-terms related to cell proliferation were enriched in one of these Pax7 positive clusters (Fig. 5c) and the top 10 differentially expressed genes included cyclin-dependent kinase 1 (Cdk1) (Fig. 5b, d). This cluster was designated proliferating, pMuSC. The second cluster comprising Pax7 expressing cells was designated MuSC. Enriched GO-terms included extracellular matrix (ECM) organi- sation (Fig. 5c), Fibronectin (Fn1) and Extracellular matrix protein 1 (Ecm1) were among the top 10 differentially expressed genes. The third cluster, dMuSC, consisted of differentiating cells characterised by MyoG expression. GO-terms were enriched for genes associated with myoﬁbril assembly, striated muscle development and contraction (Fig. 5c). The top 10 differentially expressed genes include myogenin (MyoG), Actinin alpha3 (Actn3) and titin (Ttn). In both control and TA3iSC-KO, the different populations of MuSC progeny are distributed similarly across all clusters (pMuSC: control = 188, TA3iSC-KO = 183; dMuSC: control = 112; TA3iSC-KO = 119; MuSC: control = 115; TA3iSC-KO = 115) (Fig. 5a). This is consistent with our analysis of ex vivo cultured myoﬁbers by immunostaining, where we detected no change in the proportion of cells labelled by Pax7, Myf5 or MyoG (Fig. 2). by FACS, sorted into plates and sequenced using Smart-Seq2. From three independent biological replicate samples each, for control and TA3iSC-KO MuSC, we retained 832 MuSC after quality control. Downstream scRNA-seq analysis was performed using Seurat47. The cells grouped into three clusters based on distinct transcriptional programs and expression of relevant marker genes (Fig. 5a, b). The top 40 genes identiﬁed as predominantly expressed in these three clusters are shown (Fig. 5b). A population of cells characterised by Pax7 was separated further into two distinct clusters (Fig. 5a, d). GO-terms related to cell proliferation were enriched in one of these Pax7 positive clusters (Fig. 5c) and the top 10 differentially expressed genes included cyclin-dependent kinase 1 (Cdk1) (Fig. 5b, d). This cluster was designated proliferating, pMuSC. The second cluster comprising Pax7 expressing cells was designated MuSC. Enriched GO-terms included extracellular matrix (ECM) organi- sation (Fig. 5c), Fibronectin (Fn1) and Extracellular matrix protein 1 (Ecm1) were among the top 10 differentially expressed genes. The third cluster, dMuSC, consisted of differentiating cells characterised by MyoG expression. Single cell analysis reveals deregulated signalling pathways in TA3iSC-KO MuSCs. To determine the mechanisms underlying the Fig. 4 A second injury enhances regeneration defects in TA3iSC-KO mice. a Schematic of the tamoxifen (T) and ctx injection regime. The ﬁrst injury on day eight was followed by a second cdx injection 25 days later. Analysis was performed 10 days post-second injury (10dp2ndi). b Histology shows good recovery in controls but poor recovery in TA3iSC-KO mice. c Transverse sections stained with nidogen (green) and DAPI (blue) at 10dp2ndi. Sections from non-injured (intact) and injured muscles from control and TA3iSC-KO mice are shown as indicated. d Raincloud plots show the size distribution of myoﬁber minimal Feret diameters in control (green) and TA3iSC-KO (red) mice, either non-injured or 10 days after the second injury (10dp2ndi) (ctx). e Distribution of minimal Feret diameters in control (green) and TA3iSC-KO (red) tibialis anterior muscles after repeat injury. Scale bars = 50 µm, n ≥3 mice per genotype, p < 0.05, *p < 0.01, unpaired, two-tailed t test, mean and SD, or 95% CI (conﬁdence interval) of the mean are shown. was reduced in TA3iSC-KO MuSC. This included Cilia and Flagella Associated Protein 206 (Cfap206) and TBC1 Domain family member 30 (Tbc1d30), a protein involved in regulation of cilia assembly (Fig. 5d, e). Examination of signalling pathways showed that genes involved in Hh signalling were reduced in MuSC lacking TA3, including Hedgehog interacting protein (Hhip) and the Patched 2 receptor (Ptch2) (Fig. 5f). Furthermore, expression of many Wnt ligands was reduced in TA3iSC-KO either across all three clusters (Wnt10a, Wnt5a, Wnt7b), or speciﬁcally in the Pax7 positive clusters, pMuSCs and MuSCs (Wnt2, Wnt9a) (Fig. 5d, f). We also noticed that expression of both positive and negative regulators of Wnt signalling was reduced in TA3iSC-KO MuSC progeny. Differentially expressed genes include porcupine (Porcn), which mediates Wnt palmitoylation required for activity, naked cuticle homologue 1 (Nkd1) and secreted frizzled related protein 4 (Sfrp4), which inhibit Wnt signalling. Because components of Hh and Wnt signalling were deregulated in TA3iSC-KO cells, we next tested whether pharmacological activation of these pathways could rescue muscle repair after injury. by FACS, sorted into plates and sequenced using Smart-Seq2. From three independent biological replicate samples each, for control and TA3iSC-KO MuSC, we retained 832 MuSC after quality control. Downstream scRNA-seq analysis was performed using Seurat47. The cells grouped into three clusters based on distinct transcriptional programs and expression of relevant marker genes (Fig. 5a, b). Single cell analysis reveals deregulated signalling pathways in TA3iSC-KO MuSCs. To determine the mechanisms underlying the GO-terms were enriched for genes associated with myoﬁbril assembly, striated muscle development and contraction (Fig. 5c). The top 10 differentially expressed genes include myogenin (MyoG), Actinin alpha3 (Actn3) and titin (Ttn). In both control and TA3iSC-KO, the different populations of MuSC progeny are distributed similarly across all clusters (pMuSC: control = 188, TA3iSC-KO = 183; dMuSC: control = 112; TA3iSC-KO = 119; MuSC: control = 115; TA3iSC-KO = 115) (Fig. 5a). This is consistent with our analysis of ex vivo cultured myoﬁbers by immunostaining, where we detected no change in the proportion of cells labelled by Pax7, Myf5 or MyoG (Fig. 2). Pharmacological inhibition of GSK3β-kinase restores muscle regeneration in TA3iSC-KO mice. The primary cilium is a nexus for Hh signalling and it has been shown that the co-receptor, Smoothened (Smo), which regulates downstream processing of Gli proteins to generate transcriptional activators, exhibits sig- nalling competency in the absence of ciliary accumulation48. Thus, we used purmorphamine (Pur), a pharmacological acti- vator of Smo, to determine whether this could restore the regenerative defect in TA3iSC-KO mice. We found, however, that after co-injection of Pur with ctx myoﬁber diameters remained reduced on day 10 in TA3iSC-KO muscle compared to controls (Fig. 6a–c and Supplementary Data 1). Thus, treatment was not sufﬁcient to restore muscle regeneration. We examined Despite a similar distribution of cells across each cluster, differential analysis revealed changes in gene expression between control and TA3iSC-KO. The expression of cilia associated genes COMMUNICATIONS BIOLOGY \| (2023) 6:1121 \| https://doi.org/10.1038/s42003-023-05503-9 \| www.nature.com/commsbio 6 ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-023-05503-9 OMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-023-05503-9 ARTI 5 Loss of TA3 leads to deregulation of signalling pathways in MuSC progeny. a UMAP clustering of Td-Tomato MuSC progeny isolated from ured myoﬁbers after 72 h identiﬁes equal proportions of proliferating MuSC (pMuSC), differentiating MuSC progeny (dMuSC) and MuSC po ontrol and TA3iSC-KO myoﬁbers. b Heatmap shows the top 40 most variably expressed genes between the three MuSC clusters, with the top lighted in grey. c Gene Ontology (GO) functional terms enriched in pMuSC (top), dMuSC (middle) and MuSC (bottom) populations are s pression of different marker genes across the three clusters in control and TA3iSC-KO MuSC. Markers shown are Pax7, MyoD, MyoG, Cdk1, Wnt10a. e Comparison of the expression of cilia genes and f Hedgehog (Hh) and Wnt signalling components across each of the three clu Fig. COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-023-05503-9 Fig. 6 Inhibition of GSK3β by BIO restores muscle regeneration. a, d Transverse sections 10 days post-injury (10dpi) stained for nidogen (green) and DAPI (blue). Sections are shown for control and TA3iSC-KO muscles, a co-injected with ctx and purmorphamine (Pur), or (d) co-injected with ctx and BIO (scale bar = 50 µm). b, e Histograms and c, f raincloud plots show the range of myoﬁber diameters in control (green) or TA3iSC-KO (red) mice, injected with ctx and Pur (b, c) or with ctx and BIO (e, f). g Histology of control and TA3iSC-KO transverse muscle sections at 25dpi, red shows Td-Tomato, green shows the basement membrane (BM) stained with wheat germ agglutinin (WGA), DAPI nuclei in blue. White arrowheads show MuSC identiﬁed based on Td-Tomato and their location beneath the BM. h At 25dpi similar numbers of MuSC were counted in control and mutant in presence of BIO. i Distribution of the range of myoﬁber diameters in control (green) or TA3iSC-KO (red) mice, injected with ctx and BIO at 25dpi. p ≤0.05 calculated by unpaired, two- tailed t test, n ≥3 mice, mean and SD, or 95% CI (conﬁdence interval) of the mean are shown. Fig. 6 Inhibition of GSK3β by BIO restores muscle regeneration. a, d Transverse sections 10 days post-injury (10dpi) stained for nidogen (green) and DAPI (blue). Sections are shown for control and TA3iSC-KO muscles, a co-injected with ctx and purmorphamine (Pur), or (d) co-injected with ctx and BIO (scale bar = 50 µm). b, e Histograms and c, f raincloud plots show the range of myoﬁber diameters in control (green) or TA3iSC-KO (red) mice, injected with ctx and Pur (b, c) or with ctx and BIO (e, f). g Histology of control and TA3iSC-KO transverse muscle sections at 25dpi, red shows Td-Tomato, green shows the basement membrane (BM) stained with wheat germ agglutinin (WGA), DAPI nuclei in blue. White arrowheads show MuSC identiﬁed based on Td-Tomato and their location beneath the BM. h At 25dpi similar numbers of MuSC were counted in control and mutant in presence of BIO. i Distribution of the range of myoﬁber diameters in control (green) or TA3iSC-KO (red) mice, injected with ctx and BIO at 25dpi. *p ≤0.05 calculated by unpaired, two- tailed t test, n ≥3 mice, mean and SD, or 95% CI (conﬁdence interval) of the mean are shown. predominantly β-catenin dependent Wnt signalling in response to BIO. COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-023-05503-9 expression of Hh responsive genes to assess whether TA3iSC-KO MuSC can activate Hh signalling in response to Pur. We found that Gli1 and Ptch1 expression was increased in MuSC isolated by FACS (Supplementary Fig. 6 and Supplementary Data 2). To determine whether BIO treatment restored the number of MuSC, we counted Td-Tomato positive cells in vivo on day 25 after injury (Fig. 6g, h). In presence of BIO, TA3iSC-KO muscles had similar numbers of MuSC compared to controls following ctx-induced injury. Contrary to previous observations, this suggests that—in absence of primary cilia—canonical Wnt activation promotes muscle regeneration by restoring MuSC self-renewal. GSK3β kinase is a major negative regulator of β-catenin dependent (canonical) Wnt signalling. It is highly expressed in muscle and inhibition of GSK3β enhances self-renewal of embryonic stem cells43. Given that expression of Wnt ligands was reduced in TA3iSC-KO MuSC (Fig. 5f), we examined the effect of GSK3β inhibition. Muscle regeneration was restored after co- injection of ctx with BIO (6-bromoindirubin-3′-oxime) and myoﬁber diameters were similar in TA3iSC-KO and control mice, at day 10 and 25 post-injury (Fig. 6d–f, i and Supplementary Data 1). This indicates that BIO could ameliorate the regenera- tion defect in absence of primary cilia. Since BIO-mediated GSK3β inhibition activates β-catenin dependent Wnt signalling and Hh signalling, we assessed expression of Axin2, a Wnt target gene, alongside Gli1 and Ptch1. In FACS isolated MuSC, application of BIO induced expression of Axin2 and to a lesser extent the expression of Gli1 and Ptch1 (Supplementary Fig. 6 and Supplementary Data 2). This suggests that MuSC activate Single cell analysis reveals deregulated signalling pathways in TA3iSC-KO MuSCs. To determine the mechanisms underlying the 5 Loss of TA3 leads to deregulation of signalling pathways in MuSC progeny. a UMAP clustering of Td-Tomato MuSC progeny isolated from ex vivo cultured myoﬁbers after 72 h identiﬁes equal proportions of proliferating MuSC (pMuSC), differentiating MuSC progeny (dMuSC) and MuSC populations in control and TA3iSC-KO myoﬁbers. b Heatmap shows the top 40 most variably expressed genes between the three MuSC clusters, with the top 10 genes highlighted in grey. c Gene Ontology (GO) functional terms enriched in pMuSC (top), dMuSC (middle) and MuSC (bottom) populations are shown. d Expression of different marker genes across the three clusters in control and TA3iSC-KO MuSC. Markers shown are Pax7, MyoD, MyoG, Cdk1, Tbc1d30 and Wnt10a. e Comparison of the expression of cilia genes and f Hedgehog (Hh) and Wnt signalling components across each of the three clusters. Fig. 5 Loss of TA3 leads to deregulation of signalling pathways in MuSC progeny. a UMAP clustering of Td-Tomato MuSC progeny isolated from ex vivo cultured myoﬁbers after 72 h identiﬁes equal proportions of proliferating MuSC (pMuSC), differentiating MuSC progeny (dMuSC) and MuSC populations in control and TA3iSC-KO myoﬁbers. b Heatmap shows the top 40 most variably expressed genes between the three MuSC clusters, with the top 10 genes highlighted in grey. c Gene Ontology (GO) functional terms enriched in pMuSC (top), dMuSC (middle) and MuSC (bottom) populations are shown. d Expression of different marker genes across the three clusters in control and TA3iSC-KO MuSC. Markers shown are Pax7, MyoD, MyoG, Cdk1, Tbc1d30 and Wnt10a. e Comparison of the expression of cilia genes and f Hedgehog (Hh) and Wnt signalling components across each of the three clusters. COMMUNICATIONS BIOLOGY \| (2023) 6:1121 \| https://doi.org/10.1038/s42003-023-05503-9 \| www.nature.com/commsbio 7 7 COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-023-05503-9 ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-023-05503-9 result from mutations in the Talpid3 gene (KIAA0586)13, is associated with hypotonia50,51. However, the role of primary cilia in skeletal muscle is not fully understood. Here, we use inducible deletion of an essential ciliary protein, TA3, in MuSC to show that primary cilia are critical for efﬁcient regeneration after muscle injury in vivo. Using single cell sequencing of MuSC, we found that both Hh and Wnt signalling were deregulated in absence of TA3. Importantly, we discovered that pharmacological activation of Wnt signalling, but not Hh signalling, can restore muscle repair in the absence of a primary cilium. In particular BIO- mediated inhibition of GSK3β rescued the impairment in regeneration and restored the number of MuSC, which were reduced after loss of TA3 and cilia disruption. These ﬁndings provide insights into potential avenues for treatments in muscle disease or ageing. TA3. However overall, there were few signiﬁcant differences in MuSC transcriptomes at this time point (Supplementary Fig. 5). This is not unexpected as these cells are transcriptionally not very active. The differences we do see may also be due to partial activation induced by the isolation process57–60. As primary cilia are implicated in both Hh and Wnt signalling, we examined the expression of components of these pathways in control and TA3iSC-KO MuSCs using scRNA-sequencing. Differ- ential analysis showed that both pathways were deregulated in absence of TA3 (Fig. 5f). During regeneration, canonical Wnt signalling is activated and has been implicated in regulating myogenic lineage progression61. However, β-catenin dependent Wnt signalling is only transiently active in expanding myoblasts, and its restriction is crucial for successful muscle regeneration28–30. Prevailing evidence suggests that Wnt signal- ling is particularly involved in the regulation of satellite cell dif- ferentiation and self-renewal26. For example, Wnt activates follistatin and myoblast fusion during terminal differentiation62. Myoblasts lacking β-catenin show delayed differentiation, whereas constitutively active β-catenin promotes precocious differentiation30. During embryonic development, Wnt signalling is also important for myogenesis63–65. Furthermore, Sonic hedgehog synergises with Wnt to activate myogenesis in paraxial mesoderm progenitors in both mouse and chick16–18,66. Genetic experiments targeting Smo or Gli transcriptional effectors, show that Shh acts cell-autonomously in limb muscle progenitor cells by initiating Myf5 expression and regulating directional muscle cell migration in the distal limb21,22. Thus, these signalling pathways are important in embryonic and adult myogenesis with complex and context dependent functions. COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-023-05503-9 In contrast, co-injection of ctx with an inhibitor of GSK3β kinase (BIO) restored muscle repair independently of primary cilia (Fig. 6d–f), this was associated with restored num- bers of MuSC (Fig. 6g, h). p p Molecular proﬁling of individual MuSCs showed that expres- sion of cilia related genes was signiﬁcantly reduced in cells lacking TA3 (Fig. 5e). UMAP clustering identiﬁed a population of MuSCs positive for Pax7. This population could be sub-divided into two clusters, one of which was characterised by the expression of genes related to cell division, such as Cdk1 (Fig. 5d) and com- prised mitotically active, proliferating cells. The second cluster of MuSCs positive for Pax7 was enriched for genes associated with ECM related processes. We propose that this cluster comprises MuSCs that are close to quiescence, similar to those recently described after single cell sequencing of whole hindlimb muscle53. This cluster may also include self-renewed MuSCs that have entered quiescence, as some cells express low transcript levels for myogenic regulatory factors, such as MyoD (Fig. 5e) or Myf5. During embryogenesis, Sonic hedgehog signalling regulates basement membrane components, such as laminin-11154–56. This is crucial for myotome formation and basement membranes are disrupted in the TA3 mutant13. It is therefore interesting to note that TA3 deletion led to differences in expression of ECM related genes in quiescent MuSC isolated immediately following the deletion of TA3. This included tissue inhibitor or matrix metal- loprotease 1 (TIMP1), matrillin 2 (Matn2), laminin subunit beta 1 (LAMB1), integrin alpha 8 (Itga8) and ADAM Metallopeptidase Domain 12 (ADAM12), which were all upregulated in absence of Molecular proﬁling of individual MuSCs showed that expres- sion of cilia related genes was signiﬁcantly reduced in cells lacking TA3 (Fig. 5e). UMAP clustering identiﬁed a population of MuSCs positive for Pax7. This population could be sub-divided into two clusters, one of which was characterised by the expression of genes related to cell division, such as Cdk1 (Fig. 5d) and com- prised mitotically active, proliferating cells. The second cluster of MuSCs positive for Pax7 was enriched for genes associated with ECM related processes. We propose that this cluster comprises MuSCs that are close to quiescence, similar to those recently described after single cell sequencing of whole hindlimb muscle53. This cluster may also include self-renewed MuSCs that have entered quiescence, as some cells express low transcript levels for myogenic regulatory factors, such as MyoD (Fig. 5e) or Myf5. COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-023-05503-9 Our ﬁndings indicate that modulation of signalling pathway activity by the primary cilium adds another facet to the complex regulation of regen- erative myogenesis. g g It has previously been proposed that the primary cilium may provide a potential target for regenerative therapies37,39,52 and recent work has implicated the cilium in muscle regeneration. Speciﬁcally, deletion of the intra-ﬂagellar transport protein, IFT88, in MuSCs led to a shift toward myoﬁbers with reduced cross- sectional area post-injury37. This agrees with our ﬁnding that loss of primary cilia, after conditional deletion of another ciliary pro- tein, TA3, in MuSC impairs muscle repair and that, following injury, regenerating TA3iSC-KO myoﬁbers had smaller diameters compared to controls. In addition, we found that the number of Pax7/Td-Tomato positive MuSC were reduced and that repeat injury enhanced the regeneration defect and led to a more severe phenotype (Fig. 4). This suggests, MuSC are depleted and their expansion and self-renewal is affected in absence of TA3. It is striking that MuSC lacking GLI3, which acts pre- dominantly as a repressor, display rapid cell-cycle entry, increased proliferation and expansion of the stem cell pool39, whereas loss of cilia inhibits proliferation and MuSC expansion. The latter was observed after deletion of IFT88 in MuSCs37 and in this report where, following TA3 deletion, the number of MuSC progeny was reduced on ex vivo cultured myoﬁbers (Fig. 2) and in vivo after completion of the regenerative cycle (day 25 post-injury) (Fig. 3e, f). The opposite effects observed are likely due to the dichotomy in the Hh pathway, with the GLI proteins having either activator or repressor function. This suggests the cilium is necessary to maintain an appropriate balance of GLI activator and repressor proteins38. y g It was particularly striking that Hedgehog interacting protein (Hhip), Patched2 (Ptch2) and several Wnt ligands, Wnt10a, Wnt5a, Wnt9a, Wnt2 and Wnt7b, were signiﬁcantly reduced in TA3iSC-KO MuSCs (Fig. 5f). This observation prompted us to attempt pharmacological rescue of the regeneration defect. Co- injection of ctx and purmorphamine (Pur), a smoothened agonist44, activated Hh target genes in MuSCs, however did not restore muscle repair (Fig. 6a-c, j). This agrees with a previous report, where injection of SAG1.3, another Hh pathway agonist, had no effect in mice with IFT88−/−MuSCs, either on muscle strength or MuSC proliferation37. Thus, the Hh mediated effects on MuSC during the regenerative process appear to be completely cilia dependent. COMMUNICATIONS BIOLOGY \| (2023) 6:1121 \| https://doi.org/10.1038/s42003-023-05503-9 \| www.nature.com/commsbio Discussion Quiescent muscle stem cells have a primary cilium, which is dynamically regulated during the myogenic cycle and is reas- sembled in self-renewing stem cells10,35,36,49. Myoﬁbers isolated from aged muscles have fewer MuSCs with a primary cilium, indicating that cilia might be lost with age or become less stable37. Ciliopathies, a group of genetic disorders resulting from pertur- bation of ciliary proteins in human, affect several different tissues including muscle. For example, Joubert Syndrome, which can COMMUNICATIONS BIOLOGY \| (2023) 6:1121 \| https://doi.org/10.1038/s42003-023-05503-9 \| www.nature.com/commsbio 8 ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-023-05503-9 Methods l Animal models. All mice were housed in the Disease Modelling Unit (DMU) of the University of East Anglia. Experimental procedures were performed in accordance with the Animal (Scientiﬁc Procedures) Act 1986 (ASPA) under the UK Home Ofﬁce Project Licences 70/8824 and PP3253888. All lines were maintained on a C57BL/6J background: Pax7CreERT2/+41, Talpid3ﬂox/ﬂox15. B6.Cg-Tg(Pax7-ZsGreen)1Kyba/J (kindly pro- vided by M. Kyba, University of Minnesota, USA42; and Rosa26- td-Tomato (Ai14, The Jackson Laboratory). Mice carrying the TA3 ﬂoxed allele were crossed with mice expressing the tamoxifen inducible cre-recombinase, CreERT2, under the control of the endogenous Pax7 promoter (Pax7CreERT2), generating control mice and TA3 icSC-KO in the same litter. Experimental mice were 8–12 weeks old, males and females were used. For genotyping DNA was extracted from ear notches and polymerase chain reaction (PCR) was performed as described15,41. For immunostaining of primary cilia ﬁxed ﬁbres were permeabilised with 0.5% TritonX100 and 0.5% Nonidet P40 and 2% MeOH. Primary antibodies used were directed against Arl13B (Proteintech, 1:300) and Pax7 (DSHB; 1:7.5). Secondary antibodies used were goat anti-mouse IgG-Fab2 488 and goat anti-rabbit Alexa ﬂuor 546 (1:375) (Jacksonimmuno). Microscopy and image analysis. Fluorescence images of muscle sections and muscle ﬁbres were taken using a Zeiss AxioPlan 2ie microscope. Images of muscle sections were processed in Fiji (ImageJ) after being pixel corrected in Ilastik and the minimum Feret diameter was measured. RNA extraction and real-time qPCR. RNA from the Td-Tomato positive cells was extracted using trizol and qPCR was performed in triplicates by using the RNA to CT-1-step Sybr green kit (Life Technologies Ltd). Primers to detect transcripts of Hh and Wnt targets are in Supplementary Table 1. IP injections. Tamoxifen was injected for 5 consecutive days (3 mg/40 g body weight), followed by a rest day and another tamoxifen injection on day 7. During the regeneration phase, tamoxifen was administered every other day. Fluorescence-activated cell sorting. Quiescent MuSCs (Pax7- ZsGreen+) were obtained from uninjured hindlimb muscles. For scRNAseq cells were isolated from ex vivo cultured myoﬁbers (72 h) and Td-Tomato positive MuSCs were sorted using the BD FACSMelody cell sorter (BD Biosciences, San Jose, California) directly into lysis buffer in 96-well plates. From three indepen- dent experiments we recovered two plates each from control and TA3iSC-KO myoﬁbres, generating 6 plates per genotype. Muscle injury model. Methods l Mice were anaesthetised with isoﬂuorane and muscle injury was induced with 50 µl of a 10 µM cardiotoxin (ctx) (Latoxan) solution in the right tibialis anterior muscle. The un-injured left leg served as control. Muscles were isolated at the indicated time points after injury. For double injury experiments, a second injury was induced after 25 days of recovery from the ﬁrst injury. Muscle was then analysed 10 days post repeat-injury. For the rescue experiments, purmorphamine (2 µM) or BIO (10 µM) were injected together with ctx. Single-cell RNA-seq. Ampliﬁed cDNA was generated from sor- ted cells using the SmartSeq2 protocol72. Sequencing libraries were generated using an automated, reduced-volume version of the Nextera XT protocol (Illumina) using the SPT Labtech Mosquito LV. Libraries were pooled (384-plex) and sequenced on a NovaSeq 6000 (Illumina, San Diego) in paired end, dual index mode. Raw Illumina sequencing data were analysed to obtain a single-cell expression matrix object. Subsequent analysis was performed in R using Seurat version 347. Cells showing gene counts lower than 1000 and a mitochondrial gene expression percentage higher than 5% were excluded from further analysis. Within Seurat, data were normalised using NormalizeData (normalization.method = LogNormalize, scale.factor = 10000) and data from multiple samples were merged using the FindIn- tegrationAnchors and IntegrateData commands. Muscle ﬁbre isolation. Following administration of tamoxifen extensor digitorum longus (EDL) muscles were isolated from 8- to 12-week-old mice and incubated in Collagenase I (2 mg/ml, Worthington) for 90 min at 37 °C, after which muscles were transferred to BSA (Sigma) coated dishes containing DMEM (Gibco) and PenStrep (Gibco). EDL muscles were subjected to repeated trituration through descending diameter glass Pasteur pipettes until single myoﬁbers separated from the isolated muscle. Single myoﬁbers were selected and cultured in 10% Horse Serum (HS), 1% Chick Embryo Extract (CEE) and PenStrep in DMEM. Histology and immunoﬂuorescence. To conﬁrm the degree of injury and regeneration, at the given time points mice were sacriﬁced and skeletal muscles were harvested, ﬁxed in 4% PFA, embedded in OCT after sucrose treatment and transverse cryo- sectioned (10 µm), followed by haematoxylin–eosin. Statistics and reproducibility. A minimum of three independent experiments (or animals) was used for all assays. Statistical ana- lysis was carried out using Graphpad Prism software. Multiple data points were collected per mouse to generate the population mean and represent it as raincloud plots using ggplot2 in R-studio. COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-023-05503-9 COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-023-05503-9 GSK-3β can also regulate the stability of surface Notch receptors and there is extensive cross-talk between Wnt and Notch signalling69,70. However, our transcriptomics analysis did not identify differentially expressed Notch pathway components. (0.5 µg/ml) were applied. Slides were mounted with gelvatol for imaging. Anti-wheat germ agglutinin (WGA) conjugated with Alexa Fluor 488 (Invitrogen W11261) was incubate for 15 min with a 1:200 dilution. y y p p y p In summary, we show that BIO restores muscle regeneration and that this works predominantly through activation of Wnt signalling, which can overcome the lack of TA3 and primary cilia. Thus, our study identiﬁes a therapeutic target in MuSCs that could be of interest in muscle degenerative disease or in ageing. However, more work is needed to explore cell autonomous and cell non-autonomous effects of the treatment. Muscle ﬁbres were were ﬁxed for 10’ with prewarmed 2% Paraformaldehyde (PFA) at indicated time points, washed with PBS and permeabilised by adding 0.5% TritonX100 and blocked for 2 h with 10% NGS in TBS-T at 37 °C. Primary antibodies against Pax7 (DSHB), Myf5, MyoD (Santa Cruz) and Myogenin (DSHB) were used at a 1:50 dilution in 5% NGS/TBS-T and incubated at 4 °C overnight. Samples were extensively washed with TBS-T before secondary antibodies (Jacksonimmuno) were applied together with DAPI for 2 h in the dark. Fibres were washed in TBS-T and TBS, and 10-20 ﬁbres were mounted in gelvatol on a glass slide for imaging. ARTICLE ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-023-05503-9 GSK3β kinase is highly expressed in muscle; it regulates many signalling pathways including Shh, Wnt and Notch. It has been shown that BIO mediated suppression of GSK-3 activity pro- motes Wnt/beta-catenin signalling to preserve the pluripotency of human and mouse embryonic stem cells43. GLI2, which is part of the Hh pathway, can also be phosphorylated by GSK-367,68, however, BIO did not signiﬁcantly activate the expression of Hh target genes in MuSCs (Fig. 6j). Instead, we found that in response to BIO MuSC activated expression of Axin2, a target gene of canonical Wnt signalling. However, Axin2 is also a repressor of the pathway as it inhibits β-catenin. As mentioned above, the regulation of Wnt activity levels is crucial to maintain proliferation and to control timely myogenic differentiation28–30. We hypothesise that co-injection of BIO achieved appropriate levels of Wnt activity as the regenerative response was restored in absence of cilia, both myoﬁber cross-sectional area and MuSC numbers were comparable to controls. During embryogenesis, Sonic hedgehog signalling regulates basement membrane components, such as laminin-11154–56. This is crucial for myotome formation and basement membranes are disrupted in the TA3 mutant13. It is therefore interesting to note that TA3 deletion led to differences in expression of ECM related genes in quiescent MuSC isolated immediately following the deletion of TA3. This included tissue inhibitor or matrix metal- loprotease 1 (TIMP1), matrillin 2 (Matn2), laminin subunit beta 1 (LAMB1), integrin alpha 8 (Itga8) and ADAM Metallopeptidase Domain 12 (ADAM12), which were all upregulated in absence of 9 COMMUNICATIONS BIOLOGY \| (2023) 6:1121 \| https://doi.org/10.1038/s42003-023-05503-9 \| www.nature.com/commsbio 9 References Developmental abnormalities in the head region of the Talpid mutant of the fowl. J. Embryol. Exp. Morphol. 12, 161–182 (1964). 15. Bangs, F. et al. Generation of mice with functional inactivation of talpid3, a gene ﬁrst identiﬁed in chicken. Development 138, 3261–3272 (2011). 44. Sinha, S. & Chen, J. K. Purmorphamine activates the Hedgehog pathway by targeting Smoothened. Nat. Chem. Biol. 2, 29–30 (2006). p y p p 15. Bangs, F. et al. Generation of mice with functional inactivation of talpid3, a gene ﬁrst identiﬁed in chicken. Development 138, 3261–3272 (2011). 45. von Maltzahn, J., Jones, A. E., Parks, R. J. & Rudnicki, M. A. 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Methods l These values were used to compare between control and For immunostaining transverse section were blocked with 5% Normal Goat Serum in Tris-buffered saline containing 0.1% Tween 20 (TBS-T), followed by incubation with Nidogen171 after washing Alexa Fluor 488 secondary antibody and DAPI COMMUNICATIONS BIOLOGY \| (2023) 6:1121 \| https://doi.org/10.1038/s42003-023-05503-9 \| www.nature.com/commsbio 10 ARTICLE References 1. Huangfu, D. et al. Hedgehog signalling in the mouse requires intraﬂagellar transport proteins. Nature 426, 83–87 (2003). 1. Huangfu, D. et al. Hedgehog signalling in the mouse requires intraﬂagellar transport proteins. Nature 426, 83–87 (2003). 33. Borday, C. et al. Antagonistic cross-regulation between Wnt and Hedgehog signalling pathways controls post-embryonic retinal proliferation. Development 139, 3499–3509 (2012). 2. May, S. R. et al. 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https://openalex.org/W4212993102	https://www.intechopen.com/citation-pdf-url/80272	English	null	A Home-Based Heart Failure Nursing Intervention Supporting Self-Management and Lives	IntechOpen eBooks	2,023	cc-by	5,255	Abstract In the era of a super-aged society, along with the rapid development of medical techniques to treat cardiovascular disease, there are an increasing number of aged patients with heart failure (HF). To cope with this near pandemic, coordinated-HF management combining hospital-based optimal therapy and home-based care is required. Accordingly, the concept of “living with HF” is advocated and is widely accepted as a key to improve patients’ quality of life as well as prognosis. To achieve it, home visiting nursing services are essential. But these services have some difficulties. This chapter shows the importance of promoting self-management for patients with heart failure and intervention strategies in the home care setting. Keywords: heart failure, self-management, re-hospitalization, home-based intervention, home visiting nursing Chapter A Home-Based Heart Failure Nursing Intervention Supporting Self-Management and Lives Motohiro Sano and Tomoko Majima 1. Introduction HF is the quintessential cardiovascular syndrome of aging that results from age-related cardiovascular conditions and age-associated changes in cardiovascular structure and function. The prevalence of HF among approximately 1–2% of the adult population in developed countries, rising to ≧10% among those over 70 years of age and the most common reason for hospitalization in older adults. Usually, as studies only include diagnosed HF cases, the true prevalence is likely to be higher [1, 2]. Although some progress has been made in reducing mortality in patients with HF, rates of rehos- pitalization continue to rise and approach 40–50% within 1 year after discharge [3, 4]. To reduce mortality and rehospitalization rate, it is widely recognized that, in addition to optimizing medical and device therapies for HF, attention should also be given to how HF care is delivered. Several position papers that cover non-pharmacological management, discharge planning, and standards for delivering HF care. g g g g Appropriate self-management by patients with HF plays an important role in the prevention of HF decompensation and improvement of survival and quality of life (QOL) [5]. A literature review stated that most HF treatment figure on self-man- agement intervention and focus on self-management strategies, such as to become more informed about their illness and be actively engaged in their own care, which 1 Nursing - New Insights for Clinical Care is necessary to improve the impact of self-management on long-term heart failure outcomes [6]. Therefore, nurses should play a key role in improving self-management by comprehensive patient assessment, patient-centered goal setting, evaluation of outcomes, encouraging health promotion, and self-management education. In addi- tion, self-management for patients with HF support should be a part of routine health care, and effective strategies still need to be embedded into routine care. However, a study that surveyed the knowledge of community nurses revealed that they had a basic understanding of HF but scored poorly on weight assessment, blood pressure management, and reporting to physicians of dizziness [7]. This chapter focuses on self-management and lifestyle advice for patients with HF to prevent exacerbation and rehospitalization in the home setting. 2. Types of HF Traditionally, HF has been divided into three phenotypes based on the measure- ment of left ventricular ejection fraction (LVEF). Reduced LVEF is defined as ≦40%, and left ventricular systolic dysfunction. This is designated as heart failure with reduced ejection fraction (HFrEF). Mildly reduced LVEF is defined as between 41% and 49%. Clinical features and prognosis have not yet been fully characterized. This is designated as heart failure with mildly reduced ejection fraction (HFmrEF). Those with symptoms and signs of HF, with evidence of structural and/or functional cardiac abnormalities and/or raised natriuretic peptides (NPs), and with an LVEF ≥ 50%, have heart failure with preserved ejection fraction (HFpEF). No effective treatments have been established. The simplest terminology used to describe the severity of HF is the New York Heart Association (NYHA) functional classification. The etiology of HF varies according to geography. In western countries and devel- oped countries, coronary artery disease and hypertension are predominant factors. With regard to ischemic etiology, HFmrEF resembles HFrEF, with a higher frequency of underlying CAD compared to those with HFpEF. Table 1 shows the definition of HF based on LVEF [1]. The simplest terminology used to describe the severity of HF is the New York Heart Association (NYHA) functional classification. The NYHA functional classifica- tion was developed by the New York Heart Association as a system to classify patients with heart diseases according to the severity of symptoms resulting from physical Type HFrEF HFmrEF HFpEF Criteria 1 Symptoms ± signs Symptoms ± signs Symptoms ± signs 2 LVEF ≦ 40% LVEF 41–49% LVEF ≧ 50% 3 Mainly contractile insufficiency. Many current studies include her cases of decreased LVEF under standard heart failure treatment as HFrEF The presence of elevated natriuretic peptides and other structural heart diseases Objective evidence of cardiac structural and/or functional abnormalities consistent with the presence of LV diastolic dysfunction/ raised LV filling pressures, including raised natriuretic peptides HF, heart failure; LV, left ventricular; LVEF, left ventricular ejection fraction. Table 1. Definition of HF with reduced ejection fraction, mildly reduced ejection fraction, and preserved ejection fraction. Table 1. Definition of HF with reduced ejection fraction, mildly reduced ejection fraction, and preserved ejection fraction. 2 A Home-Based Heart Failure Nursing Intervention Supporting Self-Management and Lives DOI: http://dx.doi.org/10.5772/intechopen.102509 activity and has been used in the severity classification of HF. 2. Types of HF NYHA class II patients were further classified into those with slight limitation of physical activity (IIs) and those with moderate limitation of physical activity (IIm). p y y NYHA I is “No limitation of physical activity. Ordinary physical activity does not cause severe fatigue, palpitations, dyspnea or angina.” NYHA II is “Slight or moderate limitation of physical activity. Comfortable at rest, but ordinary physical activity causes fatigue, palpitations, dyspnea or angina.” NYHA III is “Marked limitation of physical activity. Comfortable at rest, but less than ordinary activity causes fatigue, palpitations, dyspnea or angina.” NYHA IV is “Unable to carry on any physical activity without symptoms of HF, or symptoms of HF and angina at rest. Even slight activity worsens symptoms.” 3. Risk factors of exacerbation of HF HF can be prevented from developing, exacerbating, or recurring by various interventions, such as appropriate treatment and medication for risk factors for heart failure, in addition to lifestyle-related management such as diet and exercise. Smoking is a risk factor for cardiovascular disease, and smoking cessation is strongly recommended for smokers because quitting reduces the mortality rate and readmis- sion rate for cardiovascular disease, including HF [8]. Obesity and diabetes are associ- ated with the development of HF, and insulin resistance-based diabetes and metabolic syndrome are both major risk factors for cardiovascular disease and are commonly used in weight loss and exercise therapy. In addition to improving lifestyle habits, comprehensive risk management through comprehensive treatment is required [9]. To prevent the exacerbation of HF, nurses need to understand risk factors and preven- tion strategies. Table 2 shows risk factors of exacerbation of HF. Risk factors Prevention strategies Sedentary habit Regular physical activity Obesity Physical activity and healthy diet Cigarette smoking Smoking cessation Excessive alcohol No/light alcohol intake is beneficial Influenza Influenza vaccination Hypertension Lifestyle changes, antihypertensive therapy Dyslipidemia Healthy diet, statins Diabetes mellitus Physical activity and healthy diet, SGLT2 inhibitors Microbes Early diagnosis, specific antimicrobial therapy for either prevention and/or treatment CAD Lifestyle changes, statin therapy Chest radiation Cardiac function and side effect monitoring, dose adaptation Cardiotoxic drugs Cardiac function and side effect monitoring, dose adaptation, change of chemotherapy CAD, coronary artery disease; SGLT2, sodium-glucose co-transporter 2. Table 2. Risk factors of exacerbation of HF 3 Nursing - New Insights for Clinical Care 4. Importance of self-management for patients with HF HF is one of the most common causes of hospital admissions and readmissions. A recent review revealed that nurse-led hospital-to-home transitional care interventions reduced the risk of all-cause mortality and heart failure-related hospitalizations, and improved health-related quality of life (HRQOL) and heart failure knowledge compared to usual care. The narrative summary of evidence for self-care behaviors showed positive intervention effects [10, 11]. Generally, nurse-led hospital-to-home transitional care inter- ventions may play a beneficial role in decreasing mortality and improving HRQOL and self-care behaviors for patients with HF. Therefore, self-management is the cornerstone of HF management. Self-management comprises adherence to behaviors, such as maintain- ing a low sodium diet and medication regimen, as well as symptom monitoring to main- tain physiological stability and response to symptoms when they occur [12]. However, there are some concerns. First, previous studies have different contents and modes of intervention, evaluation tools, and there is a publication bias in the outcome of quality of life, which might lead to selection bias, detection bias, and attention bias. Second, due to the diverse durations of interventions and length of follow-up, these factors may affect the long-term effects of self-management interventions. As stated above, further consider- ations are needed to show the optimal self-management interventions in patients with HF. 5.1 Perception of HF This sub-concept means that patients with HF experienced the onset of HF by sudden dyspnea and abnormal changes in their bodies. As a result of seeking medical care, they subsequently learned that their abnormal changes were symptoms of HF. Sources of HF information included inpatient and outpatient settings, from medical staff and laypeople, they encountered in daily life. After hospitalization, patients with HF understand that the onset of HF and deterioration of symptoms was related to several factors, such as living habits, work and overwork, fluid retention, excessive salt intake, and untreated high blood pressure. 5. Processes of self-management and restructuring own lifestyles To promote self-management for patients with HF and support their lives, nurses need to understand patients’ perspectives. Sano et al. described the process of self- management among elderly patients with HF who had avoided re-hospitalization for over 2 years [13]. They identified three sub-concepts and one core-concept. Table 3 shows core-concept and three sub-concepts of self-management of patients with HF [13]. Core-concept Balance between preventing decompensation and preferences Begin new exercise or hobby within capabilities Adjust physical activity to reduce heart failure symptoms Modify diet to prevent decompensation. Tell friends and acquaintances about their own illness to avoid overeating or overwork. Choose how to modify their old lifestyle Make personal choices on how to avoid stress. Manage CHF without decompensation Sub-concepts Perception of HF Sudden development of heart failure symptoms. Cognition that heart failure contributes to uncomfortable feeling or cough, etc. Understanding of causes of heart failure from rehospitalization experience Encountering a new situation Understanding that heart failure can be caused by lifestyle Desire not to repeat rehospitalization or necessity for early consultation Daily activity evokes heart failure symptoms Life coordination for HF Thinking about lifestyle modification to avoid the discomfort associated with heart failure and additional treatment Limit activities to reduce heart strain Maintain a job or hobby to live meaningfully Table 3. Core-concept and sub-concepts. 4 A Home-Based Heart Failure Nursing Intervention Supporting Self-Management and Lives DOI: http://dx.doi.org/10.5772/intechopen.102509 The sub-concepts were perception of heart failure, encountering a new situation, and life coordination for heart failure. The sub-concepts yielded a single core-concept—bal- ance between preventing decompensation and preferences. Patients with HF experience each sub-concept in no particular order and acquire a lifestyle tailored to the condition of HF. Then, when the state of HF becomes stable, not only actions for preventing the worsening of HF but also their own values and preferences are taken in, and the balance between them is maintained. A conceptual model was developed to illustrate the interac- tive relationships among the sub-concepts and the core-concept (Figure 1). Figure 1 shows a conceptual framework of self-management among patients with HF [13]. Figure 1. Conceptual framework. 5.4 Balance between preventing decompensation and preferences This core-concept means that patients with HF try to maintain their agelong preferences to give their life a purpose, such as by maintaining long-term friendships and continuing to engage in interests. Instead of limiting all their activities, they stop engaging in activities that they learn would be bad for their heart and choose to continue those activities not be harmful. They seek medical guidance to determine which activities were considered good for the heart or feasible within the scope of their limited lifestyle. They begin to reassemble their life into ones that do not put a strain on the heart while making up for lost parts of their life as a result of HF. They describe taking walks, singing a song, and engaging in new physical activities or hobbies within their realm of possibility. They determine which activities caused symptoms to appear and how far they could push themselves even if these were vacation activities or when invited by acquain- tances. They make an effort to exercise at their own pace and master how to regulate their amount of exercise so as to avoid placing strain on the heart and causing symptoms. y p They also utilize the knowledge they have acquired on limiting alcohol consump- tion and on excessive fluid and salt intake, which are related to water retention and edema. In doing so, they can prevent exacerbating their HF by modifying their diet. Nonetheless, they are faced with many situations that are difficult to avoid, such as opportunities to go drinking or eating out with others. In these situations, they make an effort to mitigate factors that would exacerbate their HF by avoiding overeat- ing and overexertion, and by informing the people around them of their limits in advance. This allows patients to maintain their work and harmonious social lives while avoiding a diet that might lead to the exacerbation of HF. Although they are aware of the need to modify their lifestyle, they believe that stress is worse and that it would be better to reduce stress by eating what they liked. Thus, they believe in prioritizing values over exacerbating their condition and giving priority to their preferences to avoid stress instead of respecting their limitations. They acknowledge having chosen to avoid stress over causing HF symptoms and pain. 5.2 Encountering a new situation This sub-concept means that once the patients with HF understood how HF was related to their lifestyle, they noticed how the hospital environment was different from their own life at home in terms of activity, reducing salt intake, and management of blood pressure. They began confronting the reality that simple acts, like putting on their socks or cutting their nails, would become more difficult after hospitalization. 5 Figure 1. Conceptual framework. 5 Nursing - New Insights for Clinical Care 5.3 Life coordination This sub-concept means that patients with HF reported that they did not want to This sub-concept means that patients with HF reported that they did not want to experience that painful experience again. They believed that it had been a bad decision to just endure the pain and accept the abnormal physical changes as their condition became A h i h b l k b k h i lif l l i h This sub-concept means that patients with HF reported that they did not want to experience that painful experience again. They believed that it had been a bad decision to just endure the pain and accept the abnormal physical changes as their condition became more severe. At the same time, they began to look back on their lifestyle selections; these led to behavioral modifications to avoid both painful experience and re-hospitalization. They understand that they could not continue living as they had before hospitalization; they could not avoid re-hospitalization if they did not change their lifestyle. They also reported limiting their work. They began to adapt their lifestyle to one compatible with HF by limiting activities they had previously engaged in. Meanwhile, they were determined to continue living a worthwhile and energetic life. Table 4. Table 4. Assessment of experience and knowledge about HF. Table 4. Assessment of experience and knowledge about HF. Assessment of experience and knowledge about HF. 6. Assessments of patients with HF for promoting self-management and supporting their lives The self-management process is a process of living life such that symptoms do not worsen while balancing good choices and preferences. By trial and error, the patients with HF try to find the limits of physical capacity and dietary choices that would lead to the worsening of symptoms, while also trying to maintain quality of life to the extent possible [13]. Based on the process of self-management of patients with HF, it is possible to guide the assessment viewpoints to support self-management and their lives. It is important to understand the components of self-management. Components of self-management are “Experience and knowledge about HF,” “Self-monitoring and Early perception,” and “Life coordination for HF and to live meaningfully.” The examples of the assessment viewpoints are shown below. A Home-Based Heart Failure Nursing Intervention Supporting Self-Management and Lives DOI: http://dx.doi.org/10.5772/intechopen.102509 A Home-Based Heart Failure Nursing Intervention Supporting Self-Management and Lives DOI: http://dx.doi.org/10.5772/intechopen.102509 5.4 Balance between preventing decompensation and preferences These thoughts, actions, and lifestyle choices demonstrate the complexly of symptom management in HF even when patients want to avoid exacerbating their condition. Apparently, they accept some deterioration because they want to act on their prefer- ences and avoid the stress of compliance. In summary, nurses need to recognize and value patients’ views and experiences to support their self-care management. 6 6 6.1 Experience and knowledge about HF Acquisition and experience of knowledge about HF strongly contribute to the improvement of self-care skills, and lack of knowledge may decrease adherence and be a barrier to starting self-care behavior [12, 14]. In addition, patients with HF who understand and recognize the advice of medical professionals benefit the prevent- ing worsening symptoms and readmission that can tend to have good adherence and perform self-care behavior [15]. In the process of self-management, knowledge and understanding of heart failure affect the entire self-management process and need to be continually confirmed even after moving from hospital to home. Assessment view- points include—(1) cognition that HF, (2) understanding of causes of HF (Table 4). Assessment viewpoint Example Experience of the onset of HF, exacerbation, and hospitalization How does the patient recognize the symptoms of heart failure? How does the patient perceive the symptoms of exacerbation of heart failure? (e.g. lower limb edema, shortness of breath, reduction of urine output, nocturnal cough, etc.) Understanding causes of HF Does the patient understand the behaviors/actions that lead to the exacerbation of heart failure? (e.g. inadequate physical activities, long haul travel, overactive, etc.) Does the patient understand the lifestyles that lead to the exacerbation of HF? (e.g. excessive alcohol and/or fluid and/or salt intake, smoking) Table 4. Assessment of experience and knowledge about HF. 6.3 Life coordination for HF and living meaningfully In the stable period, excessive rest causes a decrease in exercise capacity and exac- erbates fatigue and dyspnea during exertion, so moderate exercise increases exercise capacity, and it has been clarified that it leads to improvement of symptoms and improvement of QOL [5]. Appropriate exercise tolerance, evaluation of activities of daily living, and correction of physical activity and living behavior in consideration of physi- cal function and living environment for each heart failure patient are required. Table 6 shows the viewpoint of assessment of lifestyle and life circumstance. 6.2 Self-monitoring and early perception It is said that less than half of the patients with HF regularly perform self-monitoring, such as measuring weight and confirming the degree of edema. The receiving treatment tends to be delayed due to the disagreement between the present HF symptoms and the patient’s perception, but utilizing past knowledge and experience and receiving high-qual- ity social support, it is possible to respond appropriately and early to the symptoms [16]. Symptom monitoring behavior is also a predictor of appropriate self-care management, 7 Nursing - New Insights for Clinical Care Assessment viewpoint Example Self-monitoring behaviors Does the patient regularly measure his/her own weight and blood pressure? Does the patient regularly check the symptoms of HF? (e.g. lower limb edema, shortness of breath, reduction of urine output, nocturnal cough, etc.) Self-monitoring circumstance Does the patient has a sphygmomanometer and scale? Does the patient has a habit of maintaining track of his/her own conditions? Has the patient devised a way to remember the measurement and tracking? Table 5. Assessment of self-monitoring abilities. Assessment viewpoint Example Self-monitoring behaviors Does the patient regularly measure his/her own weight and blood pressure? Does the patient regularly check the symptoms of HF? (e.g. lower limb edema, shortness of breath, reduction of urine output, nocturnal cough, etc.) Self-monitoring circumstance Does the patient has a sphygmomanometer and scale? Does the patient has a habit of maintaining track of his/her own conditions? Has the patient devised a way to remember the measurement and tracking? Table 5. Assessment of self-monitoring abilities. and regular symptom monitoring for appropriate self-management practice is recom- mended [17]. To maintain and promote self-monitoring in patients with HF, we need to support patients with HF by performing daily monitoring, integrating knowledge, and past experiences regarding HF to determine how to evaluate their own conditions and take actions. Table 5 shows the viewpoint of assessment of self-monitoring abilities. Table 6. Assessment of lifestyle and life circumstance. Assessment of lifestyle and life circumstance. Table 6. 6.4 Evaluation of exercise tolerance SAS, METs, and the NYHA classification. Ask questions about the following items and ask them to answer either “yes” or “no.” The amount of exercise (METs) of the item for which the answer “no” appears for the first time is used as an index of the minimum amount of exercise in which symptoms appear Have a good sleeping 1MET NYHA IV Feel better when lay on the bed 1MET Eat something and wash face by yourself 1.6METs Could go to the toilet by yourself 2METs NYHA III Could change clothing by yourself 2METs Could prepare food and clean room 2-3METs Could prepare the bed by yourself 2-3METs Could clean the floor 3-4METs Could take shower by yourself 3-4METs Could walk 100-200 m on the ground as normal people 3-4METs Could clean the grass in the garden 4METs Could take a bath by yourself 4-5METs NYHA II Could go upstairs as a normal people 5-6METs Could do some light farm working 5-7METs Could walk fast on the ground 6-7METs Could play tennis 6-7METs Could do jogging (8 km/h) 300–400 m 7-8METs NYHA I Could do swimming 7-8METs Could skip rope 8METs Table 7. SAS, METs, and the NYHA classification. Table 7. SAS, METs, and the NYHA classification. questionnaire [18]. METs are often used in clinical practice as a simple indicator of exercise intensity. NYHA classification is classified according to the degree of restric- tion of daily activities, and this classification is widely used for heart disease, especially in the stable period of HF. Its usefulness is high, and many reports have been made regarding its association with exercise tolerance and prognosis. The NYHA classifica- tion is simple and useful, but the content of the activity that is the basis of judgment is not clear, so SAS was developed to supplement it. SAS has tested the reproducibility and validity of the NYHA classification and METs. It is said to be more closely related to exercise tolerance and prognosis than the NYHA classification [19]. Physical activity that triggers the onset of HF symptoms is quantified by METs. Table 7 shows the cor- respondence table between SAS, METs and NYHA classification [19]. 6.4 Evaluation of exercise tolerance Specific activity scale (SAS) specifies the metabolic equivalents (METs) for each physical activity, and it is possible to estimate the physical activity level from the physical activity, and it is possible to estimate the physical activity level from the Assessment viewpoint Example Lifestyle and values What are the long-standing hobbies and/or tastes? What about housework? What about regular exercise? (e.g. frequency, intensity, times, etc.) What about social activities and/or neighborhood relationships? Scheduled long-haul travel? Life circumstance Bedroom location (1st floor or 2nd floor? Using stairs?) How about installing bedding and handrails? Type and amount of seasoning What is the transportation for shopping and going out? Is there a steep slope in the neighborhood? Assessment viewpoint Example Lifestyle and values What are the long-standing hobbies and/or tastes? What about housework? What about regular exercise? (e.g. frequency, intensity, times, etc.) What about social activities and/or neighborhood relationships? Scheduled long-haul travel? Life circumstance Bedroom location (1st floor or 2nd floor? Using stairs?) How about installing bedding and handrails? Type and amount of seasoning What is the transportation for shopping and going out? Is there a steep slope in the neighborhood? 8 A Home-Based Heart Failure Nursing Intervention Supporting Self-Management and Lives DOI: http://dx.doi.org/10.5772/intechopen.102509 A Home-Based Heart Failure Nursing Intervention Supporting Self-Management and Lives DOI: http://dx.doi.org/10.5772/intechopen.102509 Ask questions about the following items and ask them to answer either “yes” or “no.” The amount of exercise (METs) of the item for which the answer “no” appears for the first time is used as an index of the minimum amount of exercise in which symptoms appear Have a good sleeping 1MET NYHA IV Feel better when lay on the bed 1MET Eat something and wash face by yourself 1.6METs Could go to the toilet by yourself 2METs NYHA III Could change clothing by yourself 2METs Could prepare food and clean room 2-3METs Could prepare the bed by yourself 2-3METs Could clean the floor 3-4METs Could take shower by yourself 3-4METs Could walk 100-200 m on the ground as normal people 3-4METs Could clean the grass in the garden 4METs Could take a bath by yourself 4-5METs NYHA II Could go upstairs as a normal people 5-6METs Could do some light farm working 5-7METs Could walk fast on the ground 6-7METs Could play tennis 6-7METs Could do jogging (8 km/h) 300–400 m 7-8METs NYHA I Could do swimming 7-8METs Could skip rope 8METs Table 7. 7. Conclusions HF is a severe public health problem all over the world. Adequate patient self- management is essential in the effective management of HF. Patients with HF who report more effective self-management have better QOL, lower readmission rates, and reduced mortality. 9 Nursing - New Insights for Clinical Care This chapter gives an overview of the current situation of patients with HF and self-management and shows the viewpoint of promoting self-management and supporting lifestyle. Supporting patient’s life in the home setting, it is necessary to focus on their lifestyle, circumstance, and values before educational interventions. We believe that this chapter can be used not only for nurses in the home setting but also nurses in hospital settings who are involved with patients with HF at the time of discharge support. Conflict of interest The authors declares no conflict of interest. Conflict of interest The authors declares no conflict of interest. Author details Motohiro Sano* and Tomoko Majima Graduate School of Nursing, Chiba University, Chiba City, Japan Motohiro Sano* and Tomoko Majima Motohiro Sano and Tomoko Majima Graduate School of Nursing, Chiba University, Chiba City, Japan j Graduate School of Nursing, Chiba University, Chiba City, Japan *Address all correspondence to: sano-m@chiba-u.jp Conflict of interest Conflict of interest The authors declares no conflict of interest. The authors declares no conflict of interest. [6] Toback M, Clark N. Strategies to improve self-management in heart failure patients. Contemporary Nurse: A Journal for the Australian Nursing Profession. 2017;53(1):105-120 © 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 10 A Home-Based Heart Failure Nursing Intervention Supporting Self-Management and Lives DOI: http://dx.doi.org/10.5772/intechopen.102509 A Home-Based Heart Failure Nursing Intervention Supporting Self-Management and Lives DOI: http://dx.doi.org/10.5772/intechopen.102509 [8] Suskin N, Sheth T, Negassa A, Yusuf S. Relationship of current and past smoking to mortality and morbidity in patients with left ventricular dysfunction. Journal of the American College of Cardiology. 2001;37(6):1677-1682 [6] Toback M, Clark N. Strategies to improve self-management in heart failure patients. Contemporary Nurse: A Journal for the Australian Nursing Profession. 2017;53(1):105-120 References [1] McDonagh TA, Metra M, Adamo M, Gardner RS, Baumbach A, Böhm M, et al. 2021 ESC guidelines for the diagnosis and treatment of acute and chronic heart failure. European Heart Journal. 2021;42(36):3599-3726 [2] Dharmarajan K, Rich MW. Epidemiology, pathophysiology, and prognosis of heart failure in older adults. Heart Failure Clinics. 2017;13(3):417-426 [9] Wilhelmsen L, Rosengren A, Eriksson H, Lappas G. Heart failure in the general population of men— Morbidity, risk factors and prognosis. Journal of Internal Medicine. 2001; 249(3):253-261 [3] Crespo-Leiro MG, Anker SD, Maggioni AP, Coats AJ, Filippatos G, Ruschitzka F, et al. European Society of Cardiology Heart Failure Long-Term Registry (ESC-HF-LT): 1-year follow-up outcomes and differences across regions. European Journal of Heart Failure. 2016;18(6):613-625 [10] Li Y, Fang J, Li M, Luo B. Effect of nurse-led hospital-to-home transitional care interventions on mortality and psychosocial outcomes in adults with heart failure: A meta-analysis. European Journal of Cardiovascular Nursing. 2021 (Online ahead of print) [4] Shiga T, Suzuki A, Haruta S, Mori F, Ota Y, Yagi M, et al. Clinical characteristics of hospitalized heart failure patients with preserved, mid-range, and reduced ejection fractions in Japan. ESC Heart Failure. 2019;6(3):475-486 [11] Zhao Q, Chen C, Zhang J, Ye Y, Fan X. Effects of self-management interventions on heart failure: Systematic review and meta-analysis of randomized controlled trials. International Journal of Nursing Studies. 2020;110:103689 [5] Tsutsui H, Isobe M, Ito H, Okumura K, Ono M, Kitakaze M, et al. JCS 2017/JHFS 2017 guideline on diagnosis and treatment of acute and chronic heart failure—Digest version. Circulation Journal. 2019;83(10):2084-2184 [12] Jaarsma T, Cameron J, Riegel B, Stromberg A. Factors related to self- care in heart failure patients according to the middle-range theory of self- care of chronic illness: A literature update. Current Heart Failure Reports. 2017;14(2):71-77 [13] Sano M, Majima T. Self-management of congestive heart failure among elderly men in Japan. International Journal of Nursing Practice. 2018;24(Suppl 1):e12653 [7] Fowler S. Improving community health nurses' knowledge of heart failure education principles: A descriptive study. Home Healthcare Nurse. 2012;30(2):91-101 [14] Lee KS, Moser DK, Pelter MM, Nesbitt T, Dracup K. Self-care in rural residents with heart failure: What we are missing. European Journal of Cardiovascular Nursing: Journal of 11 Nursing - New Insights for Clinical Care The Working Group on Cardiovascular Nursing of the European Society of Cardiology. 2017;16(4):326-333 The Working Group on Cardiovascular Nursing of the European Society of Cardiology. References 2017;16(4):326-333 The Working Group on Cardiovascular Nursing of the European Society of Cardiology. 2017;16(4):326-333 [15] Sevilla-Cazes J, Ahmad FS, Bowles KH, Jaskowiak A, Gallagher T, Goldberg LR, et al. Heart failure home management challenges and reasons for readmission: A qualitative study to understand the Patient's perspective. Journal of general. Internal Medicine. 2018;33(10):1700-1707 [16] Riegel B. A mixed methods study of symptom perception in patients with chronic heart failure. Heart & Lung. 2018;47(2):107-114 [16] Riegel B. A mixed methods study of symptom perception in patients with chronic heart failure. Heart & Lung. 2018;47(2):107-114 [17] Lee CS, Mudd JO, Hiatt SO, Gelow JM, Chien C, Riegel B. Trajectories of heart failure self-care management and changes in quality of life. European Journal of Cardiovascular Nursing. 2015;14(6):486-494 [17] Lee CS, Mudd JO, Hiatt SO, Gelow JM, Chien C, Riegel B. Trajectories of heart failure self-care management and changes in quality of life. European Journal of Cardiovascular Nursing. 2015;14(6):486-494 [18] Goldman L, Hashimoto B, Cook EF, Loscalzo A. Comparative reproducibility and validity of systems for assessing cardiovascular functional class: Advantages of a new specific activity scale. Circulation. 1981;64(6):1227-1234 [19] Izawa H, Yoshida T, Ikegame T, Izawa KP, Ito Y, Okamura H, et al. Standard cardiac rehabilitation program for heart failure. Circulation Journal. 2019;83(12):2394-2398 12 12
https://openalex.org/W4254386707	http://rcin.org.pl/Content/146901/PDF/WA35_180745_12807-11_art-10.pdf	English	null	Note on a system of algebraical equations	Cambridge University Press eBooks	2,009	public-domain	404	39 39 715] NOTE ON A SYSTEM OF ALGEBRAICAL EQUATIONS. [From the Messenger of Mathematics, vol. vu. (1878), pp. 17, 18.] [From the Messenger of Mathematics, vol. vu. (1878), pp. 17, 18.] Assume [From the Messenger of Mathematics, vol. vu. (1878), pp. 17, 18.] [From the Messenger of Mathematics, vol. vu. (1878), pp. 17, 18.] [From the Messenger of Mathematics, vol. vu. (1878), pp. 17, 18.] [From the Messenger of Mathematics, vol. vu. (1878), pp. 17, 18.] Assume x + y + z = P, y z + zx + xy = Q, xyz = R, A = x (nyz + Q) — w2 (mx + P), B = y (nzx + Q)-w2 (my + P), C ·= z (nxy + Q) — w2 (mz + P), Θ = — mnR + PQ. Then (mz + P) B — (my + P) C = (myz + Py) (nzx + Q) — (myz + Pz) (nxy + Q) = myz (nzx + Q — nxy — Q) + Pnxyz + PQy — Pnxyz — PQz = mnxyz (z — y) — PQ (z — y) = (z — y) {mnxyz — PQ] = (y — z) Θ ; whence, identically, whence, identically, (mz + P)B - (my + P) G = (y - z) Θ, (mx + P) C — (mz + P) A = (z — x) Θ, (my + P) A — (mx + P) B = (x - y) Θ. Hence any two of the equations A = 0, B = 0, (7=0 imply the third equation. Hence any two of the equations A = 0, B = 0, (7=0 imply the third equation. www.rcin.org.pl www.rcin.org.pl [715 NOTE ON A SYSTEM OF ALGEBRAICAL EQUATIONS. 40 We have We have A = x {(π + 1) yz + zx + xy} — w2 {(m + 1) x + (y + z)} = (x2 — w2) (y + z) — x [(to + 1) w2 — (w + i) yz∖, and similarly for B and G. The three equations therefore are sc _ y + z x2 — w2 (m + 1) w2 — (n + 1) yz ’ y _ z + x y2 -w2 (to + 1) w2 — (n + 1) zx' z _ χ + y z2 — w2 (to + 1) w2 — (n + 1) xy ' and any two of these equations imply the third equation. www.rcin.org.pl
https://openalex.org/W4233008702	https://www.epj-conferences.org/10.1051/epjconf/201714800011/pdf	English	null	Introduction to Wind Energy Systems	Green energy and technology	2,018	cc-by	6,377	Introduction to wind energy systems H.-J. Wagner Ruhr-University Bochum, Energy Systems and Energy Economics P.O. Box 102148, 44721, Bochum, Germany H.-J. Wagner Ruhr-University Bochum, Energy Systems and Energy Economics P.O. Box 102148, 44721, Bochum, Germany Summary. — This article presents the basic concepts of wind energy and deals with the physics and mechanics of operation. It describes the conversion of wind energy into rotation of turbine, and the critical parameters governing the eﬃciency of this conversion. After that it presents an overview of various parts and components of windmills. The connection to the electrical grid, the world status of wind energy use for electricity production, the cost situation and research and development needs are further aspects which will be considered. Summary. — This article presents the basic concepts of wind energy and deals with the physics and mechanics of operation. It describes the conversion of wind energy into rotation of turbine, and the critical parameters governing the eﬃciency of this conversion. After that it presents an overview of various parts and components of windmills. The connection to the electrical grid, the world status of wind energy use for electricity production, the cost situation and research and development needs are further aspects which will be considered. , 00011 (2017) 1 EPJ Web of Conferences 48 LNES 2016 , 00011 (2017) 1 EPJ Web of Conferences 48 LNES 2016 DOI: 10.1051/ 71480 epjconf/201 0011 © The Authors, published by EDP Sciences . This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). - SIF 1. – Diﬀerent wind turbine types: An overview Today there are various types of wind turbines in operation, (ﬁg. 1 gives an overview). The most common device is the horizontal-axis wind turbine. This turbine consists of only a few aerodynamically optimised rotor blades, which for the purpose of regulation usually can be tumbled about their long axis (Pitch-regulation). Another cheaper way to regulate it, consists in designing the blades in such a way that the air streaming along the blades will go into turbulence at a certain speed (Stall-Regulation). These turbines can deliver power ranging from 10 kW to some MW. The largest turbine on the European DOI: 10.1051/ 71480 epjconf/201 0011 , 00011 (2017) 1 EPJ Web of Conferences 48 LNES 2016 , 00011 (2017) 1 EPJ Web of Conferences 48 Fig. 1. – Overview of the diﬀerent types of wind turbines. Fig. 1. – Overview of the diﬀerent types of wind turbines. market has a power of 8 MW, bigger machines are being tested. The eﬃciency of this type of turbine is very high. Therefore, it is solely used for electricity generation which needs “high-speed engines” to keep the gear transmission and the generator small and cheap. Another conventional (older) type of horizontal-axis rotor, is the multiblade wind turbine. It was ﬁrst built about one hundred years ago. Such wind mills have a high starting torque which makes them suitable for driving mechanical water pumps. The number of rotations is low, and the blades are made from simple sheets with an easy geometry. For pumping water, a rotation regulating system is not necessary, but there is a mechanical safety system installed to protect the turbine against storm damage. By using a so-called wind-sheet in lee direction the rotor is turned in the direction of the wind. In order to increase the number of rotations, this type of turbine had been equipped with aerodynamically more eﬃcient blades facilitating the production of electricity, here the area of a blade is smaller. The mechanical stability of such “slow-speed turbines” is very high, some have had operation periods of more than ﬁfty years. A third type of turbine is known as DARRIEUS —a vertical-axis construction. Their advantage is that they do not depend on the direction of the wind. To start, they need the help of a generator working as motor or the help of an SAVONIUS rotor in- stalled on top of the vertical axis. 1. – Diﬀerent wind turbine types: An overview In the nineteen eighties a reasonable number of DARRIEUS-turbines had been installed in California, but a further expansion into the higher-power range and in the European markets has not taken place. One reason may be that they are noisier than horizontal-axis turbines. Another disadvantage is that wind velocity increases signiﬁcantly with height, making horizontal-axis wheels on towers more economical. Nevertheless, there are some companies producing DARRIEUS-turbines in 2 , 00011 (2017) 1 EPJ Web of Conferences 48 LNES 2016 DOI: 10.1051/ 71480 epjconf/201 0011 the very low power range of a few kilowatts for decentralised electricity supply in areas without electrical grids e.g. in rural areas of developing countries. the very low power range of a few kilowatts for decentralised electricity supply in areas without electrical grids e.g. in rural areas of developing countries. The Savonius rotor is only used for research activities, e.g. as a measurement device especially for wind velocity, it is not used for power production. Therefore it will not be discussed in detail in this paper. The last technique to be dealt with is known as Up-Stream-Power-Station or thermal tower. In principle, it can be regarded as a mix between a wind turbine and a solar collector. In the top of a narrow, high tower is a wind wheel on a vertical axis driven by the rising warm air. A solar collector installed around the foot of the tower heats up the air. The design of the collector is simple; a transparent plastic foil is ﬁxed over several metres on the ground in a circle around the tower. Therefore, the station needs a lot of space and the tower has to be very high. Such a system has a very poor eﬃciency, only about one percent. World wide there has only been one Up-Stream-Power- Station built so far, it was designed by a German company. For some years it worked satisfactorily at the location of Manzarenas in Spain, but in the mid eighties it was destroyed by bad weather. This station had an electrical power of 20 kW, the tower was about 200 m high, and the collector had a diameter of approximately the same size. A second Up-Stream- Power- Station with an electrical performance of 200 MW was planned in Australia, but not yet realised until. 1. – Diﬀerent wind turbine types: An overview The tower height should be about 1000 m and the diameter of the collector area should be about 7000 m. There has no new Up-Stream-Power-Station been designed and installed so far. Since there has been tremendous technical progress over the last ten years regarding solar farm stations as well as horizontal-axis wind turbines. 2. – Physical basics 2.1. Energy content of the wind. – The following section will be used to mathematically explain where the energy in the wind comes from and what factors it depends on. Power is deﬁned as P = E t = 1 2 · A · ρa · v3, (1) P = E t = 1 2 · A · ρa · v3, (1) (1) with E: kinetic energy, A: area, ρa: speciﬁc density of the air, v: wind velocity. with E: kinetic energy, A: area, ρa: speciﬁc density of the air, v: wind velocity. Therefore, it is also proportional to the cube of the wind speed, v3. From ﬁg. 2, it can be seen that the power output per m2 of the rotor blade is not linearly proportional to the wind velocity, as proven in the theory above. This means that it is more proﬁtable to place a wind turbine in a location with occasional high winds, than in a location where there is a constant low wind speed. Measurement at diﬀerent places shows that the distribution of wind velocity over the year could be approximated by a Weibull-equitation. That means that at least about 2/3 of the produced electricity will be earned by the upper third of wind velocity. From a mechanical point of view, the power density range increases by one thousand for a wind speed change of just 10 m/s, thus producing a construction limit problem. 3 , 00011 (2017) 1 EPJ Web of Conferences 48 LNES 2016 DOI: 10.1051/ 71480 epjconf/201 0011 , 00011 (2017) 1 EPJ Web of Conferences 48 Fig. 2. – Relationship between wind velocity and power output (yearly average valid for Ger- many)(1). Fig. 2. – Relationship between wind velocity and power output (yearly average valid for Ger- many)(1). Therefore, wind turbines are constructed to harness only the power from wind speeds in the upper regions. Therefore, wind turbines are constructed to harness only the power from wind speeds in the upper regions. 2.2. Power coeﬃcients. – There is now the question of how much of the energy in the wind can be transferred to the blade as mechanical energy. (1) This ﬁgure is taken from [1]. Betz’s law and cp ⇒Slow rotating turbines have poor aerodynamic proﬁles and a high number of blades. 3. The proﬁle form ratio and the tip-speed ratio have a considerably greater inﬂuence on the power coeﬃcient than the number of blades. 3. The proﬁle form ratio and the tip-speed ratio have a considerably greater inﬂuence on the power coeﬃcient than the number of blades. ⇒The quality of an aerofoil in respect to a high speed turbine has an inferior signiﬁcance. ⇒The quality of an aerofoil in respect to a high speed turbine has an inferior signiﬁcance. Betz’s law and cp Betz’s law states that you can only convert a maximum of 59% of the kinetic energy in the wind to mechanical energy using a wind turbine. This is because the wind on the back side of the rotor must have a high enough velocity to move away and allow more wind through the plane of the rotor. The relationship between the power of the rotor blade PR and the maximum power PR max is given by the power coeﬃcient cp; PR = P1 −P2 = cp · PR max. (2) (2) The maximum power coeﬃcient is determined through the ratio v2/v1 and setting the DOI: 10.1051/ , 00011 (2017) 71480 1 s epjconf/201 48 001 DOI: 10.1051/ 71480 epjconf/201 0011 , 00011 (2017) 1 EPJ Web of Conferences 48 derivation to zero. zero. derivation to zero. cp max = 16 17 = 0.593 with v2 = 1 3 · v1. cp max = 16 17 = 0.593 with v2 = 1 3 · v1. (3) (3) Therefore, an ideal turbine will slow down the wind by 2/3 of its original speed (Betz’s law). Therefore, an ideal turbine will slow down the wind by 2/3 of its original speed (Betz’s law). The issues discussed in the theory can be summed up and related to the design of a wind energy turbine, by the so called Cooking recipe: “Cooking recipe” for the design of wind turbines. ooking recipe” for the design of wind turbines. 1. A high aerofoil form ratio leads to a high Tip-speed ratio and therefore, a large power coeﬃcient cp. 1. A high aerofoil form ratio leads to a high Tip-speed ratio and therefore, a large power coeﬃcient cp. ⇒Modern turbines with a good aerodynamic proﬁle rotate quickly. 2. Simple proﬁles with smaller proﬁle form ratios have a small Tip-speed ratio. Therefore, the area of the rotor radius that is occupied by blades must be increased in order to increase the power coeﬃcient. ⇒Slow rotating turbines have poor aerodynamic proﬁles and a high number of blades. 2. Simple proﬁles with smaller proﬁle form ratios have a small Tip-speed ratio. Therefore, the area of the rotor radius that is occupied by blades must be increased in order to increase the power coeﬃcient. increased in order to increase the power coeﬃcient. ⇒Slow rotating turbines have poor aerodynamic proﬁles and a high number of blades. 3. – Technical design of wind turbines 3.1. The design with gearbox. – The details of a design with gearbox are shown in ﬁg. 3. The main aspect of the classic design is the split shaft system, where the main shaft turns slowly with the rotor blades and the torque is transmitted through a gearbox to the high-speed secondary shaft that drives the few-pole pair generator. The transmission of torque to the generator is shut oﬀby means of a large disk brake on the main shaft. A mechanical system controls the pitch of the blades, so pitch control can also be used to stop the operation of the turbine in e.g. storm conditions. The pitch mechanism is driven by a hydraulic system, with oil as the popular medium. This system needs almost yearly maintenance and constant pressure monitoring, along with the gear box which is lubricated with oil. Due to mechanical losses in the gearbox, the oil will be treated and must be cooled. For constructions without a main brake, each blade has its pitch angle controlled by a small electric motor. This is the standard by most all wind converters. 5 , 00011 (2017) 1 EPJ Web of Conferences 48 , 00011 (2017) 1 EPJ Web of Conferences 48 LNES 2016 DOI: 10.1051/ 71480 epjconf/201 0011 Fig. 3. – The design with gearbox. Fig. 3. – The design with gearbox. To reduce weight generators with permanent magnets was developed. Some producers are equipping their converters with it last years. To reduce weight generators with permanent magnets was developed. Some producers are equipping their converters with it last years. In order to reduce vibrations by high speeds in the rotating axe, generators are often designed with more that one pole pair. This allows a reduction in the rotation velocities between the gear box and the generator. The problem with this type of conﬁgurations is that the size and weight of the generator increases with the number of pole pairs. Wind speed and direction measuring apparatus are located at the back of the hub head. A rack-and-pinion mechanism at the join of the hub and the tower, allows the hub to be rotated in to the wind direction, and out of it in storm conditions. 3.2. The design without gearbox. – Some companies e.g. the German company En- ercon, design another turbine type, without gearbox. The scheme of such a turbine is shown in ﬁg. 3. – Technical design of wind turbines 4, where the main design aspects can be clearly seen. This design has just one stationary shaft. The rotor blades and the generator are both mounted on this shaft. The generator is in the form of a large spoked wheel with e.g. forty-two pole pairs, around the outer circumference and stators mounted on a stationary arm around the wheel. The wheel is ﬁxed to the blade apparatus, so it rotates slowly with the blades. Therefore, there is no need for a gearbox, rotating shafts or a disk brake. This 6 6 , 00011 (2017) 1 EPJ Web of Conferences 48 LNES 2016 , 00011 (2017) 1 EPJ Web of Conferences 48 DOI: 10.1051/ 71480 epjconf/201 0011 Fig. 4. – The design without gearbox (Enercon E-66). Fig. 4. – The design without gearbox (Enercon E-66). minimising of mechanical parts simpliﬁes the maintenance and production of the turbine. minimising of mechanical parts simpliﬁes the maintenance and production of the turbine. The whole system is automated; pitch control and hub direction are controlled by a central computer, which operates the small directional motors. The whole system is automated; pitch control and hub direction are controlled by a central computer, which operates the small directional motors. 3.3. Aspects of design and development. – There are several critical aspects of a wind turbine that need to be considered in the design phase, to ensure the turbine will be economic and durable. The tower In principle, the tower needs to be as tall as possible, because the wind speed increases with height. However, the height is limited by costing issues; an increase in tower height of 10 m costs an extra ﬁfteen thousand Dollars, and a tower height of over 100 m requires an aircraft-warning beacon, which is again so expensive. Heat energy Large turbines (> 1 MW), have an average generator eﬃciency of 98%. Heat is also generated in the mechanical parts of the machine including the bearings and the gear box. This means that around 40 kW of power are lost to the generator heating up during operation. This heat energy needs to be controlled to prevent damage to the machine parts. A large fan system is mounted in on the back side of the hub of a turbine and used to draw cool air through the hub and remove the heat energy emitted during operation. 7 7 , 00011 (2017) 1 EPJ Web of Conferences 48 LNES 2016 DOI: 10.1051/ 71480 epjconf/201 0011 LNES 2016 Control and monitoring Control and monitoring The following aspects of a wind turbine need to be controlled and monitored to ensure eﬀective operation of a wind turbine within the legal limits: – By large turbines; vibration levels. – Speed of rotation and the pitch angle, of the rotor blades. – The natural wind speed and direction. – The voltage and frequency of the electricity produced. – The output phase angle compared to the grid phase angle. – The consistency of the electrical power output. – The acquisition and storage of electrical signals. – Signal conversion equipment for the directional motors. – Rotational speed at night, to reduce the noise levels, because the noise is propor- tional to the blade-tip speed to the power six. – Rotational speed at night, to reduce the noise levels, because the noise is propor- tional to the blade-tip speed to the power six. Mechanical stability The following forces aﬀect the stability of the mechanical system: – Gravity. – Centrifugal forces on the rotor blades. – Pressure changes on the blade due to the shadow eﬀect the tower creates. – Stochastic power output of the turbine due to wind energy levels continually chang- ing. – Stochastic power output of the turbine due to wind energy levels continually chang- ing. – Resonance of the blades. , 00011 (2017) 1 EPJ Web of Conferences 48 LNES 2016 , 00011 (2017) 1 EPJ Web of Conferences 48 LNES 2016 DOI: 10.1051/ 71480 epjconf/201 0011 Table I. – The technical ﬁgures of two diﬀerent multi-megawatt wind turbines for onshore. Enercon E-182 E3 REpower 3.2 MW Design without gearbox with gearbox Hub height 80–130 m (onshore) 100–130 m (onshore) No. of blades 3 3 Rotor speed 6–18 rpm 6.7–12 rpm Rotor diameter 82 m 114 m Material of blade Fibreglass (reinforced epoxy) Fibreglass (reinforced epoxy) Blade regulation Pitch Pitch Rated power 3 MW 3.2 MW Transmission ratio of gearbox None approx. 99 Generator Multi-pole Asynchronous, few poles Grid connection Via frequency converters Via frequency converters Table I. – The technical ﬁgures of two diﬀerent multi-megawatt wind turbines for onshore. able I. – The technical ﬁgures of two diﬀerent multi-megawatt wind turbines for onshore ures of two diﬀerent multi-megawatt wind turbines for onshore. Grid connection 3.4. Technical ﬁgures of two modern wind turbines. – The largest market introduced machine up to the year 2014, is a 8 MW turbine from the company Vestas. Table I gives an idea of the technical data of wind turbines of the 3 MW class. A design with and without gearbox have been chosen to show the diﬀerent operation of their generators. 4. – Connection to the electrical grid The main electrical grid has a constant frequency e.g. of 50 Hz or 60 Hz and a constant phase angle. Therefore, a wind turbine must produce electricity with the same constant values in order to be integrated into the main grid. The input energy of a wind turbine is proportional to the wind speed, but the wind speed is never constant. Each wind speed has a corresponding rotor rotation speed, at which the maximum power is produced. This maximum occurs for diﬀerent wind speeds at diﬀerent rates of rotation. However, the rate of rotation must be held constant in order to achieve the required constant output frequency or the wind turbine has to be connected to the grid by doubly fed asynchronous generator or by electronic frequency converters. A small turbine can be connected directly into the grid network at 0.4 kV. When the wind turbine is integrated into the grid network, there must be no voltage change, voltage oscillation or ﬂicker experienced in the homes on that network branch. The loss of voltage due to resistance in the cabling can be avoided by increasing the diameter of the cables. It is often required that a new network branch is constructed and linked to the transformer, in order to reduce the voltage disturbances. This increases the installation costs of the turbine. Wind direction set-up Wind direction set-up A wind turbine can be designed to face in to the wind (windward), or away from it (leeward). A leeward turbine has the advantage of being self orientating, but the disad- vantage of the tower disturbing the wind velocity proﬁle, before the wind has reached the plane of the rotor blades. The pressure and speed diﬀerences experienced by the blade as it passes the tower, result in stresses on the hub, which need to be alleviated by use of an extra mechanism in the hub to allow the rotor blades to move out of their usual plane of rotation. 8 9 9 , 00011 (2017) 1 EPJ Web of Conferences 48 LNES 2016 , 00011 (2017) 1 EPJ Web of Conferences 48 DOI: 10.1051/ 71480 epjconf/201 0011 LNES 2016 Fig. 5. – Load distribution measurement (Germany). Fig. 5. – Load distribution measurement (Germany). Megawatt turbines cannot be connected to the grid at the 0.4 kV stage, but have to be connected in at 10–30 kV, which is the usual level of the city electricity share distribution. In remote areas, where a 30 kV connection is not established, the connection must be created and ﬁnanced by the wind park developers. Wind parks with a lot of Megawatt turbines can also be connected into the electrical grid e.g. at the 110 kV level in Germany. As mentioned earlier the maximum power output is obtained only in few hours during the year. Figure 5 shows a typical load distribution, measured in Germany. With larger wind energy installations in future this uneven distribution leads to the need of higher regulation capacities by conventional power systems. Grid connection of oﬀshore wind farms poses a technical and economical challenge to wind turbine and grid operators. In the initial phase, the still quite limited capacity of early pilot farms enables using a conventional three-phase AC connection to the onshore grid system which is a well-known technology and inexpensive. This wind farms were also located near to the coast, the energy losses by using AC was therefore limited. Greater capacities and remote oﬀshore sites make it technically diﬃcult to connect oﬀshore wind farms to the mainland grid by using AC undersea cables. Losses, reactive power production and limited capacity of the sea cables may become important in the future. High-voltage direct current transmission to land could be a solution but it is technically more complicated and more expensive. At the moment there are some plans for connecting oﬀshore wind farms to the main- land grid. Some lines, AC and DC operated, are already into operation, some more are under realising ore planning. To protect environment on sea several wind farms are connected to one line. 10 , 00011 (2017) 1 EPJ Web of Conferences 48 LNES 2016 DOI: 10.1051/ 71480 epjconf/201 0011 Table II. – World wind power production(2). 9 Land/Region Total installed rated power up to the end of 2015 [GW] China 145 USA 74 Germany 45 Spain 23 India 25 UK 14 Italy 9 France 10 Canada 11 Brazil 9 Remaining countries 67 Total 432 To connect the oﬀshore wind farm to the onshore grid an internal grid is necessary. The produced power has to be feed to an oﬀshore transformer substation. Wind turbines are connected to it via undersea cables by a voltage of about 30 kV. After stepping-up to the transmission line voltage, the power is conveyed to shore. (2) This statistic is taken from [2]. 5. – Use of wind energy 5.1. Worldwide status. – In the nineteen eighties, it was the USA that took the lead in establishing wind farms. They set over 10000 turbines into operation, each generating between 80–200 kW. In Europe it was Denmark that was the main pioneers of wind energy. At the end of twenty-ﬁrst century, Germany has taken the world lead, producing about a third of the world’s wind produced electricity until 2008. Many governments have began to produce incentive schemes to increase the economic feasibility of wind turbines. Some incentives used include both paying more for wind produced electricity and providing a proportion of the initial construction costs. Gov- ernments of industrial countries, or those with a high power consumption, are eager to promote wind energy, because it is environmentally clean and sustainable and limits the need for fossil fuel usage (table II). The world total rated power was by the end of the year 2015 about 432 GW. This is about 1.8% of the worldwide installed capacity of power stations (water, coal, naturalgas, nuclear). 11 , 00011 (2017) 1 EPJ Web of Conferences 48 LNES 2016 DOI: 10.1051/ 71480 epjconf/201 0011 Table III. – (a) The typical investment costs of a 2 MW onshore wind power station in Germany (Amesdorf ) (Status: 2013)(3). (b) The typical operating costs of a 2 MW onshore wind power station in Germany (Wellen) (Status: 2013)(3). (a) Investment plan Costs [¤/kW] Hub height < 120 m > 120 m Wind power station, transport, installation 1150 1340 Foundation 70 Grid connection 70 Site development (lanes) 40 Planning, environmental measures, concession, others 190 Total 1520 1710 (b) Operating costs: 5.1 ct/kWh (Average over 20 years operating time) Service, reparation, others 50% Rent 20% Management (technical and business) 20% Reserve for unforeseen events 5% Insurance 5% Table III. – (a) The typical investment costs of a 2 MW onshore wind power station in Germany (Amesdorf ) (Status: 2013)(3). (b) The typical operating costs of a 2 MW onshore wind power station in Germany (Wellen) (Status: 2013)(3). Table III. – (a) The typical investment costs of a 2 MW onshore wind power station in Germany (Amesdorf ) (Status: 2013)(3). (b) The typical operating costs of a 2 MW onshore wind power station in Germany (Wellen) (Status: 2013)(3). (3) These data are taken from WindGuard GmbH, Germany. 5. – Use of wind energy It should be taken into account that wind energy have a lower capacity factor than fossil and nuclear power stations (around 1) with around 0.3 to 0.4 for onshore wind power and around 0.5 for oﬀshore wind power. 5.2. Oﬀshore wind status. – Due to high wind potential at Germany’s coastline, wind energy should play a decisive role. Therefore, Germany’s government plans to install around 6.5 GW of oﬀshore wind parks until 2020 and 15 GW until 2030. By the end of the year 2015, the wind power installed capacity in Germany for oﬀshore was around 3.4 GW and for onshore around 42 GW [3]. 5.3. Investment and operation costs. – The costs involved in installing a wind turbine vary depending on the design, size and chosen location of the new turbine. The infras- tructure costs can be minimised by constructing wind parks, where a number of new turbines are installed on the same sight. An example of the investment costs for a wind park at land in Germany is shown in tables III(a) and III(b). The money invested in an average wind park on land depreciates over about a ten year period. During this period, the set-up and installation costs are high, along with the 12 , 00011 (2017) 1 EPJ Web of Conferences 48 LNES 2016 DOI: 10.1051/ 71480 epjconf/201 0011 loan repayments and insurance costs. After this period, the costs then decrease. Over the next ten years, a ﬁnancial return can then be made on the investment, when the price for the electricity per kilowatt hour is set at a high value by the government. This means it is economic to ensure the durability of the installed turbines, so maintenance costs after the ten year period are kept low and the investors can receive a good return on their investment. The above example of investment costs for wind parks on land in Germany is non- transferable to other countries. Especially the employee’s wages and the structure of ﬁnancing wind farms are quite diﬀerent. Nevertheless power generating by wind turbines in developed countries with existing electrical grid and suﬃcient installed power, costs three to four times in comparison to power generation by conventional power stations. Wind parks on sea need higher speciﬁc investments, although the wind oﬀer is better on sea than on land. The costs of produced electricity are higher by an oﬀshore wind park (status 2012). 5. – Use of wind energy The expectation is that the cost could be reducing for new oﬀshore wind parks in next years. A lot of countries are supporting the installation of wind converters by supporting programs. 5.4. Environmental aspects. – Wind energy is a renewable energy source; therefore it holds many advantages over the fossil fuels, which have diminishing reserves. Wind energy is clean in regard to toxic emissions. Therefore, it does not add to Global warming or Acid rain problems. The wind turbines can aﬀect the environment in aesthetic and human intrusive ways. This is because they must be sited in prominent locations and through the nature the rotation of their blades; they produce optical distortions, i.e. ﬂickering shadows, and a humming noise. The land required for the sighting of a wind park can be considered large, if all the access routes are also taken into consideration. However, they very rarely require the resettlement of communities, which is a problem associated with e.g. large Hydro-Electric-Power stations. The danger to birds of the rotating blades has been questioned, but it has been found that the birds change their ﬂight paths to avoid the blades. It has also be questioned whether the reduced wind speed at ground level, aﬀects the growth of ﬂora. This is answered by the observation that many wind parks have animals grazing between the turbines. 5.5. Life cycle analysis. – Life Cycle Assessments (LCA) is an important tool for industry and policy makers, used to determine the actual emissions of a product or tech- nology throughout its whole life cycle. In case of energy production systems or power plants, analysis of energy required to produce the materials and processes; emissions resulting from various processes for materials production and processes resulting into their Cumulated Energy Demand (CED) and Global Warming Potential (GWP) become important parameters when making decisions on further research, development and de- ployment of any technology. Results and characteristics of wind energy LCA were for the ﬁrst time analyzed in the German oﬀshore wind farm alpha ventus. Alpha ventus is a test ﬁeld in the North Sea for 5 MW wind energy converters in a water depth of 30 m. 13 13 , 00011 (2017) 1 EPJ Web of Conferences 48 LNES 2016 DOI: 10.1051/ 71480 epjconf/201 0011 Results shown that within less than one year the energetic expenditure as well as the greenhouse gas emission of the entire life cycle of alpha ventus is amortized [4]. 6. – Research and development needs The ﬁeld of wind converter has been connected with high technical development activ- ities for many years. The ﬁrst report of the project “Wind Energy Thematic Network” [4] founded by the European Commission described in the year 2012 some aspects, which are also relevant in: 1. Environmental and social impacts (e.g. enhancing local incentives by developing participation models): 1. Environmental and social impacts (e.g. enhancing local incentives by developing participation models): – methods to integrate wind turbines visually in to the landscape, – reduction of noise impacts, – mitigating impacts on bird populations, habitats and ﬂight paths, – turbine design regarding life cycle analysis, – analyse social eﬀects like local employment, investment, taxes etc. 2. Wind turbine and component design issues (e.g. basic research in aerodynamics, structural dynamics, structural design and control): – new materials with higher strength like carbon ﬁbre for the blades, – feasibility studies of new wind turbines concepts and innovations, – integration of demand site requirements in the design of turbine, e.g. electrical control system interaction with grid requirement. 3. Testing, standardisation, and certiﬁcation (e.g. common accepted certiﬁcation pro- cedures for wind turbines and wind farms): – identiﬁcation of standards lacking, and initiation of appropriate actions for new standards, – identiﬁcation of standards lacking, and initiation of appropriate actions for new standards, – standards for service and maintenance concepts of oﬀshore wind converters. 4. Grid integration, energy systems and resource prediction (e.g. forecast of wind re- source): – development of scenarios for redesigning the grid system with high wind pen- etration, – increasing both: power quality and consistency, – energy management and storage systems for stand alone applications. 5. Operation and maintenance (e.g. advanced condition monitoring): – development of early failure detection and condition systems, – development in preventative maintenance, 14 , 00011 (2017) 1 EPJ Web of Conferences 48 LNES 2016 – development of central lubrication systems. From a technical perspective several conﬁgurations for wind technologies are under re- search and development in Germany (table IV). 7. – Outlook The wind energy market has grown because of the environmental advantages of har- nessing a clean and unexhaustable energy source and because of the economic incentives supplied by several governments. However, energy is required from other generation methods during the building phase of a new turbine, so in this period, Greenhouse gases and air pollution will be added to. If the life cycle of a wind turbine is looked at, more pollutants are saved on during operation, than are emitted during the building phase. A wind turbine is not a self-sustainable power station. This means that back-up power generation is needed at the times when the turbine is inoperative. This back-up is nowadays supplied by the established fossil fuel power stations. If the number of wind turbines increases, the need for extra investment in the back-up generation systems will rise, in order to maintain a stable electricity grid system. The challenges in this case are that existing power stations will deliver less electricity than before. Under this condition the electricity generation prices for new power stations are increasing due to smaller load duration time than before. However, wind energy is still one of the most important renewable energy resources for the future, because it can be harnessed in a clean and inexhaustible manner, through the application of technically advanced and eﬃcient machinery. These additional invest- ments will need in some cases subsidies. For more information especially in respects to technological and ecological aspects (life cycle aspects) see [4]. [1] Kleemann M. and Meliß M., Regenerative Energiequellen, 2.Auﬂage (Springer-Verlag, Heidelberg) 1993. , 00011 (2017) 1 EPJ Web of Conferences 48 LNES 2016 , 00011 (2017) 1 EPJ Web of Conferences 48 LNES 2016 DOI: 10.1051/ 71480 epjconf/201 0011 Table IV. – Technical developments in Wind Turbines [5]. Generators – Copper? Permanent Magnet? – Rare earths? – Superconductors? Rotor Blades – Cost reduction? – Utilization? – Legal situation? – Active control elements in rotor blades? Operations Monitoring – Drive train monitoring (CMS) for onshore plants? – Tower- and foundations vibrations – Number of Sensors, also Onshore? – Periodic Inspections- Scope, Number? Technical Uncertainties – Handling of new developments? Oﬀshore Foundations – Steel? Concrete? technologies – Gravity Foundations? – Environment, under water noise protection during the piling for the foundation? Table IV. – Technical developments in Wind Turbines [5]. – standardisation of components for easy replacement, – certiﬁcation of service and maintenance concepts, – cold and icing climates resource assessment. – standardisation of components for easy replacement, – standardisation of components for easy replacement, – certiﬁcation of service and maintenance concepts, – cold and icing climates resource assessment. certiﬁcation of service and maintenance concepts, – certiﬁcation of service and maintenance concepts, – certiﬁcation of service and maintenance concepts, – cold and icing climates resource assessment. – cold and icing climates resource assessment. – cold and icing climates resource assessment. 6. Oﬀshore wind technology (e.g. research into the control and eﬃciency of very large wind farms and more cost eﬀective foundations, transport and installation tech- niques): – monitoring of environmental impacts (eﬀects on birds, eﬀects of noise and vibration on marine life especially by installation of wind converter etc.), – development of deep water foundation structures (fundamental wind turbine design research), – oﬀshore meteorology, – special designs of systems and components for transportation, erection, access and maintenance of oﬀshore wind turbines, – investigate the use of energy storage, – improve corrosion protecting systems regarding the oﬀshore conditions. In addition to these aspects there have been done a lot of R&D needs by operating and market introduced wind turbines, e.g.: – better state of knowledge of the dynamic forces on the drive train, – improvement of the availability of gear boxes, 15 , 00011 (2017) 1 EPJ Web of Conferences 48 LNES 2016 DOI: 10.1051/ 71480 epjconf/201 0011 DOI: 10.1051/ 71480 epjconf/201 0011 – optimisation of control units and control systems, – optimisation of control units and control systems, – development of central lubrication systems. [5] Wagner Hermann-Josef, “Erneuerbare Energien Wind und Sonne - Wo geht es technisch hin?”, VGB PowerTech 1/2, 2015. [4] Wagner H.-J. and Mathur J., Introduction to Wind Energy Systems – Basics, Technology and Operation, second edition (Springer-Verlag, Berlin) 2013, ISBN 978-3-642-32975-3. [2] www.GWEC.de. [ ] [3] Ender C., “Wind Energy Use in Germany”, DEWI Magazin 02/2016. [3] Ender C., Wind Energy Use in Germany , DEWI Magazin 02/2016. [4] Wagner H.-J. and Mathur J., Introduction to Wind Energy Systems – Basics, Technology and Operation, second edition (Springer-Verlag, Berlin) 2013, ISBN 978-3-642-32975-3. [5] Wagner Hermann Josef “Erneuerbare Energien Wind und Sonne Wo geht es technisch GWEC.de. er C., “Wind Energy Use in Germany”, DEWI Magazin 02/2016. [2] www.GWEC.de. [3] Ender C “Wind Energy Use in Germany” DEWI Magazin 02/2016 REFERENCES 16
https://openalex.org/W2946982048	https://www.pure.ed.ac.uk/ws/files/87865612/s12958_019_0484_9.pdf	English	null	Analyses of bovine luteal fractions obtained by FACS reveals enrichment of miR-183-96-182 cluster miRNAs in endothelial cells	Reproductive biology and endocrinology	2,019	cc-by	4,277	General rights C i h f h General rights Copyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights. Digital Object Identifier (DOI): 10.1186/s12958-019-0484-9 Document Version: Publisher's PDF, also known as Version of record Published In: Reproductive Biology and Endocrinology Edinburgh Research Explorer Analyses of bovine luteal fractions obtained by FACS reveals enrichment of miR-183-96-182 cluster miRNAs in endothelial cells Citation for published version: Mohammed, B, Esteves, C & Donadeu, F 2019, 'Analyses of bovine luteal fractions obtained by FACS reveals enrichment of miR-183-96-182 cluster miRNAs in endothelial cells', Reproductive Biology and Endocrinology, vol. 17, 41. https://doi.org/10.1186/s12958-019-0484-9 Digital Object Identifier (DOI): 10.1186/s12958-019-0484-9 Link: Link to publication record in Edinburgh Research Explorer Document Version: Publisher's PDF, also known as Version of record Published In: Reproductive Biology and Endocrinology Abstract Our previous studies showed that the miRNA clusters, miR-183-96-182 and miR-212-132, may be critical in promoting luteal cell survival and progesterone production in both bovine and humans. To further understand their involvement in luteal development, this study aimed to establish the expression of these miRNAs in different bovine luteal cell types, namely, endothelial and steroidogenic, isolated using fluorescence-activated cell sorting (FACS). We isolated each of the two cell populations based on the presence of the endothelia surface marker, CD144, and uptake of the lipophilic dye, Nile Red, respectively. Using quantitative Polymerase Chain Reaction (qPCR) in the sorted cell fractions we confirmed that CD144 and the endothelia-specific miRNA, miR-126, were predominantly expressed in endothelial cells (CD144+), whereas HSD3B1 was expressed predominantly in steroidogenic cells (Nile RedHI). Finally, we found that whereas the miR-212-132 cluster was expressed at similar levels in luteal endothelial and steroidogenic cells, miR-183-96-182 was expressed at > 4-fold higher levels in endothelial than in steroidogenic cells (P < 0.05), suggesting that these two miRNA clusters, and particularly miR-183-96-182, may be important in functionally regulating not only steroidogenic cells but also endothelial cells in the corpus luteum (CL). Keywords: Bovine, Corpus luteum, Luteal cells, miRNAs, miR-183-96-182, miR-212-132, Nile red, Endothelial, Steroidogenic Keywords: Bovine, Corpus luteum, Luteal cells, miRNAs, miR-183-96-182, miR-212-132, Nile red, Endothelial, Steroidogenic cells, respectively) and non-steroidogenic cells (endo- thelial, pericytes, immune cells, fibroblasts); of these, endothelial cells make up about 85% of total luteal cells during the early luteal phase, a proportion that decreases to 50% as the corpus luteum matures [2–4]. Limited information is available on the molecular regulation of luteal development. miRNAs have been shown to be involved in follicular and luteal functions [5, 6]. Two miRNA clusters that are involved are miR-183-96-182 and miR-212-132 [7–9]. In a recent study, we showed that these two clusters are dramatic- ally upregulated during the follicle-luteal transition in cattle, and we further showed that one miRNA from each cluster, namely miR-96 and, to a lesser extent, miR-132, play a key role in regulating survival and ster- oid production of luteal cells of both cattle and humans [9]. Further, in situ hybridisation (ISH) analyses revealed that miR-132 is expressed in different cell compartments within the bovine CL. However, we did not succeed in Analyses of bovine luteal fractions obtained by FACS reveals enrichment of miR-183-96- 182 cluster miRNAs in endothelial cells Bushra T. Mohammed1,2, Cristina L. Esteves1 and F. Xavier Donadeu1* Bushra T. Mohammed1,2, Cristina L. Esteves1 and F. Xavier Donadeu1* © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Introduction cells, respectively) and non-steroidogenic cells (endo- thelial, pericytes, immune cells, fibroblasts); of these, endothelial cells make up about 85% of total luteal cells during the early luteal phase, a proportion that decreases to 50% as the corpus luteum matures [2–4]. The corpus luteum (CL) is a transitory structure that forms during each estrous/menstrual cycle and plays a critical role in the establishment and maintenance of pregnancy as well as in regulation of cyclic repro- ductive activity in the non-pregnant female. Develop- ment of the CL from follicular remnants after ovulation involves complex morphological and func- tional changes at the cell and tissue levels, including follicular cell differentiation, tissue remodelling and rapid development of vascular elements, with blood vessels making up to 30% of the total volume of the bovine CL [1]. The CL is a highly heterogeneous organ containing steroidogenic cells (large and small luteal, derived from follicular granulosa and theca * Correspondence: xavier.donadeu@roslin.ed.ac.uk 1The Roslin Institute and R(D)SVS, University of Edinburgh, Easter Bush, Midlothian, UK Full list of author information is available at the end of the article [9]. Further, in situ hybridisation (ISH) analyses revea that miR-132 is expressed in different cell compartme within the bovine CL. However, we did not succeed © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. * Correspondence: xavier.donadeu@roslin.ed.ac.uk 1The Roslin Institute and R(D)SVS, University of Edinburgh, Easter Bush, Midlothian, UK Full list of author information is available at the end of the article Take down policy Take down policy The University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorer content complies with UK legislation. If you believe that the public display of this file breaches copyright please contact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately and investigate your claim. Download date: 24. Oct. 2024 Mohammed et al. Reproductive Biology and Endocrinology (2019) 17:41 https://doi.org/10.1186/s12958-019-0484-9 Mohammed et al. Reproductive Biology and Endocrinology https://doi.org/10.1186/s12958-019-0484-9 Open Access Fluorescence-activated cell sorting (FACS) g ( ) Immediately after luteal digestion, 1x106cells were resus- pended in 500 μl FACS buffer (5% FBS in PBS). For CD144 staining, cells were washed with FACS buffer and centrifuged at 500 g for 5 min. After discarding the supernatants, cell pellets were blocked and incubated with CD144 antibody for 1 h at 4 °C, followed by incuba- tion with Alexa Flour 405-conjugated secondary anti- body in the dark (Table 1). Cells were also stained with Nile red (1 μg/ml) for 5 min at 4 °C before sorting. Cells were resuspended in FACS buffer and sorted in a BD FACS Aria II (BD, East Rutherford, NJ). DAPI (D1306, Thermo Fisher Scientific) was used to exclude dead cells. CD144 and DAPI were detected using Violet and Ultra- violet lasers, respectively, and the 450/50 filter, and Nile Red was resolved by using the Blue and Yellow-Green lasers and 582/15 and 586/15 filters, respectively. Sam- ples stained with secondary antibody alone were used as controls. Results were visualized using BD FACS Diva v8.0. Software. Previous studies documented the isolation of luteal endothelial cells using a variety of approaches such as density gradient centrifugation, fluorescence activated cell sorting (FACS), and lectins or antibodies ligated to magnetic beads, with variable success [10–13]. More- over, following pioneering studies by Hansel’s group [14], separation of luteal steroidogenic cell populations based on cell size/density has been extensively reported using methods including unit gravity sedimentation, elu- triation, flow cytometry or a combination of these [3, 15]. More recently, Quirk et al, (2013) used the lipo- philic dye Nile Red [16] to distinguish steroidogenic from non-steroidogenic luteal cells by flow cytometry [17]. Following from these findings, in this study we set out to optimise a procedure for simultaneous isolation of endothelial and steroidogenic cells from bovine CL using FACS. Using this method, we showed that the miR-183-96-182 cluster is enriched in endothelial rela- tive to steroidogenic cells of the bovine CL. Materials and methods Total RNA was isolated from luteal cells using TRIzol® Re- agent (Life Technologies, UK) following the manufac- turer’s instructions. RNA was quantified using Quant-iT™ RiboGreen® RNA kit (Invitrogen, UK) and reverse-transcribed using miScript II RT Kit. mRNA levels were then determined using bovine specific primers (Table 2) and the SensiFAST SYBR Lo-ROX Kit (Bioline, London, UK). miRNA levels were quantified on the same cDNA using pre-designed primers (Table 3) and miScript SYBR Green PCR Kit (Qiagen, UK) according to manufac- turer s instructions. qPCR analysis was carried out in du- plicate using Mx3005P real time PCR system (Stratagene, La Jolla, CA). Relative miRNA and mRNA abundance was Early corpora lutea (days 1 to 4 of the oestrous cycle [18]) collected from three heifers at an abattoir were processed as described before [9] to obtain cell digests. Mohammed et al. Reproductive Biology and Endocrinology (2019) 17:41 Page 2 of 6 Mohammed et al. Reproductive Biology and Endocrinology (2019) 17:41 based on preliminarily dose-response analyses to en- sure lack of cell toxicity (data not shown). The slides were visualized with a Leica DMLB fluorescence microscope and ImageJ, respectively. detecting miR-96 using this approach, possibly due to its relatively lower expression levels in the CL compared to miR-132. Establishing the relative expression of the miR-183-96-182 cluster in different luteal cell fractions, particularly steroidogenic and endothelial, would further our understanding of the role of these miRNAs in luteal development, warranting the use of approaches alterna- tive to ISH for this purpose. Fluorescence-activated cell sorting (FACS) Immunocytochemistry Luteal cells were grown on glass coverslips in DMEM/F-12 for approximately 24 h. The cells were rinsed with PBS and fixed in acetone:methanol (1:1) for 10 min at 4 °C. Following blocking, CD144 anti- body (Table 1) was diluted 1:500 in blocking buffer and incubated overnight at 4 °C. Afterwards, cells were washed and incubated with Alexa Fluor 568-conjugated secondary antibody (Table 1) and the slides were mounted with DAPI. Antibody isotype and secondary antibody-only were used as negative controls. Nile Red (N3013; Sigma-Aldrich, UK) was used to stain cells at a concentration of 1 μg/ml, Table 2 Primer pair sequences used for qPCR Genes Sequence (5′-3′) Amplicon length (bp) 18S FW GCTGGCACCAGACTTG 209 RV GGGGAATCAGGGTTCG Bovine- HSD3B1 FW GCGTTTCTCAGTGCTCAGATTT 195 RV TCAGCTTGATCTTGCTCTGGA Bovine- CD144 FW ACAGGGACACCTTCACCATC 85 RV ATGCGTTCATAGTCCAGGGG Table 1 Antibodies used in the study Antibody Catalogue no. Supplier CD144 AHP628Z AbD Serotec Donkey anti-rabbit IgG, Alexa Fluor 568 A10042 Invitrogen Goat anti-rabbit IgG, Alexa Flour 405 ab175654 Abcam Mohammed et al. Reproductive Biology and Endocrinology (2019) 17:41 Page 3 of 6 Page 3 of 6 Page 3 of 6 Table 3 Qiagen primer assays used for quantification of miRNAs miRNA Product code miRNA sequence (5′-3′) hsa-miR-132 -3p/ bta-miR-132 MS00003458 UAACAGUCUACAGCCAUGGUCG hsa-miR-212 -3p MS00003815 UAACAGUCUCCAGUCACGGCC hsa-miR-182-5p/ bta-miR-182 MS00008855 UUUGGCAAUGGUAGAACUCACACU hsa-miR-183-5p/ bta-miR-183 MS00031507 UAUGGCACUGGUAGAAUUCACUG hsa-miR-96-5p/ bta-miR-96 MS00003360 UUUGGCACUAGCACAUUUUUGCU RNU6–2 MS00033740 CGCTTCGGCAGCACATATACTA in CD144+ and Nile RedHi fractions, respectively, as ex- pected (Fig. 3a). Moreover, miR-126, a miRNA known to be highly expressed in endothelia [19], was expressed at much higher levels in CD144+ than in Nile RedHi cells, further indicating the efficacy of our sorting procedure. Of note, no attempt was made to determine the relative proportion of large and small luteal cells in the sorted Nile RedHi fraction; this was not possible due to the lim- ited number of cells obtained by FACS and available for downstream analyses. y Lastly, using this procedure we set out to determine the relative expression of the miR-212-132 and miR-183-96-182 clusters in the sorted steroidogenic and endothelial cell fractions. Our results showed that miR-132 and miR-212 were expressed at similar levels in CD144+ and Nile RedHi fractions (Fig. 3b). Con- sistent with this, our previous in situ hybridization analyses showed non-specific expression of miR-132 in the bovine corpus luteum [9]. Immunocytochemistry Moreover, the miR-212-132 cluster is known to be widely expressed across body tissues and cell types including ovarian steroidogenic cells [7, 9], and vascular cells [20]. Al- though, miR-212-132 may be expressed by both endo- thelial and steroidogenic cells, their relative expression in large and small luteal cells types and in different vascular cell components (e.g., endothelia and pericytes) was not determined in this study and should be investigated in the future. We also quanti- fied the expression of two of the miRNAs in the sec- ond cluster, miR-96 and miR-183, but not miR-182 as in our experience this miRNA is barely detectable in the bovine ovary [9]. We found that miR-96 and miR-183 were expressed in both cell fractions. Moreover, the two miRNAs were expressed at > 4-fold higher levels in CD144+ than in Nile RedHi cells (Fig. 3b). This result was unexpected in light of our previous evidence indicat- ing that miR-96 plays important roles in survival and pro- gesterone production by luteal steroidogenic cells in both cattle and humans [9]. Intriguingly, evidence of an in- volvement of the miR-183-96-182 cluster in regulation of endothelial cells is very scarce, however in light of our findings this should be investigated further, particularly in relation to the CL. Moreover, the observation that miR-96 was expressed at higher levels in endothelial than ste- roidogenic cells raises the interesting possibility that some of the reported effects on regulation of steroidogenic cells in the CL may actually be mediated by miR-96 produced by endothelial cells, a possibility that should be explored in the future. obtained using MX3005P software by extrapolating cycle threshold values from a standard curve prepared from a sample pool. Relative miRNA and mRNA levels were nor- malized using endogenous snoRNA, RNU6–2, and 18S, respectively. Gene expression data were analyzed by Stu- dent’s t tests. In all cases, statistical significance was con- sidered at P < 0.05. Results and discussion By using immunocytochemistry we confirmed that CD144 antibody and Nile Red selectively stained endothelial and steroidogenic cells, respectively, in lu- teal extracts (Fig. 1 a, b). The CD144 antibody used had been previously validated in bovine luteal endo- thelial cells by Pate’s group [13]. Accordingly, CD144 stained distinct capillary-like structures (Fig. 1a, left and middle panels), whereas Nile Red stained primar- ily large, round cells, indicative of steroidogenic cells, in the form of numerous bright dots indicating lipid globules (Fig. 1b left panel), in agreement with previ- ous results [17]. Cells stained with CD144 antibody and Nile Red were analyzed using flow cytometry and single events were selected to exclude debris and small cell clumps from the samples (Fig. 2A a,b). Live (DAPI-negative) CD144+ cells were then collected as endothelial frac- tion (Fig. 2A c1). Live CD144- cells (Fig. 2A c2) were further analyzed to select a cell fraction with high Nile Red fluorescent signal (Nile RedHi; Fig. 2A d). As expected, control cells stained with both primary and secondary antibodies showed positive staining for CD144 (Fig. 2B a) but no shift in the signal for Nile Red (Fig. 2B b). Conversely, in cells stained with Nile Red, a positive signal was observed for Nile Red (Fig. 2C b) but not for CD144 (Fig. 2C a). Authors’ contributions BTM and FXD conceived and designed the study, BTM and CLE acquired and interpreted the data, BTM and FXD drafted the manuscript and all authors revised and approved the manuscript. Funding Funding BTM was funded by College of Veterinary Medicine, University of Duhok, Kurdistan Region, Iraq. The Roslin Institutes receives funding from The Biotechnology and Biological Sciences Research Council through an Institute Strategic Programme Grant. Abbreviations CL: Corpus luteum; DAPI: 4′,6-diamidino-2-phenylindole; FACS: Fluorescence- activated cell sorting; FSA_A: Forward scatter area; ISH: in situ hybridisation; qPCR: Quantitative Polymerase Chain Reaction; SSC_A: Side scatter area; SSC- H: Side scatter height Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Conclusions DAPI was used for live/dead cell labelling Mohammed et al. Reproductive Biology and Endocrinology (2019) 17:41 Page 5 of 6 Mohammed et al. Reproductive Biology and Endocrinology (2019) 17:4 Mohammed et al. Reproductive Biology and Endocrinology Page 5 of 6 Fig. 3 Relative expression of a) known endothelial and steroidogenic cell markers and b) miRNAs under analyses in luteal cell fractions, CD144+ and Nile RedHi, obtained by FACS. Values are presented as mean + SEM and were analyzed by Student’s t test, with significant differences (P < 0.05) between cell fractions for each transcript shown by a star (), n = 3 animals Fig. 3 Relative expression of a) known endothelial and steroidogenic cell markers and b) miRNAs under analyses in luteal cell fractions, CD144+ and Nile RedHi, obtained by FACS. Values are presented as mean + SEM and were analyzed by Student’s t test, with significant differences (P < 0.05) between cell fractions for each transcript shown by a star (), n = 3 animals Fig. 3 Relative expression of a) known endothelial and steroidogenic cell markers and b) miRNAs under analyses in luteal cell fractions, CD144+ and Nile RedHi, obtained by FACS. Values are presented as mean + SEM and were analyzed by Student’s t test, with significant differences (P < 0.05) between cell fractions for each transcript shown by a star (*), n = 3 animals previous knowledge on the role of miRNAs in luteal de- velopment, specifically by providing evidence that miR-212-132 and, particularly, miR-183-96-182 may be important in functionally regulating not only steroido- genic cells but also endothelial cells in the corpus luteum (CL). Ethics approval and consent to participate Not applicable. We thanks Mrs. Shona Johnston for invaluable assistance during FACS analyses. Conclusions Expression analyses of the endothelial and steroido- genic markers, CD144 and HSD3B1, were performed using qPCR to confirm the identity of the sorted cell fractions. Each transcript was expressed predominantly In summary, we show that endothelial and steroidogenic cell fractions can be effectively isolated simultaneously from CL. In addition, our results further expand on Mohammed et al. Reproductive Biology and Endocrinology (2019) 17:41 Page 4 of 6 Fig. 1 Immunocytochemical detection of a) (from left to right) CD144 (red), CD144 DAPI and DAPI only (blue), and b) Nile Red + DAPI and DAPI only in bovine luteal cells Fig. 1 Immunocytochemical detection of a) (from left to right) CD144 (red), CD144 DAPI and DAPI only (blue), and b) Nile Red + DAPI and DAPI only in bovine luteal cells Fig. 1 Immunocytochemical detection of a) (from left to right) CD144 (red), CD144 DAPI and DAPI only (blue), and b) Nile Red + DAPI and DAPI only in bovine luteal cells Fig. 2 Representative dot-plots from Fluorescence-Activated Cell Sorting of endothelial and steroidogenic cell fractions from bovine CL. A Dot-plots showing selection of single events (individual cells) by SSC-A vs SSC-H (a), which were then visualized as FSC-A vs SSC-A (b), and sorted (c, d) by selection for CD144+ staining (c1). CD144- and DAPI- cells (c2) were further analyzed to select cells with high Nile Red (Nile RedHi) fluorescence signal (d). B, C Control cells stained with CD144 (primary and secondary) antibodies (B) and Nile Red (C) for detection of CD144 (Ba) and Nile Red (Cb) signals. “B” and “YG” in the X and Y axis stand for Blue and Yellow-Green laser, respectively. DAPI was used for live/dead cell labelling Fig. 2 Representative dot-plots from Fluorescence-Activated Cell Sorting of endothelial and steroidogenic cell fractions from bovine CL. A Dot-plots showing selection of single events (individual cells) by SSC-A vs SSC-H (a), which were then visualized as FSC-A vs SSC-A (b), and sorted (c, d) by selection for CD144+ staining (c1). CD144- and DAPI- cells (c2) were further analyzed to select cells with high Nile Red (Nile RedHi) fluorescence signal (d). B, C Control cells stained with CD144 (primary and secondary) antibodies (B) and Nile Red (C) for detection of CD144 (Ba) and Nile Red (Cb) signals. “B” and “YG” in the X and Y axis stand for Blue and Yellow-Green laser, respectively. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Author details 1 h l 1The Roslin Institute and R(D)SVS, University of Edinburgh, Easter Bush, Midlothian, UK. 2College of Veterinary Medicine, University of Duhok, Duhok, Kurdistan Region, Iraq. Received: 10 January 2019 Accepted: 15 April 2019 Received: 10 January 2019 Accepted: 15 April 2019 References 1. Stouffer RL, Hennebold JD. Chapter 23 - Structure, Function, and regulation of the Corpus luteum. Knobil and Neill's physiology of reproduction. 4th ed. San Diego: Academic Press; 2015. p. 1023–76. 1. Stouffer RL, Hennebold JD. Chapter 23 - Structure, Function, and regulation of the Corpus luteum. 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https://openalex.org/W3015965125	https://europepmc.org/articles/pmc7379447?pdf=render	English	null	Surgical considerations and audiological results of cochlear implantation in patients with otosclerosis	Turkish journal of medical sciences	2,020	cc-by	3,596	1. Introduction electrical stimulation of cochlear nerve via cochlear implant has been shown to provide adequate sound perception and communication skills [5]. Despite the fact that cochlear implantation (CI) technology improved over the years, it is still far away being “free of surgical risks” and plenty of complications have been reported in the literature [6]. Ossified cochlea in OS, is an important challenge for the surgeon, which brings along special problems that need to be addressed during or after the implantation procedure [7]. In this retrospective study, we aimed to emphasize the role of CI in the auditory rehabilitation of patients with OS and also shared our surgical experiences on this rare group of patients. Otosclerosis (OS) is a unique disease of human kind, which primarily results in progressive conductive hearing loss due to ankylosis of stapes footplate to the oval window [1]. Less often the disease may spread to the inner parts of the otic capsule and clinical picture manifest as mixed or sensorineural type profound hearing loss and/or dizziness [2]. Ultrastructural events are mainly centered around the osteoclasts of the otic capsule and their altered cytological activity. Increased bone resorption and formation in the otic capsule end up with mature calcified foci around the footplate, which results in ossicular fixation. Although hearing aids and medical therapy may offer a certain extent of solution in some patients, stapes-oriented surgical interventions are the main treatment options in the audiological rehabilitation of the patients [3,4]. In a group of patients with OS, pure tone air conduction thresholds may exceed 85 dB and these interventions are not able to satisfy patients. In approximately 20%–40% these patients, Surgical considerations and audiological results of cochlear implantation in patients with otosclerosis Tolgahan ÇATLI 1,, Taşkın TOKAT 2, Ergül Başaran BOZKURT 1, Zehra Hilal ADIBELLİ 3, Uğurtan ERGÜN 1, Enver ALTAŞ 1, Levent OLGUN 4 1Clinic of Otorhinolaryngology, University of Health Sciences Bozyaka Education and Research Hospital, İzmir, Turkey 2Clinic of Otorhinolaryngology, Sakarya University Training and Research Hospital, Sakarya, Turkey 3Clinic of Radiology, University of Health Sciences Bozyaka Education and Research Hospital, İzmir, Turkey 4Clinic of Otorhinolaryngology, Başkent University Hospital, İzmir, Turkey Tolgahan ÇATLI 1,, Taşkın TOKAT 2, Ergül Başaran BOZKURT 1, Zehra Hilal ADIBELLİ 3, Uğurtan ERGÜN 1, Enver ALTAŞ 1, Levent OLGUN 4 1Clinic of Otorhinolaryngology, University of Health Sciences Bozyaka Education and Research Hospital, İzmir, Turkey 2Clinic of Otorhinolaryngology, Sakarya University Training and Research Hospital, Sakarya, Turkey 3Clinic of Radiology, University of Health Sciences Bozyaka Education and Research Hospital, İzmir, Turkey 4Clinic of Otorhinolaryngology, Başkent University Hospital, İzmir, Turkey Received: 01.01.2020 Accepted/Published Online: 01.01.2020 Final Version: 23.06.2020 Background/aim: To emphasize the role of cochlear implantation (CI) in the auditory rehabilitation of patients with otosclerosis (OS) and share our surgical experiences on this rare group of patients. Background/aim: To emphasize the role of cochlear implantation (CI) in the auditory rehabilitation of patients with otosclerosis (OS) and share our surgical experiences on this rare group of patients. Materials and methods: Retrospective analysis of the patients who have a diagnosis of otosclerosis and implanted between January 1998–May 2019 was performed. Preoperative and postoperative clinical, radiological, audiological and surgical findings are presented. Results: Among 2195 patients who have been implanted in our institution, 12 (0.54%) met the diagnostic criteria of OS according to their preoperative (clinical, radiological, audiological) and peroperative (surgical) findings. Electrode insertion was performed via “round window membrane and cochleostomy” in 8 and 4 patients, respectively. No major complications occured. All patients showed satisfactory performances by means of audiometric scores postoperatively. Nonauditory stimulation (NAS) which manifested as “facial twitching” was a challenging problem in one patient during the surgery and subsided after the operation. Conclusion: Our experience on CI in patients with OS revealed that the implantation was a relatively safe procedure and had satisfactory impact on audiological performances. Keywords: Otosclerosis, cochlear implantation, cochlear ossification, facial twitching * Correspondence: tcatli80@hotmail.com http://journals.tubitak.gov.tr/medical/ Turkish Journal of Medical Sciences Research Article Turk J Med Sci (2020) 50: 855-859 © TÜBİTAK doi:10.3906/sag-1912-238 http://journals.tubitak.gov.tr/medical/ Turkish Journal of Medical Sciences http://journals.tubitak.gov.tr/medical/ Turkish Journal of Medical Sciences 2.1. Patient selection Between 01 January 1998 and 01 May 2019, 2195 patients received CI. Among these 2195 patients, 12 (4 females and 8 males) had an etiology of OS, representing 0.54% of all patients implanted during this time. The age at implantation ranged between 44 and 76 years (median, 62 years). The median follow-up time after implantation was 56 months (range, 12–120 months). Patient characteristics (diagnostic criteria such as family history and CT findings) are summarized in Table 2. All patients have used conventional hearing aid (HA) prior to CI surgery (via medical records and telephone questioning). Unfortunately none had a satisfactory results during years especially at the advance stages of their diseases. Concerning about the stapes oriented surgery history of the patients, 5 (S1,S3,S4,S7,S12) had a stapedotomy/ stapedectomy procedure before the implantation. However none had a detailed operation note in their medical records but their self reported information suggest that they had some degree of functional benefit from the stapes surgery which is deteriorated during the years mainly attributed to the retrofenestral progression of the disease. Among others with no history of stapes surgery, 4 patients (S2, S5, S9, S10) mentioned that they had informed about the possible stapes surgery candidacy but none had accepted the surgery mainly due to the possibility of loosing their residual hearings after the stapes oriented procedure. In the remaining 3 patients stapes surgery history could not be detailed due to lack of their medical records and/or unsatisfactory patient orientation to their medical past. Patients who had a history of clinical OS which was supported with audiological and radiological findings and received cochlear implant were included in the study. In our institution decision on CI surgery is made by a committee that composed of at least 3 otolaryngology specialists, 1 audiologist and 1 consultant radiologist when needed. In case of an inadequate amplification via conventional hearing aid (HA), CI was recommended in OS patients. Both thin-slice computed tomography (CT) and Gadolinium-enhanced magnetic resonance imaging (MRI) of the temporal bone were obtained in order to examine the middle ear cleft, cochlear bony structure, cochlear canal patency, inner ear fluids, internal auditory canal contents, cerebellopontine angle and other temporal bone anatomical subsites. CT findings were graded according to the imaging based grading system by Rotteveel et al., where CT records were available (Table 1) [8]. 2. Material and methodsh The study was carried-out in a tertiary referral center, which had CI experience in 2195 patients including children and adults, over 20 years. Retrospective analysis of the patients who have been diagnosed with OS and 855 This work is licensed under a Creative Commons Attribution 4.0 International License. ÇATLI et al. / Turk J Med Sci Wilcoxon t-test. A “P” value <0.05 was considered statistically significant. Wilcoxon t-test. A “P” value <0.05 was considered statistically significant. implanted between 01 January 1998 and 01 May 2019 was performed. Ethical commitee of the institution approved the study protocol (Protocol number: 03/18). 2.1. Patient selection All patients were evaluated by the same test batary, which included “pure tone audiometry (PTA) and tympanometry (TM)” both before and after the procedure. 3.1. HRCT findings Radiological examinations of temporal bones were performed by a 64-section CT scanner (Aquilion, Canon Medical Systems Corporation, Tochigi, Japan) with 0.6 mm axial section thickness and coronal and sagital reformations at 1 mm. All studies were performed without 2.2. Surgical procedure All procedures were performed under general anesthesia, by the senior authors of the study at the same institution. Retroauricular approach, simple mastoidectomy and posterior tympanotomy were the basic steps of the surgery. Subperiosteal pocket technique was used to maintain internal receiver stabilization. Electrode insertion routes were scala tympani via round window membrane or cochleostomy. Intraoperative and postoperative complications were also noted. 2.3. Statistical analysis Statistical analysis of the data was conducted with SPSS 21.0.0 (IBM Corp., Armonk, NY, USA). Pre- and postoperative PTA scores were compared using the Table 1. Rotteveel and colleagues’ imaging-based grading systems for otosclerosis. Type Otosclerotic lesions of the otic capsule Type 1 Solely fenestral involvement (thickened footplate and/or narrowed or enlarged windows) Type 2 Retro-fenestral with or without fenestral involvement Type 2a Double ring effect Type 2b Narrowed basal turn Type 2c Double ring and narrowed basal turn Type 3 Severe retro-fenestral (unrecognizable otic capsule), with or without fenestral involvement According to Rotteveel et al [8]. Table 1. Rotteveel and colleagues’ imaging-based grading systems for otosclerosis. Table 1. Rotteveel and colleagues’ imaging-based grading systems for otosclerosis. 856 ÇATLI et al. / Turk J Med Sci Figure 1. CT scan (axial plane) of patient (S5) shows left sided fenestral otosclerosis (yellow arrow: left sided fenestral involvement). contrast, and imaging included the entire petrous bone. In 4 of the 12 patients, radiological images were not available to examine and grading through digital screen. However in these 4 patients (S2, S3, S4, S7) radiologist had pointed out some degree of otic capsule density alterations and footplate thickenings in his written reports. According to Rotteveel grading system (16 temporal bones in total), 4 and 2 patients had bilateral “grade 1”(50%) and “grade 2c” (25%) OS, respectively (Figures 1 and 2). The remaining 2 patients had mixed type of OS grades in their ears as summarized in Table 2. In patients with bilateral grade 1 OS (S5 ,S6, S10, S12), the main radiological finding were “footplate thickening (S5, S10) and narrowed (S6 )/ enlarged (S12) windows”. In patients with bilateral grade 2c (S9, S11), the main radiological finding was “double ring appearance with basal turn narrowing” in both sides. In patients with mixed type of grades, while S1 had footplate thickening in the implanted ear and double ring appearance in the nonimplanted ear, S8 had double ring appearance in the implanted ear and basal turn narrowing in the nonimplanted ear. Figure 1. CT scan (axial plane) of patient (S5) shows left sided fenestral otosclerosis (yellow arrow: left sided fenestral involvement). Figure 2. CT scan (axial plane) of patient (S8) shows bilateral retrofenestral otosclerosis (red arrows: bilateral “double ring” ; black arrow: left sided “narrowed cochlear lumen”). 3.2. Surgical findings Electrode insertion was performed via “round window (RW) membrane and cochleostomy” in 8 and 4 (S5, S9, S10, and S12) patients respectively. Number of active electrodes for each patient and device characteristics are summarized in Table 3. In 8 of the 12 patients, electrode insertion was performed through the RW. Remaining 4 patients had varying degrees of sclerosis throughout the medial wall of the middle ear cleft (around the RW niche and promontorium) and needed to be drilled to achieve electrode insertion via cochleostomy. In patient S12, anteriorly positioned facial nerve was observed and this was noted as another factor, which has hidden the RW. No major complications occured both intra- and postoperatively. Nonauditory stimulation (NAS), which manifested as “facial twitching” during the surgery, was a challenging problem in one patient (S12) and subsided in the postoperative fitting period. None of the other patients had experienced postoperative facial twitching or other types of NAS. Figure 2. CT scan (axial plane) of patient (S8) shows bilateral retrofenestral otosclerosis (red arrows: bilateral “double ring” ; black arrow: left sided “narrowed cochlear lumen”). 4. Discussion S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 Implant model M M AB M M AB M O AB C C M Number of active electrode 5/12 11/12 12/16 12//12 7/12 15/16 9/12 20/20 16/16 22/22 22/22 11/12 Insertion routes RW RW RW RW Ch RW RW RW Ch Ch RW Ch M: Medel (Med-El, Innsbruck, Austria); AB: Advanced Bionics (Advanced Bionics, CA, U.S.A.); O: Oticon; C: Cochlear Corporation (Australia); RW: Round window; Ch: Cochleostomy. Table 3. Device characteristics, electrode status and insertion routes. M: Medel (Med-El, Innsbruck, Austria); AB: Advanced Bionics (Advanced Bionics, CA, U.S.A.); O: Oticon; C: Cochlear Corporation (Australia); RW: Round window; Ch: Cochleostomy. round window membrane and fenestral exposure was satisfactory. We suggest this could be related to “false positive” radiological evaluation or very early stage of the disease process in these 4 patients. However, we cannot make a certain conclusion due to the unavailability of the images. This might be considered as another limitation of our study. Concerning about the “false positive” results in the radiological evaluation of OS, we might consider 3 of our patients (S1, S6, and S11) had false positive results in CT imaging. Although radiologist reported “fenestral OS” (S1 and S6: Rotteveel grade 1; S11: Rotteveel grade 2c) in these 3 patients, round window exposure through the posterior tympanotomy was achieved and electrode insertion was performed via RW membrane to the inner ear. recipients both before and after the procedure. However, results of these tests are not sufficient to draw a precise statistical conclusion and this might be considered as a limitation of our study. f While unaffected neural element is the key factor for adequate electrical stimulation of the auditory pathways, patients with cochlear OS seem to have relatively favorable cochlear status compared to patients who have deseased cochleas. Therefore, excellent auditory perception skills seem to be related to the unique pathology of OS which typically spares medial aspect of the cochlea while damaging its lateral wall [9,10]. Since the diagnostic sensitivity of CT scanning of the temporal bone in OS is not so high it is not possible to exclude OS when demineralization is not present in CT [8,11]. In our study group, 8 patients showed varying degrees of otic capsule demineralization and other features of retrofenestral involvement. 4. Discussion A comparison of the PTA scores (dB) before and after the implantation had revealed that the scores were significantly lower after the surgery. While the pure tone thresholds were between 30 dB and 50 dB, discrimination scores were between 70%–90% in the postoperative period [median PTA scores were 100 dB (range, 90–110) and 43 dB (range, 30–50) before and after the implantation, respectively, P = 0.002; median discrimination scores were 16% (range, 12%–20%) and 82% (range, 70%–94%) before and after the implantation, respectively, P = 0.002]. Our experience on CI in patients with cochlear OS revealed that the procedure is relatively safe and effective by means of auditory rehabilitation. Similar to our findings, Ruckenstein et al. showed that these group of patients can express excellent audiological outcomes after the procedure. In their study (n = 8) all patients had expressed significantly higher scores on Central Institute for the Deaf (CID) sentence test after CI [9]. In our study group, we had also applied speech tests (bi-syllable open set test; language specific sentence test) to some of our 857 ÇATLI et al. / Turk J Med Sci Table 2. Diagnostic features. Table 3. Device characteristics, electrode status and insertion routes. S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 Implant model M M AB M M AB M O AB C C M Number of active electrode 5/12 11/12 12/16 12//12 7/12 15/16 9/12 20/20 16/16 22/22 22/22 11/12 Insertion routes RW RW RW RW Ch RW RW RW Ch Ch RW Ch M: Medel (Med-El, Innsbruck, Austria); AB: Advanced Bionics (Advanced Bionics, CA, U.S.A.); O: Oticon; C: Cochlear Corporation (Australia); RW: Round window; Ch: Cochleostomy. Table 2. Diagnostic features. S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 Family history + + + – + – – – + – – + CT grade (Type) (Implanted side/Other side) 1/2a – – – 1/1 1/1 – 2a/2c 2c/2c 1/1 2c/2c 1/1 According to Rotteveel et al [8]. S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 Family history + + + – + – – – + – – + CT grade (Type) (Implanted side/Other side) 1/2a – – – 1/1 1/1 – 2a/2c 2c/2c 1/1 2c/2c 1/1 According to Rotteveel et al [8]. Table 3. Device characteristics, electrode status and insertion routes. 4. Discussion Among these 8 patients, 6 (75%) and 2 patients (25%) had symmetrical (same grade) and asymmetrical involvement respectively. In their study group Rotteveel et al. reported 20% symmetrical involvement [8]. In another study, Ruckenstein et al. reported 50% ottic capsule involvement as “rarefaction of otic capsule bone, osteoneogenesis within the cochlear ducts” in their patient group. However the bilaterality and/ or symmetricity of the involvement were not clarified in the article [9]. In our remaining 4 patients, CT images were not available but radiological reports have pointed out varying degrees of demineralization and hypodensity of the otic capsule and also fenestral involvement. In these 4 patients, electrode insertion was achieved via In 4 patients (S5, S9, S10, S12), RW niche could not be identified due to the sclerotic lesions located around the niche. Therefore, electrode insertion was performed via cochleostomy. Previously, Ruckenstein et al. and Fayad et al. reported in their studies that they needed some degree of cochlear basal turn drillings in order to eradicate sclerotic lesions and achieve patent cochlear lumen [9,12]. However, we did not need drill-out procedure even in patients with narrowed cochlear lumen (S9,S11). The classical appearance of retrofenestral OS on CT is a pericochlear hypodensity named “double-ring” (aka 4th ring of Valvassori) or “halo sign”, which is highly characteristic for cochlear OS [13]. In our study group, halo sign was present bilaterally in 3 patients (S8, S9, S11) and unilaterally in 1 patient (unimplanted ear). Two (S9, S11) out of these 3 patients had required cochleostomy. 858 ÇATLI et al. / Turk J Med Sci patterns (TPP)”, “deactivation of the offending electrodes” or “prolonging the phase duration while reducing the amplitude to keep the total charge constant but limiting the current spread” [18]. In our one patient experience, we used TPP as a relatively novel option and obtained a satisfactory result. According to our experience, it is possible to conclude that if a patient has halo sign in CT imaging, a cochleostomy is more likely to be needed. This might be considered as another clinical significance of halo sign in the CI of patients with OS. There is no doubt that larger studies are necessary to make stronger and statistically significant conclusions regarding this issue.t In conclusion, our experience on CI in patients with OS revealed that the implantation is a relatively safe procedure and had a satisfactory impact on audiological performances. Acknowledgment No funding has been received for this study. Conflict of interest The authors declare no conflict of interest. Conflict of interest The authors declare no conflict of interest. 4. Discussion Diversity of the cochlear anatomy due to ossification process should be kept in mind and surgeons need to be prepared for an alternative insertion scenario during the procedure. g g Facial nerve stimulation (FNS) after CI is a rare but potentially devastating problem [14]. The reported incidence of this phenomenon in the literature varies between 0.9% to 14.6% [15,16]. FNS is more frequent when the recipient has cochlear OS. This is mainly due to the altered otic capsule bony architect after a process of demineralization and sclerosis. Thus, electric current becomes more dispersible as a result of decreased electrical impedance of the bone and the reduced distance between the electrode and the facial nerve due to bone loss and cavity formation [17]. There are some options to alleviate postoperative FNS such as “using triphasic pulse References 7. Sainz M, Garcia-Valdecasas J, Ballesteros JM. Complications and pitfalls of cochlear implantation in otosclerosis: a 6-year follow-up cohort study. Otology and Neurotology 2009; 30: 1044-1048. 1. Faranesh N, Magamseh E, Zaaroura S, Zeidan R, Shupak A. Hearing and otoacoustic emission outcome of stapedotomy: does the prosthesis diameter matter? Journal of International Advanced Otology 2017; 13: 162-170. 2. Yetiser S. Bilateral vestibulopathy due to severe cochlear otosclerosis: a well-known condition without any favorable solution. Turkish Archives of Otorhinolaryngology 2018; 56 (3): 174-176. 8. Rotteveel LJ, Snik AF, Cooper H, Mawman DJ, Van Olphen AF et al. Speech perception after cochlear implantation in 53 patients with otosclerosis: multicentre results. Audiology and Neurootology 2010; 15: 128-136. 3. West N, Brand M, Foghsgaard S, Cayé-Thomasen P. Surgical results and complications of cochlear implantation in far- advanced otosclerosis. Journal of International Advanced Otology 2017; 13: 304-307. 9. Ruckenstein MJ, Rafter KO, Montes M, Bigelow DC. Management of far advanced otosclerosis in the era of cochlear implantation. Otology and Neurotology 2001; 22: 471-474. 10. Kwok OT, Nadol JBJ. Correlation of otosclerotic foci and degenerative changes in the organ of Corti and spiral ganglion. American Journal of Otolaryngology 1989; 10: 1-12. 4. Laske RD, Röösli C, Chatzimichalis MV, Sim JH, Huber AM. The influence of prosthesis diameter in stapes surgery: a meta- analysis and systematic review of the literature. Otology and Neurotology 2011; 32: 520-528. 11. Swartz JD, Mandell DW, Wolfson RJ, Marlowe FI, Popky GL et al. Fenestral and cochlear otosclerosis: computed tomographic evaluation. American Journal of Otology 1985; 6: 476-481. 5. Semaan MT, Gehani NC, Tummala N, Coughlan C, Fares SA et al. Cochlear implantation outcomes in patients with far advanced otosclerosis. American Journal of Otolaryngology 2012; 33: 608-614. 12. Fayad J, Moloy P, Linthicum FH Jr. Cochlear otosclerosis: does bone formation affect cochlear implant surgery? American Journal of Otology 1990; 11: 196-200. 6. Bayrak F, Çatlı T, Atsal G, Tokat T, Olgun L. Waardenburg syndrome: an unusual indication of cochlear implantation experienced in 11 Patients. Journal of International Advanced Otology 2017; 13: 230-232. 13. Purohit B, Hermans R, Op de Beeck K. Imaging in otosclerosis: a pictorial review. Insights Imaging 2014; 5 (2): 245-252. 14. Shea JJ III, Domico EH. Facial nerve stimulation after successful multichannel cochlear implantation. American Journal of Otology 1994; 15: 752-756. 859
https://openalex.org/W4317107621	https://bmchealthservres.biomedcentral.com/counter/pdf/10.1186/s12913-022-08977-1	English	null	The intervention strategies and service model for pharmacist-led diabetes management: a scoping review	BMC health services research	2,023	cc-by	11,512	© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract Background There is increasing intervention activities provided during pharmacist-led diabetes management. Nevertheless, there is an unclear definition of the activities involved during the intervention. Thus, this study aimed to describe the type of intervention strategies and service model provided during pharmacist-led type 2 diabetes management and service outcomes. Methods This study utilized the scoping review methodology of the Joanna Briggs Institute Reviewers’ Manual 2015. Articles on pharmacist-led diabetes management focusing on the service content, delivery methods, settings, fre- quency of appointments, collaborative work with other healthcare providers, and reported outcomes were searched and identified from four electronic databases: Ovid Medline, PubMed, Scopus, and Web of Science from 1990 to October 2020. Relevant medical subject headings and keywords, such as “diabetes,” “medication adherence,” “blood glucose,” “HbA1c,” and “pharmacist,” were used to identify published articles. Results The systematic search retrieved 4,370 articles, of which 61 articles met the inclusion criteria. The types of intervention strategies and delivery methods were identified from the studies based on the description of activities reported in the articles and were tabulated in a summary table. Conclusion There were variations in the descriptions of intervention strategies, which could be classified into diabe- tes education, medication review, drug consultation/counseling, clinical intervention, lifestyle adjustment, self-care, peer support, and behavioral intervention. In addition, most studies used a combination of two or more interven- tion strategy categories when providing services, with no specific pattern between the service model and patient outcomes. Keywords Diabetes, Pharmacist, Non-pharmacological interventions, Patient management Hassan et al. BMC Health Services Research (2023) 23:46 https://doi.org/10.1186/s12913-022-08977-1 Hassan et al. BMC Health Services Research (2023) 23:46 https://doi.org/10.1186/s12913-022-08977-1 BMC Health Services Research Open Access Backgroundh The prevalence of diabetes is increasing worldwide [1]. The prevalence of diabetes in the developed and develop- ing countries is expected to increase by 42% and 170%, respectively, by 2030 [2]. This is particularly a concern in developing countries, as they are commonly affected by rapid population growth, an aging community, unhealthy local diet, urbanization, obesity, unhealthy lifestyle, and poor access to quality health care [3]. As these prob- lems arise, the cost of managing the illness increases. *Correspondence: Ernieda Hatah ernieda@ukm.edu.my 1 Faculty of Pharmacy, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Malaysia 2 Pharmacy Services Program, Ministry of Health Malaysia, Lot 36 Jalan Universiti, 46350 Petaling Jaya, Selangor, Malaysia © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Page 2 of 18 Hassan et al. BMC Health Services Research (2023) 23:46 The economic burden of diabetes has necessitated the development of effective interventions that simplify early diagnosis, promote effective care, and enhance primary prevention [4].h years to investigate the impact of pharmacist-led diabetic care [12–14]. Nonetheless, the studies’ primary focus was on the treatments’ impacts and results, with just a brief mention of the particular tactics and service mod- els delivered. Exploring the specifics of the intervention’s activities and strategies may provide insight into similari- ties and differences that may or may not have an impact on patient outcomes. The management of diabetes includes maintaining a healthy lifestyle, such as meal planning, physical activity, and medication adherence. Since healthcare providers are not always present, developing self-management skills is critical for diabetes management. Patient education programs have been implemented to educate patients on their active roles in disease management. These pro- grams were reported to result in a better understanding of patients’ perspectives and attitudes toward health, as well as their compliance with drug decisions, risk fac- tors, and overall quality of life [5]. Although pharma- cotherapy is an effective treatment modality to achieve optimal glycemic control and prevent the development of diabetes complications, its efficacy is often limited by poor medication adherence among patients with diabe- tes. Approximately 43.4% of diabetic patients in low- and middle-income countries do not adhere to their pharma- cotherapy treatments [6]. p A systematic review by Presley et al. (2018) on interventions to improve medication adherence among adults demonstrated the role of pharmacists in improving diabetes control [8]. In their study, the intervention by pharmacists enhanced diabetes out- comes (standardized mean difference, -0.68; 95% confidence interval, -0.79, -0.58; p < 0.001) with sub- group analysis by intervention strategy, and the type of intervention and outcome measures produced similar results. In their study, nevertheless, the intervention strategies were classified as educational, behavioral, or a combination of both, with an unclear definition of the activities involved. Since many different activities were reported to be provided during pharmacist-led diabetes management services, it is worth exploring and classifying intervention strategies based on their specific activities [8]. In addition, the effectiveness of these two broad intervention strategies was inconsist- ent between studies in which the best interventions to improve nonadherence could not be determined [8]. Thus, this study aimed to provide a review of the type of individualized intervention strategies and service model provided during pharmacist-led type 2 diabe- tes management, which includes reviewing specific service content, such as information on the activi- ties involved, delivery methods, settings, frequencies of appointments, and collaborative work with other healthcare providers and their outcomes. An increasing number and types of intervention strate- gies are being developed to complement pharmacother- apy in diabetes management. Intervention strategies that aim to promote better disease control include patient- mediated strategies through interactions with patients or via the information provided by or to patients [7]. The types of intervention strategies reported in previ- ous studies include counseling, psychological and social interventions, patient empowerment, patient-centered training, explanation of possible adverse events, nutri- tional therapy, physical activity, and health coaching [8]. Intervention strategies are introduced based on the capacity and needs at the local level and are provided in combination or as single strategies. In addition, patient- centered services facilitated by multiple healthcare pro- fessionals, including pharmacists, have shown to enhance outcomes [9–11]. Methods As the study aimed at providing an overview of the inter- vention strategies and service model provided during pharmacist-led type 2 diabetes management, scoping review methodology was deemed the most appropri- ate method to be applied. The current study utilized the scoping review methodology of the Joanna Briggs Insti- tute Reviewers’ Manual 2015 [15]. The manual was one of the latest published on scoping reviews methodol- ogy. The step-by-step approach in the manual was well described and provided clear guidance for conducting a scoping review. Articles on pharmacist-led diabetes man- agement focusing on service content, delivery methods, settings, frequencies of appointments, collaborative work with other healthcare providers, and reported outcomes were searched and identified. Pharmacists who are knowledgeable in pharmaco- therapy are well-trained in identifying patients’ pharma- ceutical care issues, such as adverse drug reactions and non-adherence. In addition, pharmacists who are work- ing in outpatient and ambulatory care who are more accessible to the community, are well-positioned to edu- cate, monitor, and support medication adherence and self-care of diabetic patients, which may contribute to the achievement of therapeutic success in diabetes man- agement. An example of a pharmacist-led diabetes man- agement service includes a review of medicines that aim to improve patients’ understanding of the disease and increase their adherence to treatment. Several system- atic evaluations have been undertaken throughout the Hassan et al. BMC Health Services Research (2023) 23:46 Hassan et al. BMC Health Services Research (2023) 23:46 Page 3 of 18 Data analysis l Data analysis was conducted through a narrative syn- thesis of the articles by evaluating and comparing the pharmacist-led interventions reported in the articles. The results were summarized according to the type of service content, delivery methods and settings, fre- quency of appointments, collaborative work with other healthcare providers, and outcomes of glycemic control and medication adherence. The types of service content and delivery methods with their definitions were identi- fied from the studies based on the description of activi- ties reported in the articles and are tabulated in the summary table. The code was initially categorized by F.H. and refined by E.H. The final coding was assessed by all team members, F.H., E.H., A.M.A., and C.W.W., and disagreement was resolved through consensus. The current study reported that the glucose control out- come as significant if at least one of the result of the glucose readings measured by the studies (random blood glucose, fasting blood glucose, HbA1c or post- prandial blood glucose) was reported significant. Study selection y All retrieved articles were imported to Mendeley, a ref- erence management system software, and duplicates were removed. Articles were included if they had been conducted as a randomized controlled trial; interven- tion conducted by pharmacists aimed at improving medication adherence and/or glycemic control, includ- ing diabetic patients aged 18 and above; and conducted in outpatient or ambulatory settings and reported gly- cemic control or medication adherence level as the out- comes. Multicomponent team-based care and programs were included if the study defined the program as a pharmacist-led program and the involvement of other healthcare providers are only to address specific issues such as prescribing and meal planning. Only studies with randomized controlled trials (RCTs) study design were included as they usually represent optimal study design and regarded as highest quality evidence. RCTs usually minimize bias in their study procedure and combining RCTs with other study designs may increase study’s het- erogeneity, hence, making comparison and conclusion difficult to be made. In addition, randomized controlled trials report usually includes a more detailed information on their intervention programs making it possible for rel- evant and accurate details to be extracted. Articles were excluded if they had been conducted in a well-controlled environment, such as in a university or academic institu- tion that did not reflect a real practice setting; full arti- cles could not be retrieved; and were not published in the English language. Data extraction F.H. performed data extraction for all articles, which was checked by E.H. Extracted data included title, year of publication, authors and location of the study, con- tents of the intervention, delivery approaches, health- care workers involved, frequency of appointments, follow-up period, and reported outcomes. C.W.W. and A.M.A. reviewed the extracted data in a table form. Discrepancies were discussed and resolved by consensus. Search criteria E.H., A.M.A., and C.W.W. to ensure the validity of the process. Any disagreements regarding whether a study should be included or excluded were resolved through consensus when the majority indicated their agreement over the matter. E.H., A.M.A., and C.W.W. to ensure the validity of the process. Any disagreements regarding whether a study should be included or excluded were resolved through consensus when the majority indicated their agreement over the matter. Articles were searched from four electronic databases: Ovid Medline, PubMed, Scopus, and Web of Science from 1990 to October 2020. Relevant Medical Sub- ject Headings (MeSH) and keywords such as “diabetes,” “medication adherence,” “blood glucose,” “HbA1c,” and “pharmacist” were used to identify published articles. The specific search strings used for the search can be found in the supplementary material. To increase the specificity and sensitivity of the identified articles, MeSH terms and keywords were combined using the Boolean operator, AND or OR, where appropriate. The reference lists of the retrieved papers were screened for potentially relevant papers that were missing during the electronic search. Resultsh The systematic search retrieved 4,370 articles located through the electronic database search. After remov- ing duplicates and titles/abstracts that were unrelated to pharmacist-led diabetes management, 140 articles were included in the full-text review. After applying the inclusion and exclusion criteria, 61 articles were included in the analysis. Figure 1 summarizes the PRISMA flow process for the identification, screening, and inclusion of the identified articles. Articles were initially screened by F.H. based on their title and abstract. The exclusion process using titles or abstracts by F.H. occurred only if the reason for exclusion was clear. If there was uncertainty, the article was not excluded, and each member of the research team (F.H., E.H., A.M.A., C.W.W.) reviewed the article. All excluded “full text” articles were independently reviewed by F.H., i All included studies were randomized controlled trials. Studies included were from Australia (n = 2), South America (n = 4), North America (n = 16), Europe Page 4 of 18 Hassan et al. BMC Health Services Research (2023) 23:46 Fig. 1 Flow chart of the search result Fig. 1 Flow chart of the search result The method of service delivery included face-to- face sessions with individual patients, which was the most common method used (n = 51), scheduled tele- phone calls (n = 27), group sessions (n = 8), and visits to patients’ homes (n = 2). Fifteen studies used face-to-face meetings as the single method to deliver interventions [16–30]. Another four studies utilized single delivery group sessions [31–33] and phone calls [34] to deliver interventions. Three studies used a combination of three intervention delivery methods [35–37]. The most com- mon combination of delivery methods was face to face meeting and phone calls (n = 22) [35, 37–56]. During the interventions, several delivery aids were used, such as videos (n = 2), printed materials consisting of a summary of important information for patients (n = 20), email reminders (n = 1), patient diaries (n = 11), and pillboxes (n = 6). (n = 7), Asia (n = 27), and Africa (n = 2). The earliest study was published in 1996, and most studies (n = 48) were published after 2010. Pharmacist interventions on diabetes management were provided in settings, such as community health centers (n = 4), community pharmacies (n = 11), outpatient clinics (n = 44), and outpatient pharmacies (n = 2). Table 1 summarizes the characteristics of the included studies. Resultsh (n = 7), Asia (n = 27), and Africa (n = 2). The earliest study was published in 1996, and most studies (n = 48) were published after 2010. Pharmacist interventions on diabetes management were provided in settings, such as community health centers (n = 4), community pharmacies (n = 11), outpatient clinics (n = 44), and outpatient pharmacies (n = 2). Table 1 summarizes the characteristics of the included studies. Most interventions were conducted by multidiscipli- nary teams of healthcare providers, including pharma- cists, doctors, nurses, dietitians, and diabetes educators (n = 37). Only 24 of the included studies contained inter- ventions performed solely by pharmacists. In such set- tings, the interventions conducted by pharmacists, which are rarely clinical interventions, include diabetes educa- tion, medication review, drug counselling, self-care rec- ommendations, and lifestyle adjustments. Page 5 of 18 Hassan et al. BMC Health Services Research (2023) 23:46 Hassan et al. BMC Health Services Research Table 1 Summary of studies and description of service model and intervention provided No. Author Country Year Location HCP involvement Method of delivery Tools/ Aids Pharmacist Multidisciplinary Tteam Face to face Phone calls Group sessions Home visits Diaries Printed materials Video Email Pill box Games 1 Castejon et al. USA 2013 Outpatient Pharmacy x x x x 2 Lim et al. Malaysia 2016 Outpatient Clinic x x x 3 Ali et al. UK 2012 Community Pharmacy x x x 4 Venkatesan et al. India 2012 Community Pharmacy x x x 5 Grant et al. USA 2003 Community Health Center x x x 6 Ramanath et al. India 2012 Outpatient Clinic x x x 7 Mahwi et al. Iraq 2013 Outpatient Clinic x x 8 Clifford et al. Australia 2002 Outpatient Clinic x x 9 Phumipa- morn et al. Thailand 2008 Outpatient Clinic x x 10 Al Mazroui et al. UAE 2009 Outpatient Clinic x x x x 11 Farsaei et al. Iran 2011 Outpatient Clinic x x x x x 12 Mehuys et al. Belgium 2011 Community Pharmacy x x 13 Jacobs et al. USA 2012 Outpatient Clinic x x 14 Jarab et al. Jordan 2012 Outpatient Clinic x x x x 15 Odegard et al. USA 2012 Community Pharmacy x x x 16 Shah et al. USA 2013 Outpatient Clinic x x 17 Chung et al. Malaysia 2014 Outpatient Clinic x x x x 18 Jahangard et al. Resultsh Iran 2015 Community Pharmacy x x x x x Page 6 of 18 Hassan et al. BMC Health Services Research (2023) 23:46 Hassan et al. BMC Health Services Research Table 1 (continued) No. Author Country Year Location HCP involvement Method of delivery Tools/ Aids Pharmacist Multidisciplinary Tteam Face to face Phone calls Group sessions Home visits Diaries Printed materials Video Email Pill box Games 19 Wishah et al. Jordan 2015 Outpatient Clinic x x x x 20 Xin et al. China 2015 Outpatient Clinic x x x x x 21 Butt et al. Malaysia 2016 Outpatient Clinic x x 22 Chen et al. Taiwan 2016 Outpatient Clinic x x x x x 23 Aguiar et al. Brazil 2016 Outpatient Clinic x x x x x 24 Chow et al. Malaysia 2015 Outpatient Clinic x x x x x 25 Renuga et al. India 2016 Outpatient Pharmacy x x x 26 Samtia et al. India 2013 Outpatient Clinic x x 27 Cani et al. Brazil 2015 Outpatient Clinic x x x 28 Chan et al. Hong Kong 2012 Outpatient Clinic x x x 29 Abuloha et al. Jordan 2016 Outpatient Clinic x x x x 30 Choe et al. USA 2005 Outpatient Clinic x x x 31 Clifford et al. Australia 2005 Outpatient Clinic x x x 32 Cohen et al. USA 2011 Outpatient Clinic x x x x 33 Doucette et al. USA 2009 Community Pharmacy x x 34 Fornos et al. Spain 2006 Community Pharmacy x x x 35 Ghosh et al. India 2010 Outpatient Clinic x x x 36 Jaber et al. USA 1996 Outpatient Clinic x x Page 7 of 18 Hassan et al. BMC Health Services Research (2023) 23:46 Hassan et al. BMC Health Services Research g ( ) Table 1 (continued) No. Author Country Year Location HCP involvement Method of delivery Tools/ Aids Pharmacist Multidisciplinary Tteam Face to face Phone calls Group sessions Home visits Diaries Printed materials Video Email Pill box Games 37 Kraemer et al. USA 2012 Community Health Centre x x 38 Mourao et al. Brazil 2013 Outpatient Clinic x x 39 Plaster et al. Brazil 2012 Outpatient Clinic x x x x 40 Sriram et al. India 2011 Outpatient Clinic x x x x x 41 Taveira et al. USA 2010 Outpatient Clinic x x 42 Jameson et al. USA 2010 Outpatient Clinic x x x 43 Odegard et al. Resultsh USA 2005 Outpatient Clinic x x x 44 Kirwin et al. USA 2010 Outpatient Clinic x x 45 Lyons et al. UK 2016 Community Pharmacy x x x 46 Malathy et al. India 2011 Outpatient Clinic x x x 47 Rothman et al. USA 2005 Outpatient Clinic x x x 48 Sarkadi & Rosenqvist et al. Sweden 2004 Community Health Center x x x x x 49 Scott et al. USA 2006 Community Health Center x x x x 50 Adepu et al. India 2007 Community Pharmacy x x x 51 Erku et al. Ethiopia 2017 Outpatient Clinic x x x 52 Korcegez et Cyprus 2017 Outpatient Clinic x x x 53 Ojieabu et Nigeria 2017 Outpatient Clinic x x x Page 8 of 18 Hassan et al. BMC Health Services Research (2023) 23:46 Hassan et al. BMC Health Services Research Table 1 (continued) No. Author Country Year Location HCP involvement Method of delivery Tools/ Aids Pharmacist Multidisciplinary Tteam Face to face Phone calls Group sessions Home visits Diaries Printed materials Video Email Pill box Games 54 Shao et al. China 2017 Outpatient Clinic x x x 55 Lauffen- burger et al. USA 2019 Outpatient x x x 56 Wu et al. USA 2018 Outpatient Clinic x x 57 Michiels et al. France 2019 Community Pharmacy x x x 58 Javaid et al. Pakistan 2019 Outpatient Clinic x x 59 Withidpan- yawong et al. Thailand 2018 Outpatient Clinic x x 60 Sarayani et al. Iran 2018 Community Pharmacy x x 61 Ayadurai et al. Malaysia 2018 Outpatient Clinic x x No. Author Country Year Location Content Outcome Diabetes education Medication review Drug counselling Clinical intervention Lifestyle adjustment Self care Peer support Behavioural intervention Glucose control Adherence 1 Castejon et al. USA 2013 Outpatient Pharmacy x x x x I > C 2 Lim et al. Malaysia 2016 Outpatient Clinic x x I > C I > C 3 Ali et al. UK 2012 Community Pharmacy x x I > C 4 Venkatesan et al. India 2012 Community Pharmacy x x x x NS 5 Grant et al. USA 2003 Community Health Center x NS NS 6 Ramanath et al. India 2012 Outpatient Clinic x I > C I > C 7 Mahwi et al. Iraq 2013 Outpatient Clinic x x I > C I > C Hassan et al. Resultsh BMC Health Services Research (2023) 23:46 Page 9 of 18 Table 1 (continued) No. Author Country Year Location Content Outcome Diabetes education Medication review Drug counselling Clinical intervention Lifestyle adjustment Self care Peer support Behavioural intervention Glucose control Adherence 8 Clifford et al. Australia 2002 Outpatient Clinic x NS 9 Phumipa- morn et al. Thailand 2008 Outpatient Clinic x x x I > C I > C 10 Al Mazroui et al. UAE 2009 Outpatient Clinic x x x I > C 11 Farsaei et al. Iran 2011 Outpatient Clinic x x x x I > C 12 Mehuys et al. Belgium 2011 Community Pharmacy x x x I > C 13 Jacobs et al. USA 2012 Outpatient Clinic x x x x I > C 14 Jarab et al. Jordan 2012 Outpatient Clinic x x x x I > C 15 Odegard et al. USA 2012 Community Pharmacy x x I > C 16 Shah et al. USA 2013 Outpatient Clinic x x I > C I > C 17 Chung et al. Malaysia 2014 Outpatient Clinic x x x I > C I > C 18 Jahangard et al. Iran 2015 Community Pharmacy x x x NS I > C 19 Wishah et al. Jordan 2015 Outpatient Clinic x x x I > C 20 Xin et al. China 2015 Outpatient Clinic x I > C I > C 21 Butt et al. Malaysia 2016 Outpatient Clinic x x x x I > C I > C 22 Chen et al. Taiwan 2016 Outpatient Clinic x x I > C 23 Aguiar et al. Brazil 2016 Outpatient Clinic x x x I > C I > C 24 Chow et al. Malaysia 2015 Outpatient Clinic x x x I > C I > C 25 Renuga et al. India 2016 Outpatient Pharmacy x I > C I > C Page 10 of 18 Hassan et al. BMC Health Services Research (2023) 23:46 ( ) No. Author Country Year Location Content Outcome Diabetes education Medication review Drug counselling Clinical intervention Lifestyle adjustment Self care Peer support Behavioural intervention Glucose control Adherence 26 Samtia et al. India 2013 Outpatient Clinic x x x NS I > C 27 Cani et al. Brazil 2015 Outpatient Clinic x x x x x NS I > C 28 Chan et al. Resultsh Hong Kong 2012 Outpatient Clinic x x I > C I > C 29 Abuloha et al. Jordan 2016 Outpatient Clinic x x x x I > C 30 Choe et al. USA 2005 Outpatient Clinic x x x x I > C 31 Clifford et al. Australia 2005 Outpatient Clinic x x x I > C 32 Cohen et al. USA 2011 Outpatient Clinic x x x x NS 33 Doucette et al. USA 2009 Community Pharmacy x x x NS 34 Fornos et al. Spain 2006 Community Pharmacy x x I > C 35 Ghosh et al. India 2010 Outpatient Clinic x x x I > C 36 Jaber et al. USA 1996 Outpatient Clinic x x x x I > C 37 Kraemer et al. USA 2012 Community Health Centre x x x NS 38 Mourao et al. Brazil 2013 Outpatient Clinic x x x x I > C 39 Plaster et al. Brazil 2012 Outpatient Clinic x x I > C 40 Sriram et al. India 2011 Outpatient Clinic x x I > C 41 Taveira et al. USA 2010 Outpatient Clinic x x x x x x I > C 42 Jameson et al. USA 2010 Outpatient Clinic x x x x NS Page 11 of 18 Hassan et al. BMC Health Services Research (2023) 23:46 Hassan et al. BMC Health Services Research No. Author Country Year Location Content Outcome Diabetes education Medication review Drug counselling Clinical intervention Lifestyle adjustment Self care Peer support Behavioural intervention Glucose control Adherence 43 Odegard et al. USA 2005 Outpatient Clinic x NS 44 Kirwin et al. USA 2010 Outpatient Clinic x x NS 45 Lyons et al. UK 2016 Community Pharmacy x NS I > C 46 Malathy et al. India 2011 Outpatient Clinic x x NS 47 Rothman et al. USA 2005 Outpatient Clinic x x x x NS 48 Sarkadi & Rosenqvist et al. Sweden 2004 Community Health Center x x x I > C 49 Scott et al. USA 2006 Community Health Center x x x x I > C 50 Adepu et al. India 2007 Community Pharmacy x x x NS 51 Erku et al. Resultsh Ethiopia 2017 Outpatient Clinic x x x I > C 52 Korcegez et Cyprus 2017 Outpatient Clinic x x x x x I > C I > C 53 Ojieabu et Nigeria 2017 Outpatient Clinic x x I > C I > C 54 Shao et al. China 2017 Outpatient Clinic x x x I > C I > C 55 Lauffen- burger et al. USA 2019 Outpatient x x NS NS 56 Wu et al. USA 2018 Outpatient Clinic x x x x NS 57 Michiels et al. France 2019 Community Pharmacy x x I > C NS 58 Javaid et al. Pakistan 2019 Outpatient Clinic x x x x I > C continued) Author Country Year Location Content Outcome Diabetes education Medication review Drug counselling Clinical intervention Lifestyle adjustment Self care Peer support Behavioural intervention Glucose control Adherence Odegard et al. USA 2005 Outpatient Clinic x NS Kirwin et al. USA 2010 Outpatient Clinic x x NS Lyons et al. UK 2016 Community Pharmacy x NS I > C Malathy et al. India 2011 Outpatient Clinic x x NS Rothman et al. USA 2005 Outpatient Clinic x x x x NS Sarkadi & Rosenqvist et al. Sweden 2004 Community Health Center x x x I > C Scott et al. USA 2006 Community Health Center x x x x I > C Adepu et al. India 2007 Community Pharmacy x x x NS Erku et al. Ethiopia 2017 Outpatient Clinic x x x I > C Korcegez et Cyprus 2017 Outpatient Clinic x x x x x I > C I > C Ojieabu et Nigeria 2017 Outpatient Clinic x x I > C I > C Shao et al. China 2017 Outpatient Clinic x x x I > C I > C Lauffen- burger et al. USA 2019 Outpatient x x NS NS Wu et al. USA 2018 Outpatient Clinic x x x x NS Michiels et al. France 2019 Community Pharmacy x x I > C NS Javaid et al. Pakistan 2019 Outpatient Clinic x x x x I > C Page 12 of 18 Hassan et al. BMC Health Services Research (2023) 23:46 No. Author Country Year Location Content Outcome Diabetes education Medication review Drug counselling Clinical intervention Lifestyle adjustment Self care Peer support Behavioural intervention Glucose control Adherence 59 Withidpan- yawong et al. Resultsh Thailand 2018 Outpatient Clinic x x I > C I > C 60 Sarayani et al. Iran 2018 Community Pharmacy x x NS I > C 61 Ayadurai et al. Malaysia 2018 Outpatient Clinic x x x I > C Table 1 (continued) No. Author Country Year Location Content Outcome Diabetes education Medication review Drug counselling Clinical intervention Lifestyle adjustment Self care Peer support Behavioural intervention Glucose control Adherence 59 Withidpan- yawong et al. Thailand 2018 Outpatient Clinic x x I > C I > C 60 Sarayani et al. Iran 2018 Community Pharmacy x x NS I > C 61 Ayadurai et al. Malaysia 2018 Outpatient Clinic x x x I > C Page 13 of 18 Hassan et al. BMC Health Services Research (2023) 23:46 Table 2 Description of intervention categorization and potential activities involved Intervention categories Description of intervention Example of activities involved References Diabetes education Provide the patient with adequate knowledge about diabetes and skills they need to manage their clinical condition and lifestyle. Educational videos, pamphlets, educational websites, power point presentations, face-to-face/group teach- ing sessions. [14–20, 22, 24, 26–34, 38–44, 46–51, 53, 56, 57, 60, 61, 63–74] Medication review Pharmacists addressed issues pertaining to medication optimization and adherence, hence enhancing the therapeutic efficacy of drugs administered to patients. Review of patients’ medications, arrangements of drug taking schedules, discussion and evaluation of medica- tion regimens, dose up-titrations per pre-established protocols without prescriber’s involvement. [10, 13, 15, 17, 21–28, 33–41, 44, 46, 48, 52, 53, 60, 64, 68, 70, 72–74] Drug counseling Aids patients in the comprehension of medications and is emphasises the importance of drug adherence. Evaluation of patients’ medication adherence, pill counts, medication diaries, pill boxes, pill reminder apps. [13, 16, 19, 26, 29, 30, 37, 49, 53, 58, 59, 63, 69, 71] Clinical review A collaborative intervention with the prescriber on drug related problems requiring clinical interventions such as regimen changes or dosage adjustment. Evaluation and adjustment of patients’ medication with the involvement of the prescribers. [10, 21, 25, 28, 34, 39, 40, 44, 72, 73] Lifestyle adjustment Focused primarily on healthy eating and encouraging patients to lead a more active lifestyle. Exercise prescription, specific diet recommendation. [15–17, 19, 20, 22–24, 26–28, 35, 36, 41–43, 46, 47, 49–52, 61, 62, 64–67, 69, 72, 73] Self-care Approaches to manage and prevent diabetes compli- cations through self-blood glucose monitoring and foot care. Discussionh The current scoping review aimed to evaluate the type of interventions and service model provided during the provision of pharmacist-led type 2 diabetes management which previously had unclear classification with no detail on the activities involved. This includes a review of the type of service content, delivery methods, settings, fre- quencies of appointments, collaborative work with other healthcare providers, and reported outcomes. Pharma- cist-led diabetes interventions were provided in six conti- nents, with most studies (n = 37) conducted in Asia. This was not surprising since the highest prevalence of diabe- tes is found in Asian countries [1]. Thirty of the studies were conducted in high-income countries, one in low- income country, 13 in lower-middle-income countries, and 17 in upper-middle-income countries. There is a lack of interest in the topic in low-income countries even though the prevalence of non-adherence towards treat- ment is high and the promotion of effective treatment plan would help reduce the burden of diabetes manage- ment in such countries [4, 6]. Most services (n = 37) were provided by a multidisciplinary health care team, and only a few (n = 24) were conducted solely by pharmacists. Nevertheless, in these studies, most showed that phar- macists also worked directly with a physician on patient issues, for example, if the patient required approval for prescription adjustment and specific diet plan such as fasting. A meta-analysis study on the multidisciplinary team approach to coordinated pharmaceutical care found that such collaborations reduced the likelihood of patients’ hospitalization and increased their quality of life [78]. The follow-up periods of the pharmacists’ interven- tions differed in each study and ranged between 1.5 [67] and 24 months [42]. One study followed up patient for 2 months [59], nine for 3 months [16, 52, 53, 56, 60, 61, 64, 70, 74], five for 4 months [21, 25, 31, 45, 68], three for 5 months [23, 50, 53], fourteen for 6 months [22, 27, 30, 36, 39, 40, 44, 46, 49, 62, 73, 75], three for 8 months [18, 55, 72], four for 9 months [29, 33, 37, 41], eighteen for 12 months [20, 24, 26, 28, 35, 38, 43, 47, 48, 51, 54, 63, 65, 66, 69, 71, 76, 77], one for 13 months [32] and another one for 16 months [10]. Resultsh The most popular strategy for this was diabetes education. The educational components of these interventions primar- ily aimed to increase the patients’ general understanding of their condition by discussing the expected degree of diabetic control, risk of complications, and ways to mini- mize these risks [57]. Patients were also informed about the types of medications used to treat their disease [58]. l d l d l most common number of follow-ups reported by the studies was three (n = 13, 21.43%). Most studies (n = 36) reported glucose control as the outcome, 24 reported glucose control and medication adherence as the outcomes, and one study reported med- ication adherence as the outcome. Most studies (n = 41) also showed significant improvement in glucose control, which was measured by glycosylated hemoglobin, fast- ing or random blood glucose levels, or a combination of these. Meanwhile, 22 of the 25 studies reported signifi- cant improvement in medication adherence measured using the eight-item Morisky Scale, Malaysian Medica- tion Adherence Scale, pill-count, self-reported adher- ence scale, dispensing history, diagnostic adherence to medication scale, or Morisky Green Levine Medication Adherence Scale. yp [ ] Only seven studies utilized a single intervention strat- egy, which included diabetes education [17, 35, 59, 60], medication review [37], and drug consultation/coun- seling [61, 62]. Most studies incorporated two or more intervention strategy categories. In particular, 17 com- bined two strategies [16, 28, 33, 34, 36, 39, 41, 45, 48, 55, 63–66], 18 combined three [18, 19, 23, 24, 26, 30, 38, 44, 46, 50, 51, 53, 54, 67–71], 16 combined four [20, 22, 25, 27, 29, 32, 37, 42, 43, 47, 49, 52, 56, 72–74], 2 combined five [75, 76], and 1 combined six [31]. The most com- monly utilized intervention strategy was diabetes educa- tion (n = 49), whereas the least utilized service content category was peer support (n = 1). Resultsh Glucose diaries, glucose monitoring device program, proper foot care program. [17–24, 28, 29, 31, 34, 35, 37, 39, 43, 44, 47–51, 53, 65–73] Peer support Promotes communications and sharing of knowledge and experience between patients that have poor con- trol of diabetes with patients that already have better experience in managing their disease. Participation of family members, friends, and other sources of social support in the intervention program. [70] Behavioural intervention Incorporate behaviour-change techniques such as goal-setting, cognitive behavioural therapy, and problem-solving. Predetermined action items, action planning, motiva- tional interviewing. [28, 29, 40, 46, 62, 71] Page 14 of 18 Hassan et al. BMC Health Services Research (2023) 23:46 Hassan et al. BMC Health Services Research (2023) 23:46 Eight intervention strategy categories were identified in the included studies. Table 2 provides a detailed descrip- tion of the categories and potential activities involved. Intervention strategies were categorized into diabetes education, medication review, drug consultation/coun- seling, clinical intervention, lifestyle adjustment, self- care, peer support, and behavioral intervention. The most popular strategy for this was diabetes education. The educational components of these interventions primar- ily aimed to increase the patients’ general understanding of their condition by discussing the expected degree of diabetic control, risk of complications, and ways to mini- mize these risks [57]. Patients were also informed about the types of medications used to treat their disease [58]. Only seven studies utilized a single intervention strat- egy, which included diabetes education [17, 35, 59, 60], medication review [37], and drug consultation/coun- seling [61, 62]. Most studies incorporated two or more intervention strategy categories. In particular, 17 com- bined two strategies [16, 28, 33, 34, 36, 39, 41, 45, 48, 55, 63–66], 18 combined three [18, 19, 23, 24, 26, 30, 38, 44, 46, 50, 51, 53, 54, 67–71], 16 combined four [20, 22, 25, 27, 29, 32, 37, 42, 43, 47, 49, 52, 56, 72–74], 2 combined five [75, 76], and 1 combined six [31]. The most com- monly utilized intervention strategy was diabetes educa- tion (n = 49), whereas the least utilized service content category was peer support (n = 1). Eight intervention strategy categories were identified in the included studies. Table 2 provides a detailed descrip- tion of the categories and potential activities involved. Intervention strategies were categorized into diabetes education, medication review, drug consultation/coun- seling, clinical intervention, lifestyle adjustment, self- care, peer support, and behavioral intervention. Discussionh The most common follow-up period for the interventions was 12 months (n = 19, 31.1%), and the mean intervention duration was 7.8 months. The frequency of follow-up varied from a minimum of a single follow-up [21, 28, 61, 69] to 24 follow-ups [42]. Five studies set a frequency of two follow-ups [44, 48, 59, 62, 68], twelve set three follow-ups [16, 20, 22, 29, 49, 52, 60, 64, 65, 70, 72, 74], five set four follow-ups [17, 18, 24, 31, 45], two set five follow-ups [53, 76], eleven set six follow-ups [19, 26, 27, 30, 33, 36, 39, 54, 67, 75, 77], four set eight follow-ups [10, 32, 37, 38], another four set nine follow-ups [40, 46, 66, 73], one set ten follow-ups [25], and six set twelve follow-ups [35, 43, 51, 56, 63, 71]. Five of the studies did not describe the number of follow-ups carried out in detail [23, 41, 47, 50, 55]. The mean num- ber of follow-ups that the patients received was six. The Face-to-face sessions were the most common method for pharmacist-led diabetes management reported in the included studies. This traditional method of service delivery is well established and generally well accepted Page 15 of 18 Hassan et al. BMC Health Services Research (2023) 23:46 Page 15 of 18 Hassan et al. BMC Health Services Research tools for improving patient clinical outcomes and safety by resolving treatment complexities. Several systematic reviews have reported that medication reviews in the care of diabetic patients have improved clinical results and provided favorable economic outcomes that are not only beneficial to the self-paying patients, but also to the healthcare system [84, 85]. Other service interven- tions include self-care content, such as self-blood glucose monitoring and foot care; lifestyle adjustments, such as eating habits and physical exercise; drug consultation or counseling that focuses on effective use of medications; clinical intervention that includes a change in regimen or dosing adjustment that was carried out after agreement with the prescriber; and behavior-change content, such as goal-setting, cognitive behavioral therapy, and prob- lem solving. The majority of the studies employed a vari- ety of terminology to name their intervention techniques. This makes determining the types of specific interven- tion delivered challenging. Discussionh A list of standardized termi- nologies and their meanings would be extremely valuable for practice harmonization and guaranteeing that future intervention program outcomes may be compared in a more methodical and meaningful manner. by patients. Most studies reported good patient reten- tion throughout the study period. However, unlike in a trial environment, patients in a real-world setting may not be able to complete proper follow-ups with frequent face-to-face appointments. In clinical trials, patients are routinely reminded to attend their subsequent appoint- ments and are often rewarded with tokens for their participation in the trials. Therefore, they might have different motivations for retaining themselves in the pro- gram compared to actual patients outside clinical trial settings. For example, in a diabetes prevention program in England involving 100,000 patients, only 22% of the participants completed the program [79]. Therefore, it is important to review an intervention program beyond the “controlled” environment and ensure its convenience for the patient. Providing more interventions through phone calls and video conferences should be explored in the future, as they are generally equally effective as face-to-face sessions [80]. Home visits may suit patients with logistics issues who require frequent clinic visits. Meanwhile, group sessions may be useful for behavioral interventions that include peer support and mentoring activities [81]. by patients. Most studies reported good patient reten- tion throughout the study period. However, unlike in a trial environment, patients in a real-world setting may not be able to complete proper follow-ups with frequent face-to-face appointments. In clinical trials, patients are routinely reminded to attend their subsequent appoint- ments and are often rewarded with tokens for their participation in the trials. Therefore, they might have different motivations for retaining themselves in the pro- gram compared to actual patients outside clinical trial settings. For example, in a diabetes prevention program in England involving 100,000 patients, only 22% of the participants completed the program [79]. Therefore, it is important to review an intervention program beyond the “controlled” environment and ensure its convenience for the patient. Providing more interventions through phone calls and video conferences should be explored in the future, as they are generally equally effective as face-to-face sessions [80]. Home visits may suit patients with logistics issues who require frequent clinic visits. Meanwhile, group sessions may be useful for behavioral interventions that include peer support and mentoring activities [81]. Discussionh g The current study found that the duration and fre- quency of pharmacist-led diabetic interventions varied between the studies. A previous review found that stud- ies with longer follow-up periods were associated with better outcomes [8]. Nevertheless, most included stud- ies had good patient retention rates throughout the study period, which could differ in actual practice; the reason for the difference could be due to the “controlled environ- ment” in the trial setting, in which patients were closely followed up. The same study also reported that pharma- cist interventions significantly improved most of the out- come measures within three follow-up visits [8]. Hence, the delivery of content should be based on the patient’s immediate needs and should not be too structured in the view that patients may not return for their next appoint- ment. In addition, no consistent pattern was found between intervention categories and patient outcomes. This would be difficult to identify because the majority of the included studies reported significant improvements in glucose control and medication adherence.h Eight categories of pharmacist-led service con- tents were identified from the reported studies, which included diabetes education, medication review, drug consultation/counseling, clinical intervention, life- style adjustment, self-care, peer support, and behavio- ral intervention. The majority of the studies combined two or more service content categories for intervention. Although the best combination of interventions for dia- betes management could not be determined due to an inconsistency in the services provided across the stud- ies, combining several types of intervention content was found to be more effective than a single intervention [8, 82]. Studies have shown that a combination of services improves patients’ medication adherence and glucose control. In the current study, diabetes education pre- vailed as the most common intervention in pharmacist- led diabetes services, with the aim of providing patients with the knowledge and skills needed to manage their clinical condition and lifestyle. Previous systematic reviews also found that diabetes education was most effective in improving diabetes control and enhancing medication adherence [8, 57, 58, 82]. During diabetes education, printed or digital materials and training or group discussions can also be considered, as they were also reported to be equally effective [8]. This study has a few limitations. Availability of data and materials 15. Peters M, Godfrey C, Mcinerney P, Soares C, Khalil H, Parker D. Methodol- ogy for JBI scoping reviews. Joanna Briggs Institute; 2015. 15. Peters M, Godfrey C, Mcinerney P, Soares C, Khalil H, Parker D. Methodol- ogy for JBI scoping reviews. Joanna Briggs Institute; 2015. All data relevant to the study are included in the article or uploaded as sup- plementary information. All data relevant to the study are included in the article or uploaded as sup- plementary information. 16. Mahwi TO, Kawa AO. Role of the Pharmaceutical Care in the manage- ment of patients with type 2 diabetes Mellitus. Int J Pharm Sci Res. 2013;14:1363–9. References 1. Cho NH, Shaw JE, Karuranga S, Huang Y, da Rocha Fernandes JD, Ohlrogge AW, et al. IDF Diabetes Atlas: global estimates of diabetes prevalence for 2017 and projections for 2045. Diabetes Res Clin Pract. 2018;138:271–81. 2. Whiting DR, Guariguata L, Weil C, Shaw J. IDF Diabetes Atlas: global estimates of the prevalence of diabetes for 2011 and 2030. Diabetes Res Clin Pract. 2011;94:311–21. 2. Whiting DR, Guariguata L, Weil C, Shaw J. IDF Diabetes Atlas: global estimates of the prevalence of diabetes for 2011 and 2030. Diabetes Res Clin Pract. 2011;94:311–21. Discussionh Firstly, since the intended purpose of a this scoping review is to gather information on type of intervention strategies provided during pharmacist-led diabetes management, as opposed to recommending a clinical practice, quality assess- ment is not undertaken hence, making it impossible for any implications for practice or policy to be graded. The components of the interventions and their catego- rization were based on the information provided in the manuscript. Some interventions were not sufficiently f In the current study, medication review was the second intervention widely used during pharmacist-led diabetes management. During medication review, pharmacists optimize patients’ medications and ensure their adher- ence [83]. Medication review is one of the most effective Hassan et al. BMC Health Services Research (2023) 23:46 Page 16 of 18 Hassan et al. BMC Health Services Research (2023) 23:46 Hassan et al. BMC Health Services Research Funding g This study received funding from the Research University Grant of Universiti Kebangsaan Malaysia reference number GUP-2021-010. Competing interests Received: 26 June 2022 Accepted: 16 December 2022 Conclusion V i i i 3. Hu FB. Globalization of diabetes: the role of diet, lifestyle, and genes. Diabetes Care. 2011;34:1249–57. Variations in intervention strategies were found in the reported studies, with the most used being diabetes edu- cation and medication review. Most studies used a com- bination of two or more intervention strategy categories when providing services, with no specific pattern found between the service model and patient outcomes. A standardization of terminologies used for future pharma- cist-led diabetes management services should be encour- aged to ensure harmonization in the service, and making it possible for more research to systematically explore the effectiveness of individual or combination of intervention strategies provided. 4. Liu X, Li C, Gong H, Cui Z, Fan L, Yu W, et al. An economic evaluation for prevention of diabetes mellitus in a developing country: a modelling study. BMC Public Health. 2013;13:729. y 5. Khunti K, Gray LJ, Skinner T, Carey ME, Realf K, Dallosso H, et al. Effective- ness of a diabetes education and self management programme (DES- MOND) for people with newly diagnosed type 2 diabetes mellitus: three year follow-up of a cluster randomised controlled trial in primary care. BMJ. 2012;344:e2333. 6. Azharuddin M, Adil M, Sharma M, Gyawali B. A systematic review and meta-analysis of non-adherence to anti-diabetic medication: evidence from low- and middle-income countries. Int J Clin Pract. 2021;75:e14717. 6. Azharuddin M, Adil M, Sharma M, Gyawali B. A systematic review and meta-analysis of non-adherence to anti-diabetic medication: evidence from low- and middle-income countries. Int J Clin Pract. 2021;75:e14717. 7. Leite RGOF, Banzato LR, Galendi JSC, Mendes AL, Bolfi F, Veroniki AA, et al. Effectiveness of non-pharmacological strategies in the management of type 2 diabetes in primary care: a protocol for a systematic review and network meta-analysis. BMJ Open. 2020;10:e034481. 7. Leite RGOF, Banzato LR, Galendi JSC, Mendes AL, Bolfi F, Veroniki AA, et al. Effectiveness of non-pharmacological strategies in the management of type 2 diabetes in primary care: a protocol for a systematic review and network meta-analysis. BMJ Open. 2020;10:e034481. y p 8. Presley B, Groot W, Pavlova M. Pharmacy-led interventions to improve medication adherence among adults with diabetes: a systematic review and meta-analysis. Res Social Administrative Pharm. 2019;15:1057–67. 8. Presley B, Groot W, Pavlova M. Pharmacy-led interventions to improve medication adherence among adults with diabetes: a systematic review and meta-analysis. Res Social Administrative Pharm. 2019;15:1057–67. Acknowledgements We would like to thank the Quality Management of Medicines Research Group, Faculty of Pharmacy, Universiti Kebangsaan Malaysia for providing support for this study. We would also like to thank Ministry of Health Malaysia and Public Services Division Malaysia for awarding study scholarship (Hadiah Latihan Persekutuan) for FH. 11. Lau BT, Ismail SZ, Ng SY, Mohmmad N. Impact of pharmacist-led diabetes program on glycated hemoglobin and diabetes-related hospitalizations in a district-level hospital: a pilot retrospective cohort study. Int J Adv Life Sci Res. 2018;1:26–36. 11. Lau BT, Ismail SZ, Ng SY, Mohmmad N. Impact of pharmacist-led diabetes program on glycated hemoglobin and diabetes-related hospitalizations in a district-level hospital: a pilot retrospective cohort study. Int J Adv Life Sci Res. 2018;1:26–36. 12. Milosavljevic A, Aspden T, Harrison J. Community pharmacist-led inter- ventions and their impact on patients’ medication adherence and other health outcomes: a systematic review. Int J Pharm Pract. 2018;26:387–97. 12. Milosavljevic A, Aspden T, Harrison J. Community pharmacist-led inter- ventions and their impact on patients’ medication adherence and other health outcomes: a systematic review. Int J Pharm Pract. 2018;26:387–97. Additional file 1. Additional file 1. 10. Lim PC, Lim K, Embee ZC, Hassali MA, Thiagarajan A, Khan TM. Study investigating the impact of pharmacist involvement on the outcomes of diabetes medication therapy adherence program Malaysia. Pak J Pharm Sci. 2016;29(2):595–601. 10. Lim PC, Lim K, Embee ZC, Hassali MA, Thiagarajan A, Khan TM. Study investigating the impact of pharmacist involvement on the outcomes of diabetes medication therapy adherence program Malaysia. Pak J Pharm Sci. 2016;29(2):595–601. Author’s contributions This study was conceived by F.H. and E.H. F.H. and E.H. conducted the searches, appraisal, extraction and analysis with input from A.M.A and C.W.W. All authors contributed to writing the draft manuscript and approved the final version. 13. Van Eikenhorst L, Taxis K, van Dijk L, de Gier H. Pharmacist-led self- management interventions to improve diabetes outcomes. A systematic literature review and meta-analysis. Front Pharmacol. 2017;8:891. 13. Van Eikenhorst L, Taxis K, van Dijk L, de Gier H. Pharmacist-led self- management interventions to improve diabetes outcomes. A systematic literature review and meta-analysis. Front Pharmacol. 2017;8:891. 14. Newman TV, San-Juan-Rodriguez A, Parekh N, Swart ECS, Klein-Fedyshin M, Shrank WH, et al. Impact of community pharmacist-led interventions in chronic disease management on clinical, utilization, and economic outcomes: an umbrella review. Res Social Administrative Pharm. 2020;16:1155–65. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Consent for publication Not applicable. explained, which may have caused limitations in the intervention categorization. However, we independently checked the assigned categories and ensured that the intervention components were identified appropriately. In addition, most included studies reported significant findings on glucose control and improvement in medica- tion adherence, making it difficult to evaluate the effec- tiveness of individual intervention categories. The risk of bias assessment was not done to evaluate the study quality, as this study aimed only to provide an overview of intervention categories according to the activities described in the study. Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s12913-022-08977-1. 9. You LX, Selvadurai S, Yee CK, Noh NB, Chin G, Joyce T, et al. Impact of pharmacist-managed diabetes medication therapy adherence clinic (DMTAC) in government health clinics. Malays J Pharm Sci. 2015;13:43–51 9. You LX, Selvadurai S, Yee CK, Noh NB, Chin G, Joyce T, et al. Impact of pharmacist-managed diabetes medication therapy adherence clinic (DMTAC) in government health clinics. Malays J Pharm Sci. 2015;13:43–51. Additional file 1. Additional file 1. Declarations 17. Clifford RM, Batty KT, Davis TME, Davis W, Stein G, Stewart G, et al. A ran- domised controlled trial of a pharmaceutical care programme in high-risk diabetic patients in an outpatient clinic. Int J Pharm Pract. 2002;10:85–9. Ethics approval and consent to participate Not applicable. Ethics approval and consent to participate Not applicable. Page 17 of 18 Hassan et al. 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https://openalex.org/W4220654987	https://jaclinicalreports.springeropen.com/track/pdf/10.1186/s40981-022-00499-3	English	null	Hyperchloremic metabolic acidosis due to saline absorption during laser enucleation of the prostate: a case report	JA clinical reports	2,022	cc-by	2,567	© The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Case presentation A male in his late seventies was scheduled to undergo holmium laser enucleation of the prostate (HOLEP) owing to benign prostate hypertrophy. He regularly con- sumed alcoholic beverages but was otherwise healthy. Since prolonged surgery was anticipated, the patient Correspondence: makiko.tabuchi@med.toho-u.ac.jp Department of Anesthesiology, Toho University Ohashi Medical Center, 2‑22‑36, Ohashi, Meguro, Tokyo 153‑8515, Japan Backgroundh pathophysiological changes from the conventional TUR syndrome. Actually, we found several cases of hyper- chloremic acidosis after the absorption of large amount of normal saline during laser enuculeation of prostate and surgical hysteroscopy in the literature [8–12]. In this report, we describe a case with hyperchloremic meta- bolic acidosis and airway edema possibly caused by the absorption of normal saline during laser-assisted TUR of the prostate, wherein we applied the simplified Stewart approach to evaluate acid-base abnormalities [13]. The absorption of non-electrolyte irrigation fluid dur- ing transurethral resection (TUR) of the prostate has been known to cause TUR syndrome [1, 2]. This syn- drome is characterized by hyponatremia, hypo-osmolar state, brain edema, and temporal neurological deficits. However, recent technological advancements in tran- surethral surgery have enabled the use of normal saline in bipolar TUR and laser enucleation. In these proce- dures, a superior coagulation profile and the use of saline considerably reduces the volume, risk of irrigating fluid absorption, and electrolyte abnormalities [3–7]. None- theless, saline absorption during TUR may cause different Abstract Background: Recent technological advancements have enabled the use of electrolyte solutions such as saline or buffered electrolyte solution during transurethral resection or laser enucleation of the prostate. However, saline absorption may cause hyperchloremic metabolic acidosis. Case presentation: A male in his late seventies underwent holmium laser enucleation of the prostate under a combination of subarachnoid block and general anesthesia. Intraoperatively, abdominal distension prompted the attending anesthesiologist to consider the possibility of SGA malposition, and the trachea was intubated. Oropharyn- geal and neck edema was observed, and laboratory examination revealed considerable acidosis with hyperchloremia. Further evaluation confirmed the absorption of a large amount of saline into the circulation via the perforated blad- der. Application of the simplified Stewart approach clearly suggested that hyperchloremia was the principal cause of metabolic acidosis. The dilution of albumin attenuated acidosis. Conclusions: Absorption of normal saline during laser enucleation of prostate caused hyperchloremic metabolic acidosis and airway edema. Keywords: Transurethral laser enucleation of the prostate, Saline, Hyperchloremia, Metabolic acidosis, Stewart approach Keywords: Transurethral laser enucleation of the prostate, Saline, Hyperchloremia, Metabolic acidosis, Stewart approach Tabuchi et al. JA Clinical Reports (2022) 8:20 https://doi.org/10.1186/s40981-022-00499-3 Open Access Correspondence: makiko.tabuchi@med.toho-u.ac.jp Department of Anesthesiology, Toho University Ohashi Medical Center, 2‑22‑36, Ohashi, Meguro, Tokyo 153‑8515, Japan Discussion h In this case, we assume absorption of normal saline dur- ing laser enucleation of prostate caused hyperchloremic metabolic acidosis and airway edema. We found two relevant publications about this complication. Grove et al. reported a case with pulmonary, facial, and upper- arm edema during hysteroscopy under general anesthe- sia using lactate Ringer solution as irrigating fluid; the authors found extensive hemodilution and concluded that the absorption of lactate Ringer caused hyperv- olemia as well as pulmonary and peripheral edema [8]. Dodd et al. also reported a case of hemodilution, facial and neck edema, and hyperchloremic metabolic acido- sis during HOLEP wherein saline was used [9]. These cases supposedly share the same pathophysiological process although the mechanism of absorption and type of irrigating fluid were different from that in our case. Typical TUR syndrome is caused by the expansion of intracellular space due to the hypo-osmotic nature of non-electrolyte irrigating fluid. Instead, the iso- osmotic solution is principally distributed to the extra- cellular space and its absorption is suspected to cause expansion of the intravascular and extracellular spaces without affecting the intracellular space [14]. Thus, the extensive hemodilution and airway edema found in After intubation, arterial blood gas analysis revealed marked hyperchloremic metabolic acidosis (Table 1). Derived parameters such as NaCl base excess effect, albumin base excess effect, and lactate base excess effect are also presented in Table 1 [13].i Based on these findings, we suspected translocation of irrigating saline to the intravascular space from the peritoneal cavity through the perforated bladder. This hypothesis was supported by the large positive difference between the infused and recovered amounts of irrigat- ing fluid and decreased hemoglobin and albumin levels without any major blood loss and was verified by fluid collection in the peritoneal space found by ultrasound examination. The balance between the used and recov- ered irrigation fluid was estimated to be +6400 ml. Table 1 Perioperative blood gas and laboratory indices and evaluation of acid-base status using the simplified Stewart approach n/a not available Hemoglobin, albumin, Na, and Cl levels were measured at the central laboratory, while other parameters were analyzed using a blood gas analyzer (Cobas B221, Roche Diagnostics, Basel, Switzerland). Tabuchi et al. JA Clinical Reports (2022) 8:20 Tabuchi et al. JA Clinical Reports (2022) 8:20 Page 2 of 4 Page 2 of 4 Owing to concerns regarding airway compromise, the patient remained intubated and mechanically ventilated in the ICU until postoperative day 1. Diuresis with furo- semide and carperitide during the ICU stay resulted in a negative fluid balance of 5200 ml and normalization of hyperchloremia (Table 1). The patient was transferred to the hospital ward without any sequel on the next day. underwent subarachnoid block with hyperbaric bupiv- acaine, followed by general anesthesia; the airway was secured with a supraglottic airway (SGA). Ninety minutes after the start of the HOLEP proce- dure, the anesthesiologist experienced progressive dif- ficulty in ventilating this patient with increased airway pressure, poor end-tidal CO2 trace, and abdominal dis- tention. These circumstances prompted the anesthesiolo- gist to consider the possibility of SGA malposition and inadvertent insufflation of the stomach via a misplaced SGA. Therefore, the anesthesiologist intended to remove the SGA and re-intubate using a standard tracheal tube. During intubation, the provider found abnormally swol- len oral and pharyngeal mucosa; nonetheless, intubation was successful. The presence of clinically relevant upper airway edema was confirmed by laryngofiberscopy per- formed by an otorhinolaryngologist immediately after intubation. Roche Diagnostics, Basel, Switzerland). NaCl base excess effect, [Na+]-[Cl-]-35 (mEq/l); albumin base excess effect, 0.25X(42-Alb (g/l)) (mEq/l); lactate base excess effect, 1-Lactate (mmol/l) (mEq/l) (from reference [13]) and Cl levels were measured at the central laboratory, while other parameters were analyzed using a blood gas analyzer (Cobas B221, f +ff umin, Na, and Cl levels were measured at the central laboratory, while other parameters were analyzed using a blood gas analyzer (Cobas s, Basel, Switzerland). NaCl base excess effect, [Na+]-[Cl-]-35 (mEq/l); albumin base excess effect, 0.25X(42-Alb (g/l)) (mEq/l); lactate base e aCl base excess effect, [Na+]-[Cl-]-35 (mEq/l); albumin base excess effect, 0.25X(42-Alb (g/l)) (mEq/l); lactate base excess effect, ce [13]) Hemoglobin, albumin, Na, and Cl levels were measured at the central laboratory, while other parameters were analyzed using a blood gas analyzer (Cobas B221, Roche Diagnostics, Basel, Switzerland). NaCl base excess effect, [Na+]-[Cl-]-35 (mEq/l); albumin base excess effect, 0.25X(42-Alb (g/l)) (mEq/l); lactate base excess effect, 1-Lactate (mmol/l) (mEq/l) (from reference [13]) Ethics approval and consent to participate Not applicable. p The underlying mechanism of metabolic acidosis was rather obvious owing to the marked hyperchloremia [15]. Physiochemical approach developed by Stewart has several advantages over traditional bicarbonate cen- tered approach to interpret hyperchloremic metabolic acidosis [16, 17]. However, standard Stewart approach requires the measurement of total protein and phos- phate. Additionally, quantitative and trend assessment of acid-base disorder is somewhat complicated. In this report, we applied the simplified Stewart approach to evaluate acid-base change [13]. In this approach, three factors are used to evaluate in base excess and expressed as equivalent to conventional base excess: the NaCl effect, albumin effect, and lactate effect. The pri- mary cause of metabolic acidosis of this case is typified by the increase of the NaCl effect. Hypoalbuminemia caused by hemodilution partially countered the NaCl effect, and the lactate effect was negligible. The effects of diuretic treatment such as normalization of chloride and albumin during the ICU stay are easily appreci- ated by the change of these parameters. We believe that semi-quantitative assessment with the simplified Stew- art approach is useful for the diagnosis and treatment of metabolic acidosis. Discussion h NaCl base excess effect, [Na+]-[Cl-]-35 (mEq/l); albumin base excess effect, 0.25X(42-Alb (g/l)) (mEq/l); lactate base excess effect, 1-Lactate (mmol/l) (mEq/l) (from reference [13]) Preoperative Intraoperative ICU admission First postoperative day Hb (g/dl) 15.7 11.7 14.2 14.9 Albumin (g/dl) 3.9 2.2 2.9 2.5 Na (mmol/l) 142 145 145 143 Cl (mmol/l) 105 120 114 108 pH n/a 7.217 7.236 7.358 HCO3 - n/a 16.7 17.6 20.8 Lactate (mmol/l) n/a 0.8 0.9 2 NaCl base excess effect 2 -10 -4 0 Albumin base-excess effect 0.8 5 3.3 4.2 Lactate base-excess effect n/a 0.2 0.1 −1 Hemoglobin, albumin, Na, and Cl levels were measured at the central laboratory, while other parameters were analyzed using a blood gas analyzer (Cobas B221, Roche Diagnostics, Basel, Switzerland). NaCl base excess effect, [Na+]-[Cl-]-35 (mEq/l); albumin base excess effect, 0.25X(42-Alb (g/l)) (mEq/l); lactate base excess effect, 1-Lactate (mmol/l) (mEq/l) (from reference [13]) Tabuchi et al. JA Clinical Reports (2022) 8:20 Page 3 of 4 these cases may be the consequences of this expansion intravascular and extracellular space.h Acknowledgements Acknowledgements We would like to thank Editage (www.editage.com) for English language editing. 12. You AH, Lee JY, Choi JH, Kim MK. Hyperchloremic metabolic acidosis during bipolar transurethral resection of the prostate: a report of two cases. J Int Med Res. 2021;49:3000605211024480. Authors’ contributions MT provided perioperative care and prepared the draft of the manuscript. KM was a major contributor in writing the manuscript. YM and DT provided perioperative care. YK provided a framework for acid-base interpretation and supervised manuscript preparation. The authors read and approved the final manuscript. 13. Story DA. Stewart acid-base: a simplified bedside approach. Anesth Analg. 2016;123:511–5. 14. Doherty M, Buggy DJ. Intraoperative fluids: how much is too much? Br J Anaesth. 2012;109:69–79. 14. Doherty M, Buggy DJ. Intraoperative fluids: how much is too much? Br J Anaesth. 2012;109:69–79. 15. Story DA, Morimatsu H, Bellomo R. Hyperchloremic acidosis in the critically ill: one of the strong-ion acidoses? Anesth Analg. 2006;103:144–8. 15. Story DA, Morimatsu H, Bellomo R. Hyperchloremic acidosis in the critically ill: one of the strong-ion acidoses? Anesth Analg. 2006;103:144–8. Consent for publication Written informed consent was obtained from the patient for publication of this case report and the accompanying images. References Metabolic acidosis with hemodilution due to massive absorption of normal saline as bladder irrigation fluid following holmium laser enucleation of prostate. J Anesth. 2016;30:1060–2. Competing interests p g MT, KM YM, and DT declare that they have no competing interests. YK received speaker fees from Edwards Lifesciences, Otsuka Pharmaceutical Laboratory, MSD, Nihon Koden Corp, GE Healthcare Japan, Covidien Japan, Baxter, and Japan Blood Products Organization. He also received an unrestricted research fund from Edwards Lifesciences and Nihon Koden Corp. Received: 26 September 2021 Revised: 19 January 2022 Accepted: 21 January 2022 Abbreviations HOLEP H l i 10. Smith CC, Brown JPR. A case of cardiac arrhythmia from absorption of nor- mal saline during hysteroscopic myomectomy. J Minim Invasive Gynecol. 2019;26:770–3. HOLEP: Holmium laser enucleation of prostate; SGA: Supraglottic airway; TUR: Transurethral resection. 11. Summers GC, Fosker SR, Faris R, Handy JM. Acute pulmonary oedema and hyperchloraemic metabolic acidosis following operative hysteroscopy using sodium chloride 0.9. Anaesth Rep. 2020;8:e12080. References 1. Hahn RG. The transurethral resection syndrome. Acta Anaesthesiol Scand. 1991;35:557–67. 1. Hahn RG. The transurethral resection syndrome. Acta Anaesthesiol Scand. 1991;35:557–67. 2. Hahn RG. Fluid absorption in endoscopic surgery. Br J Anaesth. 2006;96:8–20.l 2. Hahn RG. Fluid absorption in endoscopic surgery. Br J Anaesth. 2006;96:8–20.l 3. Shah HN, Kausik V, Hegde S, Shah JN, Bansal MB. Evaluation of fluid absorp- tion during holmium laser enucleation of prostate by breath ethanol technique. J Urol. 2006;175:537–40. 3. Shah HN, Kausik V, Hegde S, Shah JN, Bansal MB. Evaluation of fluid absorp- tion during holmium laser enucleation of prostate by breath ethanol technique. J Urol. 2006;175:537–40. 4. Bapat S, Umranikar S, Satav V, Bapat A, Joshi A, Ranade G. Comparison of fluid absorption during transurethral resection of prostate and Holmium- Yag laser enucleation of benign adenoma of prostate using breath ethanol concentration. Indian J Urol. 2007;23:126–9. 5. Hanson RA, Zornow MH, Conlin MJ, Brambrink AM. Laser resection of the prostate: implications for anesthesia. Anesth Analg. 2007;105:475–9. In conclusion, this case report documents the hyper- chloremic metabolic acidosis and upper airway edema possibly caused by saline absorption during the tran- surethral procedure. Absorption of iso-osmotic fluid such as saline is characterized by the expansion of extracellular space and accompanying facial and airway edema. Application of the simplified Stewart approach was useful for interpreting the cause of metabolic acidosis. 6. Fayad AS, Elsheikh MG, Zakaria T, Elfottoh HA, Alsergany R, Elshenoufy A, et al. Holmium laser enucleation of the prostate versus bipolar resection of the prostate: A prospective randomized study. “Pros and Cons”. Urology. 2015;86:1037–41. 7. Shigemura K, Fujisawa M. Current status of holmium laser enucleation of the prostate. Intern J Urol. 2018;25:206–11. 7. Shigemura K, Fujisawa M. Current status of holmium laser enucleation of the prostate. Intern J Urol. 2018;25:206–11. 8. Grove JJ, Shinaman RC, Drover DR. Noncardiogenic pulmonary edema and venous air embolus as complications of operative hysteroscopy. J Clin Anesth. 2004;16:48–50. 8. Grove JJ, Shinaman RC, Drover DR. Noncardiogenic pulmonary edema and venous air embolus as complications of operative hysteroscopy. J Clin Anesth. 2004;16:48–50. 9. Dodd SE, Jankowski CJ, Krambeck AE, Gali B. Metabolic acidosis with hemodilution due to massive absorption of normal saline as bladder irrigation fluid following holmium laser enucleation of prostate. J Anesth. 2016;30:1060–2. 9. Dodd SE, Jankowski CJ, Krambeck AE, Gali B. Funding 16. Seifter JL. Integration of acid-base and electrolyte disorders. New Engl J Med. 2014;371:1821–31. 16. Seifter JL. Integration of acid-base and electrolyte disorders. New Engl J Med. 2014;371:1821–31. Intramural funding. Intramural funding. 17. Magder S, Emami A. Practical approach to physical-chemical acid-base management. Stewart at the bedside. Ann Am Thorac Soc. 2015;12:111–7. 17. Magder S, Emami A. Practical approach to physical-chemical acid-base management. Stewart at the bedside. Ann Am Thorac Soc. 2015;12:111–7. Declarations Ethics approval and consent to participate Not applicable. Availability of data and materials Th d l d i hi d Availability of data and materials h d l d h d The datasets analyzed in this study are included in this published article. Page 4 of 4 Tabuchi et al. JA Clinical Reports (2022) 8:20 Tabuchi et al. JA Clinical Reports (2022) 8:20 Tabuchi et al. JA Clinical Reports Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations.
https://openalex.org/W3184135258	https://www.researchsquare.com/article/rs-38206/v1.pdf?c=1631859474000	English	null	Macromolecule Biomaterials Promote the Osteogenic Differentiation Capacity of Equine Adipose -Derived Mesenchymal Stem Cells	Research Square (Research Square)	2,020	cc-by	12,056	Macromolecule Biomaterials Promote the Osteogenic Differentiation Capacity of Equine Adipose -Derived Mesenchymal Stem Cells Page 1/29 Adipose -Derived Mesenchymal Stem Cells Mohamed I. Elashry (  mohammed.elashry@vetmed.uni-giessen.de ) Justus Liebig Universitat Giessen https://orcid.org/0000-0002-6312-2182 Nadine Baulig Institute of Veterinary Anatomy, Histology and Emryology, Justus Liebig University of Giessen Alena-Svenja Wagner clinic of small animals, c/o Institute of Veterinary Anatomy, Histology and Embryology, Justus Liebig University of Giessen, 35392 Michele C. Klymiuk Institute of Veterinary Anatomy, Histology and Embryology, Justus Liebig university of Giessen Benjamin Kruppke Institute of materials Science, Max Bergmann center of Biomaterials, Techniche Universität, Dresden Thomas Hanke Institute of Materials Sciences, Max Bergmann Center of Biomaterials, Techniche Universität, Dresden Sabine Wenisch Clinic of small Animals, c/o Institute of Veterinary Anatomy, Histology and Embryology, Justus Liebig University of Giessen Stefan Arnhold Institute of Veterinary Anatomy, Histology and Embryology, Justus Liebig University of Giessen Research Keywords: Stem cells, osteogenic differentiation, biomaterials Posted Date: July 1st, 2020 DOI: https://doi.org/10.21203/rs.3.rs-38206/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published on February 12th, 2021. See the published version at https://doi.org/10.1186/s13287-021-02146-7. Justus Liebig Universitat Giessen https://orcid.org/0000-0002-6312-218 Alena Svenja Wagner clinic of small animals, c/o Institute of Veterinary Anatomy, Histology and Embryology, Justus Liebig University of Giessen, 35392 Michele C. Klymiuk Institute of Veterinary Anatomy, Histology and Embryology, Justus Liebig university of Giessen Research Version of Record: A version of this preprint was published on February 12th, 2021. See the published version at https://doi.org/10.1186/s13287-021-02146-7. Abstract Background: Combination of mesenchymal stem cells (MSCs) and biomaterials is a rapidly growing approach in regenerative medicine particularly for chronic degenerative disorders including osteoarthritis and osteoporosis. In the present study, the effect of biomaterial bone substitutes on equine adipose derived MSCs morphology, viability, adherence, migration and osteogenic differentiation were investigated. Methods: MSCs were cultivated in conjunction with collagen CultiSpher-S Microcarrier (MC), nanocomposite xerogels B30 and B30Str biomaterials in osteogenic differentiate medium either under static or mechanical fluid shear stress (FSS) culture conditions. The data were generated by histological means, life cell imaging, cell viability, adherence and migration assays. Osteogenic differentiation was detected by semi-quantification of alkaline phosphatase (ALP) activity, matrix mineralization using Alizarin Red S (ARS) staining and quantification of the osteogenic markers; runt related transcription factor 2 (Runx2) and alkaline phosphatase (ALP) expression using RT-qPCR. All data were statistically analyzed using ANOVA. Results: The data revealed that combined mechanical stress with MC but not B30 enhanced MSCs viability and promoted their migration. Combined osteogenic medium with MC, B30 and B30Str increased ALP activity compared to cultivation in basal medium. Osteogenic induction with MC, B30 and B30Str resulted in diffused matrix mineralization by means of ARS. FSS increased the viability in the presence of the osteogenic medium with MC but not B30 or B30Str. FSS enhanced osteogenic differentiation in the presence of B30Str. Upregulation of Runx2 and ALP expression was detected with osteogenic differentiation together with B30 and B30Str regardless of static or FSS culture. Conclusions: Taken together, the data revealed that FSS in conjunction with biomaterials promoted osteogenic differentiation of MSCs. This combination may be considered as a marked improvement for clinical applications to cure bone defects. Version of Record: A version of this preprint was published on February 12th, 2021. See the published version at https://doi.org/10.1186/s13287-021-02146-7. Page 1/29 Page 1/29 1. Introduction Cell-based stem cell therapy together with biomaterials is a rapidly growing approach for tissue regeneration. The aim is to repair and restore normal tissue function by means of stem cells together with biological macromolecules as scaffold materials. Such approach may provide a new therapeutic option for the treatment of chronic degenerative disorders that are currently incurable with ordinary routine applications. Osteoporosis is a disease causing bone fragility, risk of fracture and insufficient bone healing due to deterioration of the bone microarchitecture [1, 2]. Scarcity of effective treatment has encouraged seeking for alternative therapeutic approaches [3–5]. Currently, autologous and allogenic bone grafts are the most appropriate approach to restore bone defects [6]. However, the limited bone availability and the necessity Page 2/29 Page 2/29 of an invasive and expensive surgical intervention have fostered the promotion of the development of substitute materials. Using biomaterials substitutes for bone tissue engineering allows developing biological scaffolds capable of restoring, maintaining and improving tissue function [7]. Developing a biological substitute to refill bone defects requires a combination of a proper scaffold biomaterial that maintain a suitable niche for mesenchymal stem cell differentiation [8, 9]. Certain criteria have to be considered to select the biomaterial; (1) it should mimic the target tissue components in terms of biocompatibility and biodegradability, (2) it has biomechanical and osteoinductive properties and (3) the biomaterial must be easy to establish, efficient and economical to facilitate clinical application [6, 8]. A novel biomimetic silica-collagen hybrid nanocomposites has been developed that meet the required criteria [10]. Inspired by the siliceous basal spicules of marine glass sponges, it represents a combination of the organic bovine tropocollagen type I and inorganic silicate [11]. Both ingredients of this nanocomposite are physically found in bone tissue and have been proven to exert a positive influence on bone healing. Collagen is the most abundant framework protein in mammalian tissues which provides a promising component of a biomaterial due to low antigenicity, low inflammatory and low cytotoxicity. Moreover, it has the ability to promote cell adhesion, proliferation and differentiation and it is responsible for biocompatibility, controlled biodegradation and the maintenance of signal transduction [11–13]. Silicon is one of the most abundant trace element in living organism. A group bunch of evidence demonstrated the important role of silicon for bone metabolism and formation that have been proven under in vitro and in vivo investigation [14, 15]. 1. Introduction It has been found that silicon not only increased the collagen synthesis within the extracellular matrix of human osteoblasts but also it promoted their proliferation and differentiation [16– 18]. In addition, it plays an important role as an initiator for bone mineralization and formation [19, 20]. Furthermore, Silicification reinforces the collagen fibrils leading to improved mechanical stability of the biomaterial [21]. The combined silica and collagen provides inorganic/organic composite with promising characteristics regarding chemical, structural and technological parameters for the biomaterial suitable for bone engineering [13, 21, 22]. Currently, a group of evidence have shown the effectiveness of collagen based microcarrier scaffold for MSCs proliferation and chondrogenic differentiation [23]. Similar study has combined MC with chitosan hydrogel revealed an enhanced metabolic activity and surface adhesion of chondrocytes suggesting the usefulness of the composite for tissue regeneration [24]. The criteria of cell viability, adhesion and detachement would determine the quantity of cells available for regeneration and production of the therapeutic effect. Additionally, the porosity of MC and surface adhesion support cell attachement, nutrient transfer and a reservoir system to deliver active biological molecules [25]. Mesenchymal stem cells (MSCs) are multipotent undifferentiated adult stem cells, extractable from various tissues including bone marrow and adipose tissue. These cells have the ability for self-renewal and differentiation potential into bone, fat and cartilage [26]. The osteogenic differentiation of MSCs provides a valuable tool for bone tissue engineering. Furthermore, the accessibility, straightforward isolation and immunomodulatory properties of MSCs demonstrate their usefulness in cell-based therapy for bone regeneration [27]. However, the effect of biomaterials on the cell viability, adherence, migration and osteogenic differentiation of MSCs are not fully elucidated yet. Thus, the present study aims to Page 3/29 Page 3/29 investigate the effect of biomaterial substitutes on equine adipose derived mesenchymal stem cells (MSCs) morphology, viability, adherence, migration ability and osteogenic differentiation potential. MSCs were co-cultivated in conjunction with collagen CultiSpher-S Microcarrier (MC), nanocomposite xerogels B30 and B30Str biomaterials. These combinations were allowed to differentiate into the osteogenic fate up to day 21 either under static (ST) or mechanical fluid shear stress (FSS) culture conditions. Data were generated by histological investigations as well as by evaluation of cell viability, cell adherence and the migratory potential. 2.1.1 Nanocomposite xerogels (B30 and B30Str) The silica-collagen nanocomposites were kindly provided from the Max Bergmann Center of Biomaterials and Institute of Materials Science from Dresden University of Technology and preparation was carried out as described by [11, 22]. Our investigations were performed with xerogel granules. Therefore, the silica- collagen monoliths were ground into a powder and then classified into different particle fractions. In these investigations fractions with a particle size < 0.125 mm were examined. 2.1.2 CultiSpher-S Microcarrier (MC) Preparation of the CultiSpher macroporous gelatin microcarriers was performed according to the company instruction (Percell, Biolytica AB, Sweden). Briefly, the dry MC were rehydrated in phosphate buffered saline (PBS, 1g/50 mL) for one h then were autoclaved at 121 °C for 15 min. The PBS was removed and fresh PBS was added then MC were washed three times in culture medium. Generally, 1x 105 cells were mixed with 0.1 % MC in DMEM for all experimental setups. 1. Introduction Osteogenic capacity was assessed by semi-quantification of the alizarin red S staining (ARS), alkaline phosphatase (ALP) activity and quantification of the osteogenic markers runt related transcription factor 2 (Runx2) and alkaline phosphatase (ALP) expression. Our data revealed the influence of various scaffolds biomaterials on stem cells criteria including morphology, attachment and cell migration. We show enhanced cell viability in the presence of MC and mechanical FSS. An increased ALP activity in the osteogenic differentiation together with B30 under static culture could be shown. We report that mechanical FSS together with MC improves matrix mineralization. We show that combined mechanical FSS and B30/B30Str increases osteogenic differentiation indicated by upregulated ALP and Runx2 expression. All together underpinned the effect of various biomaterials on osteogenic commitment of MSCs which suggest their usefulness for regenerative applications in veterinary medicine. 2.2. Isolation of adipose tissue derived MSCs Page 4/29 Page 4/29 Experiments were accomplished from equine adipose mesenchymal stem cells MSCs (N=8; aged 7.66 ± 1.34 years). Equine MSCs of both sex were examined as previously reported by (Elashry et al., 2018). Briefly, the adipose tissue was obtained from the subcutaneous fat from horses being slaughtered at the local abattoir and the Institute of Veterinary Pathology, Justus- Liebig University in Giessen. MSCs were transferred in cold PBS supplemented with 1 % penicillin-streptomycin (P/S, Gibco® life technologies, Germany). Adipose tissue samples were cut into 1mm2 pieces using sterile scalpel then were washed twice in PBS for 5 min. The samples were digested under shaking in 0.1 % collagenase I (Biochrom AG, Germany) in 1 % bovine serum albumin dissolved in PBS for 60 min at 37 °C. The homogenates were filtered in 70 µm cell strainer and were centrifuged at 240 g for 5 min. The supernatant was discarded and the pellets were suspended in fresh Dulbecco’s Modified Eagle’s Medium (1g/L, DMEM, Gibco® life technologies, Germany). The cells were counted using a hemocytometer, were expanded in DMEM containing 10 % foetal bovine serum (FBS, Biocell, Germany) and 1 % P/S growth medium within the T75 culture flasks. The cells were incubated at 37 °C, 5 % CO2 and 90 % humidity up to confluency. In the current study, MSCs were characterized either by flow cytometry using the surface marker CD90 also using PCR for the expression of stem cell markers CD90, CD 105 and Oct 4 as previously reported by our group [28, 29]. Upon confluency, cells were detached using TryplE (Gibco® life technologies), were counted using a hemocytometer and were stored in 1 mL aliquots at -160 °C. Medium change was carried out twice a week and cells of passage 2-4 were used for the following experiments. After expansion, 4x106 cells and 20 mg of MC and B30 biomaterials were cultivated in 10 mL growth medium using two different settings: a static culture in falcon tubes (Sarstedt, Germany) and a rotating bioreactor (Rotary cell culture systems, Synthecon Inc., Houston, TX, USA). To enable proper gas exchange, the falcon lid was perforated before incubation. In the rotating culture, the vessel was allowed to perform 11 cycles/min. Both settings were incubated at 37 °C with 5 % CO2 for four days without carrying out a medium change. 2.2. Isolation of adipose tissue derived MSCs Afterwards, the medium with biomaterial-cell- complexes (from both experimental settings) was carefully transferred to a new 15 mL falcon tube. Two washing steps followed by 5 min interval until all complexes were precipitated. The latter was suspended in 10 mL fresh medium for 5 min to select all non-attached cells using various sedimentation speed. The harvested complexes were used for the consecutive experiments 2.6. Transmission electron microscopy (TEM) To assess the ultramicrocellular morphology of the cell-biomaterial in combined culture, the complexes were fixed in cacodylate buffer solution (pH 7.2) containing 2 % PFA, 0.02 % picric acid (Fluka, Germany) and glutaraldehyde (Sigma Aldrich, Germany) for 24 hours at 4°C. After that, the complexes were post fixed in 1% osmium tetroxide (Sigma Aldrich, Germany) in 0.1 M cacodylate buffer for two hours at room temperature. The complexes were counterstained with 0.5 % uranyl acetate (Delta microscopy) for 30 min and 0.2 % lead citrate for 80 sec. Then, complexes were dehydrated and embedded in Peon (Sigma- Aldrich). Ultrathin 70nm sections were generated using an ultramicrotome (Reichert - Jung Ultracut, Leica Microsystems) and were examined using a TEM (EM 109, Zeiss, Germany). 2.4. Phalloidin staining Complexes were fixed in 4 % PFA, were washed twice for 5 min interval and were then incubated with phalloidin, an actin filaments cytoskeleton staining (2, 5 %, Sigma-Aldrich, Germany) for 30 min. The nuclei were counterstained with 0. 05 % Hoechst dye in TBS for five min (Invitrogen, USA). After the complexes were stained, they were transferred in PBS buffer on a microscopic slide and were examined using fluorescence microscope (Axio Observer.Z1, Carl Zeiss, Germany). 2.3. Histological staining The biomaterial-cell-complexes were fixed with 4 % paraformaldehyde (PFA, Roth, Germany) for 20 min at 4°C followed by two washing steps in distilled water. The cell biomaterials complexes were dehydrated in ethanol (Roth, Germany), cleared in xylene (Roth, Germany) and were processed for paraffin embedding. Sections of 6 µm were produced using a microtome(Leica, Germany), were transferred to glass slides (Roth, Germany) and were processed for hematoxylin and eosin histological staining to get an overview of cell-biomaterial interaction. The sections were mounted using Eukitt mounting media (HICO-MIC, Hirtz & Co, Germany) and were examined using light microscopy microscope equipped with a digital camera and the LAS V4.4 software (Leica, Germany). Page 5/29 2.5. Scanning electron microscopy To examine the morphology of the biomaterial surface and the appearance of attached cells, a scanning electron microscopy (SEM) was performed. Cell-biomaterial complexes were fixed in 2 % glutaraldehyde in cacodylate buffer (Merck, Germany). The specimens were dehydrated in ethanol gradient for ten min and afterwards, were coated by hexamethyldisilazane (Merck) overnight. After drying, they were sputter- coated with Au/Pd with a Balzer sputter coater (SCD 020, Balzers Union, Germany) and examined by Digital Scanning Microscope (DSM 940, Zeiss at 15kV and 16 mm working distance). The cell biomaterial complexes were photographed using a Digital Image Scanning System 5 and Digital Image Processing System 2.9 (Point Electronic, Germany). 2.9. Osteogenic differentiation induction To examine the osteogenic differentiation capacity of combined cultivation of MSCs together with biomaterial (B30, B30Str and MC), the cells were cultivated in growth medium for 48 h to facilitate cell attachment on biomaterials. The cells were allowed to differentiate in osteogenic medium containing DMEM, 5 % FBS, 0.05 mM ascorbic acid-2-phosphate (Sigma-Aldrich, Germany), 10 mM β- glycerolphosphate (Sigma-Aldrich, Germany) and 0.1 µM dexamethasone (Sigma-Aldrich, Germany) up to 21 days. Cells were provided with fresh medium twice a week. Cells were incubated in parallel either in BM (5 % FBS in DMEM and 1 % P/S) or osteogenic differentiation medium without biomaterials were served as control (NC). Osteogenic differentiation was assessed by evaluating the morphological alteration, histological examination using alizarin red S (ARS) staining, alkaline phosphatase (ALP) activity and osteogenic relative markers at day 7, 14 and 21 post induction. To examine the osteogenic differentiation capacity of combined cultivation of MSCs together with biomaterial (B30, B30Str and MC), the cells were cultivated in growth medium for 48 h to facilitate cell attachment on biomaterials. The cells were allowed to differentiate in osteogenic medium containing DMEM, 5 % FBS, 0.05 mM ascorbic acid-2-phosphate (Sigma-Aldrich, Germany), 10 mM β- DMEM, 5 % FBS, 0.05 mM ascorbic acid 2 phosphate (Sigma Aldrich, Germany), 10 mM β glycerolphosphate (Sigma-Aldrich, Germany) and 0.1 µM dexamethasone (Sigma-Aldrich, Germany) up to 21 days. Cells were provided with fresh medium twice a week. Cells were incubated in parallel either in BM (5 % FBS in DMEM and 1 % P/S) or osteogenic differentiation medium without biomaterials were served as control (NC). Osteogenic differentiation was assessed by evaluating the morphological alteration, histological examination using alizarin red S (ARS) staining, alkaline phosphatase (ALP) activity and osteogenic relative markers at day 7, 14 and 21 post induction. 2.7. Life cell imaging To investigate the cell viability and the capacity to spread out from the biomaterial after having attached on their surface, life cell imaging was carried out. Cell-biomaterial complexes were placed into a 35x10 mm culture dish (VWR, Germany) with 3 mL basic medium and then incubated for 60 min under standard culture conditions. Life cell imaging was implemented using Axio Observer Z1, Temp Module S, CO2 Module S, Zeiss, Germany). Incubation parameters were established at 37 °C and 5 % CO2. The images were taken every 15 min up to 72 h. The analysis was performed using the Axiovision Imaging Software (Zeiss, Germany). Furthermore, the complexes were seeded in 24-well plastic dishes and were incubated Page 6/29 Page 6/29 for 24 h under standard conditions with basic medium. After this, cells were fixed in 4 % PFA for 20 min and were stained in phalloidin. 2.8. MTT cell viability assay To evaluate the viability of cells already attached on the biomaterial, a colorimetric MTT assay was performed. Viable cells are able to reduce the yellow substrate MTT (3- 4, 5-Dimethylthiazol-2yl -2, 5- diphenyltetrazoliumbromid, Sigma-Aldrich, Germany) to the insoluble blue formazan, the later can be solubilized and measured by a spectrophotometer reader at a specific wavelength. Therefore, 2x106 cells with MC, B30 and B30Str were incubated in the two mentioned settings for 4 days. After washing twice in basal medium (BM), the cells and biomaterials were incubated with MTT for 12 h. The cells were lysed with 200 µL of Dimethylsulfoxid (DMSO, AppliChem, Germany) together with a tissue Lyser in oscillation frequency of 50 HZ (Quiagen, Germany) for 5 min. Hereafter, the color intensity was measured by a microplate reader at 570 nm (Tecan, Germany) and was analysed with Magellan TM – Data Analysis Software (Crailsheim, Germany). 2.10. Mechanical fluid shear stress MSCs were seeded in combination with MC, B30 and B30Str in a ratio 1x105 per well in a 24-well culture plates (VWR, Germany. After 48 h, the growth medium was replaced by the osteogenic differentiation medium (OD). The plates were divided into two experimental groups; static (ST) and mechanical fluid shear stress (FSS) culture conditions. In the latter, the cells were incubated under a regular vertical rocking pattern. To optimize the mechanical stress, a rocking angle of 10° with a frequency of 40 turns/min and a fluid depth of 3.13 mm were generated. The formula for the FSS calculation was previously described by [30]. The assumed fluid viscosity of 10-3 Pa s, the FSS was 77.21 mPa (in non SI-units 0.77 dyn/cm²) at Page 7/29 Page 7/29 the center of the dish bottom. The experimental setup guaranteed that the cells were covered with medium during plates cycling. In parallel, cells were grown in parallel in BM served as negative control. 2.11. Alizarin Red S staining (ARS) ARS specific dye with affinity to “Ca2+” used as an indicator for Ca2+ deposition in the mineralized matrix. Fixed cells in 4 % PFA were washed in distilled water three times at 5 min interval. The cells were incubated with 1 % ARS staining solution (pH 4.2) (Roth, Germany) at room temperature for 30 min. Excess of staining was washed three times in distilled water for 5 min to remove the unbound dye. The morphological alterations following each time point were examined and were photographed using light microscope equipped with a digital camera and the LAS V4. 4 Software (Leica, Germany). 2.12. Semi-quantification of ARS staining ARS stained cells were washed twice in distilled water then were incubated with a volume of 2 mL of 10 % Cetyl Pyridinium Chloride (CPC, Roth Germany) with a moderate shaking for 60 min. An equal volume from each experimental group was transferred to a 96-well microplate. The absorbance was measured in triplicates at 562 nm using microplate reader (Tecan, Germany). 2.13. Measurement of alkaline phosphatase (ALP) activity Cells were incubated with 500 µL of 1 % TritonTM X-100 in 10 mM Tris (pH 7.4) at 4 º C for 60 min. The cells/biomaterials complexes were transferred into a 1.5 mL Eppendorf tube. Cell lysates were centrifuged at 28400 g for two min at room temperature and kept on ice. P-Nitrophenylphosphate (2 mg/mL) was dissolved into buffer solution containing 1M Tris and 5 mM MgCl2 (pH 9.0). A volume of 50 µL of cell lysate were incubated with 150 µL of p-Nitrophenylphosphate solution for up to 30 min. The mixtures were loaded in triplicates into a 96-well microplates. The standard of p-nitrophenol solution was used to prepare the standard curve. ALP potency to hydrolyze p- Nitrophenylphosphate into p-Nitrophenol (PNP) was measured as previously reported [31]. The absorbance was measured at 405 nm by using a microplate reader. The rate of ALP activity was measured using the equation, Y = mx+b. Where, x= sample absorbance value and Y= pNP concentration of samples. 2.14. Real-time quantitative polymerase chain reaction (RT- PCR) Total RNA was extracted from cells cocultivated with MC, B30 and B30Str at day 7, 14 and 21 post osteogenic induction using an innuPREP RNA mini kit (Analytik, Jena AG, Germany) and was quantified using a UV-Spectrophotometer (Bio-Photometer, Eppendorf AG). Cells in parallel were examined either Page 8/29 Page 8/29 incubated in BM or osteogenic medium without biomaterials were served as controls (NC). Briefly, RNA samples were incubated with 3.9 µL per sample DNase mix containing 1.2 µL of 25 mM MgCl2, 1.2 µL DNase buffer I, 1.2 µL DNase I and 0.3 µL RNase inhibitor in 100 µL PCR tubes. The mixture was incubated at 37 °C for 25 min then at 75 °C for 5 min in a thermal cycler (Bio-Rad, Germany). RNA samples were reverse transcribed into cDNA in a master mix containing 45 µL RT-Mix, 3µL RNase inhibitor and 3 µL reverse transcriptase multiscribe (Promega). The reaction was generated at 21°C for 8 min, at 42 °C for 15 min, at 99 °C for 5 min, at 5 °C for 5 min in the Bio-Rad thermal cycler. To examine the validity of the PCR reaction and the quality of the primers, a qualitative PCR reaction was performed. All primers were purchased from (microsynth, Germany) and were listed in table (1). Glyceraldehyde-3- phosphat dehydrogenase (GAPDH) was used as an endogenous reference. PCR fragments were allowed to run in a 2 % agarose gel electrophoresis at 180 V for 20 min and were examined under UV light. Relative osteogenic markers Runx2 and ALP were quantified using RT-qPCR after 14 and 21 days. The reaction of 1 µL of cDNA template with 9 µL of a mixture composed of 5 µL 2 x SYBR Green PCR Master Mix (Bio-Rad), 0.6 µL of 10 pM forward and reverse primers mix and 3.4 µL nucleic acid free water was carried out. The samples were loaded in triplicates in a 96-well PCR plates in a real time cycler (CFX96, Real-Time PCR Detection System, Bio-Rad). The following PCR condition was followed; activation at 95 °C for 2 min, 40 cycles at 95 °C for 15 sec, and 60 °C for 30 sec, followed by a melting curve (60-95 °C for 3 sec). PCR-Mix without adding cDNA was served as a negative control. The qPCR data was relatively normalized to GAPDH expression. 2.14. Real-time quantitative polymerase chain reaction (RT- PCR) The expression of each target gene was analyzed using 2-ΔΔCt equation as reported by [32]. 3.1 Morphological evaluation of MSCs cultivated with biomaterials In order to examine the morphology and the adherence capacities of MSCs cultivation in combination with biomaterials, MSCs were seeded in growth medium with MC and B30, B30Str biomaterials. SEM of the MC and B30 granules biomaterials without MSCs could be shown in (fig. 1a, b). Equine MSCs exhibited a complete coat around the MC (fig. 1c). However, B30 -granules of different shapes and sizes were combined by a plethora of MSCs forming large cell-granule-complexes. The xerogel-granules themselves have a rather smooth surface with partially rough areas most likely formed by silica particles in conjunction with collagen fibrils. MSCs displayed cytoplasmic extensions for contacting adjacent cells and to facilitate secure attachment on the scaffold`s surface. Also, a solid network consisting of cells, cell cytoplasmic extensions with B30/B30str granules can be figured (fig. 1c, d). In contrast, the typical spindle-like fibroblast appearance of MSCs was detected in the culture dish without adding the biomaterials (fig. 1e), however, the cells seeded with MC formed a sphere-like accumulation and surface adhesion around these particles (fig. 1f). The ordinary histological sections showed MSCs invasion through the pores of MC biomaterials indicating the cell viability (fig. 1g, h). In order to examine MSCs`s capacity to spread out of the biomaterials, two culture protocols were deployed. Combined MSCs together with B30 or with MC were cultivated either under static culture (SC) and mechanical rotation (RC) culture conditions. The complex was transferred into a culture dish then a life cell imaging was recorded for 72 h. After static culture, the combined cultivation of MSCs and MC showed a homogeneous cells and biomaterial aggregates however, combined MSCs and B30 displayed scattered biomaterial fragments with attached cells (fig. 1i, j). Following the complex attachment to the culture dish, cells cultivated with MC under RC showed a comparatively a faster cell spreading out of the pellet compared to either MC under SC or B30 culture condition (fig. 1i-l). An abundant settlement of cells could be demonstrated either on the surface at single locations or in clusters for B30 and MC (fig. 1i-l). In order to examine MSCs`s capacity to spread out of the biomaterials, two culture protocols were deployed. Combined MSCs together with B30 or with MC were cultivated either under static culture (SC) and mechanical rotation (RC) culture conditions. The complex was transferred into a culture dish then a life cell imaging was recorded for 72 h. 2.15. Statistical analysis The influence of combined osteogenic induction together with MC, B30 and B30Str biomaterials on the osteogenic differentiation of MSCs were investigated. Data were collected under static (ST) and mechanical fluid shear stress (FSS) conditions at days 7, 14 and 21 post induction. In order to analyze the effect of biomaterials (MC/SC, MC/RC, B30/ST and B30/RC) on cell viability compared to BM, One way ANOVA was carried out. To evaluate the effect of biomaterials (MC vs. B30) at different time point (T0 up to 72 h) on the migration capacity of MSCs, a two-way ANOVA was examined. To assess whether various biomaterials alter the migration potential of MSCs, a two-tailed Pearson (r) correlation coefficient analysis was carried out. The effect of osteogenic induction (OD vs. BM) concurrently with biomaterials (MC, B30 and B30Str vs. NC) on cell viability, ALP activity, ARS staining and the osteogenic related markers (Runx2 and ALP) expression, a two-way ANOVA was carried out. Multiple comparisons were tested using Tukey’s and Sidak’s post hoc tests. The data from triplicates presented as the mean ± SEM and p value ≤ 0.05 was considered to be significant. All the statistical analyses were carried out using Graph Pad Prism 7.0 (La Jolla, Canada). Page 9/29 Table 1 Table 1 Table 1 Primers sequences used for RT-qPCR analysis Gene Forward Reverse Size (bp) GAPDH CCAGCAAGAGAAGGAGAAAGG AACTGTGGAGGTCAGGAGAC 93 Runx2 ACCGACAGCCCCAACTTC ACCCGCCATGACAGTAACC 132 ALP GACTGGTACTCGGACAACGAG GTTCTTGGGGAACATGTACTTC 137 3.1 Morphological evaluation of MSCs cultivated with biomaterials After static culture, the combined cultivation of MSCs and MC showed a homogeneous cells and biomaterial aggregates however, combined MSCs and B30 displayed scattered biomaterial fragments with attached cells (fig. 1i, j). Following the complex attachment to the culture dish, cells cultivated with MC under RC showed a comparatively a faster cell spreading out of the pellet compared to either MC under SC or B30 culture condition (fig. 1i-l). An abundant settlement of cells could be demonstrated either on the surface at single locations or in clusters for B30 and MC (fig. 1i-l). 3.2. Effect of biomaterials on MSCs viability and migration potential Page 10/29 To get further insight into cell-scaffold interaction, cell motility was assessed following 4 days of SC and RC conditions using life cell imaging. In this study the migration capacity can also be used as a quantitative tool for cell viability. Motility of cells that had attached to the different scaffold materials was studied at 15 min intervals over 72 h using the life cell imaging set up. Following the establishment of biomaterial/cell- cell attachment, cells started to grow out of the biomaterials and to migrate in a radial fashion into the vicinity of the scaffolds via developing long cytoplasmic processes. In addition, those cells cultivated in conjunction with MC demonstrated enhanced viability and proliferation rate under RC in comparison to SC condition (fig. 1k, l). Interestingly, within the cytoplasm of stem cells migrating away from B30 granules small particles can be detected. The quantity of internalized particles per cell is variable. Given the fact that B30/B30Str granules show a strong autofluorescence using the appropriate wave length suitable for the detection of the phalloidin staining, the assumption suggests, that the cells were indeed able to incorporate parts of the B30 granules via autophagy in order to gradually degrading them. This observation was confirmed by TEM of the cells and pellet complexes (fig. 1m, n). In some cases, after having migrated away from the scaffold material, MSCs were observed to re-invade the scaffold particles. In order to examine the effect of combined cultivation of various biomaterials on MSCs viability, MTT assay under SC and RC was carried out. After 5 days in growth medium, a significant increase in cell viability for the cells cultivated with MC under both SC and RC condition (p<0.01, p<0.001) compared to the BM without biomaterial could be observed. In contrast, combined mechanical rotation with B30 displayed a reduction in the cell viability (p<0.05) when compared to BM (fig. 1o). To assess our data, a quantification of the cross sectional area (CSA) of the biomaterial with attached MSCs at different time point was performed. The analysis revealed that significant increase in the CSA of B30/MSCs complex was observed after 36 h (p< 0.01) compared to T0. Then, a gradual increase of the size of the B30/MSCs complex was found up to 72 hours (p< 0.001) compared to 36 h. In contrast, the cell migration out of the MC showed different patterns considering the same time scale. 3.2. Effect of biomaterials on MSCs viability and migration potential MSCs/MC complex demonstrated a faster increase in the CSA already after 24 h (p< 0.01) compared to T0. Interestingly, The CSA of the MSCs/MC complex showed a significant increase at 36 h (p< 0.01) compared to 24 h which remains stable up to 72 h (fig. 1p). No significant alteration in the CSA of the pellet for B30 and MC in the initial 12 h of the complex together. Further data analysis revealed a significant linear correlation between the migration speed of MSCs out of the pellet and the cultivation time (fig. 1q). These data point out the pattern of cell migration out of the pellet in a temporo-spatial comparison. 3.3. Combined MSCs with Osteogenic differentiation under t ti lt To understand the effect of various biomaterial compounds on MSCs fate, the cells were allowed to differentiate into the osteogenic lineage using a standard osteogenic induction cocktail (OD) under static culture condition. Evaluation of the alkaline phosphatase activity (ALP) as an indicator for osteogenic differentiation was performed up to 21 days post induction. The quantification of absorbance showed a Page 11/29 Page 11/29 significant increase in the ALP activity in MSCs either combined with MC (p<0.01), B30 (p<0.05) and B30Str (p<0.01) or without adding biomaterials (NC, p<0.01) at day 7 compared to those cells grown in BM (fig. 2a). Similar observations were found in all cell-biomaterial complexes at day 21 (p<0.001) compared to the respective non induced cells in BM (fig. 2c). The MSCs/B30Str complex showed no significant change in ALP activity at day 14 compared to the control cells in BM (fig. 2b). To assess the osteogenic potential using the different biomaterials, ARS staining was semi-quantified at days 14 and 21 using the CPC assay. The analysis of CPC values showed significantly increased values in the presence of MC together with osteogenic induction medium at day 14 (p<0.05) compared to those cells cultivated in BM only. Furthermore, the combined MC and osteogenic induction revealed increased CPC values (p<0.05) compared to either osteogenic induction without biomaterials (NC) or the osteogenic induced cells cultivated with the B30 biomaterial (fig. 2d). Similarly, an increased CPC absorbance could be found in cells which were cultivated in the presence of MC and the osteogenic differentiation medium at day 21 (p<0.05) compared to the respective MSCs/MC complexes cultivated in the BM medium only (fig. 2e). Although, there was a moderate increase in CPC values for the combination of cells cultivated with B30 or B30Str in the osteogenic medium detectable, it was not statistically significant when compared with the respective biomaterials in BM. In order to confirm our data, a morphological examination of the osteogenic induced cells in combination with various biomaterials was carried out using ARS staining. Cellular aggregation-like nodules stained in red were detected using the standard OD cocktail compared to BM conditions (fig. 2f-i). By using MSCs combined with MC, B30 and B30Str revealed a diffused ARS staining at day 14 which became more disseminated at day 21 post induction (fig. 2j-o). 3.4. Combined MSCs with Osteogenic differentiation under FSS culture In order to examine whether mechanical FSS influences the osteogenic differentiation using various biomaterials, osteogenic differentiation was performed under FSS culture condition up to day 21. The analysis showed that FSS clearly increased MSCs viability in the presence of MC (p<0.01 and p<0.001) compared to B30 and B30Str in BM. Furthermore, osteogenic induction in the presence of MC biomaterials enhanced cell viability (p<0.01) compared with the combination of B30 and B30Str (p<0.01 and p<0.001) and the osteogenic differentiation medium at day 14 under FSS (fig. 3a). Similarly, the combined FSS and MC biomaterials increased the viability of MSCs in comparison to the osteogenic induction without biomaterials (NC), with B30 (p<0.01) and B30Str (p<0.001) at day 21 regardless of the cultivation medium (fig. 3b). Next, we evaluated whether FSS enhances ALP activity at day 14 using two incubation setups. After 10 min incubation, the analysis showed that osteogenic induction using osteogenic medium without biomaterials or in conjunction with B30 promoted ALP activity (p<0.001) in comparison to MC and B30Str under static condition. Furthermore, combined osteogenic differentiation together with B30Str under FSS was able to increase ALP activity compared to the respective biomaterial under BM (p<0.01). In addition, FSS together with B30Str in osteogenic medium enhanced ALP activity Page 12/29 Page 12/29 compared to NC (p<0.001), MC (p<0.01) and B30 (p<0.05) under the same condition (fig. 3c). The same trend was observed after 30 min incubation, in addition, a significant increase in ALP activity in the combined MC and osteogenic induction (p<0.05) compared to BM under static condition could be seen (fig. 3d). The superior ALP activity was measured in the presence of B30 and the standard osteogenic medium (p<0.001) compared to BM under static culture. Interestingly, however, under FSS an increased ALP activity in the presence of B30Str and osteogenic medium (p<0.001) was observed when compared with the same combination under static culture (fig. 3d). To assess the osteogenic potential of various biomaterials under FSS, ARS was quantified at day 21 using the CPC absorbance assay. By comparing to the non-induced cells, osteogenic induction using MC biomaterial increased Ca2+ deposition as evaluated using the ARS staining (p<0.001). The combined FSS and osteogenic medium including MC biomaterials increased Ca2+ deposition not only compared to standard protocol without biomaterials (NC, p<0.01) but also in comparison to cultivation protocols involving B30 (p<0.001) and B30Str (p<0.01, fig. 3e). 3.5. Evaluation of the osteogenic differentiation marker expression In order to evaluate the effect of combined biomaterials together with osteogenic medium on MSCs differentiation, osteogenic relative marker expression was analyzed at days 14 and 21 under the static and FSS experimental setup. Under static culture conditions in the presence of the osteogenic medium, an upregulation of Runx2 expression was detected in the standard osteogenic medium (NC, p<0.05), B30 (p<0.01) and B30Str (p<0.001) compared to the respective control in BM at day 14. It was clearly shown that B30Str enhanced Runx2 expression compared to NC, MC and B30 experimental condition. Runx2 expression was not detected under combined osteogenic induction and MC biomaterials (fig. 4a). Furthermore, only using the B30 biomaterial a significant Runx2 upregulation at day 21 could be achieved. At this later time point, combined MC and osteogenic induction showed a tendency of Runx2 upregulation (p=0.06) compared to the control cells in BM (fig. 4b). Expression of ALP was only detected in the osteogenic induction medium and in the presence of B30 (p<0.01) compared to matched cells in BM at day14. A similar upregulation of ALP expression using the combined osteogenic induction together with the B30 biomaterial was observed (p<0.05) in comparison to osteogenic induction with MC or without cultivation with any biomaterials added at the same time point (fig. 4c). Interestingly, however ALP upregulation at day 21 was found in the presence of control osteogenic induction without any biomaterials (p<0.01), B30 (p<0.001) and in cells cultivated in the presence with B30Str (p<0.05) in comparison to matched controls in BM. The expression of ALP was not detected in the protocol using combined MC and osteogenic medium at both day14 and day21 (fig. 4d). Under day 14 of cultivation of cells under mechanical FSS, the data revealed upregulated Runx2 expression in three out of four of the experimental groups including osteogenic induction together with MC (p<0.001), B30Str (p<0.05) and control osteogenic without biomaterial (p<0.05) compared to control in BM (fig. 4e). However, at day 21 of FSS stimulation, the level of Runx2 was only detected in cells stimulated with osteogenic induction medium together with B30 (p<0.05) and B30Str (p<0.001) in comparison to the control in BM. Page 13/29 Page 13/29 Furthermore, comparing the osteogenic induction using various biomaterials, Runx2 expression displayed two-three folds increase in the B30 and B03Str-based osteogenic induction in comparison to either osteogenic differentiation alone or with MC biomaterials (fig. 4f). 4. Discussion Mesenchymal stem cells and biological bone substitute material offer a promising approach to restore bone defects. Such combination requires a proper selection of scaffold biomaterial that maintain a suitable niche for mesenchymal stem cell proliferation and differentiation. The ideal scaffold should provide a suitable vehicle for cell viability, adhesion, migration, proliferation and differentiation. Optimization of such criteria would impact on the selection of the superior combination for regenerative medicine. In the current study the effect of biomaterial bone substitutes on equine adipose derived mesenchymal stem cells morphology, viability, adherence, migration and osteogenic differentiation were investigated. The data revealed that scaffolds including MC, B30, and B30Str demonstrate suitable delivery vehicles for MSCs in terms of cell adhesion, viable networking and migration within the pores of the biomaterials as shown by histological staining. In addition, MC biomaterial promoted cell viability compared to B30 and B30Str under both static and mechanical FSS. These data suggest that the construction of various biomaterials is a determining factor for MSCs growth and therapeutic potency. It has been found that combined cultivation of chondrocytes with microcarriers in a 3D fashion facilitates phenotype maintenance by delivering mechanical stimulation [33]. Furthermore, MC have the ability to support cell viability and cell adhesion [34]. Although biomaterials play a role for cell survival and proliferation [35, 36], it is questionable whether they also support MSCs differentiation. There are some negative examples. A study has shown that despite the silicon rubber microcarrier promoted cell growth and attachment, the functional differentiation of gastric stem cells was abolished [37]. In our study we could show that MSCs detach from the biomaterial and migrate in a radial-pattern. In some instances cells internalize biomaterial particles into their cytoplasm during the detachment process as could be shown by TEM and phalloidin staining. These data are important criteria indicating and confirming the biocompatibility and biodegradability of the scaffold material under investigation. It has been documented that combined cell culture with collagenous microcarriers are favorable for three important aspects including cell attachment, cell proliferation and cell detachment [38, 39]. In order to evaluate the cell migration out of the biomaterial, we quantified the CSA of complexes with MSCs and biomaterials in the life cell imaging set up. The analysis revealed a faster cell detachment from MC compared to the B30 biomaterial in an observation period of up to 72 h. 3.5. Evaluation of the osteogenic differentiation marker expression The effect of FSS on ALP expression was also monitored at day 14 and 21. The data showed that ALP expression was only upregulated under combined osteogenic differentiation together with B30Str at both time points either compared to BM (p<0.01) or compared to other experimental protocols (p<0.001) (fig. 4g, h). 4. Discussion These results could be referred to the morphological differences and basic construction of MC compared to B30. In the first biomaterial, the spherical form, and the micropores provide stable cell adhesion conditions so that cells spread on the Page 14/29 Page 14/29 whole surface of MC. Thus, there is a large number of cells that may detach after the adhesion of the MC/ cell conglomerate on the culture plate. In contrast, the sharp granular surface of the B30 biomaterial leads to an unstable attachment and thus an impaired cell detachment. These data are congruent with the enhanced cell viability on MC. In agreement with our hypothesis, it has been documented that MC should provide a suitable cell attachment to tolerate mechanical stress but also not to impair cell detachment at the end of the regenerative process as previously reviewed [40]. Thus, our data point out the suitable support of cell migration out of the biomaterial, which should be the basis for the differentiation fate and an efficient tissue regeneration. whole surface of MC. Thus, there is a large number of cells that ma MC/ cell conglomerate on the culture plate. In contrast, the sharp gr leads to an unstable attachment and thus an impaired cell detachm the enhanced cell viability on MC. In agreement with our hypothesis should provide a suitable cell attachment to tolerate mechanical str detachment at the end of the regenerative process as previously rev the suitable support of cell migration out of the biomaterial, which s differentiation fate and an efficient tissue regeneration. Our data revealed no significant alteration in the CSA of the combined cell pellet together with B30 and MC in the first 12 h of the cultivation period. This aspect might refer to the establishment of the proper cell niche with a stable and secure cell adhesion on the surface of the appropriate biomaterials before the induction of further cell networking and cell proliferation. P 15/29 The data of the presented study revealed that the ALP activity at day 7 and day 21 is increased by the osteogenic induction under static culture conditions in all experimental setups with biomaterials. The osteogenic commitment was indicated by ARS semi-quantification particularly in the presence of combined MC and osteogenic medium. 4. Discussion In another study from the same group reported about an enhancement of the osteogenic potential of human MSCs by a collagen-strontium based hydroxyapatite scaffold. In the latter material the included strontium activates bone formation via the β-catenin signaling pathway [45]. The data revealed that the combination of B30Str and the osteogenic medium enhanced the ALP activity indicating the osteogenic commitment. This could even be enhanced under mechanical FSS. These results indicate a double osteoinductive effect in which the applied FSS might mimic the invivo bone formation under mechanical loading and the additional presence of strontium promotes Ca2+ deposition in the vicinity of cells. These findings confirm reports suggest that strontium promotes osteoblasts viability and reduces the osteoclasts activity as previously reviewed [46]. Moreover, strontium has shown to promote the matrix mineralization by increasing the Ca2+ level in the extracellular microenvironment which modulates the activity of a Ca2+ sensory receptor [47]. Surprisingly, the expression of Runx2 and ALP was only weak under conditions of the osteogenic induction using MC under static culture conditions. However, the combined osteogenic medium with MC under FSS increased Ca2+ deposition as shown by CPC analysis not only compared to standard protocol without biomaterials but also in comparison to a B30 and B30Str based protocol. The data clearly indicate that FSS enhances the osteogenic capacity of MC compared to static culture condition. This is also underlined by the increased Runx2 expression under the stimulation of FSS in most experimental setups independent of the biomaterial used. In this context previous studies by our and other groups have shown that the ectopic stimulation using Ca2+ [48] and/or mechanical FSS [49] promoted osteogenic differentiation of MSCs. 4. Discussion These data are in the same line with previous report that showed a rapid osteogenic differentiation, matrix mineralization and upregulation of Runx2 and ALP expression in periodontal stromal stem cells after cultivated with alginate-based MC [41]. The osteoinductive effect of MC could deliver stable cell adhesion via the gelatin coat and induction of matrix mineralization. In a similar study comparing the effect of enzymatic harvested and MC bound cells regarding the osteogenic differentiation of human fetal MSCs, there was an upregulation of the osteogenic markers as well as matrix mineralization of MC bound cells [42]. Additionally, our data point out that there might be a link between the osteogenic commitment of cells and the structure of MC. It has been reported that gelatin coated surfaces or MC fully generated from collagen enhance the osteogenic differentiation and matrix mineralization of human bone marrow MSCs as indicated by enhanced ALP activity, ARS staining and upregulated expression of ALP and bone sialoprotein [42]. On the other hand, the analysis of the osteogenic specific markers have shown that under static culture conditions in the presence of an osteogenic induction medium, B30Str induced an early upregulation of Runx2 and ALP expression at day 14. However, MSCs cultivated in combination with B30 biomaterial showed a later upregulation of Runx2 at day 21. These data highlight the potential of biomaterials on the osteogenic induction of MSCs in a time wise comparison. In agreement with our data, an earlier study has shown that calcium phosphate based hydroxyapatite and a composite of degradable gel (BONITmatrix) have promoted the osteogenic differentiation of human bone marrow derived MSCs even without the addition of any osteogenic induction medium as could be shown by an enhanced ALP activity and an upregulation of osteogenic markers [43]. The authors of this study concluded that the surface interaction of MSCs with a certain and suitable biomaterial already act as an osteogenic stimulant even in the absence of an osteogenic induction medium. Furthermore, by integrating strontium within the B30 biomaterial, the osteogenic commitment was even promoted as shown by the upregulation of Runx2 and ALP expression. In the same line, several reports revealed that strontium enhances the osteogenic differentiation and matrix Page 15/29 Page 15/29 Ca2+ deposition which can be a useful tool for bone repair [44]. 5. Conclusion The present study investigates the impact of various biomaterials on the osteogenic differentiation potential of equine adipose tissue MSCs under static and mechanically stimulated culture conditions. We provide evidence that especially the collagenous MC promoted cell viability, migration as well as matrix mineralization suggesting the usefulness of MC as a scaffold for cell-based therapy. All biomaterial constructs promoted ALP activity in the presence of osteogenic medium already under static non- stimulated culture conditions. We found that FSS increases cell viability of those cells cultivated on MC in the osteogenic medium which might be an important factor to enhance tissue regeneration. Our data revealed that mechanical FSS enhanced osteogenic differentiation of MSCs as shown by increased ALP activity and upregulated Runx2 and ALP osteogenic markers expression which could be a target to promote bone regeneration. The upregulation of the osteogenic marker Runx2 was found to be B30 and B30Str biomaterials dependent. Authors’ contributions MIE, MCK and FG have collected the raw data set, analyzed the results and wrote the original draft of the manuscript. SA has interpreted the results, revised and finalized the submitted version of the manuscript. Funding This work receives no funding to report or Not applicable. Availability of data and material The data collected and the analysis performed to generate the manuscript results are available from the corresponding author on reasonable request. Consent for publication Not applicable Acknowledgements We thank the Education, Audiovisual and Culture Executive Agency of the European Commission for partially funding the project in the framework of the Erasmus Mundus Master Program EUCOMOR. Further thanks given to Manuela Heimann, Eva Kammer, Kathrin Wolf-Hofmann and Susanne Schubert- Porth for technical support. 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Shell K, Raabe O, Freitag C, Ohrndorf A, Christ H-J, Wenisch S, Arnhold S. Comparison of Equine Adipose Tissue-Derived Stem Cell Behavior and Differentiation Potential Under the Influence of 3% and 21% Oxygen Tension. Journal of Equine Veterinary Science. 2013;33:74–82. doi:10.1016/j.jevs.2012.05.002. 29. Arnhold S, Elashry MI, Klymiuk MC, Geburek F. Investigation of stemness and multipotency of equine adipose-derived mesenchymal stem cells (ASCs) from different fat sources in comparison with lipoma. Stem Cell Res Ther. 2019;10:309. doi:10.1186/s13287-019-1429-0. 30. Zhou X, Liu D, You L, Wang L. Quantifying fluid shear stress in a rocking culture dish. J Biomech. 2010;43:1598–602. doi:10.1016/j.jbiomech.2009.12.028. 30. Zhou X, Liu D, You L, Wang L. Quantifying fluid shear stress in a rocking culture dish. J Biomech. 2010;43:1598–602. doi:10.1016/j.jbiomech.2009.12.028. 31. BESSEY OA, LOWRY OH, BROCK MJ. A method for the rapid determination of alkaline phosphates with five cubic millimeters of serum. J Biol Chem. 1946;164:321–9. Page 19/29 Page 19/29 32. Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc. 2008;3:1101–8. doi:10.1038/nprot.2008.73. 33. Caron MMJ, Emans PJ, Coolsen MME, Voss L, Surtel DAM, Cremers A, et al. Redifferentiation of dedifferentiated human articular chondrocytes: comparison of 2D and 3D cultures. Osteoarthr Cartil. 2012;20:1170–8. doi:10.1016/j.joca.2012.06.016. 34. Sharfstein ST, Kaisermayer C. Microcarrier Culture. In: Flickinger MC, editor. Encyclopedia of industrial biotechnology: Bioprocess, bioseparation, and cell technology. Oxford: Wiley-Blackwell; 2010. doi:10.1002/9780470054581.eib432. 35. Howard D, Buttery LD, Shakesheff KM, Roberts SJ. Tissue engineering: strategies, stem cells and scaffolds. J Anat. 2008;213:66–72. doi:10.1111/j.1469-7580.2008.00878.x. 36. Rana D, Zreiqat H, Benkirane-Jessel N, Ramakrishna S, Ramalingam M. Development of decellularized scaffolds for stem cell-driven tissue engineering. J Tissue Eng Regen Med. 2017;11:942–65. doi:10.1002/term.2061. 37. 2011;29:981–91. doi:10.1002/stem.646. 2011;29:981–91. doi:10.1002/stem.646. 2011;29:981–91. doi:10.1002/stem.646. 46. Marie PJ, Ammann P, Boivin G, Rey C. Mechanisms of action and therapeutic potential of strontium in bone. Calcif Tissue Int. 2001;69:121–9. doi:10.1007/s002230010055. 46. Marie PJ, Ammann P, Boivin G, Rey C. Mechanisms of action and therapeutic potential of strontium in bone. Calcif Tissue Int. 2001;69:121–9. doi:10.1007/s002230010055. 47. Canalis E. The divalent strontium salt S12911 enhances bone cell replication and bone formation in vitro. Bone. 1996;18:517–23. doi:10.1016/8756-3282(96)00080-4. 47. Canalis E. The divalent strontium salt S12911 enhances bone cell replication and bone formation in vitro. Bone. 1996;18:517–23. doi:10.1016/8756-3282(96)00080-4. 48. Elashry MI, Baulig N, Heimann M, Bernhardt C, Wenisch S, Arnhold S. Osteogenic differentiation of equine adipose tissue derived mesenchymal stem cells using CaCl2. Res Vet Sci. 2018;117:45–53. doi:10.1016/j.rvsc.2017.11.010. 48. Elashry MI, Baulig N, Heimann M, Bernhardt C, Wenisch S, Arnhold S. Osteogenic differentiation of equine adipose tissue derived mesenchymal stem cells using CaCl2. Res Vet Sci. 2018;117:45–53. doi:10.1016/j.rvsc.2017.11.010. 49. Elashry MI, Gegnaw ST, Klymiuk MC, Wenisch S, Arnhold S. Influence of mechanical fluid shear stress on the osteogenic differentiation protocols for Equine adipose tissue-derived mesenchymal stem cells. Acta Histochem. 2019;121:344–53. doi:10.1016/j.acthis.2019.02.002. 49. Elashry MI, Gegnaw ST, Klymiuk MC, Wenisch S, Arnhold S. Influence of mechanical fluid shear stress on the osteogenic differentiation protocols for Equine adipose tissue-derived mesenchymal stem cells. Acta Histochem. 2019;121:344–53. doi:10.1016/j.acthis.2019.02.002. References Alkhatib R, Hilal-Alnaqbi A, Naciri M, Al-Majmaie R, Saseedharan P, Karam SM, Al-Rubeai M. 3D culture of mouse gastric stem cells using porous microcarriers. Front Biosci (Schol Ed). 2017;9:172– 9. doi:10.2741/s481. 38. Bock A, Sann H, Schulze-Horsel J, Genzel Y, Reichl U, Möhler L. Growth behavior of number distributed adherent MDCK cells for optimization in microcarrier cultures. Biotechnol Prog. 2009;25:1717–31. doi:10.1002/btpr.262. 39. Forestell SP, Kalogerakis N, Behie LA, Gerson DF. Development of the optimal inoculation conditions for microcarrier cultures. Biotechnol Bioeng. 1992;39:305–13. doi:10.1002/bit.260390308. 40. Derakhti S, Safiabadi-Tali SH, Amoabediny G, Sheikhpour M. Attachment and detachment strategies in microcarrier-based cell culture technology: A comprehensive review. Mater Sci Eng C Mater Biol Appl. 2019;103:109782. doi:10.1016/j.msec.2019.109782. 41. Čebatariūnienė A, Jarmalavičiūtė A, Tunaitis V, Pūrienė A, Venalis A, Pivoriūnas A. Microcarrier culture enhances osteogenic potential of human periodontal ligament stromal cells. J Craniomaxillofac Surg. 2017;45:845–54. doi:10.1016/j.jcms.2017.03.009. 42. Guo L, Fan Y, Kawazoe N, Fan H, Zhang X, Chen G. Fabrication of gelatin-micropatterned surface and its effect on osteogenic differentiation of hMSCs. J Mater Chem B. 2018;6:1018–25. doi:10.1039/c7tb03165c. 43. Müller P, Bulnheim U, Diener A, Lüthen F, Teller M, Klinkenberg E-D, et al. Calcium phosphate surfaces promote osteogenic differentiation of mesenchymal stem cells. J Cell Mol Med. 2008;12:281–91. doi:10.1111/j.1582-4934.2007.00103.x. 44. Yang F, Tu J, Yang D, Li G, Cai L, Wang L. Osteogenic differentiation of mesenchymal stem cells could be enhanced by strontium. Conf Proc IEEE Eng Med Biol Soc. 2010;2010:823–6. doi:10.1109/IEMBS.2010.5626774. Page 20/29 45. Yang F, Yang D, Tu J, Zheng Q, Cai L, Wang L. Strontium enhances osteogenic differentiation of mesenchymal stem cells and in vivo bone formation by activating Wnt/catenin signaling. Stem Cells. Figures Page 21/29 Figure 1 Page 22/29 Figure 1 Morphological evaluation of MSCs cultivated with biomaterials (a-d) Scanning electron microscopy (SEM) of equine adipose derived mesenchymal stem cells cultivated in growth medium in conjunction with MC and B30 biomaterials for 48 h. (a, b) SEM of native cultiSpher-S MC and B30 biomaterials without MSCs. (c, d) SEM of combined cultivation of MSCs together with MC and B30 show cell adhesion, aggregation and intercellular contacts. (e, f) Phase contrast (PC) images of MSCs seeded in a Figure 1 Page 22/29 Morphological evaluation of MSCs cultivated with biomaterials (a-d) Scanning electron microscopy (SEM) of equine adipose derived mesenchymal stem cells cultivated in growth medium in conjunction with MC and B30 biomaterials for 48 h. (a, b) SEM of native cultiSpher-S MC and B30 biomaterials without MSCs. (c, d) SEM of combined cultivation of MSCs together with MC and B30 show cell adhesion, aggregation and intercellular contacts. (e, f) Phase contrast (PC) images of MSCs seeded in a Page 22/29 monolayer or in combination with MC. (g, h) Histological paraffin section of the MSCs pellet in conjunction with MC stained with H&E shows cell migration within the pores of the MC. (i-l) Combined cultivation of MSCs in conjunction with B30 (I, j) and MC (k, l) after static (SC) and mechanical rotation (RT) culture for 72 h show the pattern of cell adhesion and detachment from the pellet using phalloidin staining (green). Hoechst counterstain visualizes cell nuclei (blue). (m) TEM of combined MSCs and B30 biomatrerial section show the phagocytic properties of MSCs to the biomaterials using podoytes extension (star). (n) TEM of MSCs section demonstrates various sized of B30 granules within the cell cytoplasm (black arrow). (o) Combined cultivation of MSCs with MC and B30 biomaterials under both SC and mechanical rotation. Average quantification of cell viability using a microplate reader at 570 nm. Enhanced cell viability in the presence of MC compared to B30 can be shown. Cells cultivated in basal medium (BM) without any biomaterials were used as negative control. (p) Average CSA measurement (µm2) for combined MSCs and biomaterial pellet up to 72h. (q) Linear correlation between the cultivation time point and the size of the MSCs/biomaterial. All data presented as mean ± SEM. = p<0.05, = p<0.01, *= p<0. 001). Scale bar in (a, b)= 200 nm, (c, d)= 2000 nm, (e, f)= 100 µm, (g, h)= 70 µm, (i-l) =20 µm and (m,n)= 2500 nm. monolayer or in combination with MC. (g, h) Histological paraffin section of the MSCs pellet in conjunction with MC stained with H&E shows cell migration within the pores of the MC. (i-l) Combined cultivation of MSCs in conjunction with B30 (I, j) and MC (k, l) after static (SC) and mechanical rotation (RT) culture for 72 h show the pattern of cell adhesion and detachment from the pellet using phalloidin staining (green). Hoechst counterstain visualizes cell nuclei (blue). Figure 1 (m) TEM of combined MSCs and B30 biomatrerial section show the phagocytic properties of MSCs to the biomaterials using podoytes extension (star). (n) TEM of MSCs section demonstrates various sized of B30 granules within the cell cytoplasm (black arrow). (o) Combined cultivation of MSCs with MC and B30 biomaterials under both SC and mechanical rotation. Average quantification of cell viability using a microplate reader at 570 nm. Enhanced cell viability in the presence of MC compared to B30 can be shown. Cells cultivated in basal medium (BM) without any biomaterials were used as negative control. (p) Average CSA measurement (µm2) for combined MSCs and biomaterial pellet up to 72h. (q) Linear correlation between the cultivation time point and the size of the MSCs/biomaterial. All data presented as mean ± SEM. = p<0.05, = p<0.01, = p<0. 001). Scale bar in (a, b)= 200 nm, (c, d)= 2000 nm, (e, f)= 100 µm, (g, h)= 70 µm, (i-l) =20 µm and (m,n)= 2500 nm. Page 23/29 Figure 2 Page 24/29 Figure 2 Assessment the effect of biomaterial scaffold on the osteogenic differentiation of MSCs under static culture condition MSCs were cultivated in the presence of MC, B30 and B30Str biomaterials under standard culture condition for 48h. Cells were induced to osteogenic differentiation (OD) using 5% FBS in DMEM, 0.05 mM ascorbic acid-2-phosphate, 10 mM β-glycerolphosphate and 0.1 µM dexamethasone up to 21 days. (a, b, c) Spectrophotometric quantification of alkaline phosphatase (ALP) activity at day 7, 14 Figure 2 Assessment the effect of biomaterial scaffold on the osteogenic differentiation of MSCs under static culture condition MSCs were cultivated in the presence of MC, B30 and B30Str biomaterials under standard culture condition for 48h. Cells were induced to osteogenic differentiation (OD) using 5% FBS in DMEM, 0.05 mM ascorbic acid-2-phosphate, 10 mM β-glycerolphosphate and 0.1 µM dexamethasone up to 21 days. (a, b, c) Spectrophotometric quantification of alkaline phosphatase (ALP) activity at day 7, 14 Page 24/29 Page 24/29 and 21 shows promoted ALP activity post osteogenic induction. ALP activity was detected by measuring the metabolism of p- Nitrophenylphosphate into p-Nitrophenol (PNP) in the presence of ALP. The values of PNP was measured at 405 nm absorbance. MSCs were grown in basal medium (BM) composed of 5 % FBS in DMEM served as negative control. Cells were cultivated in osteogenic medium without adding biomaterials was used as negative control (NC). (d, e) Semi-quantification of the solubilized bound alizarin red s (ARS) using 10 % Cetyl Pyridinium Chloride (CPC, Roth Germany) at day 14 and 21 post osteogenic induction. Analysis of CPC revealed improved matrix mineralization as shown with MC biomaterial scaffold. (f-o) Representative ARS stained images (red) for MSCs cultivated in basal medium (BM, f, g), osteogenic differentiation medium (OD, h, i), OD+MC (j, k), OD+B30 (l, m) and OD+B30Str (n, o) under static conditions at day 14 and day 21. Osteogenic nodules could be shown in the presence of OD medium compared to BM. Diffused ARS staining was observed in the presence of OD+MC and scattered mineralization with OD+B30 and OD+B30/Str. All data presented as mean ± SEM. = p<0.05, = p<0.01, = p<0. 001). Scale bar = 100 µm. and 21 shows promoted ALP activity post osteogenic induction. ALP activity was detected by measuring the metabolism of p- Nitrophenylphosphate into p-Nitrophenol (PNP) in the presence of ALP. The values of PNP was measured at 405 nm absorbance. MSCs were grown in basal medium (BM) composed of 5 % FBS in DMEM served as negative control. Cells were cultivated in osteogenic medium without adding biomaterials was used as negative control (NC). (d, e) Semi-quantification of the solubilized bound alizarin red s (ARS) using 10 % Cetyl Pyridinium Chloride (CPC, Roth Germany) at day 14 and 21 post osteogenic induction. Analysis of CPC revealed improved matrix mineralization as shown with MC biomaterial scaffold. Figure 2 (f-o) Representative ARS stained images (red) for MSCs cultivated in basal medium (BM, f, g), osteogenic differentiation medium (OD, h, i), OD+MC (j, k), OD+B30 (l, m) and OD+B30Str (n, o) under static conditions at day 14 and day 21. Osteogenic nodules could be shown in the presence of OD medium compared to BM. Diffused ARS staining was observed in the presence of OD+MC and scattered mineralization with OD+B30 and OD+B30/Str. All data presented as mean ± SEM. = p<0.05, = p<0.01, = p<0. 001). Scale bar = 100 µm. Page 25/29 Fi 3 Figure 3 Page 26/29 Evaluation the effect of mechanical FSS and biomaterial scaffold on the osteogenic differentiation capacity of MSCs MSCs were grown in conjunction with MC, B30 and B30Str biomaterials under standard culture condition for 48h to establish adhesion. The cells/biomaterial complex were induced to the osteogenic differentiation (OD) fate under mechanical FSS in comparison to static (ST) culture up to 21 days. In parallel, MSCs were grown in basal medium BM was served as negative control. Cells were Evaluation the effect of mechanical FSS and biomaterial scaffold on the osteogenic differentiation capacity of MSCs MSCs were grown in conjunction with MC, B30 and B30Str biomaterials under standard culture condition for 48h to establish adhesion. The cells/biomaterial complex were induced to the osteogenic differentiation (OD) fate under mechanical FSS in comparison to static (ST) culture up to 21 days. In parallel, MSCs were grown in basal medium BM was served as negative control. Cells were Page 26/29 Page 26/29 cultivated in osteogenic medium without adding biomaterials was used as negative control (NC). (a, b) Average cell viability at day 14 and day 21 using MTT assay show enhanced cell viability in the presence of OD together with MC compared to MC, B30, B30Str and BM condition. (c, d) Spectrophotometric measurement of ALP activity at 405 nm absorbance following 10 min and 30 min incubation. (e) Semi- quantification of the solubilized bound alizarin red s (ARS) using 10 % CPC at day 21 post osteogenic induction. Analysis of CPC revealed improved matrix mineralization as shown with MC biomaterial and control osteogenic condition without biomaterial (NC). All data presented as mean ± SEM. = p<0.05, = p<0.01, = p<0. 001). cultivated in osteogenic medium without adding biomaterials was used as negative control (NC). (a, b) Average cell viability at day 14 and day 21 using MTT assay show enhanced cell viability in the presence of OD together with MC compared to MC, B30, B30Str and BM condition. (c, d) Spectrophotometric measurement of ALP activity at 405 nm absorbance following 10 min and 30 min incubation. (e) Semi- quantification of the solubilized bound alizarin red s (ARS) using 10 % CPC at day 21 post osteogenic induction. Analysis of CPC revealed improved matrix mineralization as shown with MC biomaterial and control osteogenic condition without biomaterial (NC). All data presented as mean ± SEM. = p<0.05, = p<0.01, = p<0. 001). Figure 3 cultivated in osteogenic medium without adding biomaterials was used as negative control (NC). (a, b) Average cell viability at day 14 and day 21 using MTT assay show enhanced cell viability in the presence of OD together with MC compared to MC, B30, B30Str and BM condition. (c, d) Spectrophotometric measurement of ALP activity at 405 nm absorbance following 10 min and 30 min incubation. (e) Semi- quantification of the solubilized bound alizarin red s (ARS) using 10 % CPC at day 21 post osteogenic induction. Analysis of CPC revealed improved matrix mineralization as shown with MC biomaterial and control osteogenic condition without biomaterial (NC). All data presented as mean ± SEM. = p<0.05, = p<0.01, = p<0. 001). Page 27/29 Figure 4 Figure 4 Figure 4 Evaluation of the osteogenic marker expression MSCs were cultivated in osteogenic differentiation (OD) medium in the presence of MC, B30, B30Str biomaterials under static and mechanical FSS culture condition. Cells were osteogenic induced without adding biomaterials served as control (NC). Cells grown in parallel in basal medium (BM) were used as non induced control. The levels of Runx2 and alkaline phosphatase expression (N=6) at day 14 and day 21 were quantified using RT-qPCR. (a) Relative Runx2 Evaluation of the osteogenic marker expression MSCs were cultivated in osteogenic differentiation (OD) medium in the presence of MC, B30, B30Str biomaterials under static and mechanical FSS culture condition. Cells were osteogenic induced without adding biomaterials served as control (NC). Cells grown in parallel in basal medium (BM) were used as non induced control. The levels of Runx2 and alkaline phosphatase expression (N=6) at day 14 and day 21 were quantified using RT-qPCR. (a) Relative Runx2 Page 28/29 Page 28/29 expression at day 14 under static culture condition. (b) Relative Runx2 expression at day 21 under static culture condition. (c) Relative ALP expression at day 14 under static culture condition. (d) Relative ALP expression at day 21 under static culture condition. (e) Relative Runx2 expression at day 14 under FSS condition. (f) Relative Runx2 expression at day 21 under FSS culture condition. (g) Relative ALP expression at day 14 under FSS culture condition. (h) Relative ALP expression at day 21 under FSS culture condition. The expression was normalized to non-induced cells in BM using 2^-∆∆cq method [32]. GAPDH was used as an endogenous reference. The data presented as mean ± SEM. = p<0.05, = p<0.01, *= p<0. 001). Elashryetal.2020coverletter.docx Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Page 29/29
https://openalex.org/W3134337570	https://www.nature.com/articles/s41598-021-85097-6.pdf	English	null	RETRACTED ARTICLE: MicroRNA-205-5p inhibits skin cancer cell proliferation and increase drug sensitivity by targeting TNFAIP8	Scientific reports	2,021	cc-by	14,332	MicroRNA‑205‑5p inhibits skin cancer cell proliferation and increase drug sensitivity by targeting TNFAIP8 OPEN ICL Xinhong Ge1,2,5, Suryakant Niture2,5, Minghui Lin3, Patrice Cagle2, P. Andy Li4 & Deepak Kumar2 RTIC Tumor necrosis factor-α-induced protein 8 (TNFAIP8) is a member of the TIPE/TNFAIP8 family which regulates tumor growth and survival. Our goal is to delineate the detailed oncogenic role of TNFAIP8 in skin cancer development and progression. Here we demonstrated that higher expression of TNFAIP8 is associated with basal cell carcinoma (BCC), squamous cell carcinoma (SCC), and melanoma development in patient tissues. Induction of TNFAIP8 expression by TNFα or by ectopic expression of TNFAIP8 in SCC or melanoma cell lines resulted in increased cell growth/proliferation. Conversely, silencing of TNFAIP8 decreased cell survival/cell migration in skin cancer cells. We also showed that miR-205-5p targets the 3′UTR of TNFAIP8 and inhibits TNFAIP8 expression. Moreover, miR-205-5p downregulates TNFAIP8 mediated cellular autophagy, increased sensitivity towards the B-RAFV600E mutant kinase inhibitor vemurafenib, and induced cell apoptosis in melanoma cells. Collectively our data indicate that miR-205-5p acts as a tumor suppressor in skin cancer by targeting TNFAIP8. TED ART Skin cancer is currently one of the most common types of malignancy1,2. In the United States alone, more than 9500 people are diagnosed with skin cancer every day and more than two people die of the disease every hour (Skin cancer facts and statistics: 2019, https://skincancer.org/skin-cancer-information/skin-cancer-facts/: Accessed November 14, 2019)3,4. Caucasians are 20 times more likely to be diagnosed with skin cancer than African Americans. Skin cancers exist in several forms and therefore early diagnosis and treatment is a significant challenge5. Skin cancers include malignant melanoma (MM) and non-melanoma skin cancers (NMSC)6, and the most common non-melanoma skin cancers are squamous cell carcinoma (SCC) and basal cell carcinoma (BCC)6. Ultraviolet radiation (UV), environmental pollutants, chemical carcinogens, and work-related exposures are known to increase the risk of skin cancer7–9. A variety of molecular mechanisms associated with the development of skin cancer also have been described10,11. Exposure of UVA induces the expression of Autophagy Receptor Adaptor p62 protein in melanocytes and p62 acts as an oncogene in UVA-associated melanoma development and progression12. Analysis of whole-genome landscapes of major melanoma subtypes has identified several key gene mutations in cutaneous melanoma, including B-RAF, CDKN2A, NRAS, in acral melanoma TP53, B-RAF, NRAS and NF1 and SF3B1 in mucosal melanoma13. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Scientific Reports \| (2021) 11:5660 MicroRNA‑205‑5p inhibits skin cancer cell proliferation and increase drug sensitivity by targeting TNFAIP8 OPEN ICL The study further releveled that these mutations are not cor- related with alternative telomere lengthening but associated with greater telomere length and also modulates the MAPK and PI3K pathway in melanomas13. Moreover, during melanoma development, several somatic alterations activate the MAPK and PI3K pathway, upregulate telomerase activity, modulate chromatin landscape, override the G1/S checkpoint, the ramp-up of MAPK signaling, and disrupt the p53 pathway14. In melanoma, activation of several oncogenes including RAS, B-RAF, Cyclin D1, CDK2, c-MYC, HDM2, MAPK1/2, ERBB4, GRIN2A, GRM3, RAC1, and PREX2 were reported previously15,16. TRACTE 1Department of Dermatology, General Hospital of Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China. 2Julius L. Chambers Biomedical Biotechnology Research Institute (BBRI), North Carolina Central University, 1801 Fayetteville St., Durham, NC 27707, USA. 3Department of Respiratory Diseases, The Forth People’s Hospital of Ningxia Hui Autonomous Region, Yinchuan 750021, Ningxia Hui Autonomous Region, China. 4Department of Pharmaceutical Sciences, Bio‑Manufacturing Research Institute and Technology Enterprise (BRITE), College of Health and Sciences, North Carolina Central University, Durham, NC 27707, USA. 5These authors contributed equally: Xinhong Ge and Suryakant Niture. email: sniture@nccu.edu; dkumar@ nccu.edu \| https://doi.org/10.1038/s41598-021-85097-6 Scientific Reports \| (2021) 11:5660 www.nature.com/scientificreports/ microRNAs (miRNAs) have been shown to regulate key pathways in skin cancer. miRNAs are small single- stranded non-coding RNAs that modulate post-transcriptional gene expression by binding to the 3′ untranslated regions (3′UTRs) of target mRNAs. The binding of miRNAs to 3′UTRs of target mRNA regulates both the stability and translation of mRNA transcripts and thus affects gene expression17. Reports suggest that by directly targeting key gene expression, miRNAs modulate various cellular processes such as cell proliferation/survival, cell-cycle control, cell apoptosis, the stress response, cell metabolism, development, and differentiation18,19. In melanoma, the expression of several miRNAs are upregulated, for example, miR-214, miR-30b, miR-30d, miR-506, miR-514, miR-21, miR-155, and miR-221. These microRNAs promote melanoma cell growth and proliferation by acting as oncogenes20–24. On the other hand, studies also demonstrate that miR-29c, miR-34b, miR-375, and miR-205, are downregulated in melanoma and behave as tumor suppressors19,25–28. E g pp Tumor necrosis factor-α-induced protein 8 (TNFAIP8) is also known as SCC-S2, GG2-1, and NDED. TNFAIP8 is a member of the TNFAIP8/TIPE family which has three other members designated as TNFAIP8- like protein 1 (TIPE1), TNFAIP8-like protein 2 (TIPE2), and TNFAIP8-like protein 3 (TIPE3)29–32. TNFAIP8 is a tumor necrosis factor-alpha (TNFα) inducible protein33–35. Results i Expression of TNFAIP8 is higher in skin cancer tissues. To investigate the pathological role of TNFAIP8 in skin cancer progression, we first analyzed the expression of TNFAIP8 in BCC, SCC, and melanoma by immunohistochemistry in human skin cancer patient tissue samples. The expression of TNFAIP8 was found to be significantly increased in BCC (n = 6) and low/medium differentiated SCC tissue (n = 4) compared with normal skin tissues (n = 6) (Fig. 1A,B). The expression of TNFAIP8 was not significantly altered in highly dif- ferentiated SCC compared with normal skin tissue. Interestingly, the expression of TNFAIP8 was significantly increased (~ two fold) in melanoma tissues compared with normal skin tissues (Fig. 1A,B). Also, we analyzed the TNFAIP8 mRNA expression between cutaneous melanoma (n = 45) and melanoma precursor (n = 18) sam- ples as reported in the Oncomine dataset (https://www.oncomine.org/resource/login.html accessed October 17, 2020)48 (Fig. 1C). The data suggest that the expression of TNFAIP8 transcripts is 2.55-fold higher in melanoma precursor tissues compared with cutaneous melanoma (Fig. 1C). Collectively, our data suggest that TNFAIP8 expression is higher in skin cancer and melanoma tissues. CTED AR g To establish the role of TNFAIP8 in skin cancer, we compared the expression of TNFAIP8 in normal human skin cell line HaCaT (B-RAF wild) to human skin cancer cell lines, including the epidermoid squamous cell car- cinoma A431 (B-RAF wild) and melanoma cell lines A375, A2058 (B-RAFV600E mutant) and SK-MEL-2 (B-RAF wild) cells. First, we analyzed the expression of B-RAF wild and B-RAFV600E mutant proteins in these cell lines (Fig. 1D). Consistent with earlier reports, immunoblotting data from our laboratory indicate that all the skin cancer cell lines and normal skin cells used in this study expressed moderate levels of either B-RAF wild-type protein in HaCaT, A431, SK-MEL-2 cells, or B-RAFV600E mutant protein in A375 and A2058 cells (Fig. 1D)49,50. Further, we analyzed the expression of TNFAIP8 protein and transcripts in normal skin cells and B-RAF wild type or B-RAFV600E mutant skin cancer cells (Fig. 1E,F). Immunoblotting and RT/qPCR data demonstrated that TNFAIP8 expression was higher in SK-MEL-2 (melanoma cells), A431 (SCC cells), and HaCaT (normal skin cells) which express B-RAF wild type protein, and lower expression of TNFAIP8 was observed in A375 and A2058 melanoma cells that expressed B-RAFV600E mutant protein (Fig. 1E,F). MicroRNA‑205‑5p inhibits skin cancer cell proliferation and increase drug sensitivity by targeting TNFAIP8 OPEN ICL Moreover, the expression of TNFAIP8 is also controlled by several transcriptional factors including nuclear factor-κB (NF-κβ), androgen receptor (AR), p53, and orphan nuclear receptor chicken ovalbumin upstream promoter transcription factor I (COUP-TFI)32,35–37. TNFAIP8 also regulates inflammation, immunity, and involved in several human diseases36. TNFAIP8 is known to regulate several genes associated with cell proliferation (IL-24, FAT3, LPHN2, EPHA3), fatty-acid oxida- tion (ACADL), and several oncogenes, for example, NFAT5, MALAT1, MET, FOXA1, KRAS, S100P, OSTF135. TNFAIP8 has been shown to inhibit cell apoptosis and increase cell proliferation in numerous cancers including lung cancer38–41, liver cancer42,43, gastric cancer44, esophageal cancer (ESCC)45,46, pancreatic cancer47, and prostate cancer32,34,35. Here we extend the analysis of the oncogenic role of TNFAIP8 and investigate the role TNFAIP8 in skin cancer cell proliferation/survival and progression. Our data suggest that overexpression of TNFAIP8 promotes cell survival and cell colony formation/cell growth in skin cancer cells. Further, we demonstrated that miR-205-5p controls TNFAIP8 expression and reduces skin cancer cell survival, and increases sensitivity to the B-RAFV600E mutant kinase inhibitor vemurafenib in melanoma cells. ARTICLE Results i (B) Immunohistochemical expression levels of TNFAIP8 protein from normal skin (n = 6), BCC (n = 6), H-SCC (n = 4), M-SCC (n = 4), L-SCC (n = 4), nevus (n = 6), and melanoma (n = 6) tissues were quantified using ImageJ software (https://imagej.nih.gov/ij/) and plotted. The data represent mean ± SEM from 4 to 6 skin cancer tissues. P < 0.05 relative to normal skin tissues. (C) Expression of TNFAIP8 transcripts between cutaneous melanoma (n = 45) and melanoma precursor (n = 18) samples were analyzed and presented from the Oncomine dataset (https://www.oncomine.org/resource/login.html accessed October 17, 2020). **P < 0.001 relatives to cutaneous melanoma tissues. (D, E) Expression of wild-type B-RAF, mutant B-RAFV600E, and TNFAIP8 in HaCaT, SK-MEL-2, A431, A375, and A2058 cells were analyzed by western blotting. Fifty micrograms of lysates from normal and skin cancer cells were immunoblotted with B-RAF, TNFAIP8, GAPDH, and β-actin antibodies. Immunoreactive bands were visualized using ECL chemiluminescence detection reagents after exposing the blots on X-ray films (D) or by scanning the blots with an Odyssey CLx Imager (E). The immunoblot scans were converted into grayscale and presented. (F) Expression of TNFAIP8 mRNA from indicated normal skin cells and skin cancer cells were analyzed by RT/qPCR as described in the “Materials and methods” section. ACTED AR melanoma cells expressed isoform two predominantly but not in normal HaCaT cells. Normal HaCaT cells, A431, A375, A2058 cells expressed isoform one, whereas expression of isoform one is not observed in SK-MEL-2 cells suggesting that, skin cancer cells expressed isoform two predominantly (Fig. 2B) and the involvement of TNFAIP8 variant/isoform 2 in lung cancer and liver cancer development and progression has been reported earlier37,43. TRA Further, to test whether TNFα induced expression of TNFAIP8 in skin cancer cells, we treated normal and skin cancer cells with TNFα (10–50 ng/ml), and the induction of TNFAIP8 protein and mRNA were analyzed (Fig. 2C,D). Treatment of TNFα (10–20 ng/ml) induced the expression of TNFAIP8 protein in normal HACaT skin cells as well as in skin cancer cell lines (Fig. 2C). Consistent with immunoblotting data, the relative mRNA expression of TNFAIP8 was also increased by 1–2.7 fold in HaCaT, A431, A375, and A2058 cell lines when cells treated with TNFα (10–20 ng/ml), whereas, the expression of TNFAIP8 was decreased at higher concentration of TNFα (50 ng/ml) (Fig. 2D). Results i TRACT p p g B-RAF protein is a serine-threonine kinase and mutation of B-RAFV600E increases approximately 500-fold kinase activity compared with wild-type B-RAF in 27–70% of melanoma cancer. B-RAFV600E mutant protein is constitutively active51 and earlier studies demonstrated that B-RAFV600E mutant dysregulates NF-κB/Snail/ RKIP/PTEN circuit as well as activates MEK/ERK kinases that promote the epithelial to mesenchymal transi- tion (EMT) and melanoma cell growth51,52. Since NF-κB regulates the expression of TNFAIP8, the exact role of B-RAFV600E mutant in the regulation of TNFAIP8 expression transcriptionally or post-transcriptionally through NF-κB is not clear, however, our data suggest that mutant B-RAFV600E downregulates the expression of TNFAIP8 (mRNA) as well as decreases the level/stability of TNFAIP8 protein in A375 and A2058 melanoma cells. This needs to be further investigated. T TNFα regulates the expression of TNFAIP8 and skin cancer cell survival. The classical model of TNFAIP8 regulation suggests that cytokine TNFα binds with TNFα 1/2 receptors and induced cellular inflam- mation. This leads to dissociations of transcription factor NF-κB from its inhibitor IKκB, NF-κB translocates into the nucleus, binds with TNFAIP8 promoter, and induced TNFAIP8 expression32. Reports also suggest that TNFAIP8 gene expressed several protein variants/isoforms in cancer cell lines34,35, and therefore first we ana- lyzed the expression TNFAIP8 isoforms in normal and skin cancer cells by RT/PCR (Fig. 2A,B). SCC-A431 and https://doi.org/10.1038/s41598-021-85097-6 Scientific Reports \| (2021) 11:5660 \| www.nature.com/scientificreports/ Figure 1. TNFAIP8 expression is upregulated in BCC, SCC, and melanoma patient skin tissues. (A) Relative immunohistochemical TNFAIP8 expression in indicated skin cancer tissues is presented. (B) Immunohistochemical expression levels of TNFAIP8 protein from normal skin (n = 6), BCC (n = 6), H-SCC (n = 4), M-SCC (n = 4), L-SCC (n = 4), nevus (n = 6), and melanoma (n = 6) tissues were quantified using ImageJ software (https://imagej.nih.gov/ij/) and plotted. The data represent mean ± SEM from 4 to 6 skin cancer tissues. P < 0.05 relative to normal skin tissues. (C) Expression of TNFAIP8 transcripts between cutaneous melanoma (n = 45) and melanoma precursor (n = 18) samples were analyzed and presented from the Oncomine dataset (https://www.oncomine.org/resource/login.html accessed October 17, 2020). **P < 0.001 relatives to cutaneous melanoma tissues. (D, E) Expression of wild-type B-RAF, mutant B-RAFV600E, and TNFAIP8 in HaCaT, SK-MEL-2, A431, A375, and A2058 cells were analyzed by western blotting. Results i Fifty micrograms of lysates from normal and skin cancer cells were immunoblotted with B-RAF, TNFAIP8, GAPDH, and β-actin antibodies. Immunoreactive bands were visualized using ECL chemiluminescence detection reagents after exposing the blots on X-ray films (D) or by scanning the blots with an Odyssey CLx Imager (E). The immunoblot scans were converted into grayscale and presented. (F) Expression of TNFAIP8 mRNA from indicated normal skin cells and skin cancer cells were analyzed by RT/qPCR as described in the “Materials and methods” section. ACTED ARTICLE ARTICLE CLE CLE d d l k ( ) ARTICL Figure 1. TNFAIP8 expression is upregulated in BCC, SCC, and melanoma patient skin tissues. (A) Relative immunohistochemical TNFAIP8 expression in indicated skin cancer tissues is presented. (B) Immunohistochemical expression levels of TNFAIP8 protein from normal skin (n = 6), BCC (n = 6), H-SCC (n = 4), M-SCC (n = 4), L-SCC (n = 4), nevus (n = 6), and melanoma (n = 6) tissues were quantified using ImageJ software (https://imagej.nih.gov/ij/) and plotted. The data represent mean ± SEM from 4 to 6 skin cancer tissues. P < 0.05 relative to normal skin tissues. (C) Expression of TNFAIP8 transcripts between cutaneous melanoma (n = 45) and melanoma precursor (n = 18) samples were analyzed and presented from the Oncomine dataset (https://www.oncomine.org/resource/login.html accessed October 17, 2020). **P < 0.001 relatives to cutaneous melanoma tissues. (D, E) Expression of wild-type B-RAF, mutant B-RAFV600E, and TNFAIP8 in HaCaT, SK-MEL-2, A431, A375, and A2058 cells were analyzed by western blotting. Fifty micrograms of lysates from normal and skin cancer cells were immunoblotted with B-RAF, TNFAIP8, GAPDH, and β-actin antibodies. Immunoreactive bands were visualized using ECL chemiluminescence detection reagents after exposing the blots on X-ray films (D) or by scanning the blots with an Odyssey CLx Imager (E). The immunoblot scans were converted into grayscale and presented. (F) Expression of TNFAIP8 mRNA from indicated normal skin cells and skin cancer cells were analyzed by RT/qPCR as described in the “Materials and methods” section. ACTED AR and melanoma patient skin tissues. (A) AR Figure 1. TNFAIP8 expression is upregulated in BCC, SCC, and melanoma patient skin tissues. (A) Relative immunohistochemical TNFAIP8 expression in indicated skin cancer tissues is presented. Results i To clarify the exact role of TNFAIP8 in skin cancer cell proliferation, we transiently transfected normal HaCaT skin cells and A431, A375, and A2058 skin cancer cells with empty vector (EV) or TNFAIP8-Myc plas- mid (Fig. 3A). Immunoblotting data demonstrated the overexpression of TNFAIP8-Myc tagged protein in cells transfected with TNFAIP8-Myc tagged plasmid but not in an empty vector (EV) transfected cells (Fig. 3A). The MTT assay demonstrated that overexpression of TNFAIP8 increased cell survival significantly; by ~ 55%, 82%, 68% in A431, A375, and A2058 skin cancer cells, respectively (Fig. 3B). However, no significant effect on cell survival/proliferation was observed in HaCaT normal skin cells transfected with TNFAIP8 (Fig. 3B). We also examined the effect of TNFAIP8 overexpression on skin cancer cell colony formation ability. TNFAIP8 expres- sion promoted cell colony formation in A431 cells by 3.1-fold, A375 cells by 4.2-fold, and A2058 cells by 5.1-fold compared with empty vector-transfected cells (Fig. 3C, upper and lower panels). Similarly, no significant effect of TNFAIP8 overexpression on cell colony formation was observed in normal skin HaCaT cells (Fig. 3C, upper and lower panels). These results suggest that TNFAIP8 overexpression promotes cell growth in skin cancer cells but not in normal skin cells. TRAC In parallel, we used siRNA to knockdown endogenous TNFAIP8 protein in normal and skin cancer cells (Fig. 3D). Transfection of TNFAIP8 siRNA significantly reduced endogenous TNFAIP8 levels in all cell lines compared with the control siRNA transfected cells (Fig. 3D). The effect of TNFAIP8 knockdown on cell survival was then analyzed by MTT assay. MTT assay demonstrated that TNFAIP8 knockdown reduced cell survival in A431 cells by 22%, in A375 cells by 55.2%, and in A2058 cells by 45.3% compared with control siRNA transfected cells (Fig. 3E). Similarly, TNFAIP8 knockdown also significantly decreased cell colony formation ability in A431 cells by 18.4%, in A375 cells by 53.2%, and in A2058 cells by 40.3% compared with control siRNA transfected cells (Fig. 3F, upper and lower panels). Also, we evaluated the effect of TNFAIP8 knockdown on skin cancer cell migration using wound healing migration assay. The wound healing migration assay revealed that TNFAIP8 knockdown significantly inhibited cell migration and wound closure in A431 (15.1%), A375 (52.5%), and A2058 (70.4%) compared with control siRNA transfected cells (Fig. 3G, upper and lower panels). Results i The immunoblot scans were converted into grayscale and presented. (D) Similarly, normal and skin cancer cells were treated with vehicle or TNFα (10-50 ng/ml) for 30 h, and TNFAIP8 mRNA expression was analyzed by RT/ qPCR as described in the “Materials and methods” section. P < 0.05, P < 0.01, P < 0.001 relative to control/ vehicle-treated cells. (E) HaCaT, A431, A375 and, A2058 cells were treated with vehicle or TNFα (10–50 ng/ ml) for 48 h and, relative cell survival was analyzed by MTT assay as described in the “Materials and methods” section. Results are representative of three independent experiments. P < 0.05, **P < 0.001 relative to control/ vehicle-treated cells. CTED ARTICLE E E ARTICLE ll ( ) h ARTICLE Figure 2. TNFα-induced TNFAIP8 expression in skin cancer cells (A) Schematic represents TNFAIP8 isoform-specific forward and reverse primer design. (B) The expression of different variants/isoforms of TNFAIP8 in normal HaCaT and skin cancer cells was analyzed by RT-PCR. NC–negative control (no cDNA). (C) HaCaT, A431, A375, and A2058 cells were treated with vehicle or TNFα (10–50 ng/ml) for 30 h, and cell lysates were immunoblotted with TNFAIP8 or β-actin antibodies. Immunoreactive bands were visualized using ECL chemiluminescence detection reagents and the blots were scanned using an Odyssey CLx imager. The immunoblot scans were converted into grayscale and presented. (D) Similarly, normal and skin cancer cells were treated with vehicle or TNFα (10-50 ng/ml) for 30 h, and TNFAIP8 mRNA expression was analyzed by RT/ qPCR as described in the “Materials and methods” section. P < 0.05, P < 0.01, P < 0.001 relative to control/ vehicle-treated cells. (E) HaCaT, A431, A375 and, A2058 cells were treated with vehicle or TNFα (10–50 ng/ ml) for 48 h and, relative cell survival was analyzed by MTT assay as described in the “Materials and methods” section. Results are representative of three independent experiments. P < 0.05, **P < 0.001 relative to control/ vehicle-treated cells. CTED AR Figure 2. TNFα-induced TNFAIP8 expression in skin cancer cells (A) Schematic represents TNFAIP8 isoform specific for ard and re erse primer design (B) The e pression of different ariants/isoforms of AR skin cancer cells (A) Schematic represents TNFAIP AR Overexpression of TNFAIP8 increases skin cancer cell survival, colony formation and migra‑ tion. Results i Similarly, no significant effect of TNFAIP8 overexpression on cell colony formation was observed in normal skin HaCaT cells (Fig. 3C, upper and lower panels). These results suggest that TNFAIP8 overexpression promotes cell growth in skin cancer cells but not in normal skin cells. In parallel, we used siRNA to knockdown endogenous TNFAIP8 protein in normal and skin cancer cells (Fig. 3D). Transfection of TNFAIP8 siRNA significantly reduced endogenous TNFAIP8 levels in all cell lines compared with the control siRNA transfected cells (Fig. 3D). The effect of TNFAIP8 knockdown on cell survival was then analyzed by MTT assay. MTT assay demonstrated that TNFAIP8 knockdown reduced cell survival in Figure 2. TNFα-induced TNFAIP8 expression in skin cancer cells (A) Schematic represents TNFAIP8 isoform-specific forward and reverse primer design. (B) The expression of different variants/isoforms of TNFAIP8 in normal HaCaT and skin cancer cells was analyzed by RT-PCR. NC–negative control (no cDNA). (C) HaCaT, A431, A375, and A2058 cells were treated with vehicle or TNFα (10–50 ng/ml) for 30 h, and cell lysates were immunoblotted with TNFAIP8 or β-actin antibodies. Immunoreactive bands were visualized using ECL chemiluminescence detection reagents and the blots were scanned using an Odyssey CLx imager. The immunoblot scans were converted into grayscale and presented. (D) Similarly, normal and skin cancer cells were treated with vehicle or TNFα (10-50 ng/ml) for 30 h, and TNFAIP8 mRNA expression was analyzed by RT/ qPCR as described in the “Materials and methods” section. P < 0.05, P < 0.01, P < 0.001 relative to control/ vehicle-treated cells. (E) HaCaT, A431, A375 and, A2058 cells were treated with vehicle or TNFα (10–50 ng/ ml) for 48 h and, relative cell survival was analyzed by MTT assay as described in the “Materials and methods” section. Results are representative of three independent experiments. P < 0.05, **P < 0.001 relative to control/ vehicle-treated cells. RETRACTED ARTICLE Figure 2. TNFα-induced TNFAIP8 expression in skin cancer cells (A) Schematic represents TNFAIP8 isoform-specific forward and reverse primer design. (B) The expression of different variants/isoforms of TNFAIP8 in normal HaCaT and skin cancer cells was analyzed by RT-PCR. NC–negative control (no cDNA). (C) HaCaT, A431, A375, and A2058 cells were treated with vehicle or TNFα (10–50 ng/ml) for 30 h, and cell lysates were immunoblotted with TNFAIP8 or β-actin antibodies. Immunoreactive bands were visualized using ECL chemiluminescence detection reagents and the blots were scanned using an Odyssey CLx imager. Results i Interestingly, B-RAF mutant A375 and A2058 melanoma cell lines which expressed lower levels of TNFAIP8, upon a dose-dependent treatment with TNFα (10–50 ng/ml) induced TNFAIP8 pro- tein expression (Fig. 2C) suggesting that TNFα induced TNFAIP8 expression in normal and skin cancer cell lines irrespective B-RAF background. Since TNFAIP8 modulates cell survival/proliferation in several cancer cell lines, the role of TNFα mediated induction of TNFAIP8 in skin cancer cell survival was analyzed by MTT assay (Fig. 2E). The data suggest that induction of TNFAIP8 by TNFα (10–20 ng/ml) increased cell survival in normal HaCaT cells as well as in A431 cells and A375, A2058 melanoma cell lines significantly, whereas, a higher concentration of TNFα (50 ng/ml) treatment decreased cell survival in A431, A375 and A2058 skin cancer cell lines (Fig. 2E). We also analyzed the time-dependent effect of TNFα (20 ng/ml) on the expression of TNFAIP8 and cell survival (Supplementary Fig. S1A,B). Our data suggest that treatment of TNFα (20 ng/ml) increased TNFAIP8 expression and cell survival after 12–24 h and significantly decreased the TNFAIP8 expression and cell survival after 48 h treatment (Supplementary Fig. S1A,B) suggesting that, TNFα regulates the expression of TNFAIP8 and cell survival in skin cancer cells with a time-dependent manner. TR https://doi.org/10.1038/s41598-021-85097-6 Scientific Reports \| (2021) 11:5660 \| www.nature.com/scientificreports/ Overexpression of TNFAIP8 increases skin cancer cell survival, colony formation and migra‑ tion. To clarify the exact role of TNFAIP8 in skin cancer cell proliferation, we transiently transfected normal HaCaT skin cells and A431, A375, and A2058 skin cancer cells with empty vector (EV) or TNFAIP8-Myc plas- mid (Fig. 3A). Immunoblotting data demonstrated the overexpression of TNFAIP8-Myc tagged protein in cells transfected with TNFAIP8-Myc tagged plasmid but not in an empty vector (EV) transfected cells (Fig. 3A). The MTT assay demonstrated that overexpression of TNFAIP8 increased cell survival significantly; by ~ 55%, 82%, 68% in A431, A375, and A2058 skin cancer cells, respectively (Fig. 3B). However, no significant effect on cell survival/proliferation was observed in HaCaT normal skin cells transfected with TNFAIP8 (Fig. 3B). We also examined the effect of TNFAIP8 overexpression on skin cancer cell colony formation ability. TNFAIP8 expres- sion promoted cell colony formation in A431 cells by 3.1-fold, A375 cells by 4.2-fold, and A2058 cells by 5.1-fold compared with empty vector-transfected cells (Fig. 3C, upper and lower panels). Results i Overexpression of miR-205-5p mimic significantly decreased both TNFAIP8 mRNA and protein expression in HaCaT, A431 and A2058 cell lines (Fig. 4E,F). We also performed immunocytochemistry and analyzed endogenous TNFAIP8 protein expression by immunofluorescence after delivery of Cy3 labeled miR- 205-5p or Cy3 labeled NC mimic into HaCaT, A431 and A2058 cells (Fig. 4G). TNFAIP8 immunofluorescence data suggest that transfection of Cy3-miR-205-5p in all three cell lines decreased cytosolic TNFAIP8-associated fluorescence (merge panel) compared with Cy3-labeled-NC mimic transfected cells (Fig. 4G) suggesting that miR-205-5p targets the TNFAIP8-3′UTR and inhibits TNFAIP8 protein expression. ARTIC miR‑205‑5p inhibits cell survival and TNFAIP8‑mediated autophagy induction. Since miR- 205-5p inhibits TNFAIP8 expression and TNFAIP8 promotes cell proliferation in skin cancer cells, we further examined the role of miR-205-5p mimic in the regulation of skin cancer cell survival/cell colony formation. No significant effect on cell proliferation/survival was observed in HaCaT cells transfected of miR-205-5p or the NC mimic control, however, a significant decrease in cell proliferation was observed in A431 and A2058 cell lines (Fig. 5A). Also, we analyzed the effect of miR-205-5p on cell colony formation in normal skin cell line HaCaT, and A431 and A2058 cell lines (Fig. 5B). Similar to the effects were seen in the cell survival assay, transfection with miR-205-5p mimic or the NC mimic control did not affect cell colony formation in HaCaT cells (Fig. 5B, left panel). Interestingly, cell colony formation was decreased by 32.5% in A431 cells and 38.5% in A2058 cells when cells were transfected with miR-205-5p compared with NC transfected cells (Fig. 5B, middle and right panels). ACTED A In a previous study, we showed that TNFAIP8 induced autophagy in prostate, breast, and liver cancer cells and increased resistance to specific anti-cancer drugs34,43. Consistent with these findings, we demonstrate here that overexpression of TNFAIP8 also induced autophagy in HaCaT, A431, and A2058 cell lines based on induction of autophagy bio-marker LC3β II (Fig. 5C). Conversely, inhibition of TNFAIP8 expression by miR-205-5p mimic decreased autophagy biomarkers LC3β II and p62 expression in A431 and A2058 cells compared with NC mimic control transfected cells (Fig. 5D). To support this, we also performed immunocytochemistry and analyzed the effect of miR-205-5p mimic on LC3β-related puncta formation in skin cancer cells. Immunocytochemistry data showed that miR-205-5p mimic transfection significantly decreased LC3β related puncta formation in A431 and A2058 cells as well as in HaCaT cells (Fig. Results i In contrast, no sig- nificant effects on cell survival, cell colony formation, and migration were observed in HaCaT cells transfected https://doi.org/10.1038/s41598-021-85097-6 Scientific Reports \| (2021) 11:5660 \| www.nature.com/scientificreports/ with TNFAIP8 siRNA compared with control siRNA transfected cells (Fig. 3E–G). Indeed, the data presented in Fig. 3 suggests that TNFAIP8 promotes cell proliferation and cell migration in skin cancer cells. with TNFAIP8 siRNA compared with control siRNA transfected cells (Fig. 3E–G). Indeed, the data presented in Fig. 3 suggests that TNFAIP8 promotes cell proliferation and cell migration in skin cancer cells. miR‑205‑5p targets the TNFAIP8‑3′UTR and inhibits TNFAIP8 expression. In a previous study, it was reported that miR-205-5p targets the 3′-UTR of either transcription factor E2F1 or transcription factor E2F5 and suppresses the cell proliferation in melanoma cells28. Since our data show that TNFAIP8 promotes skin cancer cell survival and migration, we searched for miRNAs that possibly target TNFAIP8 gene expression. TargetScan, an online software tool (http://www.targetscan.org/vert_72/: accessed June 10, 2019), was used to predict and identify possible 3′TNFAIP8-UTR binding miRNAs. TargetScan analysis revealed that miR-205-5p is predicted to bind with the 3′ UTR of TNFAIP8 gene (61–68) (Fig. 4A). To analyze the effect of miR-205-5p on TNFAIP8 expression, first, we determined the endogenous expression levels of miR-205-5p in HaCaT cells and skin cancer cells. RT/qPCR data showed that miR-205-5p expression was higher in HaCaT cells and SCC-A431 cells, whereas, the expression of miR-205-5p was not detected in melanoma A375, A2058, and SK- MEL-2 (Fig. 4B). To determine whether miR-205-5p binds directly with the TNFAIP8-3′UTR, we performed a TNFAIP8-3′UTR luciferase assay. A431 and A2058 cells were co-transfected with TNFAIP8-3′UTR-Luc plasmid for 18 h and then cells were transfected miR-205-5p mimic or mutant miR-205-5p mimic for 30 h and luciferase activity was determined. The data showed that the miR-205-5p mimic significantly inhibited luciferase activity compared with negative control (NC) or mutant miR-205-5p mimic transfected both cell lines (Fig. 4C). These results support the model where the miR-205-5p mimic binds with the TNFAIP8-3′UTR. ICLE pp p To determine whether the miR-205-5p mimic represses the endogenous expression of TNFAIP8 mRNA and protein, we transfected normal HaCaT cells, A431 (SCC) and A2058 melanoma cells with miR-205-5p mimic or negative control (NC). Overexpression of miR-205 mimic in HaCaT, A431, and A2058 cell lines was analyzed by RT/qPCR (Fig. 4D). Results i (C) The effect of overexpression of TNFAIP8-Myc on cell colony formation was analyzed and plotted (upper and lower panels) as described in the “Materials and methods” section. P < 0.05 relative to EV transfected cells. (D) HaCaT, A431, A375 and, A2058 cells were transfected with control siRNA or TNFAIP8 siRNA (100 nM) for 30 h, and cell lysates were western blotted with anti-TNFAIP8 and β-actin antibodies. Western blots were developed using ECL chemiluminescence detection reagents and the blots were scanned using an Odyssey CLx imager. The immunoblot scans were converted into grayscale and presented. TNFAIP8 and β-actin protein levels were quantified using ImageJ software (https://imagej.nih.gov/ij/) and presented below. (E) The effect of TNFAIP8 knockdown by siRNA on cell survival was analyzed by MTT assay. The experiments were independently performed three times. P < 0.05 relative to control siRNA transfected cells. (F) Control siRNA and TNFAIP8 siRNA-transfected HaCaT, A431, A375 and A2058 cells (3000 cells/well) were re-plated in 6-well plates in triplicate for 7–10 days and skin cancer cell colony formation was analyzed and plotted (upper and lower panels) as described in the “Materials and methods” section. P < 0.05 relative to control siRNA transfected cells. (G) Wound healing assay: HaCaT, A431, A375 and A2058 cells were transfected with control siRNA or TNFAIP8 siRNA for 48 h. The effect of the TNFAIP8 knockdown on cell migration was analyzed using the wound healing assay. Representative images of the wound healing assay (top) and the calculated scratch area (bottom) are shown. P < 0.05 relative to control siRNA transfected cells. ◂ TICLE increased sensitivity to vemurafenib treatment after transfection with miR-205-5p compared with B-RAF wild protein-expressing cell lines. Thus, by targeting TNFAIP8, miR-205-5p could further increase drug sensitivity in the B-RAFV600E mutant melanoma cell lines (Fig. 6B). Moreover, transfection of miR-205-5p in A375 (B-RAFV600E mutant) cells induced the expression of cell apoptotic marker cPARP compared with NC mimic transfected cells (Fig. 6C, lane 1 & 2). Furthermore, transfection of miR-205-5p combined with vemurafenib treatment increased cPARP expression compared with NC mimic transfected and vemurafenib treated cells (Fig. 6C, lane 3 & 4). On the other hand, overexpression of TNFAIP8 decreased paclitaxel and vemurafenib-mediated cPARP expression compared with EV transfected A375 cells (Fig. 6D, compare lanes 1–3 and 4–6). Also, we generated vemurafenib drug-resistant A375R and A2058R melanoma cell lines and utilized in the study. Results i 5E, upper and lower panels) suggesting that miR-205-5p inhibits cell proliferation and TNFAIP8-mediated autophagy induction in skin cancer cells. TRAC miR‑205‑5p increases the sensitivity of skin cancer cells to vemurafenib. Currently, vemurafenib and paclitaxel drugs are used as combine chemotherapy for the treatment of skin cancer patients affected by advanced melanoma which express B-RAFV600E mutant protein 53. We treated HaCaT and SK-MEL-2 cells which possess wild type B-RAF protein, and A375 and A2058 cells which express mutant B-RAFV600E with vemurafenib and relative drug sensitivity were analyzed by cell survival MTT assay (Fig. 6A). Consistent with the clinical data, a dose-dependent treatment of vemurafenib (0.1–25 µM) decreased cell survival in A375 and A2058 which possesses B-RAFV600E mutant, whereas, SK-MEL-2 cells and HaCAT cell lines which express wild type B-RAF protein showed significant resistance to vemurafenib (Fig. 6A). T p gi ( g ) Since the expression miR-205-5p was not detected in melanoma A375 and A2058 cell lines (Fig. 4B), and miR-205-5p targets TNFAIP8, we further examined the potential role of miR-205-5p in vemurafenib sensitiv- ity. We transfected normal and skin cancer cells with miR-205-5p and treated with vemurafenib as indicated and relative drug sensitivity was analyzed (Fig. 6B). Compared with control NC mimic transfected cells, miR- 205-5p transfection alone decreased cell survival in A375, and A2058 cells but not in HaCaT or SK-MEL-2 cells significantly (Fig. 6B). Transfection with miR-205-5p and treatment with vemurafenib further decreased cell survival compared with vemurafenib alone in skin cancer lines SK-MEL-2, A375, and A2058 cells (Fig. 6B, left and right panels). Interestingly, the data also indicated that B-RAFV600E mutant cell lines A375 and A2058 showed https://doi.org/10.1038/s41598-021-85097-6 Scientific Reports \| (2021) 11:5660 \| ture.com/scientificreports/ TRACTED ARTICLE www.nature.com/scientificreports/ RETRACTED ARTICLE LE LE LE ARTICLE ACTED ARTICL T T RETRACTED ARTIC CTED AR TRACT https://doi.org/10.1038/s41598-021-85097-6 Scientific Reports \| (2021) 11:5660 \| www.nature.com/scientificreports/ Figure 3. TNFAIP8 regulates cell proliferation and migration in skin cancer cells. (A) Western blotting analysis of TNFAIP8-Myc tagged protein expression in HaCaT and skin cancer cells. Immunoreactive bands were developed using ECL chemiluminescence detection reagents and the blots were scanned using an Odyssey CLx imager. The immunoblot scans were converted into grayscale and presented. (B) Effect of TNFAIP8-Myc protein overexpression on HaCaT, A431, A375 and, A2058 on cell survival was measured by MTT assay. Results are representative of three independent experiments. P < 0.05 relative to EV transfected cells. Results i A higher expression of TNFAIP8, autophagy marker LB3β II, and increased cell proliferation were observed in A375R or A2058R vemurafenib drug-resistant cells compared with parent A375 or A2058 melanoma cells (Fig. 6E,F). ED ART Collectively the data presented in Fig. 6 suggest that (A) expression of miR-205-5p in melanoma cells increases sensitivity towards vemurafenib particularly in B-RAFV600E mutant cells (B) increased expression of TNFAIP8 reduces drug-mediated cell apoptosis, and (C) increased expression of TNFAIP8 due to vemurafenib drug- resistance increases autophagy and cell proliferation. Indeed, our data suggest that TNFAIP8 might be involved in drug resistance in melanoma cell lines by inducing autophagy as reported in prostate and liver cancer cell lines previously34,43, whereas miR-205-5p controls TNFAIP8/autophagy axis and increased sensitivity towards skin cancer drug vemurafenib. ACTED Scientific Reports \| (2021) 11:5660 \| Discuss A In the current study and for the first time, we demonstrate a molecular and mechanistic role of TNFAIP8 in the regulation of skin cancer cell survival/proliferation, colony formation, migration, and drug resistance. Our data was generated by comparison of the normal skin cell-derived cell line (HaCaT) to melanoma cell lines A375, A2058, SK-MEL-2, and the epidermoid carcinoma A431 cell line. TNFAIP8 has been shown to play a role in oncogenesis in several cancers32,54 and its expression is induced by inflammatory cytokine TNFα34. In the cur- rent study, we demonstrate that exposure of skin cancer cells with TNFα induced expression of TNFAIP8 that modulates cell proliferation. Similarly, overexpression or knockdown of TNFAIP8 affected cell proliferation in skin cancer cell lines. Mechanistically, TNFAIP8 contains a death effector domain that modulates the caspase-8 activity and negatively regulates apoptosis 32. Our preliminary data indicate that TNFAIP8 expression is down- regulated in B-RAFV600E mutant A375, A2058 melanoma cells compared with A431, SK-MEL-2, or normal HaCaT cells which express wild-type B-RAF kinase. However, in skin melanoma patient tissues the expression of TNFAIP8 is higher compared with BCC, SCC, or normal skin tissues indicating that the B-RAF background might play an important role in TNFAIP8 expression in skin cancer and this needs to be further investigated.hi TRA g p y p p g This study identified miR-205-5p as a negative regulator of TNFAIP8 gene expression. Other studies have shown that the expression of several miRNAs with established oncogenic roles is upregulated in melanoma cells20–24, whereas particularly miR-205 expression in skin cancer cells is down-regulated also reported in previous studies28,55,56. We found that melanoma cell lines show undetectable levels of miR-205-5p expression as well as higher expression of TNFAIP8 in SK-MEL-2 melanoma cells and melanoma tissue. Further, we demonstrated that miR-205-5p binds with the 3′UTR of TNFAIP8 gene and inhibits TNFAIP8 expression and TNFAIP8-mediated autophagy in skin cancer cells. Previously, we showed that TNFAIP8 mediated induction of autophagy is required for drug resistance and cell survival in prostate cancer cells34 as well as liver cancer cells43, and similarly, in the current study we demonstrated that miR-205-5p controls the expression of TNFAIP8 and TNFAIP8 mediated- autophagy in skin cancer cells. miR-205-5p expression in melanoma cell lines not only controlled the expression of TNFAIP8 but also increased sensitivity towards vemurafenib, a skin cancer drug currently used for the treat- ment of patients affected by advanced melanoma with B-RAFV600E mutant kinase57. Discuss A Scientific Reports \| (2021) 11:5660 https://doi.org/10.1038/s41598-021-85097-6 ww.nature.com/scientificreports/ Figure 4. miR-205-5p targets the 3′UTR of TNFAIP8 and inhibits TNFAIP8 expression. (A) The binding site of miR-205-5p in the 3′UTR of TNFAIP8 was analyzed by TargetScan (http://www.targetscan.org/vert_72/) and presented. We also generated a wild type and mutated binding nucleotides of miR-205-5p to the 3′UTR of TNFAIP8. Wild-type miR-205-5p and mutant miR-205-5p mimics are shown. (B) Relative expression of miR-205-5p in HaCaT, A431, A375, A2058, and SK-MEL-2 cell lines was analyzed by RT/qPCR as described in the “Materials and methods” section. (C) Luciferase reporter assay: A431 and A2058 cells were transfected with TNFAIP8-3′UTR-Luciferase reporter construct (0.5 µg) for 18 h and then transfected with NC mimic or wild-type miR-205-5p mimic or mutant miR-205-5p mimic for an additional 24 h. Transfected cells were lysed, and luciferase activity was measured. Results are representative of two independent experiments. P < 0.01, P < 0.001 compared with NC or mutant-type miR-205-5p mimic. (D, E) Normal or skin cancer cells were transfected with NC or miR-205-5p mimic for 48 h and the overexpression of miR-205-5p mimic and TNFAIP8 transcripts were analyzed by RT/qPCR. *P < 0.001 compared with NC-transfected cells. P < 0.05 compared with NC-transfected cells. ns-no significance. (F) HaCaT, A431 and A2058 cell lines were transfected with NC mimic or miR-205-5p mimic for 40 h and expression of endogenous TNFAIP8 protein were analyzed by western blotting. Western blots were developed using ECL chemiluminescence detection reagents, the blots were exposed to X-Ray films, scans were converted into grayscale and presented. (G) HaCaT, 431 and A2058 cell lines were grown on a coverslip and transfected with Cy3-labeled NC mimic or Cy3-labeled miR-205-5p mimic and the effect of Cy3-labeled miR-205-5p mimic on endogenous TNFAIP8 expression (green) was visualized by immunofluorescence as described in the “Materials and methods” section TRACTED ARTICLE www.nature.com/scientificreports/ D ARTICLE D ARTICLE E D ARTICLE D ARTICLE D Figure 4. miR-205-5p targets the 3′UTR of TNFAIP8 and inhibits TNFAIP8 expression. (A) The binding site of miR-205-5p in the 3′UTR of TNFAIP8 was analyzed by TargetScan (http://www.targetscan.org/vert_72/) and presented. We also generated a wild type and mutated binding nucleotides of miR-205-5p to the 3′UTR of TNFAIP8. Wild-type miR-205-5p and mutant miR-205-5p mimics are shown. (B) Relative expression of miR-205-5p in HaCaT, A431, A375, A2058, and SK-MEL-2 cell lines was analyzed by RT/qPCR as described in the “Materials and methods” section. Discuss A (C) Luciferase reporter assay: A431 and A2058 cells were transfected with TNFAIP8-3′UTR-Luciferase reporter construct (0.5 µg) for 18 h and then transfected with NC mimic or wild-type miR-205-5p mimic or mutant miR-205-5p mimic for an additional 24 h. Transfected cells were lysed, and luciferase activity was measured. Results are representative of two independent experiments. P < 0.01, P < 0.001 compared with NC or mutant-type miR-205-5p mimic. (D, E) Normal or skin cancer cells were transfected with NC or miR-205-5p mimic for 48 h and the overexpression of miR-205-5p mimic and TNFAIP8 transcripts were analyzed by RT/qPCR. *P < 0.001 compared with NC-transfected cells. P < 0.05 compared with NC-transfected cells. ns-no significance. (F) HaCaT, A431 and A2058 cell lines were transfected with NC mimic or miR-205-5p mimic for 40 h and expression of endogenous TNFAIP8 protein were analyzed by western blotting. Western blots were developed using ECL chemiluminescence detection reagents, the blots were exposed to X-Ray films, scans were converted into grayscale and presented. (G) HaCaT, 431 and A2058 cell lines were grown on a coverslip and transfected with Cy3-labeled NC mimic or Cy3-labeled miR-205-5p mimic and the effect of Cy3-labeled miR-205-5p mimic on endogenous TNFAIP8 expression (green) was visualized by immunofluorescence as described in the “Materials and methods” section. TRACTED A An earlier study revealed that constitutive activation of B-RAFV600E mutant activates the MEK/ERK pathway and enhances melanoma cell proliferation whereas inhibition of the B-RAFV600E mutant by vemurafenib decreases melanoma cell proliferation58. Irrespective of prior treatment with MEK inhibitors or BRAF inhibitor, a recent phase I clinical study demonstrates that vemurafenib, carboplatin, and paclitaxel combination chemotherapy is more effective and tolerable in patients with advanced melanoma with B-RAFV600E mutant protein53. We also showed that inhibition on TNFAIP8 by expression of miR-205-5p drastically decreased vemurafenib-mediated cell proliferation in melanoma cell lines. On the other hand, overexpression of TNFAIP8 reduced vemurafenib or paclitaxel-mediated melanoma cell apoptosis suggests that, TNFAIP8 increased drug resistance in melanoma cell lines by inducing autophagy and by inhibiting cell apoptosis. T y g p gy y g p p Not only the mutation of key oncogenes (RAS, B-RAF, MAPK1/2) involved in skin cancer progression, but reports also suggest that chronic inflammatory pathways regulate the development of melanoma59,60. Dur- ing chronic inflammation, activation of several transcription factors such as NF-kB, STAT3, HIF-1-alpha, and up-regulation of their downstream targets including prostaglandins, cyclooxygenase-2 (COX-2), and cytokine TNF-α was reported59,60. Discuss A TNF-α activates NF-kβ that binds with the TNFAIP8 promoter and induces TNFAIP8 expression32,34. Similarly, our data suggest that the induction of TNFAIP8 by TNFα increases cell proliferation and drug resistance in skin cancer cells. https://doi.org/10.1038/s41598-021-85097-6 Scientific Reports \| (2021) 11:5660 ww.nature.com/scientificreports/ Fi iR TNFAIP d d TNFAIP di d ll lif i d h RACTED ARTICLE www.nature.com/scientificreports/ Figure 5. miR-205-5p targets TNFAIP8 and reduces TNFAIP8-mediated cell proliferation and autophagy. (A) HaCaT, A431 and A2058 cells were transfected with NC mimic or miR-205-5p mimic for 48 h and relative cell survival was analyzed by MTT assay. The experiments were independently repeated three times. P < 0.05 compared with NC mimic transfected cells. (B) NC mimic or miR-205-5p mimic transfected cells were re-plated in 6-well plates for 7–10 days and cell colony formation was analyzed and plotted (upper and lower panels) as described in the “Materials and methods” section. The experiment was repeated twice in triplicates. P < 0.05 relative to NC mimic transfected cells. (C) HaCaT, A431 and A2058 cells were transfected EV or TNFAIP8-Myc plasmids for 30 h and lysates were western blotted with anti-Myc, anti-LC3β I/II, and anti-β-actin antibodies. Western blots were developed using ECL chemiluminescence detection reagents, the blots were exposed on X-ray films, converted into grayscale, and presented. (D) HaCaT, lanoma A431 and A2058 cells were transfected NC mimic or miR-205-5p mimic for 30 h and lysates were western blotted with anti-TNFAIP8, anti-LC3β I/II, anti-p62, and anti-β-actin antibodies. Western blots were developed using ECL chemiluminescence detection reagents, exposed on X-Ray films, converted into grayscale, and presented. (E) HaCaT, A431 and A2058 cells were grown on a coverslip transfected with Cy3-labeled NC mimic or Cy3-labeled miR-205-5p mimic and the effect of Cy3-labeled miR-205-5p mimic on endogenous LC3β I/II expression (green) and related puncta formation were visualized by immunofluorescence (upper panels) as described in the “Materials and methods” RETRACTED ARTICLE LE LE ED ARTICLE T Figure 5. miR-205-5p targets TNFAIP8 and reduces TNFAIP8-mediated cell proliferation and autophagy. (A) HaCaT, A431 and A2058 cells were transfected with NC mimic or miR-205-5p mimic for 48 h and relative cell survival was analyzed by MTT assay. The experiments were independently repeated three times. P < 0.05 compared with NC mimic transfected cells. Discuss A (B) HaCaT cells and skin cancer SK-MEL-2, A375 and A2058 cell lines were transfected with NC mimic or miR-205-5p mimic for 24 h and treated with vemurafenib or vehicle for an additional 48 h and relative cell survival were measured by MTT assay as described in the “Materials and methods” section. P < 0.05, P < 0.01, P < 0.001 compared to NC mimic vehicle-treated cells or NC mimic and vemurafenib-treated cells. ns-no significance. (C) Melanoma A375 cells were transfected with NC mimic or miR-205-5p mimic for 18 h and then cells were treated with vemurafenib for an additional 24 h. Fifty micrograms of lysates were immunoblotted with anti- cleaved-PARP and anti-β-tubulin antibodies. Western blots were developed using ECL chemiluminescence detection reagents, the blots were scanned using Azure C-500 Biosystem, scans were converted into grayscale and presented. (D) Melanoma A375 cells were transfected with EV of TNFAIP8-Myc plasmid for 18 h first and then cells were treated with vemurafenib or paclitaxel for 24 h as indicated and fifty micrograms lysates were immunoblotted with anti-Myc, anti-cleaved-PARP, and anti-β-actin antibodies. Western blots were developed using ECL chemiluminescence detection reagents and the blots were scanned using an Odyssey CLx imager. The immunoblot scans were converted into grayscale and presented. (E) Fifty micrograms of lysates from A375 and A2058 cells or A375R and A2058R (vemurafenib resistant) cells were immunoblotted with anti-TNFAIP8, anti- LC3β I/II, and anti-β-actin antibodies. Western blots were developed using ECL chemiluminescence detection reagents, the blots were exposed on X-Ray films, scans were converted into grayscale and presented. (F) Relative cell proliferation/cell survival rate of A375 and A2058 cells, or A375R and A2058R (vemurafenib resistant) cells were measured by using WST-1 reagent (Cayman Chemical) (upper panel) or MTT assay reagent (lower panel) and plotted. P < 0.01, *P < 0.001 compared to A375 and A2058 parent cells. Results are representative of three independent experiments. Vem. Vemurafenib. TRACTED A Since disruption of the p53 pathway was reported in melanoma14, and recently the role of TNFAIP8 in p53 signaling was analyzed in lung cancer cells and the study revealed that TNFAIP8 variant 2 (v2) represses tumor suppressor wild-type p53 function in lung cancer A549 cells37. Silencing of TNFAIP8 v2 induces p53-independent inhibition of DNA synthesis, widespread p53 binding, the initiation of p53-dependent cell-cycle arrest, and the sensitization of cells to doxorubicin37. Discuss A (B) HaCaT cells and skin cancer SK-MEL-2, A375 and A2058 cell lines were transfected with NC mimic or miR-205-5p mimic for 24 h and treated with vemurafenib or vehicle for an additional 48 h and relative cell survival were measured by MTT assay as described in the “Materials and methods” section. P < 0.05, P < 0.01, P < 0.001 compared to NC mimic vehicle-treated cells or NC mimic and vemurafenib-treated cells. ns-no significance. (C) Melanoma A375 cells were transfected with NC mimic or miR-205-5p mimic for 18 h and then cells were treated with vemurafenib for an additional 24 h. Fifty micrograms of lysates were immunoblotted with anti- cleaved-PARP and anti-β-tubulin antibodies. Western blots were developed using ECL chemiluminescence detection reagents, the blots were scanned using Azure C-500 Biosystem, scans were converted into grayscale and presented. (D) Melanoma A375 cells were transfected with EV of TNFAIP8-Myc plasmid for 18 h first and then cells were treated with vemurafenib or paclitaxel for 24 h as indicated and fifty micrograms lysates were immunoblotted with anti-Myc, anti-cleaved-PARP, and anti-β-actin antibodies. Western blots were developed using ECL chemiluminescence detection reagents and the blots were scanned using an Odyssey CLx imager. The immunoblot scans were converted into grayscale and presented. (E) Fifty micrograms of lysates from A375 and A2058 cells or A375R and A2058R (vemurafenib resistant) cells were immunoblotted with anti-TNFAIP8, anti- LC3β I/II, and anti-β-actin antibodies. Western blots were developed using ECL chemiluminescence detection reagents, the blots were exposed on X-Ray films, scans were converted into grayscale and presented. (F) Relative cell proliferation/cell survival rate of A375 and A2058 cells, or A375R and A2058R (vemurafenib resistant) cells were measured by using WST-1 reagent (Cayman Chemical) (upper panel) or MTT assay reagent (lower panel) and plotted. P < 0.01, P < 0.001 compared to A375 and A2058 parent cells. Results are representative of three independent experiments. Vem. Vemurafenib. TRACTED A Figure 6. miR-205-5p targets TNFAIP8 in skin cancer cells and increase sensitivity to vemurafenib. (A) HaCaT cells, and skin cancer SK-MEL-2, A375 and A2058 cell lines were treated with increasing concentrations of vemurafenib for 48 h and relative cell survival/proliferation was measured by MTT assay. The experiments were independently repeated three times. P < 0.01, **P < 0.001 compared to vehicle-treated cells. Discuss A (B) NC mimic or miR-205-5p mimic transfected cells were re-plated in 6-well plates for 7–10 days and cell colony formation was analyzed and plotted (upper and lower panels) as described in the “Materials and methods” section. The experiment was repeated twice in triplicates. P < 0.05 relative to NC mimic transfected cells. (C) HaCaT, A431 and A2058 cells were transfected EV or TNFAIP8-Myc plasmids for 30 h and lysates were western blotted with anti-Myc, anti-LC3β I/II, and anti-β-actin antibodies. Western blots were developed using ECL chemiluminescence detection reagents, the blots were exposed on X-ray films, converted into grayscale, and presented. (D) HaCaT, lanoma A431 and A2058 cells were transfected NC mimic or miR-205-5p mimic for 30 h and lysates were western blotted with anti-TNFAIP8, anti-LC3β I/II, anti-p62, and anti-β-actin antibodies. Western blots were developed using ECL chemiluminescence detection reagents, exposed on X-Ray films, converted into grayscale, and presented. (E) HaCaT, A431 and A2058 cells were grown on a coverslip transfected with Cy3-labeled NC mimic or Cy3-labeled miR-205-5p mimic and the effect of Cy3-labeled miR-205-5p mimic on endogenous LC3β I/II expression (green) and related puncta formation were visualized by immunofluorescence (upper panels) as described in the “Materials and methods” section (upper panel). LC3β I/II and related puncta were measured from individual cells (n = 10) and plotted (lower panels). P < 0.01, P < 0.001 relatives to Cy3 labeled-NC mimic transfected cells. TRA https://doi.org/10.1038/s41598-021-85097-6 Scientific Reports \| (2021) 11:5660 \| www.nature.com/scientificreports/ ww.nature.com/scientificreports/ Since disruption of the p53 pathway was reported in melanoma14, and recently the role of TNFAIP8 in p53 signaling was analyzed in lung cancer cells and the study revealed that TNFAIP8 variant 2 (v2) represses tumor suppressor wild-type p53 function in lung cancer A549 cells37. Silencing of TNFAIP8 v2 induces p53-independent inhibition of DNA synthesis, widespread p53 binding, the initiation of p53-dependent cell-cycle arrest, and the Figure 6. miR-205-5p targets TNFAIP8 in skin cancer cells and increase sensitivity to vemurafenib. (A) HaCaT cells, and skin cancer SK-MEL-2, A375 and A2058 cell lines were treated with increasing concentrations of vemurafenib for 48 h and relative cell survival/proliferation was measured by MTT assay. The experiments were independently repeated three times. P < 0.01, *P < 0.001 compared to vehicle-treated cells. Discuss A (B) HaCaT cells and skin cancer SK-MEL-2, A375 and A2058 cell lines were transfected with NC mimic or miR-205-5p mimic for 24 h and treated with vemurafenib or vehicle for an additional 48 h and relative cell survival were measured by MTT assay as described in the “Materials and methods” section. P < 0.05, P < 0.01, P < 0.001 compared to NC mimic vehicle-treated cells or NC mimic and vemurafenib-treated cells. ns-no significance. (C) Melanoma A375 cells were transfected with NC mimic or miR-205-5p mimic for 18 h and then cells were treated with vemurafenib for an additional 24 h. Fifty micrograms of lysates were immunoblotted with anti- cleaved-PARP and anti-β-tubulin antibodies. Western blots were developed using ECL chemiluminescence detection reagents, the blots were scanned using Azure C-500 Biosystem, scans were converted into grayscale and presented. (D) Melanoma A375 cells were transfected with EV of TNFAIP8-Myc plasmid for 18 h first and then cells were treated with vemurafenib or paclitaxel for 24 h as indicated and fifty micrograms lysates were immunoblotted with anti-Myc, anti-cleaved-PARP, and anti-β-actin antibodies. Western blots were developed using ECL chemiluminescence detection reagents and the blots were scanned using an Odyssey CLx imager. The immunoblot scans were converted into grayscale and presented. (E) Fifty micrograms of lysates from A375 and A2058 cells or A375R and A2058R (vemurafenib resistant) cells were immunoblotted with anti-TNFAIP8, anti- LC3β I/II, and anti-β-actin antibodies. Western blots were developed using ECL chemiluminescence detection reagents, the blots were exposed on X-Ray films, scans were converted into grayscale and presented. (F) Relative cell proliferation/cell survival rate of A375 and A2058 cells, or A375R and A2058R (vemurafenib resistant) cells were measured by using WST-1 reagent (Cayman Chemical) (upper panel) or MTT assay reagent (lower panel) and plotted. P < 0.01, P < 0.001 compared to A375 and A2058 parent cells. Results are representative of three independent experiments. Vem. Vemurafenib. RETRACTED ARTICLE LE ARTICLE A Figure 6. miR-205-5p targets TNFAIP8 in skin cancer cells and increase sensitivity to vemurafenib. (A) HaCaT cells, and skin cancer SK-MEL-2, A375 and A2058 cell lines were treated with increasing concentrations of vemurafenib for 48 h and relative cell survival/proliferation was measured by MTT assay. The experiments were independently repeated three times. P < 0.01, ***P < 0.001 compared to vehicle-treated cells. Materials and methods In short, cell lysates (50 µg) were separated on NuPAGE 4–12% Bis–Tris-SDS gels (Invitrogen) and then transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA). The membranes were blocked in 1X blocking buffer (Sigma-Aldrich, St. Louis, MO) and incubated with primary antibodies overnight at 4 °C. The following anti- bodies were obtained from Cell Signaling Technology (Danvers, MA): anti-B-RAF, anti-LC3β I/II, anti-p62, anti-Myc tag, anti-Cleaved-PARP, anti-β-tubulin, and anti-GAPDH. Anti-β-actin antibody from Sigma (St. Louis, MO) and anti-TNFAIP8 antibody from Proteintech Group (Rosemont, IL) was also purchased. All pri- mary antibodies were used as per the manufacturer’s suggestions, typically at a 1:1000 dilution. After washing the membranes three times, the membranes were incubated in the appropriate secondary antibody conjugated with HRP (1:10,000 dilution) (Jackson ImmunoResearch, PA) or anti-rabbit secondary antibody conjugated with IRdye-680RD (LI-COR) for 1 h at room temperature, and immunoreactive bands were visualized using ECL chemiluminescence detection reagents (Signagen Laboratories, Rockville, MD) or by scanning the blots with an Odyssey CLx imager. The immunoblot scans were converted into grayscale and presented. The original immunoblot scans were presented as Supplementary Figs. S2–S7 with the manuscript. ACTED ART Cell transfection. Empty vector (EV) pcDNA3.1 or TNFAIP8 (human tumor necrosis factor-alpha- induced protein eight transcript variant 1)-Myc-DDK-tagged ORF cDNA plasmid was obtained from Origene (Rockville-MD, Cat # RC202729). Skin cancer A431, A375, and A2058 or HaCaT cell lines were grown in 6-well plates (1 × 105 cells/wells) for 24 h before transfection and 2 μg of EV or TNFAIP8-Myc tagged plasmid DNA was transfected using Lipofectamine LTX Plus transfection reagent (Invitrogen). After 30–40 h of transfection, cells were harvested, and the expression of the TNFAIP8-Myc tagged protein was examined by immunoblotting. For siRNA transfection, control siRNA and human TNFAIP8 siRNA were obtained from Dharmacon (Lafayette, CO). Skin cancer A431, A375, and A2058 or normal skin cells were grown in antibiotic-free medium overnight and transfected with 100 nM control or TNFAIP8 siRNA using Lipofectamine RNAiMAX reagent (Invitrogen). For miRNA transfection, negative control (NC) and miR-205-5p mimic miRNAs were obtained from Origene (Rockville-MD) and skin cancer cells or normal skin cells were transfected with NC or miR-205-5p mimic microRNAs (100 nM) using Lipofectamine-2000 reagent (Invitrogen), After transfection of 30 h, cells were harvested and expression of miR-205-5p or TNFAIP8 was analyzed. TRAC RT/PCR and RT/qPCR. Materials and methods Materials and methods Cell culture. Skin cancer A431, A375, A2058, SK-MEL-2 cell lines, and the normal skin HaCaT cell line were obtained from ATCC (Manassas, VA). Cells were grown in DMEM medium (Invitrogen, Carlsbad, CA) contain- ing 5% fetal bovine serum (FBS; Access Biologicals, Vista, CA) and 1% penicillin/streptomycin. Cell lines were maintained at 37 °C in a humidified cell culture incubator containing 5% CO2. We also generated B-RAFV600E mutant inhibitor vemurafenib resistance A375R and A2058R Cells. A375 and A2058 Cells were treated with 0.5–10 µM of vemurafenib. The cells were passaged twice every week to induce B-RAFV600E mutant inhibitor resistance and drug resistance cells were maintained in 1 µM vemurafenib. All cell lines were grown for at least 24 h and used for experimentation when they reached 70–80% confluence. Immunohistochemical analysis (IHC) of human skin cancer tissue samples. Skin cancer tissue samples used in the study were gifted from the Department of Pathology, General Hospital of Ningxia Medical University, Yinchuan, Ningxia Hui Autonomous Region, China. The samples included normal skin tissue (n = 6), basal cell carcinoma (BCC) (n = 6), high differentiated squamous cell carcinoma (H-SCC) (n = 4), medium dif- ferentiated squamous cell carcinoma (M-SCC) (n = 4), low differentiated squamous cell carcinoma (M-SCC) (n = 4), skin nevus tissues (n = 6), and skin melanoma tissues (n = 6). Paraffin-embedded four-micron tissue sec- tions were prepared and stained with a TNFAIP8 antibody (1:50 dilution) by incubation at 4 °C overnight. TNFAIP8 expression was detected using a Vector Labs Elite ABC Kit with 3,3′-diaminobenzidine as the chromo- gen and hematoxylin as the counterstain (Innovex Biosciences). Slides were observed under a light microscope and photographed. The relative expression of TNFAIP8 from normal skin tissue and skin cancer tissues was quantified using Image-J software (https://imagej.nih.gov/ij/) and an average TNFAIP8 expression density was plotted. RTICLE Western blot. Skin cancer A431, A375, A2058, SK-MEL-2 cell lines, and the normal skin HaCaT cell line were grown for 24 h and treated or transfected with indicated plasmids. Transfected or treated cells were lysed in cell lysis buffer (Cell Signaling Technology, Danvers, MA) containing a protease inhibitor cocktail (Roche, Indianapolis, IN) and after centrifugation (10 min at 10,000 rpm) cell supernatant lysates were collected. The protein concentrations were measured from the supernatants using the Bio-Rad protein assay reagent (Bio- Rad, Hercules, CA). Western blotting was performed as described previously34. Discuss A Mutant p53 (p53-K120) binds the TNFAIP8 locus at a cryptic p53 response element that is not occupied or bound by wild‐type p53 and thus increases TNFAIP8 expression, which promotes lung cancer cell survival/proliferation39. However, the exact role of TNFAIP8 in p53 regulation and signaling in melanoma and other skin cancers remains unknown.i Indeed, for the first time, we report here that TNFAIP8 enhances skin cancer cell proliferation and cell colony formation and migration. By induction of autophagy and by inhibiting cell apoptosis, TNFAIP8 also increased drug resistance in skin cancer cells. Interestingly, we also demonstrated that the expression of miR-205-5p controls the expression of TNFAIP8 and sensitizes melanoma cells to the B-RAFV600E mutant kinase inhibitor vemurafenib. https://doi.org/10.1038/s41598-021-85097-6 Scientific Reports \| (2021) 11:5660 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ were isolated using the mirVana microRNA Isolation Kit (Thermo Fisher Scientific). Total miRNAs (10 ng) were reverse transcribed using primers specific for miR-205-5p and U44 (Assay IDs 000509 and 001094, Applied Bio- systems, Carlsbad, CA) and TaqMan Reverse Transcription reagents (Applied Biosystems). Expression of miR- 205-5p and U44 was quantified by RT/qPCR using TaqMan PCR master mixture and Taqman expression assay primers. U44 expression was used as an internal control. The PCR reactions were run on a QuantStudio-3 PCR system (Applied Biosystems) and relative quantitation was analyzed according to the manufacturer’s protocols. were isolated using the mirVana microRNA Isolation Kit (Thermo Fisher Scientific). Total miRNAs (10 ng) were reverse transcribed using primers specific for miR-205-5p and U44 (Assay IDs 000509 and 001094, Applied Bio- systems, Carlsbad, CA) and TaqMan Reverse Transcription reagents (Applied Biosystems). Expression of miR- 205-5p and U44 was quantified by RT/qPCR using TaqMan PCR master mixture and Taqman expression assay primers. U44 expression was used as an internal control. The PCR reactions were run on a QuantStudio-3 PCR system (Applied Biosystems) and relative quantitation was analyzed according to the manufacturer’s protocols. Cell survival MTT assay. Skin cancer A431, A375, A2058 and, normal skin HaCaT cells (1 × 104 cells/well) were grown in 96 plates for 18 h. Cells were treated with different concentrations of TNFα for 48 h and relative cell survival was analyzed by MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) rea- gent (MP Biochemicals, Santa Ana, CA). Similarly, in other experiments, skin cancer cells or normal skin cells (1 × 105) were seeded onto 6-well plates for 24 h and transfected with EV of TNFAIP8-Myc plasmids or control siRNA and TNFAIP8 siRNA separately for 24 h. The transfected cells were harvested, and ten thousand cells seeded onto 96-well plates 48 h. Cells were incubated with 5 µl/well of MTT reagent (5 mg/ml) for 1 h at 37 °C in a cell culture incubator. Cells were washed with PBS, formazan crystals were dissolved in DMSO, and cell survival was quantified by reading the plates at 570 nm using a Fluostar Omega plate reader (BMG Lab Tech, Cary, NC). CLE Colony formation assay. Immunofluorescence. T Immunofluorescence. HaCaT cells and skin cancer cell lines A431 and A2058 (1.5 × 105) were grown on coverslips in 6-well plates and transfected with custom made Cy3-labeled NC or miR-205-5p mimic (Bioneer, Oakland, CA) for 48 h. After washing with PBS, cells were fixed with paraformaldehyde (4%) for 15 min, per- meabilized with 0.25% Triton X-100 in PBS, and blocked with 2% bovine serum albumin in PBS for 1 h at room temperature. Cells were washed with PBS two times (15 min each) and incubated with anti-TNFAIP8 or anti- LB3β I/II rabbit primary antibodies (diluted 1:500) at 4 °C for 18 h. Cells were washed again twice with PBS and incubated with Alexa-Fluor 488-conjugated fluorescein-labeled anti-rabbit secondary antibody (Invitrogen). After the immunostaining, cells were washed three times with PBS mounted with Vectashield-DAPI mount- ing medium (Vector Laboratories, Burlingame, CA, Cat # H-1500-10). Slides were observed under a confocal microscope ZEISS LS 800 and images were captured. RACTED Luciferase assay. For luciferase assay, first A431 and A2058 melanoma cells were transfected with 1 µg of TNFAIP8-3′UTR-Luciferase reporter construct (OriGene) in 96 well plates. After 18 h of transfection, cells were further transfected with 100 nM of miR-205-5p (wild-type), or miR-205-5p (mutant) mimic or NC mimic miRNAs for an additional 24 h. Transfected cells were washed with PBS and mixed with luciferase substrate (Switchgear Genomics, Carlsbad, CA, Cat # LS010). Plates were covered with aluminum foil to protect from light and incubated at room temperature for 30 min. The plates were read by using Fluostar Omega plate reader (BMG Lab Tech, Cary, NC) and relative luciferase activity was measured and plotted. TRA Luciferase assay. For luciferase assay, first A431 and A2058 melanoma cells were transfected with 1 µg of TNFAIP8-3′UTR-Luciferase reporter construct (OriGene) in 96 well plates. After 18 h of transfection, cells were further transfected with 100 nM of miR-205-5p (wild-type), or miR-205-5p (mutant) mimic or NC mimic miRNAs for an additional 24 h. Transfected cells were washed with PBS and mixed with luciferase substrate (Switchgear Genomics, Carlsbad, CA, Cat # LS010). Plates were covered with aluminum foil to protect from light and incubated at room temperature for 30 min. The plates were read by using Fluostar Omega plate reader (BMG Lab Tech, Cary, NC) and relative luciferase activity was measured and plotted. TRA Statistical analysis. Results are from independent duplicate or triplicate experiments and presented as means ± SEM. www.nature.com/scientificreports/ Skin cancer A431, A375, A2058, and normal skin HaCaT cells were (1 × 105 cells/ well) were grown in six plates for 18 h and cells were transiently transfected with TNFAIP8-Myc plasmid or empty-vector (EV) (2 µg/well in 6-well plates), or TNFAIP8 siRNA or control siRNA as indicated in different figures. In other experiments, cells were transfected with NC mimic or miR-205-5p mimic miRNAs for 24 h. Transfected cells were trypsinized and counted and live cells (3000 cells/well) were re-plated in 6-well plates in triplicate and allowed to grow for 7–10 days. Cells were washed with PBS, fixed with cold methanol, and stained with 0.1% crystal violet for 1 h. Cells were washed with distilled water and allowed to dry. Blue colonies were counted and plotted. ARTIC Migration assay. The effect of miR-205-5p overexpression on the migratory ability of skin cancer cells was analyzed by a wound healing migration assay as described previously17. Cells were transfected with miR- 205-5p or NC mimic and transfected cells were plated, and the cell monolayer was scraped using a micropipette tip (A0). At 24 h post-wounding (A24), cells were photographed, and the migration gap length was calculated using ImageJ software (https://imagej.nih.gov/ij/). The percent wound closure was calculated using the formula [(A0 – A24)/A0] × 100. ED AR Materials and methods Total RNA from skin cancer SK-MEL-2, A431, A375, A2058, and HaCaT cell lines was isolated using TRIZOL reagent (Life Technologies), and after cDNA synthesis, the expression of TNFAIP8 isoforms was amplified by PCR by using isoform-specific primers (Supplementary Table S1) followed by RT/ PCR as described previously34. The PCR products were electrophoresed on a 1.5% agarose gel stained with ethidium bromide. The PCR-amplified bands were extracted from the agarose gel and sequenced to confirm the identity of the isoforms. In other experiments, skin cancer A431, A375, A2058, and normal skin HaCaT cells were treated with TNFα (20 ng/ml) for 30 h and total RNA from cells was isolated using TRIZOL reagent. Equal amounts of RNA (1 µg) was reverse transcribed using a High Capacity cDNA Reverse Transcription kit (Applied Biosystems), and cDNA was incubated with Power SYBR Green PCR master mix (Applied Biosystems) and mixed with forward and reverse primers of TNFAIP8 (Supplementary Table S1). GAPDH primers were used as an internal control. For miR-205-5p expression analysis, total microRNAs from normal or skin cancer cells https://doi.org/10.1038/s41598-021-85097-6 Scientific Reports \| (2021) 11:5660 \| www.nature.com/scientificreports/ Immunofluorescence. T Differences between groups were analyzed using either two-tailed Student’s t test or one-way ANOVA followed by Tukey HSD post-hoc test. 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CTE g We gratefully acknowledge the Grants U01CA194730, U54MD012392, and R01MD012767 from the National Institutes of Health awarded to D.K and National Natural Science Foundation of China Grant 81641106 to XG. TE Author contributions CT X.G. and S.N. planned, designed, and performed the experiments and wrote the manuscript. M.L. and P.C. performed the experiments and P.A.L. edited the manuscript and involved in the discussion. D.K. super- vised, planned, and designed the research and wrote the manuscript. All authors read and approved the final manuscript. ACT www.nature.com/scientificreports/ Identification of coexistence of BRAF V600E mutation and EZH2 gain specifically in melanoma as a promising for combination therapy J Transl Med 15 243 https://doi org/10 1186/s12967-017-1344-z (2017) CLE V600E activity in BRAF mutant cells. Biomol. Ther. (Seoul) 23, 320 326. https://doi.org/10.4062/biomolther.2015.007 (2015). 0. Yu, H. et al. 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Induction of resistance to BRAF inhibitor is associated with the inability of Spry2 to inhib V600E activity in BRAF mutant cells Biomol Ther (Seoul) 23 320–326 https://doi org/10 4062/biomolther 2015 007 ( LE V600E activity in BRAF mutant cells. Biomol. Ther. (Seoul) 23, 320 326. https://doi.org/10.4062/biomolther.2015.007 (20 50. Yu, H. et al. Additional informationh TR Additional information Supplementary Information The online version contains supplementary material available at https://doi.org/ 10.1038/s41598-021-85097-6. TR Additional information Supplementary Information The online version contains supplementary material available at https://doi.org/ 10.1038/s41598-021-85097-6. TR Additional information Supplementary Information The online version contains supplementary material available at https://doi.org/ 10.1038/s41598-021-85097-6. TR Correspondence and requests for materials should be addressed to S.N. or D.K. 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https://openalex.org/W3097823548	https://arts.units.it/bitstream/11368/2989693/1/OJCOMS3034747.pdf	English	null	Energy-Constrained Uncoordinated Multiple Access for Next-Generation Networks	IEEE open journal of the Communications Society	2,020	cc-by	10,677	Energy-Constrained Uncoordinated Multiple Access for Next-Generation Networks F. BABICH (Senior Member, IEEE), G. BUTTAZZONI (Member, IEEE), F. VATTA (Member, IEEE), AND M. COMISSO (Member, IEEE) Department of Engineering and Architecture, University of Trieste, 34127 Trieste, Italy CORRESPONDING AUTHOR: F. BABICH (e-mail: babich@units.it) This work was supported in part by the Italian Ministry of University and Research within project FRA 2018 (University of Trieste, Italy) “UBER-5G: Cubesat 5G networks—Access layer analysis and antenna system development.” ABSTRACT This article analyzes the performance of a distributed joint power/packet diversity random access scheme in the presence of energy requirements. In particular, the two main approaches separately developed in the recent years for exploiting the Interference Cancellation (IC) capabilities of modern receivers, that is, Non-Orthogonal Multiple Access (NOMA) and Packet Repetition (PR), are firstly compared and then combined by imposing a constraint on the total available power. This constraint, which is alternative to the commonly adopted one based on the maximum power allowed for a transmission, results much more practical, since it enables to better infer the energy efficiency of a scheme, be it a pure NOMA, PR or combined NOMA/PR one. Both theoretical derivations and numerical simulations are carried out to evaluate the success probability and the throughput of the considered schemes by accounting for evolved reception criteria and different fading scenarios. Furthermore, the influence of several nonidealities, including imperfect IC and packet overhead, is discussed together with the impact of the system parameters, such as the user rate, the average signal to noise ratio, and the number of slots that compose a random access frame. INDEX TERMS Random access, slotted Aloha, NOMA, packet diversity, energy constraints. INDEX TERMS Random access, slotted Aloha, NOMA, packet diversity, energy constraints. I. INTRODUCTION performance may be in fact significantly improved by introducing energy diversity and Interference Cancellation (IC), whose combination enables to design different Non- Orthogonal Multiple Access (NOMA) schemes [6]–[14]. More precisely, the NOMA concept consists in allowing a set of users, which rely on the same modulation and code rate, to transmit using different energy levels selected in a set of L elements. This concept may be used both in centralized and distributed scenarios. In a centralized system, such as a cellular one, the coordinating authority, represented by the base station, assigns the different levels to the different users. Instead, in a distributed system, each user randomly selects its level by knowing the allowed ones, but not knowing those selected by the other users. In particular, the levels are cho- sen so that, if at most L users send their packets in a given slot using different levels, all the packets can be decoded. Beside NOMA, another approach has been developed in the last years for increasing the SA throughput. This approach, which combines Packet Repetition (PR) and IC, relies on R ANDOM access represents a fundamental issue for all kinds of networks that cannot rely on a central- ized authority for managing the communications [1]–[4]. Its importance will further grow in the forthcoming fifth and sixth-generation (5G/6G) systems, whose final aim will be that of creating a pervasive space/terrestrial wireless scenario implementing the Internet of Things (IoT) paradigm. To this purpose, 5G/6G technologies will have to reliably support a large number of possible distributed applications, cover- ing management, automation, safety, and security services, whose simultaneous execution will generate sporadic and heterogeneous traffic. R This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ Digital Object Identifier 10.1109/OJCOMS.2020.3034747 A. RELATED WORK The above studies have highlighted the benefits of com- bined NOMA/PR strategies in an ideal scenario, but their actual applicability to real contexts requires further consider- ations. Firstly, the impact of imperfect IC and unideal power control should be better quantified [9], since the residual interference and the fading effects may significantly affect the evolution of the IC process. Secondly, for cost and safety reasons, ultra-dense 5G/6G networks will be characterized by stringent energy saving constraints [24], [25], whose influ- ence on NOMA/PR strategies has not been even deeply addressed. Thirdly, the detection criterion should properly match the behavior of a real receiver, in which a success- ful reception does not occur only for the single uncollided or cleaned (after IC) slot (single-slot erasure model). In a practical receiver, in fact, even a collision may lead to decod- able packets, as long as the Signal to Interference-plus-Noise Ratio (SINR) of one of them lies beyond the reception threshold [26]–[28]. Besides, when PR schemes are used, suitable headers must be added to each packet replica for enabling the receiver to identify, within the Random Access Frame (RAF), the positions of the other replicas of the same packet [29]. These headers enable, on one hand, the cancel- lation of the replicas of an already decoded packet, and, on the other hand, the study of more evolved multi-slot capture models, such as Chase combining, in the presence of PR, or joint decoding, in the presence of incremental redun- dancy [30]–[34]. Preliminary simulations concerning the impact of the reception model and of the energy requirement on the performance of a combined power/packet diversity scheme have been presented in [35], but ideally assuming perfect IC and absence of fading. II. ENERGY LEVEL DESIGN Consider a distributed wireless network in which H contend- ing users send packets to a common destination. The packet arrival is described by a Poisson process and the time domain is subdivided into RAFs of K slots. Each user, assumed frame- and slot-synchronous, sends at most one packet in each RAF by randomly selecting, among the K slots, those in which M replicas or M encoded segments are inserted. The term ‘segment’ is from now on adopted in general to identify the possibility of including in the analysis either repetition or incremental redundancy, in which the content of a slot may be designed to be individually decodable. A. RELATED WORK Current 2-4G cellular networks manage the distributed load by adopting the well known Slotted Aloha (SA) proto- col [5], for which several extensions, suitable for the more challenging 5G/6G scenario, have been proposed. The SA 1808 VOLUME 1, 2020 the transmission of a certain number M of replicas of a packet [15]–[19]. Two fixed repetitions are adopted in the Contention Resolution Diversity SA (CRDSA) protocol [15], while a random number of them is used in the Irregular Repetition SA (IRSA) scheme [16], already included in the last update of the DVB-RCS2 standard [17], and in its pri- oritized extension [18]. A coded strategy is alternatively adopted in the Coded SA (CSA) algorithm [19], where the packets are firstly divided into segments and subsequently encoded. Some useful proposals have also investigated the combination of NOMA and PR to jointly exploit energy and packet diversities [20]–[23]. In particular, in [20], the benefits of PR in a NOMA scheme are analyzed by intro- ducing a multichannel SA system, while, in [21] and [22], the NOMA/CRDSA scheme is specifically discussed and an analytical expression for the packet error probability is derived. The NOMA/IRSA case is instead addressed in [23], where the degree-distributions originally presented in [16] are recalculated for the joint scheme. imperfect IC. To this aim, the NOMA energy levels and the transmission power of each packet replica are analyt- ically derived in agreement with the rate and the residual interference due to unideal IC. Subsequently, tight upper bounds on the success probability in slow and fast fading scenarios are theoretically estimated for different reception criteria, including the single-slot erasure model and the multi-slot capture ones represented by Chase combining and joint packet decoding. In particular, closed-form expressions for the success probability of a NOMA scheme are obtained for any number of energy levels in a fading-less channel. All theoretical results are validated by extensive Monte Carlo simulations, which are also carried out to investigate the dependance of the throughput from the average Signal to Noise Ratio (SNR) and the header carried by each replica. This article is organized as follows. Section II describes the energy design. Section III derives the upper bounds on the success probability. Section IV discusses the obtained results. Finally, Section V summarizes the main conclusions. A. RELATED WORK In particu- lar, the pure SA and NOMA schemes [6], are characterized by M = 1, since they do not use repetition. Instead, the pure CRDSA and the joint NOMA/CRDSA schemes [15], are characterized by M = 2, while, in the pure IRSA and the joint NOMA/IRSA ones, M is a random variable (r.v.) whose optimized Probability Density Functions (PDFs) have been determined in [23]. Beside the packet encoding in segments, in NOMA, NOMA/CRDSA, and NOMA/IRSA schemes, each user can select, among L possible equally likely energy levels, that adopted for the transmission of its segments. The other schemes, i.e., SA, CRDSA, and IRSA, of course adopt L = 1. At the destination, according to the IC mechanism, the segments, possibly collided but referred to already success- fully decoded packets, are cancelled from the corresponding slots, thus enabling further packet decodings. To realistically account for the nonideality of IC, define as ϵ (0 ≤ϵ ≤1) the fraction of the residual interference after each cancel- lation cycle and consider the presence of a specific header in each segment. This header, whose effect on the final performance of each scheme will be specifically addressed in Section IV.D, contains specific pointers to the positions in the RAF of the other segments referred to the same packet. This enables the destination to infer the slots in which IC must VOLUME 1, 2020 B. CONTRIBUTION In light of these considerations, this article investigates the performance of distributed joint NOMA/PR schemes under total power constraints, adopting both single- and multi-slot detection techniques in the presence of Rayleigh fading and 1809 VOLUME 1, 2020 BABICH et al.: ENERGY-CONSTRAINED UNCOORDINATED MULTIPLE ACCESS FOR NEXT-GENERATION NETWORKS TABLE 1. NOMA/IRSA: Probability of selecting M segments as a function of the number L of energy levels. TABLE 2. Eav/N0 thresholds for R = 1 information bits/symbol. TABLE 1. NOMA/IRSA: Probability of selecting M segments as a function of the number L of energy levels. be performed. For such system, the instantaneous channel load can be expressed (in packets per slot) as [15]: G = χ H K , (1) (1) where 0 ≤χ ≤1 is the activation probability. All users are assumed to adopt the same bi-dimensional modulation of order ς and the same code rate ϱ. To jointly account for these two quantities, one may usefully consider the rate [32]: TABLE 2. Eav/N0 thresholds for R = 1 information bits/symbol. a / 0 y R = ϱ log2 ς, (2) (2) which identifies the number of information bits carried by each transmission, channel use or symbol. For the com- munication channel, three propagation environments are considered: a fading-less one, suitable to investigate the performance of each scheme in a not mobile scenario, and two characterized by Rayleigh fading. These ones may be slow, if the fading level in a RAF remains constant for all segments of a packet, or fast, if, instead, the segments experience different fading levels in the same RAF. where α1 = mf α accounts for a possible margin mf . More precisely, (5) identifies, for the generic l-th normalized energy level, the condition to meet for ensuring the cor- rect reception of a packet using that level, when, for all the other packets, lower and different levels are adopted. By using (6), the condition (5) is satisfied when mf ≥1. Furthermore, mf may be used to obtain a specific value given L. Actually, given the equal probability assumption, the normalized average energy can be expressed as: According to the above introduced scenario, the following of this section presents the technique conceived to design the energy levels of a generic NOMA/PR scheme. The here developed approach generalizes those in [38], [39], which may be viewed as special cases of the here proposed one. A. NOMA/PR UNDER PERFECT IC The design of the energy levels may be suitably addressed by moving from the Shannon bound to relate the rate R to the minimum SINR α required to correctly receive a packet. In fact, by inverting the Shannon formula, one can immediately obtain: As a possible example, Table 1 reports the probabilities, derived according to [23, Table 1], of selecting M segments as a function of the number of energy levels. Using these values and fixing L, E[M], and Eav/N0, the minimum normal- ized average energies (i.e., the SINR thresholds) required for the correct reception of a segment can be obtained using (7). Table 2 summarizes some values for R = 1, which, from (3), implies α = 1. As a possible example, Table 1 reports the probabilities, derived according to [23, Table 1], of selecting M segments as a function of the number of energy levels. Using these values and fixing L, E[M], and Eav/N0, the minimum normal- ized average energies (i.e., the SINR thresholds) required for the correct reception of a segment can be obtained using (7). Table 2 summarizes some values for R = 1, which, from (3), implies α = 1. α = 2R −1. (3) (3) Define now as El/N0 the energy El used by the l-th (l = 1, . . . , L) NOMA level normalized to the noise spec- tral density N0. Let us assume R ≥1 so that, for a given energy level, only one successful packet reception is allowed. According to these definitions, the normalized average Signal to Noise Ratio (SNR) can be determined as: B. CONTRIBUTION = Eav/N0 E[M] = (1 + α1)L −1 L ≥(1 + α)L −1 L . (7) (7) B. NOMA/PR UNDER IMPERFECT IC Consider now the imperfect IC condition with a residual interference ϵ. In the presence of this residual, the normalized energy levels become [39]: = Eav/N0 E[M] , (4) El N0 = EL N0 βL−l, l = 1, . . . , L, (8) (4) (8) where Eav/N0 denotes the normalized average energy and E[M] represents the average number of repetitions. Recalling the fundamental NOMA concept under perfect IC, the correct detection of a packet when all transmitters use differ- ent energy levels requires that the following constraint be met [6]: where Eav/N0 denotes the normalized average energy and E[M] represents the average number of repetitions. Recalling the fundamental NOMA concept under perfect IC, the correct detection of a packet when all transmitters use differ- ent energy levels requires that the following constraint be met [6]: where: β = 1 + α1ϵ 1 + α1 , (9) (9) and: EL N0 = α1(1 −ϵ) 1 −ϵ −(1 + α1ϵ) 1 −βL−1. (10) (10) El/N0 1 + l−1 i=1 Ei/N0 ≥α, l = 1, . . . , L. (5) (5) In the sequel of this article, the energy levels defined by (8) with ϵ > 0 will be referred to as robust design, while the levels obtained by (6) with ϵ = 0 will be referred to as basic design. More precisely, by adopting a value ϵ > 0 in the energy design (robust design), a packet using the l-th level is It may be shown that (5) is satisfied when the l-th normalized energy level is given by: El/N0 = α1 (1 + α1)l−1, l = 1, . . . , L, El/N0 = α1 (1 + α1)l−1, l = 1, . . . , L, (6) (6) 1810 VOLUME 1, 2020 TABLE 3. Number of energy levels L as a function of the rate R and of the residual interference ϵ. TABLE 3. Number of energy levels L as a function of the rate R and of the residual interference ϵ. correctly received when all the other packets use lower and different energy levels, and when the residual interference amount after IC does not exceed ϵ. When ϵ = 0 instead (basic design), perfect IC is required. B. NOMA/PR UNDER IMPERFECT IC The usage of the term ‘robust’ hence arises from the objective of making the system capable to correctly receive a packet when, realistically, a residual interference is present, a situation in which the basic design, relying on an ideal IC, would instead experience a poor performance. By recalling (4) and using (8)-(10), the normalized SNR can be expressed as: be expressed as a function of the offered load G, of the rate R, and of the success probability Ps as [29]: = 1 L 1 −βL βL −ϵ , (11) S = G Ps R. (17) (11) (17) Observe that G Ps represents the number of packets success- fully received in a slot, thus it identifies the multiplexing capability of the access scheme, while the rate R takes into account the transmission mode, i.e., the modulation/code pair. In (17), the key quantity for obtaining a proper through- put estimation is the success probability. Its theoretical analysis is analytically tractable when R > 1 and mf ∼= 1 in two cases: for a pure NOMA scheme in a fading-less envi- ronment, but accounting for any traffic load, and for a general NOMA/PR scheme in low traffic conditions (G << 1), but accounting for the presence of Rayleigh fading. These two cases are respectively analyzed in the following subsections. which enables to reformulate the definition of β in (9) when ϵ, L, and the values of given by (4) are assigned. Accordingly, by inverting (11), one obtains: β = 1 + ϵL 1 + L 1/L , (12) (12) (12) and then, by further inverting (9), one can derive: α1 = 1 −β β −ϵ . (13) (13) Now that the NOMA levels in the presence of IC are avail- able, it is worth to investigate their implications on the rate. In particular, R must meet the following condition: R < log2(1 + α1) = log2 1 −ϵ β −ϵ , (14) A. NOMA PERFORMANCE IN FADING-LESS SCENARIO (14) A. NOMA PERFORMANCE IN FADING LESS SCENARIO According to the proposed model, there are two kinds of successful events in a fading-less situation. To clarify them, assume to observe the result of the receptions from the point of view of a specific user that is attempting to send a packet (target user). The first kind of events, in which all users experience a successful decoding, involves h = k + 1 ≤L users (the target one and the other k concurrent ones), all choosing different energy levels. The second kind of events is still referred to h = k + 1 users, but is just partly successful, since it includes some energy levels selected by more users, which leads, given the assumption R ≥1, to collisions, and some other levels, selected by just one user, which may be successful. Identify by s the highest collision level. In such a situation, some receptions may be successful only if there is at least one idle energy level in a position t satisfying the inequality 1 ≤s < t ≤L. More precisely, the k + 1 transmitted segments may be subdivided into four groups (Fig. 1): the ns successful ones (1 ≤ns ≤L −t = np), the nu = t −s −1 unsuccessful ones that individually select an energy level between s and t, the nc ≥2 ones that collide in the s-th level, and the nf = h −ns −nu −nc ones that select an energy level between 1 and s −1 when s > 1. Following this classification, the success probability given k which, for →∞, becomes: which, for →∞, becomes: R < log2 1 −ϵ ϵ1/L −ϵ , (15) (15) by using (12). This latter expression is interesting because it enables to derive an upper bound on the number of energy levels as: L < log ϵ log 2−R(1 −ϵ) + ϵ . (16) (16) As possible examples, Table 3 reports the maximum number of energy levels as a function of R and ϵ. As expected, the number of levels, which holds both for pure NOMA and joint NOMA/PR algorithms, decreases with the increase of ϵ. BABICH et al.: ENERGY-CONSTRAINED UNCOORDINATED MULTIPLE ACCESS FOR NEXT-GENERATION NETWORKS FIGURE 1. Collision model. TABLE 4. Closed-form NOMA success probabilities for the ﬁrst four L values. FIGURE 1. Collision model. signal level below the decoding threshold, but may also have a positive effect in case of collision, since it may reduce the interference level due to some segments. In low load conditions, this positive effect is however negligible. For this reason, the estimators presented below are considered approximating upper bounds. When the fading statistic is introduced in the analysis, the normalized SNR defined by (4) may be viewed as its average value. Hence, assuming a Rayleigh fading channel, the actual SNR γ is described by an exponential r.v., whose Cumulative Distribution Function (CDF) is given by: TABLE 4. Closed-form NOMA success probabilities for the ﬁrst four L values. ABLE 4. Closed-form NOMA success probabilities for the ﬁrst four L values. TABLE 4. Closed-form NOMA success probabilities for the ﬁrst four L values. TABLE 4. Closed-form NOMA success probabilities for the ﬁrst four L values. F(γ, ) = 1 −exp(−γ/) 1R+ 0 (γ ), (21) (21) where R+ 0 is the set of nonnegative real numbers, and 1X(x) is the indicator function, that is, 1X(x) = 1 if x ∈X and 1X(x) = 0 if x ̸∈X. For a NOMA/PR system, it is useful to consider, for comparison purposes, both the commonly adopted single-slot model and the more advanced multi-slot one, which enables to apply Chase combining or, if incre- mental redundancy is used, joint decoding. Besides, it is interesting to consider slow and fast fading conditions, and also the possibility to transmit the segments with indepen- dent or identical energy levels. The complete analysis of all these possibilities leads to four different upper bounds for the success probability of each decoding model (single- slot, multi-slot with Chase combining, multi-slot with joint decoding). Observe that, for the PR schemes in single-slot scenario, it is sufficient to consider only the best segment. In an equal energy scenario with slow fading, all the seg- ments experience the same conditions, while in independent energy scenarios and/or in the presence of fast fading, a transmission attempt fails only when all the segments fail. In the Chase and code combining scenarios, instead, all the segments should be considered. The following paragraphs explain these aspects in more detail by presenting the success probability upper bounds for each receiving model. 1) SINGLE-SLOT DETECTION As a first step, it is useful to define the probability pl that the fading level exceeds the threshold of correct reception for the l-th energy level without exceeding the threshold of the (l + 1)-th one. This probability is given by: Ps = ∞ k=0 Ps\|k Gk k! exp(−G). (20) (20) pl = F α, El N0 −F α, El+1 N0 , l = 1 · · · L, (22) (22) This latter formula may be evaluated in closed-form for different L values. Some expressions are reported in Table 4. Observe that, for L = 1, we obtain the well known success probability for the SA protocol. where the term EL+1/N0 = ∞is introduced for math- ematical purposes. Assuming a slow fading scenario, the probability of a single attempt failure when the fading level lies between the l-th and the (l+1)-th energy levels is given by pf\|l = l/L. Considering a constant level for the M seg- ments, l/L represents also the failure probability for all of them. Thus, the success probability is upper bounded by: BABICH et al.: ENERGY-CONSTRAINED UNCOORDINATED MULTIPLE ACCESS FOR NEXT-GENERATION NETWORKS can be evaluated according to (18), reported at the bottom of the previous page, where: tmax = min(L −1, h −nc + s), (19a) ns max = min np, h −nc −nu , (19b) nc min = max(2, h −L + 2), (19c) tmax2 = min(L −1, 1 + h −nc), (19d) tmax = min(L −1, h −nc + s), (19a) ns max = min np, h −nc −nu , (19b) nc min = max(2, h −L + 2), (19c) tmax2 = min(L −1, 1 + h −nc), (19d) and u(x) denotes the unit step function (u(x) = 1 if x > 0, u(x) = 0 otherwise). In (18), the first addendum corre- sponds to the collision scenario when s > 1, the second addendum models the collision scenario when s = 1, while the third addendum corresponds to the fully successful sce- nario. Assuming a Poisson traffic, from (18) it is possible to determine the average successful probability as: and u(x) denotes the unit step function (u(x) = 1 if x > 0, u(x) = 0 otherwise). In (18), the first addendum corre- sponds to the collision scenario when s > 1, the second addendum models the collision scenario when s = 1, while the third addendum corresponds to the fully successful sce- nario. Assuming a Poisson traffic, from (18) it is possible to determine the average successful probability as: III. ANALYSIS This section presents a theoretical analysis for evaluating the success probability Ps when the energy levels are defined according to (8). The derivation of Ps is relevant since it enables to directly obtain the throughput S, which is mea- sured as the number of information bits per transmission that are correctly decoded at the receiver. This latter quantity can Ps\|k = 1 Lk+1 ⎡ ⎣ k nc=2 L−2 s=2 tmax t=s+1 ns max ns=1 k! nc! np ns ns (s −1)nf nf ! + k nc=nc min tmax2 t=2 k! nc! np ns ns + u(L −k) L! (L −k −1)! ⎤ ⎦ (18) (18) LUME 1, 2020 1811 1811 1811 VOLUME 1, 2020 B. NOMA/PR LIMITING PERFORMANCE IN FADING SCENARIO At very low traffic, the collision is an unlikely event. This simplifies the analysis developed in the previous subsec- tion and enables to include fading effects in the general NOMA/PR scenario. Observe that, from the point of view of the success probability, fading may reduce the received Ps ≤1 − L l=1 pl l L. (23) (23) VOLUME 1, 2020 1812 If, instead, the transmitter uses the same energy level for the m segments, the success probability is upper bounded by: If, instead, independent levels are considered, the reception fails if all the M segments fail. Given the energy level, this event occurs with probability (l/L)m. Therefore, the success probability becomes upper bounded by: If, instead, independent levels are considered, the reception fails if all the M segments fail. Given the energy level, this event occurs with probability (l/L)m. Therefore, the success probability becomes upper bounded by: Ps ≤1 −1 L M m=1 P(m) L l=1 F α m, El N0 . (31) (31) Ps ≤1 − L l=1 pl M m=1 l L m P(m), (24) (24) For the fast fading scenario with equal energy levels, let’s preliminarily define the CDF of the sum of m exponential r.v.s with equal average value as: where P(m) is the probability of generating m segments per packet. More precisely, for CRDSA, in which M = 2, we have P(1) = 0 and P(2) = 1, while, for IRSA, in which M is a r.v., the corresponding probabilities are those reported in Table 1. Q(ξ, , m) = 1 −exp −ξ m−1 k=0 1 k! ξ k 1R+ 0 (ξ), (32) which represents the Erlang distribution. Exploiting this def- inition, the upper bound on the success probability can be evaluated as: For a fast fading scenario, it is useful to preliminarily define the probability: ql = F α, El N0 , (25) Ps ≤1 −1 L M m=1 P(m) L l=1 Q α, El N0 , m . (33) (25) (33) that the attempt using the l-th level fails. When equal energy levels are used, the success probability is upper bounded by: If, instead, the users adopt independent energy levels, it is necessary to first consider the vector m = [ 1, . . . B. NOMA/PR LIMITING PERFORMANCE IN FADING SCENARIO , m] of the possible different average values and then evaluate the CDF: Ps ≤1 −1 L M m=1 P(m) L l=1 qm l . (26) (26) Q(ξ, m, m) = 1 − m k=1 exp −ξ k m−1 k h̸=k( k − h) × 1R+ 0 (ξ), (34) When, instead, independent levels are used, the upper bound becomes: (34) Ps ≤1 − M m=1 P(m)pm f1, (27) Ps ≤1 − M m=1 P(m)pm f1, (27) pf1 = 1 L L l=1 ql, (28) (27) which holds for 1 ̸= 2 ̸= · · · ̸= m, but allows the eval- uation of all the other combinations through the extension by continuity of (34). The upper bound for the success prob- ability can now be evaluated by taking into account all the combinations Elm of energy values as: where: pf1 = 1 L L l=1 ql, (28) (28) Ps ≤1 − M m=1 P(m) Lm Elm Q α, Elm N0 , m . (35) (35) represents the probability that a single attempt fails. B. SINGLE-SLOT MODEL Consider now the throughput achievable by the different schemes. To this aim, let’s initially focus on the basic design in a fading-less scenario with perfect IC. Figs. 2 and 3 illus- trate the throughput and the success probability, respectively, for pure NOMA and NOMA/PR schemes, where the termi- nation ‘-I’ identifies the usage of independent energy levels (also identified by filled markers), while the termination ‘-E’ identifies the usage of equal energy levels (also identified by empty markers). The first of these two figures shows that joint NOMA/CRDSA has a slightly better performance at low loads, while pure NOMA is better at high loads. Joint NOMA/IRSA has a slightly worse performance, given the limited energy and the low number of slots available in the RAF. For both joint PR schemes, the independent energy design parallels the equal energy one. Fig. 3 puts instead into evidence the most important benefit of the repe- tition schemes with respect to pure NOMA: the capability to 3) MULTI-SLOT DETECTION WITH JOINT DECODING For this latter quantity, a general formula holding for any M value is difficult to derive. However, a closed-form expression may be calculated for the relevant M = 2 case as: Pf (Rm) = Pf (R1, R2) = 1 −exp ξ2 + 4δα −ξ 2δ , (39) in which ξ = (El1 + El2)/N0 and δ = El1El2/N2 0. When the transmitters use the same energy level, instead, the upper bound of the success probability may be more simply evaluated by recalling (36), so as to obtain: FIGURE 2. Throughput as a function of the channel load in a fading-less scenario with perfect IC. success probability. This confirms the acceptability of the adopted approximation, holding in low traffic conditions, which consists in the negligibility of the positive effects of fading to possibly increase the received power for a segment. Some case by case comparisons may be also useful to put into evidence some general aspects. In particular, given the decoding model and the energy policy, fast fading always leads to larger Ps values with respect to slow fading, since, in the first case, the propagation channel introduces a higher diversity. For similar reasons, given the fading scenario and the decoding model, the adoption of independent energy levels is preferable to the usage of equal ones. Finally, as expected, when the fading scenario and the energy policy are fixed, the more sophisticated the decoding model the higher the success probability. The same agreement between the- ory and simulations has been observed for the NOMA/IRSA scheme and for the robust design. Ps ≤1 −1 L M m=1 P(m) L l=1 T R m, El N0 . (40) (40) For the fast fading scenario, the CDF of the sum of the equivalent rates corresponding to m segments is too complex and has to be determined by numerical techniques. This implies that also the corresponding bounds for the success probability with independent and equal energy levels must be numerically estimated. For the fast fading scenario, the CDF of the sum of the equivalent rates corresponding to m segments is too complex and has to be determined by numerical techniques. This implies that also the corresponding bounds for the success probability with independent and equal energy levels must be numerically estimated. 3) MULTI-SLOT DETECTION WITH JOINT DECODING When multi-slot detection is applied, the decision jointly involves all segments. This is significantly different from the previously analyzed single-slot case, where the decision was based on the best segment, i.e., that with the highest SINR. In particular, adopting a capture-based reception crite- rion in combination with Chase combining, the performance depends on the sum of the SINR values of the M segments. For code combining, the performance depends on the sum of the equivalent rates of the individual segments. The concept of equivalent rate has been defined and deeply discussed in [32]. It substantially represents the average value of the rates of the segments associated to a packet and depends on the fading statistic. More precisely, in a slow Rayleigh fading scenario when the Shannon bound is adopted, the CDF of the equivalent rate r is given by [32]: Let’s again start with the slow fading scenario. If a trans- mitter uses m independent energy levels lm = [l1, l2, . . . , lm] for its segments, one can determine the corresponding failure probability as: T(r, ) = F 2r −1, . (36) (36) If the transmitters use independent energy levels, we can then identify the vector of the equivalent rates as: If the transmitters use independent energy levels, we can then identify the vector of the equivalent rates as: Pf (lm) = F ⎛ ⎝α, 1 N0 l∈lm El ⎞ ⎠. (29) (29) Rm = log2 1 + Elm N0 . (37) (37) According to this definition, the upper bound for the success probability can be expressed as: Exploiting these definitions, the upper bound of the success probability may be evaluated as: Ps ≤1 − M m=1 P(m) Lm lm Pf (lm). (30) Ps ≤1 ≤1 − M m=1 P(m) Lm lm Pf (lm). (30) Ps ≤1 − M m=1 P(m) Lm lm Pf (lm). (30) Ps ≤1 −1 Lm M m=1 P(m) Rm Pf (Rm), (38) (38) (30) VOLUME 1, 2020 1813 1813 BABICH et al.: ENERGY-CONSTRAINED UNCOORDINATED MULTIPLE ACCESS FOR NEXT-GENERATION NETWORKS TABLE 5. Parameter values. FIGURE 2. Throughput as a function of the channel load in a fading-less scenario with perfect IC. FIGURE 2. Throughput as a function of the channel load in a fading-less scenario with perfect IC. where Pf (Rm) denotes the failure probability as a function of the rate vector. IV. RESULTS The default parameters adopted to derive the results are reported in Table 5. These values have been chosen because they represent sensible scenarios, with limited energy, lim- ited delay, and realistic IC factors. In particular, the unity rate R is the limiting value for the NOMA throughput estimation developed in Section III.A. The impact of each parameter on the network performance has been investigated by moving from the initial values in the table. In all the evaluations, L has been selected as the highest integer complying with (7). Besides, according to the system model in Section II, the input traffic is generated according to a Poisson process. Both the theoretical analysis and the simulations are implemented in MATLAB. In particular, each point of a simulated curve is obtained through Monte Carlo simulations by averaging the results derived over 1000 realizations. A. BOUND VALIDATION Success probability as a function of the channel load in a fading-less scenario with perfect IC. A comparison, still in a slow fading scenario, between the basic (ϵ = 0) and the robust (ϵ = 0.05) designs is reported in Fig. 6. This figure reveals that, in realistic conditions, the performance of the robust design parallels that of the basic one in terms of maximum throughput. One may how- ever specifically note that, in low traffic conditions, the robust design is preferable to the basic one, while, for higher loads, the opposite choice should be carried out. The better performance of the robust design for low traffic loads is con- firmed by Table 7, which focuses on the success probability by considering the simulated values and the theoretical upper bounds in both slow and fast fading scenarios. In particular, the values in this table extend to the fast fading scenario the observations so far formulated on the suitability of the robust design in low traffic conditions. almost completely deliver the packets from low to moderate loads. This represents a general, basic difference between pure NOMA and both pure and joint PR-based techniques, which will remain observable for all the situations addressed in the remaining of this article. A comparison, still in a slow fading scenario, between the basic (ϵ = 0) and the robust (ϵ = 0.05) designs is reported in Fig. 6. This figure reveals that, in realistic conditions, the performance of the robust design parallels that of the basic one in terms of maximum throughput. One may how- ever specifically note that, in low traffic conditions, the robust design is preferable to the basic one, while, for higher loads, the opposite choice should be carried out. The better performance of the robust design for low traffic loads is con- firmed by Table 7, which focuses on the success probability by considering the simulated values and the theoretical upper bounds in both slow and fast fading scenarios. In particular, the values in this table extend to the fast fading scenario the observations so far formulated on the suitability of the robust design in low traffic conditions. Consider now the robust design, which has been specif- ically conceived to operate in the presence of a residual interference ϵ. Note that this characteristic does not imply its sole usage under imperfect IC. A. BOUND VALIDATION The first group of results aims to prove the correctness of the analysis by comparing the theoretical upper bounds on the success probability calculated in Section III.B with the cor- responding Ps values obtained through simulations. Table 6 reports the results of this comparison for NOMA/CRDSA (M = 2) using the basic design. One may immediately notice that each bound is very tight to the corresponding actual VOLUME 1, 2020 1814 TABLE 6. Theoretical upper bounds and simulated success probabilities for NOMA/CRDSA with L = 7 using the basic design (ϵ = 0) and considering different fading scenarios, decoding criteria, and energy level selection policies. FIGURE 3. Success probability as a function of the channel load in a fading-less scenario with perfect IC. FIGURE 4. Throughput as a function of the channel load in a fading-less scenario using a robust design. TABLE 6. Theoretical upper bounds and simulated success probabilities for NOMA/CRDSA with L = 7 using the basic design (ϵ = 0) and considering different fading scenarios, decoding criteria, and energy level selection policies. FIGURE 3. Success probability as a function of the channel load in a fading-less scenario with perfect IC. FIGURE 4. Throughput as a function of the channel load in a fading-less scenario using a robust design. TABLE 6. Theoretical upper bounds and simulated success probabilities for NOMA/CRDSA with L = 7 using the basic design (ϵ = 0) and considering different fading scenarios, decoding criteria, and energy level selection policies. TABLE 6. Theoretical upper bounds and simulated success probabilities for NOMA/CRDSA with L = 7 using the basic design (ϵ = 0) and considering different fading scenarios, decoding criteria, and energy level selection policies. bounds and simulated success probabilities for NOMA/CRDSA with L = 7 using the basic design (ϵ = 0) and considering different fading nd energy level selection policies. TABLE 6. Theoretical upper bounds and simulated success probabilities for NOMA/CRDSA with L = 7 using the basic design (ϵ = 0) an scenarios, decoding criteria, and energy level selection policies. FIGURE 4. Throughput as a function of the channel load in a fading-less scenario using a robust design. FIGURE 3. Success probability as a function of the channel load in a fading-less scenario with perfect IC. FIGURE 4. Throughput as a function of the channel load in a fading-less scenario using a robust design. FIGURE 3. VOLUME 1, 2020 BICH et al.: ENERGY-CONSTRAINED UNCOORDINATED MULTIPLE ACCESS FOR NEXT-GENERATION NETWORKS Furthermore, when R is limited within a specific range, for example forcing the usage of a Quadrature Phase-Shift Keying (QPSK) modulation, which leads to R ≤2 the performance of pure CRDSA becomes FIGURE 5. Throughput as a function of the channel load in slow fading scenario with perfect IC using a basic design. FIGURE 7. Throughput for different rates R in ideal conditions with perfect IC. FIGURE 5. Throughput as a function of the channel load in slow fading scenario with perfect IC using a basic design. FIGURE 7. Throughput for different rates R in ideal conditions with perfect IC. FIGURE 7 Th h t f diff t t R i id l diti ith f t IC FIGURE 5. Throughput as a function of the channel load in slow fading scenario with perfect IC using a basic design. FIGURE 7. Throughput for different rates R in ideal conditions with perfect IC. FIGURE 6. Throughput as a function of the channel load in slow fading scenario with perfect IC. Comparison between basic and robust designs. C. IMPACT OF THE PARAMETERS This section investigates in more detail the effects of the parameters on the performance. To this purpose, consider first the rate R. This quantity must meet the condition (14), which depends on the access technique and on its param- eters L, M, ϵ, Eav/N0. Besides, by (17), the throughput directly depends on R. To this regard, note that, in this article, the condition R ≥1 has been always assumed in order to exclude the situations in which more than one packet is successfully detected in a collision. To discuss the effects of R on the performance, initially consider the access techniques with a single energy level (L = 1), such as pure SA (M = 1) and CRDSA (M = 2) for Eav/N0 = 16 dB (Table 5). In such cases, R must meet the condition R < log2(1 + ), which leads, for SA, to R < 5.35 and S ≈5.35/e ≈1.97 (both given in information bits/transmission). For CRDSA, instead, one obtains, still in information bits/transmission, R < 4.39 and a maxi- mum throughput S = 2.19 by assuming G Ps = 0.5, a value that can be approached when K →∞. These values are comparable with the maximum throughput of the joint NOMA/CRDSA access technique, shown in Fig. 2. Thus, for the single-slot detection model, the performance of pure CRDSA parallels that of joint NOMA/CRDSA, although the performance of the latter may be improved by increasing K. A higher throughput may be obtained by using IRSA with a sufficiently large K value. Furthermore, when R is limited within a specific range, for example forcing the usage of a Quadrature Phase-Shift Keying (QPSK) modulation, which leads to R ≤2, the performance of pure CRDSA becomes much lower. The effects of this limitation for pure NOMA and NOMA/CRDSA are addressed in Fig. 7 by consider- ing different L values and designing the R ones to comply with (14) when ϵ = 0. This figure shows that the maxi- mum offered load increases with the decrease of R, but the maximum throughput remains almost constant. The effect FIGURE 6. Throughput as a function of the channel load in slow fading scenario with perfect IC. Comparison between basic and robust designs. techniques have also a slightly higher maximum throughput, and the advantage is significant at low loads, while pure NOMA has a higher throughput at high loads. A. BOUND VALIDATION It might be in fact even used with perfect IC, but at the expense of a certain performance reduction. To this aim, Fig. 4 quantifies the throughput deriving from adopting the robust design in NOMA and NOMA/CRDSA for different ϵ values. This figure highlights the impact of this latter parameter on the performance, whose reduction with the increase of ϵ is significant. The subsequent group of results in Fig. 5 outlines the impact of fading by showing the throughput for the slow fading case when the basic design is adopted. The curves confirm that the independent energy design outperforms the fixed energy one, a result already observed in Table 6 from the analysis of the theoretical and numerical upper bounds. As a summary of the results obtained for the single-slot model, one may notice that the major advantage of the joint NOMA/PR techniques is their higher success probability at low to moderate loads (Fig. 3). In fact, Ps approaches 1 in fading-less conditions and remains satisfactorily close to this value in different fading scenarios (Table 6). Joint NOMA/PR 1815 BICH et al.: ENERGY-CONSTRAINED UNCOORDINATED MULTIPLE ACCESS FOR NEXT-GENERATION NETWORKS BICH et al.: ENERGY-CONSTRAINED UNCOORDINATED MULTIPLE ACCESS FOR NEXT-GENERATION NETWORKS BABICH et al.: ENERGY-CONSTRAINED UNCOORDINATED MULTIPLE ACCESS FOR NEXT-GENERATION NETWORKS FIGURE 5. Throughput as a function of the channel load in slow fading scenario with perfect IC using a basic design. FIGURE 6. Throughput as a function of the channel load in slow fading scenario with perfect IC. Comparison between basic and robust designs. TABLE 7. Theoretical upper bounds and simulated success probabilities for NOMA/CRDSA obtained with independent energy levels using the single-slot decoding model in the presence of different fading scenarios and designs. techniques have also a slightly higher maximum throughput FIGURE 7. Throughput for different rates R in ideal conditions with perfect IC. C. IMPACT OF THE PARAMETERS This section investigates in more detail the effects of the parameters on the performance. To this purpose, consider first the rate R. This quantity must meet the condition (14), which depends on the access technique and on its param- eters L, M, ϵ, Eav/N0. Besides, by (17), the throughput directly depends on R. To this regard, note that, in this article, the condition R ≥1 has been always assumed in order to exclude the situations in which more than one packet is successfully detected in a collision. To discuss the effects of R on the performance, initially consider the access techniques with a single energy level (L = 1), such as pure SA (M = 1) and CRDSA (M = 2) for Eav/N0 = 16 dB (Table 5). In such cases, R must meet the condition R < log2(1 + ), which leads, for SA, to R < 5.35 and S ≈5.35/e ≈1.97 (both given in information bits/transmission). For CRDSA, instead, one obtains, still in information bits/transmission, R < 4.39 and a maxi- mum throughput S = 2.19 by assuming G Ps = 0.5, a value that can be approached when K →∞. These values are comparable with the maximum throughput of the joint NOMA/CRDSA access technique, shown in Fig. 2. Thus, for the single-slot detection model, the performance of pure CRDSA parallels that of joint NOMA/CRDSA, although the performance of the latter may be improved by increasing K. A higher throughput may be obtained by using IRSA with a sufficiently large K value. C. IMPACT OF THE PARAMETERS In ideal con- ditions, the not necessary usage of the robust design may cause a reduction of the throughput, but, in realistic condi- tions, it allows a higher success probability, being able to sustain a given degree of imperfect IC. techniques have also a slightly higher maximum throughput, and the advantage is significant at low loads, while pure NOMA has a higher throughput at high loads. In ideal con- ditions, the not necessary usage of the robust design may cause a reduction of the throughput, but, in realistic condi- tions, it allows a higher success probability, being able to sustain a given degree of imperfect IC. VOLUME 1, 2020 1816 FIGURE 8. Throughput for different average signal to noise ratios Eav/N0 in ideal conditions with perfect IC for R ≈1.15. FIGURE 9. Throughput for different RAF lengths K in ideal conditions with perfect IC. FIGURE 9. Throughput for different RAF lengths K in ideal conditions with perfect IC. FIGURE 8. Throughput for different average signal to noise ratios Eav/N0 in ideal conditions with perfect IC for R ≈1.15. FIGURE 9. Throughput for different RAF lengths K in ideal conditions with perfect IC. packet decoding. Therefore, a header must be enclosed in each segment to determine the positions of the other seg- ments of the same packet. This header may have a strong impact on the throughput, especially for large M values. Hence, before presenting the achievable performance, it is worth to briefly summarize the header characteristics. of the reduction of R is substantially that of increasing the performance of the scheme at the higher loads at the cost of a reduction at the lower ones. Consider now the effects of the energy on the performance by analyzing Fig. 8, which is obtained for different normal- ized average energies Eav/N0 when the rate is still designed according to (14) for ϵ = 0. As expected, the increase of the energy and hence of the number of levels leads to a higher diversity, thus improving the performance. More precisely, the figure puts into evidence that the throughput improve- ment experienced by the joint NOMA/CRDSA scheme is higher than that provided by the pure NOMA one. y When M replicas are generated and the RAF consists of K slots, b = ⌈log2 K⌉bits are required to specify the position of each segment belonging to the same packet. C. IMPACT OF THE PARAMETERS Therefore, the header should include at least b(M −1) bits, even if header compression may be used to reduce the overhead. To enable packet reception and IC, at least one among the M headers should be correctly decoded. This operation depends on the decoding strategy. For the single-slot model, the header and the packet adopt the same coding rate, being the success condition identical for them. For the multi-slot decoding strategies the situation differs, since at least one header must be correctly received before performing packet decoding. If Chase combining is adopted, the condition for correct packet decoding can be given in terms of the rate R as: Finally, let’s observe the impact of the RAF length on the throughput. Fig. 9 puts into evidence that both analyzed NOMA/PR schemes benefit from the increase of the num- ber of available slots, even if the highest gain in terms of maximum throughput when the RAF length increases from K = 20 to K = 100 is obtained by the joint NOMA/IRSA scheme. Summarizing, the pure techniques, such as SA, CRDSA, and IRSA, may obtain a good performance if the modulation can be adaptively selected. In particular, CRDSA and IRSA achieve their limiting throughput by using large K values, a choice that however implies large delays. Assuming instead some constraints on the choice of the modulation, joint techniques may be preferable, having the maximum throughput of NOMA/CRDSA only a weak dependence on R. R < log2 1 + M m=1 γm , (41) (41) where γm is the actual SINR of the m-th segment. Concerning the M headers, the one with the best SINR is character- ized by γh = maxm(γm). Hence, from (41), we can infer the following requirement: γh ≥1 M M m=1 γm, (42) VOLUME 1, 2020 V. CONCLUSION Next generation networks will support a large amount of uncoordinated sporadic traffic coming from a large number of nodes that attempt to simultaneously communicate with a common receiver. For such kind of traffic, SA represents a suitable access technique, but, in its basic behavior, may have a not satisfactory performance. Multiple energy levels provided by NOMA may be used to obtain a first kind of diversity for improving the throughput. Besides, PR schemes (IRSA and CRDSA) may be adopted to introduce a second kind of diversity, which allows one to improve the system reliability by exploiting an increased delay and complexity. y y p g y p y This article has explored the joint utilization of multiple energy levels and repetition schemes to increase both the throughput and the reliability of the random access scheme. This task has been carried out by assuming suit- able energy constraints and realistic operation conditions, including imperfect IC and packet overhead. The results have confirmed that one important benefit of repetition schemes is the delivery of almost all packets at low to moderate loads, while for schemes not adopting repetitions this is not the case. However, to fully exploit the repetition schemes capa- bilities, a large RAF duration should be used, and advanced detection schemes, such as Chase combining or joint decod- ing, should be adopted. To this end, a suitable, self-decodable header should be added to each segment of a packet to infer the position in the RAF of the other segments referred to the same packet. The influence of this header has been investigated for the more diffused NOMA/CRDSA scheme by showing that the introduced overhead has an acceptable impact on the throughput, specially for low M values. A final and positive aspect that is worth to consider when combined NOMA/PR protocols are conceived is that the complexity derived by the joint introduction of two diversity techniques is mainly at charge of the common receiver, while FIGURE 11. Throughput as a function of the channel load in a slow fading scenario with imperfect IC assuming a robust design. If, instead, code combining is used, the header rate must satisfy this other constraint: Rh ≤R M . (44) (44) The above discussion shows that the effect of the header on the performance may be determined by considering the packet information size, the number of slots per RAF, and the number of segments. BABICH et al.: ENERGY-CONSTRAINED UNCOORDINATED MULTIPLE ACCESS FOR NEXT-GENERATION NETWORKS BABICH et al.: ENERGY-CONSTRAINED UNCOORDINATED MULTIPLE ACCESS FOR NEXT-GENERATION NETWORKS FIGURE 10. Throughput as a function of the channel load in ideal conditions and perfect IC assuming a robust design. the higher the throughput for the NOMA/CRDSA scheme with independent and equal energy levels from low to mod- erate loads. Conversely, pure NOMA, being characterized by the transmission of a unique packet copy, is not capable to exploit the benefits of multi-slot decoding in the same traffic conditions, but becomes again preferable when the load gets significant. This trend is confirmed by the final figure, still derived for the robust design, but in slow fading conditions with imperfect IC (Fig. 11). In this situation, the fading has a positive effect on the NOMA performance and a slightly negative effect on the NOMA/CRDSA one. However, the advantage of the joint system remains significant for the code combining detection model. This advantage may be also improved by increasing the RAF duration. Note that, to fairly compare the three decoding techniques, the robust design has been adopted in all the evaluations of this sub- section, although this specific design is mandatory only for the results presented in the last figure, which addresses the realistic situation in which a residual interference remains present after cancellation. FIGURE 10. Throughput as a function of the channel load in ideal conditions and perfect IC assuming a robust design. FIGURE 11. Throughput as a function of the channel load in a slow fading scenario with imperfect IC assuming a robust design. D. CHASE COMBINING AND JOINT DECODING (42) Consider now, beside the single-slot model (maximum SINR), the more advanced multi-slot ones represented by Chase combining (sum of SINRs) and joint decoding (sum of equivalent rates) [36]. For properly exploiting the bene- fits of these detection techniques, the position of all the M segments should be known in advance, before performing which, in turn, leads to the following constraint for the corresponding header rate: Rh ≤log2 1 + 2R −1 M . (43) (43) 1817 VOLUME 1, 2020 REFERENCES Comisso and F. Babich, “Coverage analysis for 2D/3D millimeter wave peer-to-peer networks,” IEEE Trans. Wireless Commun., vol. 18, no. 7, pp. 3613–3627, Jul. 2019. [7] Y. Liu, Z. Qin, M. Elkashlan, Z. Ding, A. Nallanathan, and L. Hanzo, “Nonorthogonal multiple access for 5G and beyond,” Proc. IEEE, vol. 105, no. 12, pp. 2347–2381, Dec. 2017. [28] T. Bai and R.W. Heath Jr., “Coverage and rate analysis for millimeter- wave cellular networks,” IEEE Trans. Wireless Commun., vol. 14, no. 2, pp. 1100–1114, Feb. 2015. pp [8] Z. Ding et al., “Application of non-orthogonal multiple access in LTE and 5G networks,” IEEE Commun. Mag., vol. 55, no. 2, pp. 185–191, Feb. 2017. [29] F. Babich and M. 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Lin, “Decentralized power control for random access with successive interference cancel- lation,” IEEE J. Sel. Areas Commun., vol. 31, no. 11, pp. 2387–2395, Nov. 2013. [26] F. Babich and M. Comisso, “Segmented framed slotted Aloha (SFSA) with capture and interference cancellation,” in Proc. IEEE 87th Veh. Technol. Conf. (VTC Spring), Porto, Portugal, 2018, pp. 1–5. [27] M. V. CONCLUSION More precisely, this effect is heavy on NOMA/IRSA (large M values) and less significant for NOMA/CRDSA (M = 2). For this reason and for its wider usage in current satellite systems [17], the results presented in this section are focused on NOMA/CRDSA. In particu- lar, Fig. 10 compares the performance of the three different decoding techniques in ideal conditions for the robust design. As expected, the more sophisticated the reception criterion, VOLUME 1, 2020 1818 the transmitters (sensors, actuators) maintain their desired technological simplicity. [18] L. Toni and P. Frossard, “Prioritized random MAC optimization via graph-based analysis,” IEEE Trans. Commun., vol. 63, no. 12, pp. 5002–5013, Dec. 2015. [19] E. Paolini, G. Liva, and M. Chiani, “Coded slotted ALOHA: A graph- based method for uncoordinated multiple access,” IEEE Trans. Inf. Theory, vol. 61, no. 12, pp. 6815–6832, Dec. 2015. REFERENCES McCann, “Modulation and multiple access for 5G networks,” IEEE Commun. Surveys Tuts., vol. 20, no. 1, pp. 629–646, 1st Quart., 2018. [34] A. Mengali, R. De Gaudenzi, and P. D. Arapoglou, “Enhancing the physical layer of contention resolution diversity slotted ALOHA,” IEEE Trans. Commun., vol. 65, no. 10, pp. 4295–4308, Oct. 2017. [13] S. M. R. Islam, M. Zeng, O. A. Dobre, and K. Kwak, “Resource allocation for downlink NOMA systems: Key techniques and open issues,” IEEE Wireless Commun., vol. 25, no. 2, pp. 40–47, Apr. 2018. [35] F. Babich, G. Buttazzoni, F. Vatta, and M. Comisso, “Energy- constrained NOMA with packet diversity for slotted ALOHA systems,” in Proc. IEEE Mediterranean Commun. Comput. Netw. Conf. (MedComNet), Arona, Italy, 2020, pp. 1–8. [14] W. Shin, M. Vaezi, B. Lee, D. J. Love, J. Lee, and H. V. Poor, “Non-orthogonal multiple access in multi-cell networks: Theory, performance, and practical challenges,” IEEE Commun. Mag., vol. 55, no. 10, pp. 176–183, Oct. 2017. [36] F. Babich and A. Crismani, “Cooperative coding schemes: Design and performance evaluation,” IEEE Trans. Wireless Commun., vol. 11, no. 1, pp. 222–235, Jan. 2012. [15] E. Casini, R. De Gaudenzi, and O. del Rio Herrero, “Contention resolution diversity slotted ALOHA (CRDSA): An enhanced ran- dom access schemefor satellite access packet networks,” IEEE Trans. Wireless Commun., vol. 6, no. 4, pp. 1408–1419, Apr. 2007. [37] P. Kota and C. Schlegel, “A wireless packet multiple access method exploiting joint detection,” in Proc. IEEE Int. Conf. Commun. (ICC), Anchorage, AK, USA, 2003, pp. 2985–2989. [16] G. Liva, “Graph-based analysis and optimization of contention resolu- tion diversity slotted ALOHA,” IEEE Trans. Commun., vol. 59, no. 2, pp. 477–487, Feb. 2011. [38] J. G. Andrews and T. H. Meng, “Optimum power control for suc- cessive interference cancellation with imperfect channel estimation,” IEEE Trans. Wireless Commun., vol. 2, no. 2, pp. 375–382, Mar. 2003. [17] Second Generation DVB Interactive. Satellite System (DVB-RCS2); Part 2: Lower Layers for Satellite Standard, Standard ETSI EN 301 545-2 V1.2.1, 2014. [39] A. Agrawal, J. G. Andrews, J. M. Cioffi, and T. H. Meng, “Iterative power control for imperfect successive interference cancellation,” IEEE Trans. Wireless Commun., vol. 4, no. 3, pp. 878–884, May 2005. 1819 VOLUME 1, 2020
https://openalex.org/W4280582274	https://www.cambridge.org/core/services/aop-cambridge-core/content/view/FA9D43B9CB3A14956B06F1A051B589F9/S2057563722000128a.pdf/div-class-title-prototype-warfare-innovation-optimisation-and-the-experimental-way-of-warfare-div.pdf	English	null	‘Prototype warfare’: Innovation, optimisation, and the experimental way of warfare	European journal of international security	2,022	cc-by	12,190	European Journal of International Security (2022), 7, 322–336 doi:10.1017/eis.2022.12 RESEARCH ARTICLE © The Author(s), 2022. Published by Cambridge University Press on behalf of the British International Studies Association. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. 1US Deputy Secretary of Defense, ‘Establishment of an Algorithmic Warfare Cross Functional Team (Project Maven)’, Memorandum (25 April 2017), available at: {https://www.govexec.com/media/gbc/docs/pdfs_edit/establishment_of_th- e_awcft_project_maven.pdf} accessed 22 March 2021. 2Scott Shane and Daisuke Wakabayasi, ‘“The business of war”: Google employees protest work for the Pentagon’, New York Times (4 April 2018). 3Patrick Tucker, ‘Google wants more work from the defense department’, Defense One (6 November, 2019), {available at: https:// www.defenseone.com/technology/2019/11/google-we-want-more-work-defense-department/161133/} accessed 8 December 2021. ‘Prototype warfare’: Innovation, optimisation, and the experimental way of warfare Marijn Hoijtink* Department of Political Science and Public Administration, Vrije Universiteit Amsterdam, The Netherlands *Corresponding author. Email: m.hoijtink@vu.nl (Received 1 April 2021; revised 13 April 2022; accepted 13 April 2022; first published online 17 May 2022) Keywords: Artificial Intelligence; Experimentation; Military; Technology; Innovation Keywords: Artificial Intelligence; Experimentation; Military; Technology; Innovation Abstract In recent years, the concept of ‘prototype warfare’ has been adopted by Western militaries to accelerate the experimental development, acquisition, and deployment of emerging technologies in warfare. Building on schol- arship at the intersection of Science and Technology Studies and International Relations investigating the broader discursive and material infrastructures that underpin contemporary logics of war, and taking a specific interest in the relationship between science, technology, and war, this article points out how prototype warfare captures the emergence of a new regime of warfare, which I term the experimental way of warfare. While warfare has always been defined by experimental activity, what is particular in the current context is how experimen- tation spans across an increasingly wide range of military practices, operating on the basis of a highly speculative understanding of experimentation that embraces failure as a productive force. Tracing the concept of prototype warfare across Western military discourse and practice, and zooming in on how prototype warfare takes experi- mentation directly into the battlefield, the article concludes by outlining how prototype warfare reconfigures and normalises military intervention as an opportunity for experimentation, while outsourcing the failures that are a structural condition of the experimental way of warfare to others, ‘over there’. 1US Deputy Secretary of Defense, ‘Establishment of an Algorithmic Warfare Cross Functional Team (Project Maven)’, Memorandum (25 April 2017), available at: {https://www.govexec.com/media/gbc/docs/pdfs_edit/establishment_of_th- e_awcft_project_maven.pdf} accessed 22 March 2021. 2Scott Shane and Daisuke Wakabayasi, ‘“The business of war”: Google employees protest work for the Pentagon’, New York Times (4 April 2018) 1US Deputy Secretary of Defense, ‘Establishment of an Algorithmic Warfare Cross Functional Team (Project Maven)’, Memorandum (25 April 2017), available at: {https://www.govexec.com/media/gbc/docs/pdfs_edit/establishment_of_th- e_awcft_project_maven.pdf} accessed 22 March 2021. Introduction In April 2017, the US Department of Defense (DoD) published a memo announcing the Algorithmic Warfare Cross-Functional Team, codenamed Project Maven, to accelerate the incorporation of ‘artificial intelligence and machine learning across [DoD] operations’.1 The work of Project Maven focused on the development and deployment of machine learning algo- rithms to automate the extraction of ‘objects of interests’ and relevant activity across US surveil- lance footage. In the summer of 2018, Project Maven came under public scrutiny when it was revealed that the US DoD had contracted Google to help military analysts detect objects in video images. In a letter addressed to Google CEO Sundar Pichai, over four thousand Google employees stated that Google ‘should not be in the business of war’ and demanded the contract to be cancelled.2 After months of protest, Google ended its work on Project Maven, although it maintained that it was ‘eager to do more’ for the US DoD in other fields of activity.3 323 European Journal of International Security The case of Project Maven has become central within academic and wider public debates related to the role of artificial intelligence (AI) in war and the emergence of so-called lethal autonomous weapons systems specifically.4 Promoters of Project Maven have claimed that the project merely renders existing practices of data analysis more efficient and precise. Critical voices, however, point out that such investments ‘are poised to automate the process of identify- ing targets, including people’ and may well serve as the basis for the development of autonomous weapons.5 Similarly, critical scholarship on security has situated Project Maven against the back- ground of an already problematic targeting regime that operates on the basis of algorithmic cal- culations and ‘pattern-of-life’ analysis, and which has become entangled with pre-existing and more affective registers of race and gender.6 As pointed out by Lucy Suchman, the automation of data analysis as pursued within Project Maven ‘can only serve to exacerbate military operations that are at once discriminatory and indiscriminate in their targeting’.7 y g g My own interest in Project Maven derives from a different observation, related to how the pro- ject represents a wider ambition on the part of Western militaries to rethink and revise our very way of warfare. As explained by Air Force Lt Gen. Jack Shanahan, in charge of the project, Project Maven served a wider purpose beyond the development of computer vision algorithms. Introduction It was supposed to be ‘that pilot project – that pathfinder – that spark that kindles the flame front for artificial intelligence across the rest of the department’.8 Understood as such, Project Maven marked the beginning of what Shanahan called ‘prototype warfare’.9 g g p yp f Prototype warfare, as described by Shanahan, centres on an overarching imperative of military innovation set against the backdrop of an increasingly fierce technological competition on the global stage.10 At its core, prototype warfare represents a shift in how Western militaries develop 4For an overview of this debate, see, for example, Christof Heyns, UN Doc. A/HRC/23/47, ‘Report of the Special Rapporteur on Extrajudicial, Summary or Arbitrary Executions’, United Nations (9 April 2013); Paul Scharre, Army of None: Autonomous Weapons and the Future of War (New York, NY: W. W. Norton & Company, 2018); Noel Sharkey, ‘Saying “no!” to lethal autonomous targeting’, Journal of Military Ethics, 9:4 (2010), pp. 369–83. For scholarship on the (potential) legality of lethal autonomous weapons systems and their ethical challenges, see Peter Asaro, ‘On banning autono- mous weapon systems: Human rights, automation, and the dehumanization of lethal decision-making’, International Review of the Red Cross, 94:886 (2013), pp. 678–709; Denise Garcia, ‘Future arms, technologies and international law: Preventive security governance’, European Journal of International Security, 1:1 (2016), pp. 94–111; Heather M. Roff, ‘Lethal autono- mous weapons and jus ad bellum proportionality’, Case Western Reserve Journal of International Law, 47 (2015), pp. 37– 52; Robert Sparrow, ‘Robots and respect: Assessing the case against autonomous weapon systems’, Ethics & International Affairs, 30:1 (2016), pp. 93–116. For more on the concept of ‘meaningful human control’, see, for example, Rebecca Crootof, ‘A meaningful floor for meaningful human control’, Temple International & Comparative Law Journal, 30:1 (spring 2016), pp. 53–62. pp 5Lucy Suchman, Lilly Irani, and Peter Asaro, ‘Google’s march to the business of war must be stopped’, The Guardian (16 May 2018), available at: {https://www.theguardian.com/commentisfree/2018/may/16/google-business-war-project-maven} accessed 22 March 2021. 6On ‘algorithmic warfare’ and algorithmic ‘pattern-of-life’ analysis, see Lucy Suchman, ‘Algorithmic warfare and the reinvention of accuracy’, Critical Studies on Security, 8:2 (2020), pp. 175–82; Louise Amoore, ‘Algorithmic war: Everyday geographies of the war on terror’, Antipode, 41:1 (2009), pp. 49–69; Katharine Hall Kindervater, ‘The emergence of lethal surveillance: Watching and killing in the history of drone technology’, Security Dialogue, 47:3 (2016), pp. 4For an overview of this debate, see, for example, Christof Heyns, UN Doc. A/HRC/23/47, ‘Report of the Special Rapporteur on Extrajudicial, Summary or Arbitrary Executions’, United Nations (9 April 2013); Paul Scharre, Army of None: Autonomous Weapons and the Future of War (New York, NY: W. W. Norton & Company, 2018); Noel Sharkey, ‘Saying “no!” to lethal autonomous targeting’, Journal of Military Ethics, 9:4 (2010), pp. 369–83. For scholarship on the (potential) legality of lethal autonomous weapons systems and their ethical challenges, see Peter Asaro, ‘On banning autono- mous weapon systems: Human rights, automation, and the dehumanization of lethal decision-making’, International Review of the Red Cross, 94:886 (2013), pp. 678–709; Denise Garcia, ‘Future arms, technologies and international law: Preventive security governance’, European Journal of International Security, 1:1 (2016), pp. 94–111; Heather M. Roff, ‘Lethal autono- mous weapons and jus ad bellum proportionality’, Case Western Reserve Journal of International Law, 47 (2015), pp. 37– 52; Robert Sparrow, ‘Robots and respect: Assessing the case against autonomous weapon systems’, Ethics & International Affairs, 30:1 (2016), pp. 93–116. For more on the concept of ‘meaningful human control’, see, for example, Rebecca Crootof, ‘A meaningful floor for meaningful human control’, Temple International & Comparative Law Journal, 30:1 (spring 2016), pp. 53–62. g p 8Cited in Jack Corrigan, ‘Three-star general wants AI in every new weapon system’, Defense One (3 November 2017), avail- able at: {https://www.defenseone.com/technology/2017/11/three-star-general-wants-artificial-intelligence-every-new-weapon- system/142239/?oref=d-riv} accessed 22 March 2021. 9Jack Shanahan, ‘Interview with Lieutenant General Jack Shanahan: Part 2’, Over the Horizon (OTH) (14 April 2018), available at: {https://othjournal.com/2018/04/04/interview-with-lieutenant-general-jack-shanahan-part-2/} accessed 22 March 2021. 10Matthew A. Horning, Robert E. Smith, and Shaheen Shidfar, ‘Mission engineering and prototype warfare: Operationalizing technology faster to stay ahead of the threat’, in Proceedings of the Ground Vehicle Systems Engineering and Technology Symposium (GVSETS) (13–15 August 2019). pp 5Lucy Suchman, Lilly Irani, and Peter Asaro, ‘Google’s march to the business of war must be stopped’, The Guardian (16 May 2018), available at: {https://www.theguardian.com/commentisfree/2018/may/16/google-business-war-project-maven} accessed 22 March 2021. Introduction 223–8; Lauren Wilcox, ‘Embodying algorithmic war: Gender, race and the posthuman in drone warfare’, Security Dialogue, 48:1 (2017), pp. 11–28. 7Suchman, ‘Algorithmic warfare’, p. 175. 8 8Cited in Jack Corrigan, ‘Three-star general wants AI in every new weapon system’, Defense One (3 November 2017), avail- able at: {https://www.defenseone.com/technology/2017/11/three-star-general-wants-artificial-intelligence-every-new-weapon- system/142239/?oref=d-riv} accessed 22 March 2021. y 9Jack Shanahan, ‘Interview with Lieutenant General Jack Shanahan: Part 2’, Over the Horizon (OTH) (14 April 2018), available at: {https://othjournal.com/2018/04/04/interview-with-lieutenant-general-jack-shanahan-part-2/} accessed 22 March 2021. 10Matthew A. Horning, Robert E. Smith, and Shaheen Shidfar, ‘Mission engineering and prototype warfare: Operationalizing technology faster to stay ahead of the threat’, in Proceedings of the Ground Vehicle Systems Engineering and Technology Symposium (GVSETS) (13–15 August 2019). 10Matthew A. Horning, Robert E. Smith, and Shaheen Shidfar, ‘Mission engineering and prototype warfare: Operationalizing technology faster to stay ahead of the threat’, in Proceedings of the Ground Vehicle Systems Engineering and Technology Symposium (GVSETS) (13–15 August 2019). Marijn Hoijtink 324 and acquire technology and weaponry. It promotes moving away from a focus on the mass pro- duction of high-end weapons to the development, rapid fielding, and testing of prototypes in spe- cific operational environments, ‘where units can fail early and fail small, to scale fast and learn fast’.11 As formulated by the UK army as part of its own strategic conceptualisation of prototype warfare, prototype warfare this way involves more than the modification of existing acquisition processes.12 It represents a broader ‘mindset’ or ‘new approach’ to military activity, which ‘seeks to mimic the pace and intensity of wartime transformation by prioritizing experimentation and adaption’ and encourage warfighters ‘to take calculated risks with a higher than normal tolerance for failure, enabling them to learn, succeed and exploit catalytic ideas that deliver asymmetric advantage’.13 Tracing recent conceptions of ‘prototype warfare’ across Western military discourse and prac- tice, my objective in this article is to map the contours of a new regime of warfare, which is emer- ging against the background of an overarching imperative of military innovation and speculative understanding of the potential role of ‘AI’ in warfare. Broadly defined, regimes of warfare are con- stellations of discourses, practices, interactions, technologies, and rationalities that constitute and organise war in a specific and (largely) shared way. 17As explained in the below, my thinking here is based on some of the recent work on ‘experimentality’ as a form of ‘government-by-experiment’. See Vinh-Kim Nguyen, ‘Government-by-exception: Enrolment and experimentality in mass HIV treatment programmes in Africa’, Social Theory & Health, 7:3 (2009), pp. 196–217. For a recent intervention within security studies that is also based on this line of thinking and that has shaped my work, see Claudia Aradau, ‘Experimentality, surplus data and the politics of debilitation in borderzones’, Geopolitics, online first (2020), pp. 1–21. 11Arnel P. David, Jason Buckley, and Toby Whitmarsh, ‘Prototype warfare: Inculcating a culture of adaption in the British Army’, Wavell Room Contemporary British Military Thought (4 June 2019), available at: {https://wavellroom.com/2019/06/04/ prototype-warfare-inculcating-a-culture-of-adaptation-in-the-british-army/#easy-footnote-bottom-6-13179} accessed 9 December 2021. 16With governmental rationality, I refer to ‘any relatively systematic way of thinking about government’, in this case the government of war. See Mitchell Dean, Governmentality: Power and Rule in Modern Society (London, UK: SAGE Publications, 2010), p. 268. See also Graham Burchell, Colin Gordon, and Peter Miller (eds), The Foucault Effect: Studies in Governmentality (Chicago, IL: The University of Chicago Press, 1991). 13Toby Whitmarsh and Arnel David, ‘If you are not first you are last: Gaining an adaptive edge through prototype warfare’, War Room, US Army War College (21 May 2019), available at: {https://warroom.armywarcollege.edu/articles/if-you-are-not- first-you-are-last-gaining-an-adaptive-edge-through-prototype-warfare/} accessed 9 December 2021. 14Antoine Bousquet, The Scientific Way of Warfare: Order and Chaos on the Battlefields of Modernity (New York, NY: Columbia University Press, 2010). 12Website of the British Army, available at: {https://www.army.mod.uk/news-and-events/events/army-combat-power-dem- onstration/} accessed 22 December 2021. 15Bousquet, The Scientific Way of Warfare, p. 3. Introduction As Michelle Murphy writes, this kind of experimentation legitimises continued intervention and is largely self-perpetuating, hailing ‘life as composed of composed of potential, of chances, of pos- sibilities for becoming, of manipulable relations that can be triggered and altered, if only the right protocol and technique can be employed’.18 As I will argue, this speculative understanding of experimentation in the context of war has serious political implications. Driven by this kind of experimental rationality, the experimental way of warfare is largely immune to failure, or uninterested in the costs or aftermath of the experi- ments being deployed. Any result represents valuable data gained, and so failures only serve to advance further experimentation. This way, the experimental way of warfare is largely insulated from critique, as militaries can always point to lessons learned or to the future potential of the ‘next’ experiment. As part of this development, war and conflict become reconfigured mainly as an opportunity to experiment. Our wars become endless and everywhere, not because of the (discursive representation of) the enemy, but because of the broader experimental infrastruc- ture that is created and that needs to be maintained. In developing these arguments, empirically, my analysis traces the emergence of conceptions of prototype warfare across Western military discourse and practice, with a particular focus on how prototype warfare is taking experimentation directly into the battlefield, specifically when it comes to experimental practices of data collection and analysis, which are seen as foundational to the role of AI in warfare.19 In so doing, I acknowledge, following Dan Öberg, that ‘Western military discourse is by no means unitary’, but ‘traversed by logics depending on service branches as well as on power structures and debates within them.’20 At the same time, tracing prototype warfare across different national contexts and levels of engagement, I do find that an overarching regime of warfare defined by experimentation can be identified. In analysing this regime or experimental way of warfare, I rely on a variety of publicly available sources, such as official policy documents, speeches, blogs, defence magazine articles, project descriptions, newspaper articles, video recordings of panels at defence trade shows, and information coming from freedom of information requests. Introduction As argued by Antoine Bousquet, historically, such shared ways of thinking about and organising the conduct of war have strongly been informed by an overarching scientific rationality.14 What Bousquet defines as ‘the scientific way of warfare’ refers to this systematic and extensive deployment of scientific ideas ‘to inform thinking about the very nature of combat and the forms of military organization best suited to prevail in it’.15 As the regimes that Bousquet identifies are connected to the prevalence of a spe- cific set of scientific ideas and technologies in a given period, this means that they are historically variable and always subject to change. My argument in this article is that prototype warfare represents such a shift and is reflective of a broader transformation, or new regime of warfare. Specifically, I argue that the concept of prototype warfare captures the emergence of a profoundly experimental way of warfare, which is driven by an overarching experimental rationality,16 and largely conducted ‘by-experiment’.17 While warfare has always been underpinned by experimental activity, what is particular in the current context is how experimentation spans across an increasingly wide range of military prac- tices, into the actual (or simulated) battlefield, and across novel military-corporate assemblages. What explains this spatialisation of experimental practice and its ubiquity in contemporary war- fare is a particular, highly speculative, understanding of experimentation that is increasingly 11Arnel P. David, Jason Buckley, and Toby Whitmarsh, ‘Prototype warfare: Inculcating a culture of adaption in the British Army’, Wavell Room Contemporary British Military Thought (4 June 2019), available at: {https://wavellroom.com/2019/06/04/ prototype-warfare-inculcating-a-culture-of-adaptation-in-the-british-army/#easy-footnote-bottom-6-13179} accessed 9 December 2021. 12Website of the British Army, available at: {https://www.army.mod.uk/news-and-events/events/army-combat-power-dem- onstration/} accessed 22 December 2021. 13 13Toby Whitmarsh and Arnel David, ‘If you are not first you are last: Gaining an adaptive edge through prototype warfare’, War Room, US Army War College (21 May 2019), available at: {https://warroom.armywarcollege.edu/articles/if-you-are-not- first-you-are-last-gaining-an-adaptive-edge-through-prototype-warfare/} accessed 9 December 2021. y g g p g g p yp 14Antoine Bousquet, The Scientific Way of Warfare: Order and Chaos on the Battlefields of Modernity (New York, NY: Columbia University Press, 2010). European Journal of International Security 325 detached from a more narrow interpretation of experimentation as a scientific practice of repli- cation and controlled testing and that embraces uncertainty and failure as a productive force. 18Michelle Murphy, The Economization of Life (Durham, NC: Duke University Press, 2017), p. 81. 19Recent scholarship on the practices, technologies, and rationalities of machine learning points out how these technolo- gies are themselves experimental. For example, building on the work of N. Katherine Hayles, Louise Amoore explains how the inductive or abductive logics of machine learning allow ‘mathematics to be practiced as an experimental science’ (p. 5). For Amoore, to view mathematics as an experimental science ‘opens the way to understanding entangled and collaborative human and machine inferences that feel their way towards a solution’, or that intuitively determine relevant patterns across large amounts of data (p. 5). Resonating with the anticipatory vision of the experimentation deployed within the experimen- tal way of warfare that I define in this article, these intuitive and experimental practices of machine learning likewise thrive on uncertainty, contingency, and speculation: ‘the experimental design of machine learning algorithms seems to profit from instability and uncertainty, [precisely] because these conditions yield data to the corpus of learning.’ See Louise Amoore, ‘Introduction: Thinking with algorithms: Cognition and computation in the work of N. Katherine Hayles’, Theory, Culture & Society, 36:2 (2019), pp. 3–16; see also Louise Amoore and Rita Raley, ‘Securing with algorithms: Knowledge, deci- sion, sovereignty’, Security Dialogue, 48:1 (2017), pp. 3–10; Luciana Parisi, ‘Critical computation: Digital automata and gen- eral artificial thinking’, Theory, Culture & Society, 36:2 (2019), pp. 89–121. 20Dan Öberg, ‘“Warfare as design”: Transgressive creativity and reductive operational planning’, Security (2018), pp. 493–509 (p. 496). 18Michelle Murphy, The Economization of Life (Durham, NC: Duke University Press, 2017), p. 81. 24Carol Cohn, ‘Sex and death in the rational world of defense intellectuals’, Signs: Journal of Women in Culture and Society, 12:4 (1987), pp. 687–718; Hugh Gusterson, Nuclear Rites: A Weapons Laboratory at the End of the Cold War (Berkeley, CA: University of California Press, 1998); Marijn Hoijtink and Matthias Leese (eds), Technology and Agency in International Relations (London, UK: Routledge, 2019); Allison Howell, Madness in International Relations: Psychology, Security, and the Global Governance of Mental Health (New York, NY: Routledge, 2011); Donald A. Mackenzie, Inventing Accuracy: A Historical Sociology of Nuclear Missile Guidance (Cambridge, MA: MIT Press, 1990); Nisha Shah, ‘Gunning for war: Infantry rifles and the calibration of lethal force’, Critical Studies on Security, 5:1 (2017), pp. 81–104; Bousquet, Grove, and Shah, ‘Becoming war’. 22Here, I contribute to the abovementioned scholarship that has developed around the concept of ‘algorithmic warfare’, but also to existing work on ‘remote warfare’. See, for example, Jolle Demmers and Lauren Gould, ‘The remote warfare paradox: Democracies, risk aversion and military engagement’, in Alasdair McKay, Abigail Watson, and Megan Karlshoj-Pedersen (eds), Remote Warfare: Interdisciplinary Perspectives (Bristol, UK: E-International Relations, 2021), pp. 34–47 (p. 34). On ‘remote warfare’, see also Jolle Demmers and Lauren Gould, ‘An assemblage approach to liquid warfare: AFRICOM and the “hunt” for Joseph Kony’, Security Dialogue, 49:5 (2018), pp. 364–81; Thomas Waldman, ‘Vicarious warfare: The counterpro- ductive consequences of modern American military practice’, Contemporary Security Policy, 39:2 (2018), pp. 181–205. Introduction Zooming in on how prototype warfare takes experimentation into the battlefield, my focus is on examples from the US, UK, and The Netherlands, which can be con- sidered important drivers of prototype warfare, although my arguments are also relevant for other Western militaries. My objective is not to provide a full mapping of existing prototypes in this field, as this would be extremely difficult given the evolving and experimental character of these projects, as well as because of a more general lack of oversight of military practice and operations, but, rather, to map the broader contours of prototype warfare and the experimental way of warfare that it is shaping by relying on the sources mentioned. 326 Marijn Hoijtink My argument proceeds in four steps. I first examine the emergence of the experimental way of warfare as a distinct technoscientific regime of warfare, drawing on recent literature at the inter- section of Science and Technology Studies (STS) and International Relations (IR) that examines how wars are made and made possible, with a specific focus on the entanglements of science, technology, and war. Next, I trace the concept of prototype warfare from its inception in the early 1990s to its current invocations across Western military discourse and practice, focusing on how the experimental way of way of warfare takes shape under this very term within the US, UK, and The Netherlands. Third, I zoom in on how prototype warfare takes experimentation into the actual or simulated battlespace, focusing on the case of the Land Information Manoeuvre Centre (LIMC), an ‘experimental data center’ established by the Dutch Army in March 2020 to collect and assess data to help crisis response and fight disinformation during the COVID-19 pandemic.21 I conclude by calling for a more sustained research focus on the ways in which the experimental way of warfare contributes to the notion that our wars are endless and every- where and accelerates and deepens existing vulnerabilities and asymmetries in war.22 25Bousquet, The Scientific Way of Warfare, p. 3. 21Karel Berkhout, Esther Rosenberg, and Bram Petraeus, ‘Hoe Defensie Zijn Eigen Bevolking in de Gaten Houdt’, NRC Handelsblad (16 November 2020). All translations from Dutch to English are by the author, who is a native Dutch speaker. 23Antoine Bousquet, Jairus Grove, and Nisha Shah, ‘Becoming war: Towards a martial empiricism’, Security Dialogue, 51:2–3 (2020), pp. 99–118 (p. 103). 25Bousquet, The Scientific Way of Warfare, p. 3. 26Ibid. 27Ibid., p. 4. The experimental way of warfare This article builds on scholarship at the intersection of IR and STS investigating the broader ‘con- figurations of people, things and processes that come together to make war possible and shape its mutable characteristics’.23 Specifically, I am following a growing field of scholarship that focuses on the role of science and technology in assembling and executing warfare.24 Antoine Bousquet has examined this relationship between war, science, and technology in detail in his book The Scientific Way of Warfare, starting from the premise that ‘an ever more intimate symbiosis between science and warfare has established itself, with the increasing reliance on the develop- ment and integration of technology within complex social assemblages of war.’25 In his book, Bousquet’s interest is not so much with the direct role of science or scientists in building military technology, but with the diverse and historically situated ways ‘in which scientific ideas have been systematically recruited to inform thinking about the very nature of combat and the forms of military organization best suited to prevail in it’.26 What he terms ‘the scientific way of warfare’ refers to the ‘array of scientific rationalities, techniques, frameworks of interpretation, and intel- lectual dispositions which have characterised the approach to the application of socially organised violence in the modern era’.27 327 European Journal of International Security According to Bousquet, while the overarching role of science in shaping warfare has remained stable since the eighteenth century, the specific ways in which science and technology have informed our thinking about and conduct of warfare are historically variable and dependent on the prevalence of a particular set of scientific ideas and technologies in a given period. Bousquet identifies four distinct regimes of the scientific way of warfare, which are mechanistic, thermodynamic, cybernetic, and chaoplexic warfare.28 At the core of each regime is a specific set of scientific ideas and a paradigmatic technology, respectively the clock, the engine, the computer and the network. Each of these technologies has shaped military organisation and the conduct of war in considerable ways. At the same time, all of these technologies could only become thinkable or possible as part of a broader regime of warfare and a distinct set of scientific rationalities. 31David Arnold, Colonizing the Body: State Medicine and Epidemic Disease in Nineteenth Century India (Berkeley and Los Angeles, CA and London, UK: University of California Press, 1993); Helen Tilley, Africa as a Living Laboratory: Empire, Development, and the Problem of Scientific Knowledge (Chicago, IL: University of Chicago Press, 2011); Fouzieyha Towghi and Kalindi Vora, ‘Bodies, markets and the experimental in South Asia’, Ethnos, 79:1 (2014), pp. 1–18. 33Sharon Weinberger, The Imagineers of War: The Untold History of DARPA, the Pentagon Agency that Changed the World (New York, NY: Alfred A. Knopf, 2017). 32Katja Lindskov Jacobsen, The Politics of Humanitarian Technology: Good Intentions, Unintended Consequences and Insecurity (London, UK: Routledge, 2015). 34Annie Jacobsen, The Pentagon’s Brain: An Uncensored History of DARPA, America’s Top-Secret Military Research Agency (London, UK: Hachette UK, 2015). 35Dan Öberg, ‘Warfare as design’. 30I am not denying that AI has a much longer historical role in warfare, and that developments within the broader field of AI have always been closely connected to military funding and a strongly militarised vision of what AI can do, but the cur- rent, widescale investment in AI technology within the military domain is unmistaken. For more on the role of AI as a mili- tary technology, see Paul N. Edwards, The Closed World: Computers and the Politics of Discourse in Cold War America (Cambridge, MA: MIT Press). 28Ibid. 29Antoine Bousquet, ‘Chaoplexic warfare or the future of military organization’, International Affairs, 84:5 (2008), pp. 915–29. 30I am not denying that AI has a much longer historical role in warfare, and that developments within the broader field of AI have always been closely connected to military funding and a strongly militarised vision of what AI can do, but the cur- rent, widescale investment in AI technology within the military domain is unmistaken. For more on the role of AI as a mili- tary technology, see Paul N. Edwards, The Closed World: Computers and the Politics of Discourse in Cold War America (Cambridge, MA: MIT Press). 31David Arnold, Colonizing the Body: State Medicine and Epidemic Disease in Nineteenth Century India (Berkeley and Los Angeles, CA and London, UK: University of California Press, 1993); Helen Tilley, Africa as a Living Laboratory: Empire, Development, and the Problem of Scientific Knowledge (Chicago, IL: University of Chicago Press, 2011); Fouzieyha Towghi and Kalindi Vora, ‘Bodies, markets and the experimental in South Asia’, Ethnos, 79:1 (2014), pp. 1–18. 32Katja Lindskov Jacobsen, The Politics of Humanitarian Technology: Good Intentions, Unintended Consequences and Insecurity (London, UK: Routledge, 2015). 33Sharon Weinberger, The Imagineers of War: The Untold History of DARPA, the Pentagon Agency that Changed the World (New York, NY: Alfred A. Knopf, 2017). 34Annie Jacobsen, The Pentagon’s Brain: An Uncensored History of DARPA, America’s Top-Secret Military Research Agency (London, UK: Hachette UK, 2015). 35Dan Öberg, ‘Warfare as design’. Ibid. 29Antoine Bousquet, ‘Chaoplexic warfare or the future of military organization’, International Affairs, 84:5 (2008), pp. 915–29. 30I am not denying that AI has a much longer historical role in warfare, and that developments within the broader field of ne Bousquet, ‘Chaoplexic warfare or the future of military organization’, International Affairs, 84:5 (200 . The experimental way of warfare As Bousquet clarifies elsewhere, ‘technology [does not] appear ex nihilo: it is the product of a par- ticular human engagement with the world.’29 g g Building on the concept of the technoscientific regime of warfare, in this article I identify another transformation of warfare – or an emergent regime of warfare – that is linked to a highly speculative understanding of the current role of AI and digital technologies in warfare,30 and defined by an overarching experimental rationality. Of course, the conduct of war has always been underpinned by experimental activity. There is a long history of experimentation with tech- nology ‘out in the field’ of war and conflict, or as part of colonial, interventionist projects.31 For instance, much has been written about medical experimentation on populations in war and on the production of gendered and racialised bodies for experimentation.32 Popular histories of the US Defense Advanced Research Projects Agency (DARPA) or the Central Intelligence Agency (CIA), such as Sharon Weinberger’s The Imagineers of War33 or Annie Jacobsen’s The Pentagon’s Brain,34 have similarly shown how war and experimentation with technology and weaponry have a long and entangled history. More recently, experimentation has been linked to counterinsurgency warfare and the deployment of future-oriented action (scenarios, exercises, gaming) and creative design thinking in the context of the Western wars in Afghanistan and Iraq.35 q What is specific within the current context, however, is how experimentation has become truly distributed, informing an increasingly wide range of military practices and spanning across novel military-corporate assemblages. As such, experimentation has become centre stage in military thinking about the organisation of warfare, what I refer to here as the experimental way of war- fare. The experimental way of warfare In conceptualising the experimental way of warfare, I draw on recent work within anthro- pology and associated disciplines on ‘experimentality’ as a mode of governing that operates Marijn Hoijtink 328 ‘by-experiment’ and that is driven by an overarching experimental rationality.36 In her analysis of experimentality in 1970s Bangladesh, Michelle Murphy describes how experimental practice became ubiquitous, spreading across a distributed field of experts and producing a ‘transnation- ally attached, locally organized patchwork of unevenly distributing funds, technologies, practices, infrastructure, experts, services, collectivities, and workers’.37 Observing a ‘creative explosion of Bangladeshi trials, pilot projects, and experiments’ over the course of the 1970s, Murphy points out how experimentation transitioned from being a more or less centralised and spatially con- fined scientific practice limited to the lab, clinic, or field site, to a broader way of governing life and population as part of development practices.38 As part of this process, Bangladesh became a key site for experimentation with new techniques and technologies, as well as a global model for this kind of governing by-experiment. What makes experimentality in this and other contexts so generative, argues Murphy, is that it operates on the basis of a highly speculative or anticipatory vision of the experiments being deployed.39 This speculative understanding of experimentation is what makes experimentality as a mode of governing largely self-perpetuating. As Vinh-Kim Nguyen argues, experimentality inverts ‘the classical model whereby evidence of efficacy permits intervention’, in the sense that, ‘in this case intervention drives the need for self-validating evidence (that is, the intervention was effective).’40 Even more so, experimentality distinguishes itself in the way in which it thrives on uncertainty or failure. Writes Murphy, ‘if an experiment does not work, [this only means that] a better experiment is needed.’41 The outcome is the reproduction of ‘the infrastructure of experi- ment itself’.42 Extending these insights on experimentality to the context of war, I am approaching the experimental way of warfare as an emerging technoscientific regime that relies on experimenta- tion as a shared way of thinking about and organising warfare today. 36Nguyen, ‘Government-by-exception’. See also Murphy, The Economization of Life; Towghi and Vora, ‘Bodies’; Aradau, ‘Experimentality’; Melinda Cooper, ‘The pharmacology of distributed experiment: User-generated drug innovation’, Body & Society, 18:3–4 (2012), pp. 18–43. 37Murphy, The Economization of Life, p. 87. 38Ibid., p. 79. 39On this point, see also Cooper, ‘The pharmacology’. 40Nguyen, ‘Government-by-exception’, p. 196. 41Murphy, The Economization of Life, p. 91. 42Ibid. 43Andrew Merrill, ‘Start-Up War: Silicon Valley, the Pentagon and Wars Yet-to-Come’ (PhD manuscript [in progress], University of Toronto). See also Emily Gilbert, ‘Military geoeconomics: Money, finance and war’, in Rachel Woodward (ed.), A Research Agenda for Military Geographies (Cheltenham, UK: Edward Elgar Publishing, 2019), pp. 100–14. 44D bbi Li l ‘F ili ? S i it d th bi th f b d t h l ’ E J l f I t ti l 43Andrew Merrill, ‘Start-Up War: Silicon Valley, the Pentagon and Wars Yet-to-Come’ (PhD manuscript [in progress], University of Toronto). See also Emily Gilbert, ‘Military geoeconomics: Money, finance and war’, in Rachel Woodward (ed.), A Research Agenda for Military Geographies (Cheltenham, UK: Edward Elgar Publishing, 2019), pp. 100–14. 44Debbie Lisle, ‘Failing worse? Science, security and the birth of a border technology’, European Journal of International 36Nguyen, ‘Government-by-exception’. See also Murphy, The Economization of Life; Towghi and Vora, ‘Bodies’; Aradau, ‘Experimentality’; Melinda Cooper, ‘The pharmacology of distributed experiment: User-generated drug innovation’, Body & Society, 18:3–4 (2012), pp. 18–43. 37Murphy The Economization of Life p 87 sle, ‘Failing worse? Science, security and the birth of a border technology’, European Journal of International (2018), pp. 887–910. The experimental way of warfare As part of this development, experimentation increasingly spans across all levels of military engagement and brings together a diverse group of actors, including the military, tech start-ups, and venture capitalists, into experi- mental assemblages and informal partnerships.43 The experimental way of warfare operates on a particular understanding of experimentation that is increasingly detached from a more narrow interpretation of experimentation as a scientific practice of replication and controlled testing, favouring a more speculative and entrepreneurial vision of experimentation (dominant within the surroundings of Silicon Valley) that is about acting fast, taking risks, and accepting, or adapt- ing to, failure.44 It is this speculative vision of the experiment that makes the experimental way of warfare largely immune to criticism linked to effectiveness or failure, as one can always point to lessons learned and a process of ‘moving forward’ to deflect from failures. p g Strategic conceptions of prototype warfare, as they are currently being pushed by Western militaries, capture these aspects, while also taking experimentation as an embodied form into the battlefield. For example, a leading UK defence company defines prototype warfare as ‘experi- mentation in contact’, which ‘centers on a willingness to use experimental technologies, tools and European Journal of International Security 329 techniques at an earlier stage of readiness in live environments as part of a continuous cycle of learning and optimization’.45 Similarly, a UK General conceptualises prototype warfare as ‘a new, democratized approach to experimentation and innovation’, which ‘will extend to the battlespace, with deployed operations becoming the ultimate laboratories for innovation’.46 Below, I will fur- ther examine this extension of prototype warfare into the battlespace, where experimentation serves primarily as a proof of concept or vehicle for constant learning (by doing). The next sec- tion, however, first traces the emergence of prototype warfare as a strategic concept across Western military discourse and practice, examining how the experimental way of warfare is taking shape under this concept. 45Qinetiq, ‘Deploying Prototype Warfare’, available at: {https://www.qinetiq.com/en/insights/deploying-prototype-warfare} accessed 22 March 2021, emphasis added. 46Mark Carleton-Smith, ‘Foreword’, Defence Global (November, 2018), available at: {https://edition.pagesuite-professional.co. uk/html5/reader/production/default.aspx?pubname=&edid=fc50d232-0df5-4759-bfaa-05e5761e5701} accessed 21 December 2021, emphasis added. 47Robert R. Leonhard, The Principles of War for the Information Age (New York, NY: Presidio/Ballantine Books, 1998), p. 33. 48Ibid., p. 69. 49Ibid., p. 198. 50Robert Kozloski, ‘The path to prototype warfare’, War on the Rocks (17 July 2017), available at: {https://warontherocks. com/2017/07/the-path-to-prototype-warfare/} accessed on 24 March 2021. 51Arthur K. Cebrowski and John J. Gartska, ‘Network centric warfare: Its origins and future’, Proceedings of the U.S. Naval Institute, 124:1 (1998), pp. 1–139. 52Leonhard, The Principles of War, p. 124. p p yp 51Arthur K. Cebrowski and John J. Gartska, ‘Network centric warfare: Its origins and future’, Proceedings of Institute, 124:1 (1998), pp. 1–139. 52 p 50Robert Kozloski, ‘The path to prototype warfare’, War on the Rocks (17 July 2017), available at: {https://warontherocks. com/2017/07/the-path-to-prototype-warfare/} accessed on 24 March 2021. 52Leonhard, The Principles of War, p. 124. 45Qinetiq, ‘Deploying Prototype Warfare’, available at: {https://www.qinetiq.com/en/insights/deploying-prototype-warfare} accessed 22 March 2021, emphasis added. 46Mark Carleton-Smith, ‘Foreword’, Defence Global (November, 2018), available at: {https://edition.pagesuite-professional.co. uk/html5/reader/production/default.aspx?pubname=&edid=fc50d232-0df5-4759-bfaa-05e5761e5701} accessed 21 December 2021, emphasis added. 47Robert R. Leonhard, The Principles of War for the Information Age (New York, NY: Presidio/Ballantine Books, 1998), p. 33. 48Ibid., p. 69. 49Ibid., p. 198. 50 , p 50Robert Kozloski, ‘The path to prototype warfare’, War on the Rocks (17 July 2017), available at: {https://warontherocks. com/2017/07/the-path-to-prototype-warfare/} accessed on 24 March 2021. 51Arthur K. Cebrowski and John J. Gartska, ‘Network centric warfare: Its origins and future’, Proceedings of the U.S. Naval com/2017/07/the-path-to-prototype-warfare/} accessed on 24 March 2021. 51Arthur K. Cebrowski and John J. Gartska, ‘Network centric warfare: Its origins and future’, Proceedings of the U.S. Naval Institute, 124:1 (1998), pp. 1–139. , p 50Robert Kozloski, ‘The path to prototype warfare’, War on the Rocks (17 July 2017), available at: {https://warontherocks. com/2017/07/the-path-to-prototype-warfare/} accessed on 24 March 2021. 51Arthur K. Cebrowski and John J. Gartska, ‘Network centric warfare: Its origins and future’, Proceedings of the U.S. Naval Institute, 124:1 (1998), pp. 1–139. 52Leonhard, The Principles of War, p. 124. 49Ibid., p. 198. Tracing prototype warfare While the concept of prototype warfare has recently gained traction across Western military discourse, it was first coined in 1998 by a US Lieutenant Colonel, Robert R. Leonhard. In his book The Principles of War for the Information Age, Leonhard aimed to revise military doc- trine ‘as warfare progressed through the Industrial Age into the Information Age’.47 He argued that in order to create technological advantage in the Information Age, militaries needed to adapt to prototype warfare as a radically different approach to warfighting. Leonhard observed that while technological change in the Industrial Age developed at a moderate tempo and that, as a result, ‘mass production was the sine qua non of Industrial Age warfare’, future warfare would ‘feature a constant myriad of technological advances that [would] come at a tempo that disallows mass production’.48 This meant that a new approach to technology development was needed that would favour the deployment of limited-production prototypes tailored to spe- cific missions. In turn, such an approach would entail a new and more ‘dynamic relationship among economy, organization, and training’,49 as well as a more general disposition towards experimentation, as part of which ‘military forces [would] become comfortable with deploying novel weapons, supported by novel tactics, in hopes of achieving an operational advantage against an opponent’.50 g Clearly, Leonhard’s thinking on prototype warfare at the time was shaped by existing debates on the so-called Revolution of Military Affairs of the late 1990s. Tracing prototype warfare His ideas were also informed by military strategic thinking as it developed within the doctrine of network-centric warfare, as part of which military strategists proposed to decentralise military organisation, drawing from insights and the vocabulary offered by complexity theory and the information sciences.51 Leonhard writes for instance that ‘[o]ne of the most exciting technologies of twenty-first century warfare is that of “situational awareness”’, which is to be attained through a ‘network of computers’ through which a military commander and his subordinates would see each other digitally.52 This network of computers would free militaries ‘forever from the need for mass-based control’ and allow them ‘to come at the enemy in a patternless fashion that defies anticipation and complicates Marijn Hoijtink 330 the enemy’s understanding’.53 Prototype warfare would entail a shift away from mass as a means of control and towards ‘patternless maneuver’ or ‘precision maneuver’ enabled by digital commu- nications and a decentralised and networked military command structure.54 y Tracing its origins this way, we can see how prototype warfare does not present a clear epis- temological break with conceptions of decentralised control and information superiority as they have been central to the doctrine of network-centric warfare. Indeed, to a large extent, the emer- gence of the prototype warfare concept appears to be about fulfilling earlier promises of a fully legible and malleable battlefield, this time by taking advantage of the technological advances that AI and new digital technologies appear to afford. Understood as such, the emergence of prototype warfare as a concept within Western military discourse and practice should be situated against the background of a highly performative and speculative rhetoric around the transforma- tive potential of AI, specifically in relation to its proclaimed ability to improve and accelerate mili- tary decision-making,55 or the way in which it can ‘sift through today’s data-rich environment’, as a report by the North Atlantic Treaty Organization (NATO) puts it.56 At the same time, proto- type warfare is rooted in the widely shared belief across Western militaries that existing military innovation and acquisition strategies are excessively cumbersome and slow, and insufficiently capable of incorporating the latest technological advances. This is seen as particularly problematic in the context of what is presented as a wider technological competition in which competitors such as China and Russia are contesting Western technological superiority, or are at least narrow- ing the technology gap. 53Leonhard, The Principles of War. 54Ibid., pp. 125–6. 55Marijn Hoijtink and Anneroos Planqué-van Hardeveld, ‘Machine learning and the platformization of the military: A study of google’s machine learning platform TensorFlow’, International Political Sociology (2021), pp. 1–19. 56NATO Parliamentary Assembly Science and Technology Committee, Sub-Committee on Technology Trends and Security (STCTTS), ‘Artificial Intelligence: Implications for NATO’s Armed Forces, 149 STCTTS 19 E rev. 1 fin (13 October 2019), p. 4, available at: {https://www.nato-pa.int/download-file?filename=sites/default/files/2019-10/REPORT% 20149%20STCTTS%2019%20E%20rev.%201%20fin-%20ARTIFICIAL%20INTELLIGENCE.pdf} accessed 24 March 2021. 57Rob Murray, ‘Building a resilient innovation pipeline for the alliance’, NATO Review (1 September 2020), available at {https://www.nato.int/docu/review/articles/2020/09/01/building-a-resilient-innovation-pipeline-for-the-alliance/index.html} accessed 24 March 2021. 58See, for example, Kozloski, ‘The path to prototype warfare’. 59US Under Secretary of Defense, ‘Implementation Directive for Better Buying Power 3.0: Achieving Dominant Capabilities through Technical Excellence and Innovation’, Memorandum (9 April 2015), p. 13, available at: {https://www acq.osd.mil/fo/docs/betterBuyingPower3.0(9Apr15).pdf} accessed 24 March 2021. 60Chuck Hagel, ‘Defense Innovation Days’, Opening Keynote (Southeastern New England Defense Industry Alliance) Secretary of Defense Speech (3 September 2014), available at: {https://www.defense.gov/News/Speeches/Speech/Article/ 605602/} accessed 5 January 2022. pp 55Marijn Hoijtink and Anneroos Planqué-van Hardeveld, ‘Machine learning and the platformization of the military: A study of google’s machine learning platform TensorFlow’, International Political Sociology (2021), pp. 1–19. 58See, for example, Kozloski, ‘The path to prototype warfare’. 59 53Leonhard, The Principles of War. Ibid., pp. 125 6. 55Marijn Hoijtink and Anneroos Planqué-van Hardeveld, ‘Machine learning and the platformization of the military: A study of google’s machine learning platform TensorFlow’, International Political Sociology (2021), pp. 1–19. 56NATO Parliamentary Assembly Science and Technology Committee, Sub-Committee on Technology Trends and Security (STCTTS), ‘Artificial Intelligence: Implications for NATO’s Armed Forces, 149 STCTTS 19 E rev. 1 fin (13 October 2019), p. 4, available at: {https://www.nato-pa.int/download-file?filename=sites/default/files/2019-10/REPORT% 20149%20STCTTS%2019%20E%20rev.%201%20fin-%20ARTIFICIAL%20INTELLIGENCE.pdf} accessed 24 March 2021. 57Rob Murray, ‘Building a resilient innovation pipeline for the alliance’, NATO Review (1 September 2020), available at: {https://www.nato.int/docu/review/articles/2020/09/01/building-a-resilient-innovation-pipeline-for-the-alliance/index.html} accessed 24 March 2021. 58S f l K l ki ‘Th th t t t f ’ 54Ibid., pp. 125–6. 55Marijn Hoijtink and Anneroos Planqué-van Hardeveld, ‘Machine learning and the platformization 56NATO Parliamentary Assembly Science and Technology Committee, Sub-Committee on Technology Trends and Security (STCTTS), ‘Artificial Intelligence: Implications for NATO’s Armed Forces, 149 STCTTS 19 E rev. 1 fin (13 October 2019), p. 4, available at: {https://www.nato-pa.int/download-file?filename=sites/default/files/2019-10/REPORT% 20149%20STCTTS%2019%20E%20rev.%201%20fin-%20ARTIFICIAL%20INTELLIGENCE.pdf} accessed 24 March 2021. 57Rob Murray, ‘Building a resilient innovation pipeline for the alliance’, NATO Review (1 September 2020), available at: {https://www.nato.int/docu/review/articles/2020/09/01/building-a-resilient-innovation-pipeline-for-the-alliance/index.html} accessed 24 March 2021. p p p yp 59US Under Secretary of Defense, ‘Implementation Directive for Better Buying Power 3.0: Achieving Dominant Capabilities through Technical Excellence and Innovation’, Memorandum (9 April 2015), p. 13, available at: {https://www. acq.osd.mil/fo/docs/betterBuyingPower3.0(9Apr15).pdf} accessed 24 March 2021. 60Chuck Hagel, ‘Defense Innovation Days’, Opening Keynote (Southeastern New England Defense Industry Alliance), Secretary of Defense Speech (3 September 2014), available at: {https://www.defense.gov/News/Speeches/Speech/Article/ 605602/} accessed 5 January 2022. Tracing prototype warfare Set against the backdrop of this global ‘technological adoption race’,57 prototype warfare, as it is being articulated by Western militaries, represents and calls for a dis- tinct approach to military innovation that centres on the rapid deployment of, and ongoing experimentation with, a smaller quantity of tailored systems. The main rationale is that prototyp- ing enables militaries to adopt new technologies quicker, produce them at a lower cost, and increase the level of surprise.58 Experimentation underpins this practice of prototyping as it ‘puts prototypes into the Warfighter’s hands for assessment in an operational context’ and allows for an ongoing process of adaptation and learning.59 g g p p g Within the US context, prototyping and experimentation first appeared as central strategic concepts within the context of the so-called Third Offset Strategy that was announced by the US DoD under President Barack Obama to sustain US military dominance within the broader field of AI.60 Under the Third Offset, the US DoD launched a range of new programmes and offices, including Better Buying Power 3.0, a set of policies aimed at strengthening and streamlining US DoD innovation, which focused on ‘increas[ing] the use of prototyping and p p p yp 59US Under Secretary of Defense, ‘Implementation Directive for Better Buying Power 3.0: Achieving Dominant Capabilities through Technical Excellence and Innovation’, Memorandum (9 April 2015), p. 13, available at: {https://www. acq.osd.mil/fo/docs/betterBuyingPower3.0(9Apr15).pdf} accessed 24 March 2021. 59US Under Secretary of Defense, ‘Implementation Directive for Better Buying Power 3.0: Achieving Dominant Capabilities through Technical Excellence and Innovation’, Memorandum (9 April 2015), p. 13, available at: {https://www. acq.osd.mil/fo/docs/betterBuyingPower3.0(9Apr15).pdf} accessed 24 March 2021. q y g ( p ) p } 60Chuck Hagel, ‘Defense Innovation Days’, Opening Keynote (Southeastern New England Defense Industry Alliance), Secretary of Defense Speech (3 September 2014), available at: {https://www.defense.gov/News/Speeches/Speech/Article/ 605602/} accessed 5 January 2022. q y g p p 60Chuck Hagel, ‘Defense Innovation Days’, Opening Keynote (Southeastern New England Defense Industry Alliance), Secretary of Defense Speech (3 September 2014), available at: {https://www.defense.gov/News/Speeches/Speech/Article/ 605602/} accessed 5 January 2022. Tracing prototype warfare European Journal of International Security 331 experimentation’,61 and the Defense Innovation Unit Experimental (DIUx, now DIU), which was meant to be ‘accelerating the adoption of commercial technology throughout the military and growing the national security innovation base’.62 In so doing, DIU partners with the private sec- tor ‘including firms and academic institutions outside DoD’s traditional orbit’,63 and operates on the basis of a new type of contracting, called Other Transactions, that can be used for prototyping activities. This allows the DIU to award ‘prototype projects without the onerous rules and regula- tions of the traditional defense acquisition process’.64 In parallel with these strategic initiatives at DoD level, different service branches within the US military have established and expanded initiatives to increase the use of prototyping and experi- mentation. For instance, the US Army is investing, prototyping and experimenting under Army Futures Command, a new command structure founded in 2018, based in Houston, Texas. Army Futures Command hosts the annual Army Expeditionary Warrior Experiment, which is described as a ‘live prototype assessment’ with soldiers and units, and a ‘perfect laboratory to bring indus- try, army labs, academia and the acquisition community together’.65 Army Futures Command also leads Project Convergence, described as ‘the centerpiece of the Army’s campaign of learning’,66 as part of which the Army organises experiments every two weeks, culminating in an annual demonstration, to ‘take the service’s big ideas for future warfare and test them in the real world’.67 Similarly, the US Air Force established the Research Laboratory’s Strategic Development Planning and Experimentation Office in 2016 to ‘institutionalize experiments as regular practice in the Air Force’.68 Part of the Air Force Laboratory is AFWERX, a dedicated innovation unit that serves to facilitate the integration of commercial technology into the Air Force and that operates its own investment fund, AFVentures, to attract private investment and target early stage companies working on emerging technology.69 In the UK, the prototype warfare concept has also gained traction and informed a wide range of new activities and programmes. y 63Cheryl Pellerin, ‘Hagel Announces New Defense Innovation, Reform Efforts’, US Department of Defense (15 November, 2014), available at: {https://www.defense.gov/News/News-Stories/Article/Article/603658/igphoto/2001773969/} accessed 5 January 2022. y p p 62Defense Innovation Unit website, available at: {https://www.diu.mil/about} accessed 5 January 2022. 63 { p // y / J g _ } J y 66Will Reinier, ‘Campaign of Learning: US Army, AFC Introduce Project Convergence, US Army (10 September 2020), available at: {https://www.army.mil/article/238896/campaign_of_learning_u_s_army_afc_introduce_project_convergence} accessed 5 January 2022. 67 J y 64Fred Kaplan, ‘The Pentagon’s innovation experiment’, MIT Technology Review (14 January 2016), available at: {https:// www.technologyreview.com/2016/12/19/155246/the-pentagons-innovation-experiment/} accessed 24 March 2021. 61US Under Secretary of Defense, ‘Implementation Directive’, p. 12. 69Theresa Hitchens, ‘Air force to pump new tech startups with $10M awards’, Breaking Defense (25 February 2020), avail- able at: {https://breakingdefense.com/2020/02/air-force-to-pump-new-tech-startups-with-10m-awards/} accessed 24 March 2021. 68Rachel S. Cohen, ‘Experiments take root across the Air Force’, Air Force Magazine (30 January 2020 {https://www.airforcemag.com/experiments-take-root-across-the-air-force/} accessed 5 January 2022. 65Available at: {https://www.youtube.com/watch?v=tJzgc_IffM0} accessed 5 January 2022. 66 67Congressional Research Service, ‘The Army’s Project Convergence’ (27 September 2021), available at: {https://sgp.fas.org/ crs/weapons/IF11654.pdf} accessed 5 January 2022. 68R h l S C h ‘E i k h Ai F ’ Ai F M i ( J ) il bl h Army website, available at: {https://www.army.mod.uk/news-and-events/events/army-combat-power-demons ssed 5 January 2022. nder Secretary of Defense, ‘Implementation Directive’, p. 12. 61US Under Secretary of Defense, ‘Implementation Directive’, p. 12. 62Defense Innovation Unit website, available at: {https://www.diu.mil/about} accessed 5 January 2022. 63Cheryl Pellerin, ‘Hagel Announces New Defense Innovation, Reform Efforts’, US Department of Defense (15 November, 2014), available at: {https://www.defense.gov/News/News-Stories/Article/Article/603658/igphoto/2001773969/} accessed 5 January 2022. 64Fred Kaplan, ‘The Pentagon’s innovation experiment’, MIT Technology Review (14 January 2016), available at: {https:// www.technologyreview.com/2016/12/19/155246/the-pentagons-innovation-experiment/} accessed 24 March 2021. 65Available at: {https://www.youtube.com/watch?v=tJzgc_IffM0} accessed 5 January 2022. 66Will Reinier, ‘Campaign of Learning: US Army, AFC Introduce Project Convergence, US Army (10 September 2020), available at: {https://www.army.mil/article/238896/campaign_of_learning_u_s_army_afc_introduce_project_convergence} accessed 5 January 2022. 67Congressional Research Service, ‘The Army’s Project Convergence’ (27 September 2021), available at: {https://sgp.fas.org/ crs/weapons/IF11654.pdf} accessed 5 January 2022. 68Rachel S. Cohen, ‘Experiments take root across the Air Force’, Air Force Magazine (30 January 2020), available at: {https://www.airforcemag.com/experiments-take-root-across-the-air-force/} accessed 5 January 2022. 69Theresa Hitchens, ‘Air force to pump new tech startups with $10M awards’, Breaking Defense (25 February 2020), avail- able at: {https://breakingdefense.com/2020/02/air-force-to-pump-new-tech-startups-with-10m-awards/} accessed 24 March 2021. 70British Army website, available at: {https://www.army.mod.uk/news-and-events/events/army-combat-power-demonstra- tion/} accessed 5 January 2022. 71British Army website, available at: {https://www.army.mod.uk/news-and-events/events/army-combat-power-demonstra- tion/} accessed 5 January 2022. 70British Army website, available at: {https://www.army.mod.uk/news-and-events/events/army-combat-pow tion/} accessed 5 January 2022. Tracing prototype warfare The British Army embraces prototype warfare as their main new approach to warfighting, defining it as ‘a pathway to deliberately enable research, innovation, experimentation, demonstration then delivery – rapidly’ and as ‘an opportunity designed to secure competitive advantage via trial and error and adaptation’.70 Prototype warfare within the British Army is driven by the Army Rapid Innovation and Experimentation Laboratory, an innovation unit that ‘aims to bring expertise and cutting-edge technologies into direct contact with Army users’, thereby ‘challenging perceived barriers to the rapid prototyping and exploitation’.71 rmy website, available at: {https://www.army.mod.uk/news-and-events/events/army-combat-power-demonstra- d 5 January 2022. Marijn Hoijtink 332 In 2022, the Army will open its new British Army Battle Lab, which will feature as the physical manifestation of the Army Rapid Innovation and Experimentation Laboratory, or as an ‘engineer- ing workshop’ where soldiers and industry meet in a shared space to develop ideas and experiment with technology.72 Experimentation will also take place as part of the Army’s newly established Enhanced Light Force Battalion (ELFB), a unit that sits ‘at the vanguard of the Army’s approach to prototype warfare and develop capabilities to be tested on exercises and operations’.73 Stationed in Cyprus, the main purpose of the unit is to be ‘“supercharging experimentation”’ by trialling new technologies and integrating them into actual Army operations and way of fighting.74 g g g y p y g g To give a final example, in The Netherlands, the Defense Innovation Strategy, which was pub- lished by the Dutch Ministry of Defense (MoD) in 2018, strongly relies on prototyping and experimentation as a means of innovation.75 Among the range of initiatives announced by the strategy is the deployment of Concept Development & Experimentation (CD&E), described as a specific approach to innovation that is based on the implementation of small-scale experiments and shorter innovation cycles.76 In operationalising this CD&E approach, the Dutch MoD has established a variety of so-called field labs, where the military implements and tests specific ideas and technologies in collaboration with private partners, such as start-ups and (technical) universities. The Dutch Army has taken the lead in this space, creating, among other ‘labs’, the Robots and Autonomous Systems (RAS) unit and the Land Information Manoeuvre Center (LIMC), further discussed in the below. 72‘British Army “Battle Lab” to lead technology innovation for battlefield edge’, Defence Connect (27 August 2020), avail- able at: {https://www.defenceconnect.com.au/land-amphibious/6712-british-army-battle-lab-to-lead-technology-innovation- for-battlefield-edge} accessed 5 January 2022. 73British Army, ‘New Experimentation Unit to Supercharge Army Transformation’ (27 March 2021), available at: {https:// www.army.mod.uk/news-and-events/news/2021/03/experimentation-battlegroup/} accessed 5 January 2022. 74Ibid. 75Ministerie van Defensie, ‘Samen Sneller Innoveren: Innovatiestrategie Defensie’ (16 November 2018), available at: {https://www.defensie.nl/downloads/publicaties/2018/11/16/defensie-innovatie-strategie-2018} accessed 5 January 2022. 76The Dutch MoD uses the term ‘kort-cyclish innoveren’ (short-cyclical innovation) as an alternative for prototyping. In recent years, the term has become a real buzzword. 77Stew Magnuson, ‘Army’s Project convergence continues on 10-year learning curve’, National Defense (17 December 2021), available at: {https://www.nationaldefensemagazine.org/articles/2021/12/17/armys-project-convergence-continues- on-10-year-learning-curve} accessed 11 January 2022, emphasis added. 72‘British Army “Battle Lab” to lead technology innovation for battlefield edge’, Defence Connect (27 August 2020), avail- able at: {https://www.defenceconnect.com.au/land-amphibious/6712-british-army-battle-lab-to-lead-technology-innovation- for-battlefield-edge} accessed 5 January 2022. 73 www.army.mod.uk/news and events/news/2021/03/experimentation battlegroup/} accessed 5 January 2022. 74Ibid. 75Ministerie van Defensie, ‘Samen Sneller Innoveren: Innovatiestrategie Defensie’ (16 November 2018), available at: {https://www.defensie.nl/downloads/publicaties/2018/11/16/defensie-innovatie-strategie-2018} accessed 5 January 2022. 76The Dutch MoD uses the term ‘kort-cyclish innoveren’ (short-cyclical innovation) as an alternative for prototyping. In recent years, the term has become a real buzzword. 77Stew Magnuson, ‘Army’s Project convergence continues on 10-year learning curve’, National Defense (17 December 2021), available at: {https://www.nationaldefensemagazine.org/articles/2021/12/17/armys-project-convergence-continues- on-10-year-learning-curve} accessed 11 January 2022, emphasis added. my, ‘New Experimentation Unit to Supercharge Army Transformation’ (27 March 2021), available at: {https:// d.uk/news-and-events/news/2021/03/experimentation-battlegroup/} accessed 5 January 2022. Battlefield learning: The case of the Land Information Manoeuvre Centre This section examines the extension of prototype warfare into the (simulated) battlespace, focusing on how the performed need to constantly learn from the deployment of new technology and opti- misation of military operations enables and justifies ongoing experimentation on the actual battle- field. My arguments are embedded in a close analysis of recent innovation efforts within the Dutch military, which has been outspoken in its ambition to lead information- and innovation-led warfare and develop niche capabilities in technological innovation as an alternative way to protect national interests and exercise international influence. Specifically, I focus on the case of the Land Information Manoeuvre Centre (LIMC), ‘an experimental data center’, established by the Dutch Army in March 2020 for the purpose of engaging in open-source data collection and analysis, based on machine learning technology, to help crisis response and fight disinformation within the context of the COVID-19 pandemic. The work of LIMC had a particularly wide scope, exploring ‘all available and relevant information, from open and semi-closed sources’ to map networks, sentiments and behavioural dynamics on ‘everything COVID-19-related that is also relevant for the military’.79 This included data on anti-vaccination movements in The Netherlands, but also on protest movements, such as QAnon, 5G, and even the Dutch Black Lives Matter movement. The Dutch public first learned about LIMC in November 2020, when national newspaper NRC Handelsblad published an article about the centre, which was titled ‘How the Dutch Army Keeps an Eye on Its Own Population’.80 Following the initial NRC Handelsblad article, it soon became clear that the Army did not have a legal basis for this kind of data collection on Dutch citizens, and that many within the military itself, including then-Defense Minister Ank Bijleveld, had been unaware of the (full scope) of the centre’s activities. This, combined with criticism from the Dutch Parliament, impelled Bijleveld to temporarily pause all LIMC activities and to order an independent investigation into LIMC’s methods and legal basis. Tracing prototype warfare Both have embraced prototyping and experimen- tation to gain ‘real-world’ experience with new digital and autonomous technologies and demon- strate their value to the Dutch armed forces, seizing opportunities and taking action outside of formal frameworks of military acquisition and often without formal permission. In summary, while not a new concept, prototype warfare has in recent years been gaining trac- tion across Western military discourse and practice. Driven by a highly speculative rhetoric around the potential of AI technology and the problematisation of existing military innovation and acquisition strategies in the context of a performative global technological competition, prototype warfare operates as the strategic concept under which the experimental way of warfare is taking shape, assembling a range of new and experimental practices, technologies, and inter- actions. Prototype warfare is also the concept under which experimental practice and experimen- tation with new technologies is taken more explicitly into the (simulated) battlespace, or near the edge of battle. As a US Army Colonel puts it, ‘the idea is the sooner we can bring things out here, get them in the dirt, get them in soldiers hands, the sooner we can learn’.77 Again, while the mili- tary has a much longer history of engaging in experimental learning in actual operations or as part of simulated exercises, gaming and scenario planning, what is different in today’s context is how experimentation becomes very much the end goal. In prototype warfare, the aim is to experiment on a constant basis, with the purpose of constant learning, optimising, and innovat- ing. In this context, experimentation is supposed to happen throughout, in peacetime training and in actual combat, as part of a continuous cycle of learning and optimisation, and uninterrupted European Journal of International Security 333 by failure.78 By extension, I argue that this means that military intervention becomes itself recon- figured – and justified – as a learning experience and opportunity for further experimentation. Politically, much is at stake here: if warfighting becomes in and of itself experimental and failure instructive, how can we then resist military intervention in the experimental way of warfare? 78Carleton-Smith, ‘Foreword’. 79Berkhout, Rosenberg, and Petraeus, ‘Hoe Defensie’. 80Ibid. 81These documents are available via this website: {https://www.tweedekamer.nl/kamerstukken/brieven_regering/detail? id=2021Z18543&did=2021D39896} accessed 14 January 2022. 82Cited in Berkhout, Rosenberg, and Petraeus, ‘Hoe Defensie’. 80Ibid. 81These documents are available via this website: {https://www.tweedekamer.nl/kamerstukken/brieven_regering/detail? id=2021Z18543&did=2021D39896} accessed 14 January 2022. 82Cited in Berkhout, Rosenberg, and Petraeus, ‘Hoe Defensie’. 78Carleton-Smith, ‘Foreword’. 79Berkhout, Rosenberg, and Petraeus, ‘Hoe Defens 80 87Hans van Dalen, ‘On Mind War: Manoevreren op het Internetslagveld’, Militaire Spectator, 189:9 (2020), pp. 405–17, available at: {https://www.militairespectator.nl/thema/operaties/artikel/mind-war} accessed 18 January 2022; Paul Ducheine, Corstiaan de Haan, and Norbert Moerkens, ‘Informatiemanoeuvre en Nieuwe Traditieverbanden in de Koninklijke Landmacht’, Militaire Spectator, 190:5 (2021), pp. 259–67, available at: {https://www.kvbk.nl/thema/geschiede- nis-operaties/artikel/informatiemanoeuvre-en-nieuwe-traditieverbanden-de-koninklijke} accessed 18 January 2022. Battlefield learning: The case of the Land Information Manoeuvre Centre After completion of the investi- gation in late 2021 and a subsequent Freedom of Information request on behalf of NRC Handelsblad, a collection of internal Ministry of Defense communications and documents was released, which reveal a lengthy discussion among military and policymaker units on the practices and legality of open-source data collection and analysis by LIMC.81 These documents also pro- vide us further insights into how the COVID-19 pandemic and crisis context was seized by those responsible within the Army as an opportunity to gain experience with open-source intelligence methods through real-world experimentation and the collection of real-world data. g p Indeed, this broader objective of LIMC to experiment with new digital technologies and learn about them in a real-world setting was clearly articulated by the responsible Lieutenant Colonel, who instructed his subordinates on the first day of the new centre by saying: ‘Prepare for experi- ments. We are reinventing ourselves.’82 It was also formulated by the Commander of the Armed Forces, who authorised the establishment of LIMC on 19 March 2021 in his day order by giving LIMC as a formal assignment ‘to provide the Armed Forces and other public authorities with new Marijn Hoijtink 334 insights in an experimental form in the fight against COVID-19’.83 More specifically, in his day order the Commander of the Armed Forces stated that LIMC was supposed to serve two main aims: insights in an experimental form in the fight against COVID-19’.83 More specifically, in his day order the Commander of the Armed Forces stated that LIMC was supposed to serve two main aims: To evaluate all available and relevant information, from of open and semi-closed sources, about the Covid crisis, to generate situational awareness and understanding … Hereby, mili- tary and civil decision making processes will be informed by insights and where possible an action perspective.84 p 84Commando Landstrijdkrachten (CLAS), Fragmentary Order 003 Day Order 2020-710 (COVID-19) (19 March 2020), available at: {https://www.tweedekamer.nl/kamerstukken/brieven_regering/detail?id=2021Z18543&did=2021D39896} accessed 19 January 2022. 85Ibid 83Cited in ibid., emphasis added. 86Royal Dutch Army, ‘Informatie als Wapen’, Studie Delphi (2017). 83Cited in ibid., emphasis added. 84Commando Landstrijdkrachten (CLAS), Fragmentary Order 003 Day Order 2020-710 (COVID-19) (19 March 2020), available at: {https://www.tweedekamer.nl/kamerstukken/brieven_regering/detail?id=2021Z18543&did=2021D39896} accessed 19 January 2022. 85Ibid. 86Royal Dutch Army, ‘Informatie als Wapen’, Studie Delphi (2017). 87Hans van Dalen, ‘On Mind War: Manoevreren op het Internetslagveld’, Militaire Spectator, 189:9 (2020), pp. 405–17, available at: {https://www.militairespectator.nl/thema/operaties/artikel/mind-war} accessed 18 January 2022; Paul Ducheine, Corstiaan de Haan, and Norbert Moerkens, ‘Informatiemanoeuvre en Nieuwe Traditieverbanden in de Koninklijke Landmacht’, Militaire Spectator, 190:5 (2021), pp. 259–67, available at: {https://www.kvbk.nl/thema/geschiede- nis-operaties/artikel/informatiemanoeuvre-en-nieuwe-traditieverbanden-de-koninklijke} accessed 18 January 2022. And: [T]o improve military operations with the aid of new technologies and better use of infor- mation. This also means that new threats can be countered. With the LIMC, the Royal Netherlands Army can gain experience in bringing together information maneuver capabil- ities that offer insight, foresight and continuous action perspective in three dimensions (cog- nitive, virtual, physical).85 From this perspective, LIMC is part a wider ambition on the part of the Dutch military, and the Army specifically, to develop new capabilities in what it calls information-driven warfare, as an alternative to protect national interests and exercise international influence as part of coalition fighting. This ambition can be traced back to a report published by the Army in 2016 on ‘Information as a Weapon, Means And End’, which articulated the need to invest in ‘in-house’ intelligence capabilities.86 Frustrated with the workings of the Military Intelligence and Security Service (MIVD), which the Army saw as being too much focused on strategic intelligence to support Army commanders in making tactical or operational decisions, the Army approached their intelligence branch, the Joint Intelligence, Surveillance, Target Acquisition & Reconaissance Commando (JISTARC), to meet this need. Seeking to leverage the potential of ‘information as a weapon’, JISTARC established unit 109 in 2019, with the aim to engage in open-source data collection and analysis and experiment with ‘information maneuvering’.87 However, even before the new unit was officially launched, it was already caught up in a legal debate about the use of open-source data as training data in these experiments. Unlike the MIVD, the Army has no legal basis to conduct open-source data collection and analysis, which means that JISTARC 109 is constrained by the General Data Protection Regulation (GDPR). This severely hampered the Army’s ambition to collect with real-world data and experiment with it in a live-setting, even if JISTARC continued to look for a legal mandate to carry out those tasks. g y Then came the COVID-19 crisis, which allowed the Army to move ahead with open-source intelligence, founding LIMC only a few days after The Netherlands went into a first lockdown. Although LIMC was established as a new and separate unit, it was almost entirely staffed by for- mer JISTARC 109 members and initially based in the same location as JISTARC. Soon after its establishment, LIMC started reaching out to the Dutch COVID response task force offering its European Journal of International Security 335 services. 88Berkhout, Rosenberg, and Petraeus, ‘Hoe Defensie’. 89This speaks to existing work that has studied how technologies are being developed, used, and tested in ‘laboratories’ in/ of the Global South before they move back to the Global North, but in this context, interestingly, that relationship is being reversed. Still, the Dutch Army has been forthcoming about its ambition to experiment with new technology in conflict set- tings (for example, as part of Operation Flintlock in the Sahel region), not in the least because this provides the Army with more legal room. For a discussion on the concept of the ‘laboratory’ within critical security studies, see, for example, Claudia Aradau, Marijn Hoijtink, and Matthias Leese, ‘Technology, agency, critique: An interview with Claudia Aradau’, in Hoijtink and Leese (eds), Technology and Agency in International Relations, pp. 188–203; Mike Bourne, Heather Johnson, and Debbie Lisle, ‘Laboratizing the border: The production, translation and anticipation of security technologies’, Security Dialogue, 46:4 (2015), pp. 307–25. And: As part of the COVID response task force, LIMC began to collect data from a range of sources, including information from military liaisons in hospitals and nursing homes, to predict logistical challenges and needs, such as material shortages and ICU occupancy rates. At the same time, LIMC started tracking public behaviour, sentiments, and networks based on open-source and semi-closed data on anti-vaccination and other protest movements. These data were pro- cessed, analysed, and visualised after which they were being disseminated to other organisations such as the national police and the National Coordinator for Counter Terrorism and Security (NCTV). LIMC continued doing so without a legal basis, until the publication by NRC Handelsblad revealed its existence in November 2020. Overall, the case of LIMC reveals how much the imperative to experiment in ‘real-life’ settings shapes the Dutch Army’s activities, with the COVID-19 pandemic merely serving as the ‘perfect crisis’ to move ahead with training and experimentation in the field of open-source intelligence gathering and analysis. ‘Successful’ experimentation with these technologies in the context of the COVID-19 crisis, the Army believed, would serve as a proof of concept and demonstrate the value of open-source intelligence and the importance of the Army possessing these capabilities within the domain of information-driven warfare, while lending credibility to the Army as an important player in this field. But the COVID-19 crisis also provided LIMC staff more directly with an opportunity to gain ‘actual’ experience and practice with open-source intelligence meth- ods, using real data. This option was not available to JISTARC 109 prior to the COVID-19 crisis, and legally speaking it was not available to LIMC even during the crisis. However, the urgency of the pandemic and the performed need to act fast, take risks, and learn-by-doing allowed for this kind of experimentation and experimental data collection. The emphasis on LIMC’s experimental character eventually also became a way to ward off criticism. When the publication by NRC Handelsblad raised a series of questions in Parliament, Bijleveld responded by saying that the centre was merely ‘experimental in character’, and, therefore, that a legal basis for these kinds of activities was apparently irrelevant.88 Bijleveld also stressed that she remained committed to continue the work of LIMC to advance the incorp- oration of new technologies into the Dutch military and to experiment with them in simulated and ‘in the field’ tests. And: Indeed, part of the reason why the case of LIMC is particularly revealing is that Bijleveld admitted that the Army used the pandemic as a pretext to collect and model data of Dutch citizens, because it could not experiment with these technologies and learn about them in actual combat at the time.89 The suggestion, however, that this would have been the preferred option raises important questions about how the experimental way of warfare is or will be driving and justifying military intervention, and with what effects for those on the receiving end of the technologies deployed. These questions become all the more important in the light of the chal- lenge of critiquing the experimental way of warfare as I will argue in the below conclusions. Conclusions This article has examined how Western militaries are increasingly experimenting with new digital and autonomous technologies, situating these developments against the background of an emer- ging and distinct regime of warfare, called ‘prototype warfare’. As I have argued, prototype Marijn Hoijtink 336 warfare is not simply a new doctrinal concept or reflective of how much conceptual and language innovation the military is producing, but rather denotes a wider and more structural transform- ation of how warfare is shaped and made possible. Specifically, I have argued that the concept of prototype warfare represents an experimental way of warfare, which is driven by an overarching experimental rationality and governed by-experiment, or constant experimentation and optimisa- tion. It operates on the basis of a highly speculative understanding of the experiments being deployed, and, as such, it explicitly incorporates failure as part of an ongoing process of learning or ‘moving forward’. g Understood as such, prototypes such as Project Maven or LIMC do not need to produce any new knowledge or work successfully to create effects. To the contrary, any demonstrated limita- tion or vulnerability of these kinds of projects and the technologies they deploy are grounds for further investment and experimentation. If an experiment fails, or the outcomes are uncertain, this only means that more experimentation or a better kind of experiment is needed. Again, as Murphy writes in her analysis of experimentality, ‘[n]o matter the result of the experiment, no matter if results are achieved as intended or if no change is measured (as often happened), what is reproduced is the infrastructure of the experiment itself.’90 It is this speculative vision of the experiment that makes the experimental way of warfare described here largely immune to criticism linked to effectiveness. But even if we want to raise critique based on other grounds, such as on legal grounds as hap- pened in the example of the Dutch experimental data centre, or on ethical grounds such as in the case of Project Maven, such criticisms are often fenced off on grounds that these projects are ‘mere’ experiments. However, more than ‘mere’ experiments, the objective of this article has been to show that such projects are structural and part of a broader regime of experimentation that deserves our critical attention. 90Murphy, The Economization of Life, p. 91. Cite this article: Hoijtink, M. 2022. ‘Prototype warfare’: Innovation, optimisation, and the experimental way of warfare. European Journal of International Security 7, 322–336. https://doi.org/10.1017/eis.2022.12 Acknowledgements. The author would like to thank Lauren Gould, Ingvild Bode, Marieke de Goede, Anneroos Planqué-van Hardeveld, and two anonymous reviewers for their helpful comments on a previous version of this article. She would also like to thank Jack Davies, Martine Jaarsma, Aoife Keogh, Lauren Gould, and Jolle Demmers for our collaborative work on the concept of prototype warfare and the Dutch LIMC case. This work was supported by the Dutch Research Council (NWO), Grant No. 16.Veni.195.3816845. Conclusions Specifically, we should pay attention to how, as part of the experimental way of warfare, military intervention and violence become reconfigured and nor- malised as an opportunity for experimentation and learning, and with what effects for those who are who are commonly on the receiving end of the technologies with which our militaries are experimenting. This means highlighting how the risks and failures that are a structural con- dition of the experimental way of warfare are being outsourced to others, ‘over there’, and shed- ding light on the costs and aftermath of the experiments that are being deployed in the name of mere optimisation. Acknowledgements. The author would like to thank Lauren Gould, Ingvild Bode, Marieke de Goede, Anneroos Planqué-van Hardeveld, and two anonymous reviewers for their helpful comments on a previous version of this article. She would also like to thank Jack Davies, Martine Jaarsma, Aoife Keogh, Lauren Gould, and Jolle Demmers for our collaborative work on the concept of prototype warfare and the Dutch LIMC case. This work was supported by the Dutch Research Council (NWO), Grant No. 16.Veni.195.3816845. Disclosure statement. No potential conflict of interest was reported by the author. Marijn Hoijtink is Assistant Professor in International Relations at Vrije Universiteit Amsterdam. Her research interests include emerging security technologies, their circulation and relation to risk, militarism, and weapons research. She is the editor of Technology and Agency in International Relations (with Matthias Leese), published by Routledge in 2019. Her cur- rent research project, funded by the Dutch Research Council (NWO), focuses on the politics of AI and examines how novel AI applications shape and transform current military practices and forms of decision-making.
https://openalex.org/W4393388002	https://www.cambridge.org/core/services/aop-cambridge-core/content/view/1E9151CA630EC7B73C1537787FB10D09/S0272263124000214a.pdf/div-class-title-a-comparison-of-lab-and-web-based-elicited-imitation-insights-from-explicit-implicit-l2-grammar-knowledge-and-l2-proficiency-div.pdf	English	null	A comparison of Lab- and Web-based elicited imitation: Insights from explicit-implicit L2 grammar knowledge and L2 proficiency	Studies in second language acquisition	2,024	cc-by	11,842	Abstract Elicited imitation (EI) tasks are a practical tool for measuring second language (L2) knowledge and skills. In this study, we implemented a web-based EI task that measures English morphosyntactic knowledge and compared its measurement properties to a tradi- tional laboratory-based EI. A cohort of 149 L2 English learners engaged in the web-based EI task, and 151 participants completed a traditional lab-based EI counterpart. Correlation analyses revealed a significant, comparable relationship between English proficiency and the two EI versions, with the ungrammatical items showing less consistency that neither improved nor harmed the overall EI effectiveness. Factor analyses corroborated the validity of web-programmed EI, with both EI versions relating similarly to time-pressured, implicit knowledge and untimed, explicit knowledge measures. Our results suggest the potential for utilizing web-based EI to substitute lab-based tasks, enabling larger-scale, more diverse sampling. We end with implications for future web-based EI task users and include a coding guideline for customized web-based EI use. Studies in Second Language Acquisition (2024), 1–22 doi:10.1017/S0272263124000214 Studies in Second Language Acquisition (2024), 1–22 doi:10.1017/S0272263124000214 A comparison of Lab- and Web-based elicited imitation: Insights from explicit-implicit L2 grammar knowledge and L2 proficiency A comparison of Lab- and Web-based elicited imitation: Insights from explicit-implicit L2 grammar knowledge and L2 proficiency 1Boston University; 2Western New England University; 3University of Hawaiʻi at Maˉnoa; 4University of Tübingen g Corresponding author: Xiaobin Chen; Email: xiaobin.chen@uni-tuebingen.de (Received 07 July 2023; Revised 01 February 2024; Accepted 14 February 2024) Kathy Minhye Kim et al. Kathy Minhye Kim et al. Kathy Minhye Kim et al. 2 The goal of this study is to establish the validity of a low-fee, web-programmed EI that measures English morphosyntactic knowledge. To this end, we examined the comparability, in terms of construct validity, of an EI task delivered in a traditional in-person laboratory setting against an equivalent EI task administered on the web. Drawing on theoretical models and empirical work in L2 psycholinguistics and assessment, we formed two hypotheses. From a view of psycholinguistics, L2 grammar knowledge is composed of at least two types of knowledge: explicit (conscious) and implicit (unconscious). While the exact nature of the EI construct has long been debated (see Godfroid & Kim, 2021; Suzuki et al., 2023), the meaning-focused and time-pressured element of EI requires largely automatic processing of sentences, relying on implicit knowledge (R. Ellis, 2005; Godfroid & Kim, 2021) or more autom- atized version of explicit knowledge (Suzuki & DeKeyser, 2015; Suzuki et al., 2023). If the web-based (WB) EI were to be as valid as the lab-based (LB) version, we expect to obtain a similar factor structure and loadings for EI scores when situated among several other measures of implicit and explicit morphosyntactic knowledge. y g Second, L2 morphosyntactic knowledge, which is measured through the EI task in the current study, is a central component of L2 proficiency (Bachman & Palmer, 1996; Canale & Swain, 1980; Hulstijn, 2015); as such, we expected a significant and compa- rable relationship between both EIs and L2 proficiency. In reference to Hulstijn’s language proficiency model (2011, 2015), EI may pertain mostly to basic language cognition (BLC) while standardized proficiency tests, which were used as a measure of L2 proficiency in this paper, may extend beyond BLC to higher language cognition (HLC). The EI test, for instance, typically uses words and structures that frequently appear in communicative situations and is largely free of differences among L2 speakers in cognition factors such as phonological short-term memory capacity (Kim et al., 2016; Park et al., 2020). It is therefore common for native speakers of the target language to achieve near-perfect scores in EI, reflective of their proficiency in these linguistic elements. Introduction Elicited imitation (EI) has a long, productive history in second language acquisition (SLA) as a means of assessing morphosyntactic knowledge (Erlam, 2006; Godfroid & Kim, 2021; Suzuki et al., 2023) and second language (L2) global proficiency (Kostromitina & Plonsky, 2022; Yan et al., 2016). The primary objective of EI is to measure one’s ability to reconstruct spoken language. The reconstruction process involves the use of working memory, comprehension, and knowledge of syntax, collectively reflecting individuals’ developing L2, known as their interlanguage. This interlanguage system is what researchers aim to measure through EI, serving as a proxy for implicit (or automatized explicit) linguistic knowledge in psycholinguistics and global L2 proficiency in the field of language assessment. © The Author(s), 2024. Published by Cambridge University Press. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/4.0), which permits unrestricted re-use, distribution and reproduction, provided the original article is properly cited. https://doi.org/10.1017/S0272263124000214 Published online by Cambridge University Press https://doi.org/10.1017/S0272263124000214 Published online by Cambridge University Press Kathy Minhye Kim et al. Standardized proficiency tests, on the other hand, require the use of HLC that contains more complex words and structures that need not be spoken, pertain to topics other than everyday matters (e.g., academic topics), and is subject to individual differences in education, literacy, and other cognitive abilities (Hulstijn, 2011, pp. 230– 231). Importantly, because BLC is situated as a core component within HLC proficiency and is entirely encompassed by it, we expect a good amount of shared variance between standardized L2 proficiency test scores and EI. y Heeding calls to probe the validity of measures more rigorously in SLA research (Chapelle, 2021; Révész & Brunfaut, 2021; Sudina, 2021), we address this issue in the context of a web-programmed spoken production task. While WB spoken data collection brings many benefits, it does raise questions about its comparability to LB studies. Among these are construct-irrelevant factors, such as environmental distrac- tions or suboptimal and/or varying audio playback conditions, which may threaten construct validity. Also, even when LB and WB versions of an instrument tap into the same construct, how similarly the construct is measured might still vary, influencing the interpretation of scores and study outcomes. Acknowledging that establishing strict psychometric equivalence (or concordance; see Zumbo, 2021) across different forms or administration conditions of a test is a difficult endeavor generally requiring carefully planned studies with large samples, our goal in the current study is to preliminarily compare the construct measurement of WB and LB EI scores by using existing data to examine (1) relationships with conceptually related constructs and (2) a relationship with a criterion variable (American Educa- tional Research Association et al., 2014). For trait-type constructs, such as implicit A comparison of lab- and web-based elicited imitation 3 morphosyntactic knowledge, a critical juncture in evaluating the validity of a test is what Chapelle (2021) describes as an explanation inference. This inference has to do with whether test scores are reflective of a well-defined target construct. Evidence to support such an inference can come from examining a nomological network of related concepts, among other sources. In our case, there is theoretical support to suggest that EI scores reflect implicit morphosyntactic knowledge that is distinguishable from explicit knowledge. Theory also suggests that (implicit) morphosyntactic knowledge ought to have a positive correlation of at least moderate strength with language proficiency (Hulstijn, 2011). Kathy Minhye Kim et al. This relationship should hold for subparts of an EI test composed of grammatical and ungrammatical items (see section, Elicited Imitation in SLA Research), although the relationship may be relatively stronger for grammatical items (Kostromitina & Plonsky, 2022; Yan et al., 2016). Importantly, we wish to know whether these hypothesized relationships bear out in a similar fashion across WB and LB EI tests. Positive evidence of this would suggest that the scores from each version similarly indicate standing on the theoretical construct being measured, namely, implicit morphosyntactic knowledge. p p y g As such, to investigate the comparability of the LB and WB EI tasks, we examined evidence (1) regarding relationships with conceptually related constructs and (2) of a relationship with a relevant criterion variable. In particular, we empirically tested the extent to which an EI administered in one’s home yielded relationships to measures of implicit (i.e., oral production [OP], timed grammaticality judgement test [TGJT]) and explicit (i.e., untimed grammaticality judgement test [UGJT] and metalinguistic knowledge test [MKT])] grammar knowledge and L2 proficiency (i.e., standardized language test) similar to those of an EI administered in a lab setting. https://doi.org/10.1017/S0272263124000214 Published online by Cambridge University Press Elicited imitation in SLA research However, delving further into the literature reveals skepticism of the inconsistency in error correction, especially in the absence of explicit correction instructions, poten- tially undermining the effectiveness of ungrammatical sentences in EI tasks. In response to these concerns, Yan et al. (2016) conducted a meta-analysis of studies that included ungrammatical items (k = 9), and found that the grammaticality of EI stimuli had little impact on measurement: Both measures, with and without ungrammatical stimuli, were similarly sensitive measures of morphosyntactic knowledge thought to be implicit in nature. However, there was a trend of ungrammatical items being less discriminating across proficiency levels than grammatical items (g = 1.16 vs. g = 1.34), but these differences were comparable (i.e., Q[1] = 2.87, p = 0.09), suggesting that the inclusion of ungrammatical items neither improved nor harmed the sensitivity of EI. Kostromitina and Plonsky (2022) echoed the concerns about the validity of ungrammatical items and expanded Yan et al.’s meta-analysis by including 4 additional studies (k = 15). They found ungrammatical items to be less discriminating than grammatical ones: mean effect sizes of studies that included and excluded ungram- matical items fall outside their respective confidence intervals (CIs) (r = .56, CI [0.46, 0.65] vs. r = .71, CI [0.66, 0.76]). The authors concluded that the repetition of ungrammatical sentences did not aid in EI’s capacity to assess learner proficiency, particularly when clear correction instructions are not provided. Their research adds another layer to the debate, highlighting the practical challenges in the administration of EI tasks with ungrammatical items. In the realm of psycholinguistics, a series of factor analytic studies involving multiple measures of L2 morphosyntactic knowledge (R. Ellis, 2005; R. Ellis & Loewen, 2007; Godfroid & Kim, 2021; Kim & Nam, 2017; Spada et al., 2015), including measures designed to target implicit and those designed to target explicit knowledge, has yielded two key findings. First, EI-based measures of morphosyntax correlate more strongly with other measures of morphosyntax thought to tap into implicit knowledge (e.g., timed grammaticality judgments and oral narratives) compared to measures that clearly access explicit knowledge (e.g., metalinguistic knowledge tests). Elicited imitation in SLA research Among several means of measuring L2 implicit knowledge, EI has a long, productive history in SLA (R. Ellis, 2005; Erlam, 2006; Godfroid & Kim, 2021; Isbell & Rogers, 2020). As described by Vinther (2002), R. Ellis (2005), Erlam (2006), and others, EI tasks are characterized by the following elements: • Stimuli in the form of spoken sentences, designed to vary in overall length or complexity, or to target specific (morpho)syntactic features • Stimuli in the form of spoken sentences, designed to vary in overall length or complexity, or to target specific (morpho)syntactic features p y g p ( p ) y • A pause between stimuli playback and subject response • Scoring based on the accuracy of the repeated stimuli • Scoring based on the accuracy of the repeated stimuli The EI task format has been adapted to measure global L2 oral proficiency (e.g., Ortega et al., 2002) and, in line with the focus of the current study, L2 (morpho)syntactic knowledge. Because of its fleeting audio stimuli and time constraints on production that require largely automatic processing of language with minimal opportunity to apply metalinguistic knowledge, EI tasks meet R. Ellis’ (2005) criteria for operationalizing implicit knowledge. p g A common design feature of EI tasks intended to measure morphosyntactic knowl- edge more narrowly is the introduction of ungrammatical stimuli, which are expected to be corrected in a test taker’s repetition as (further) evidence of automatic—and even subconscious—application of target L2 grammatical representations (Erlam, 2006; Erlam & Akakura, 2015). Markman et al. (1975) provided early insights by emphasizing the difference between simple memory-based replication and the application of an “internalized grammar factor” (p. 37). They claim that learners often adjust linguistic Kathy Minhye Kim et al. 4 structures in ways that align with their inherent understanding of a language’s gram- mar. Corrected imitation of ungrammatical sentences, then, becomes evidence of its assimilation into their internal grammar or interlanguage system, suggesting that learners are actively processing and understanding the content, rather than merely replicating it. Therefore, when a learner, armed with an accurate internal grammar system of the target language, confronts these items, their capacity to recognize and rectify them becomes a potent indicator of their deep-seated understanding of the language. https://doi.org/10.1017/S0272263124000214 Published online by Cambridge University Press Elicited imitation in SLA research Second, EI measures load primarily on factors interpreted as reflecting implicit knowledge, though some researchers motivated by skill acquisition theory have argued that EI may be tapping into a third construct referred to automatized explicit knowledge (AEK) rather than truly implicit knowledge (Suzuki, 2017; see also Suzuki & DeKeyser, 2015; Suzuki et al., 2023). Even so, the rapid and efficient access associated with AEK is thought to be largely indistinguishable from implicit knowledge when it comes to successful L2 communication. Although research has generally supported EI as a measure of implicit L2 knowl- edge, until this point, most EI tasks have been administered in controlled lab settings, either on paper or by computer (see Erlam & Akakura, 2015), in part due to the LB research tradition in the field of SLA and the complexity of setting up custom environments for running EI tests. Technically, such an environment requires the development of a web application that is, for instance, capable of user authentication, A comparison of lab- and web-based elicited imitation 5 audio playback, and recording file storage/transmission between the user interface and the backend server as well as interactive interfaces for test takers and administrators. Recent advances in online experiment platforms and Internet communications tech- nology more generally have ameliorated these difficulties, making precisely timed and faithfully recorded EI tasks possible online. Table 1. Benefits and costs of web experimentation Benefits • Fewer logistical concerns (no physical presence needed) • Wider and larger participant access • Fewer motivational confounds (no extra-credit incentives) • Reduction in research, equipment, and employment costs • Reduction of experimenter effects (“clues” that influence participants’ choices) • Ease of replicability (standardized and optimized testing procedures) • Increased ecological validity (experimental setting closer to a natural setting) Web- and lab-based experimentation Benefits and costs of web experimentation Benefits • Fewer logistical concerns (no physical presence needed) • Wider and larger participant access • Fewer motivational confounds (no extra-credit incentives) • Reduction in research, equipment, and employment costs • Reduction of experimenter effects (“clues” that influence participants’ choices) • Ease of replicability (standardized and optimized testing procedures) • Increased ecological validity (experimental setting closer to a natural setting) • Higher dropout rates Offer rewards, such as immediate feedback or monetary incentives • Limited control over experimental access, such as multiple submissions Restrict IP addresses Collect personal identification • Limited interaction with staff Add contact information Provide test tutorials in advance • Higher risk of distractions Include progress markers Include screening tasks • Noisy data Increase sample size • Higher dropout rates Offer rewards, such as immediate feedback or monetary incentives • Limited control over experimental access, such as multiple submissions Restrict IP addresses • Limited control over experimental access, such as multiple submissions Restrict IP addresses • Limited interaction with staff Add contact information Provide test tutorials in advance • Higher risk of distractions Include progress markers Include progress markers Include screening tasks • Noisy data Increase sample size Costs and possible solutions Web- and lab-based experimentation WB experimentation was first introduced in the field of psychology on auditory perception (Welch & Krantz, 1996) and has been used with increasing frequency across disciplines (Birnbaum, 2000). The transition from traditional lab settings to online experimentation has offered researchers practical advantages (Table 1). For one, lifting restrictions on time and location allows data to be drawn from larger and more diverse sample pools, which may circumvent restrictions of traditional experiments, such as low statistical power and convenience sampling. WB experimentation is also more cost-efficient. Without the need for dedicated lab space and supervising and hiring staff, there is a significant reduction in cost. The flexibility and efficiency of online data collection, however, have raised questions about the validity of web-solicited data. Some claim that web-collected data are inher- ently different from LB data (Bauer et al., 2012), because a WB format may subvert the purpose of an experiment. It could also limit research approaches due to the differences in processing and response speed (Parsons et al., 2018) and environmental distractors that may add noise to the data (Gagné & Franzen, 2023). Despite some differences worthy of concern, accumulating findings suggest WB tasks are reliable alternatives to LB instruments. Comparable performances have been reported between lab- and web-delivered cognitive measures, such as working memory capacity and Table 1. https://doi.org/10.1017/S0272263124000214 Published online by Cambridge University Press https://doi.org/10.1017/S0272263124000214 Published online by Cambridge University Press Kathy Minhye Kim et al. 6 declarative memory (Ruiz et al., 2019), cognitive functioning (Backx et al., 2020; Houben & Wiers, 2008), and psycholinguistics measures, such as lexical decision task (Hilbig, 2016). declarative memory (Ruiz et al., 2019), cognitive functioning (Backx et al., 2020; Houben & Wiers, 2008), and psycholinguistics measures, such as lexical decision task (Hilbig, 2016). In response to technological advances coupled with COVID-19 and the replication crisis (Klein et al., 2014; McManus, 2021), online experimentation has only recently bloomed in L2 research. From recruitment to implementation, various topics of research have been carried out online. In L2 speech perception, for instance, researchers inves- tigated the effectiveness of high variability phonetic training in which both the perception and production data were collected online (Saito et al., 2022). In some studies, researchers crowdsourced listeners through Amazon Mechanical Turk (AMT) and sampled their transcription and speech ratings remotely (Loukina et al., 2015; Nagle, 2019; Nagle & Huensch, 2020). In psycholinguistics, topics such as bilinguals’ agreement processing (Lee & Phillips, 2022), pragmatic inferencing by autistic learners (Van Teil & Kissine, 2018), and implicit L2 learning (Kerz et al., 2017), were investigated on the web, enabling the collection of both accuracy and reaction time data. For implementation, many studies haveused a rangeofcommercial services, such asPsychoPy, Prolific, Gorilla,LimeSurvey, and Unity. Alternatively, some programmed tasks created customized systems for web delivery by utilizing technologies such as HTML, CSS, JavaScript, SQL, and server-side programming. For crowdsourcing purposes, AMT, Prolific, and Appen (known as CrowdFlower until 2019), served to promote remote recruitment. ) p Despite the increased transition to WB research, few studies have directly explored the validity of web-solicited data. Hilbig (2016) investigated the reaction time effects of the lexical decision task by software (E-Prime vs. JavaScript) and environment (webvs. lab). To dissect such effects, participants were assigned to one of three conditions: 1) the lab using commercial software, E-Prime; 2) the lab using the JavaScript version; or 3) the web using the same JavaScript version. All conditions yielded the desired word frequency effects, demonstrating the comparability of effects across different conditions and environments. Likewise, results from Ruiz and colleagues (2019) suggested that the web version of a paired associates subtest of the Modern Language Aptitude Test yielded a strong correlation to its LB counterpart (r = .82), affirming its comparability to the web-version. https://doi.org/10.1017/S0272263124000214 Published online by Cambridge University Press Lastly, in SLA, NagleandRehman(2021)aimedtodevelopareliableonlinemethodforrecruitinglisteners to rate L2 speech samples for previous research (Nagle, 2019). In their approach, they included both traditional local labs and the digital domain of AMT. Within AMT, participants were either exposed to the specific target dialect or a broader range of dialects. Withqualitycontrolsincluded(i.e.,prescreeningandtasktimers), thestudyconfirmedthat three groups of raters (local lab and two AMT groups) had similar speech ratings, suggesting the high comparability between WB and LB participant recruitment with proper quality validation. Collectively, these results bring prospects for administering language research, including reaction time– and accuracy-based, cognitive, and speech- rating tasks on the web. g To our knowledge, no studies in SLA have explored the validity of web-delivered speech production tasks on the web. With the increased possibility of collecting spoken responses remotely using custom web platforms and services, empirical validation is needed to ensure accurate and reliable use of the web version of EI. Methods This study drew on data from Kim’s dissertation (n = 149; Kim, 2020; Kim & Godfroid, 2023) and Godfroid et al.’s project (n = 151; Godfroid & Kim, 2021; Godfroid et al., 2018). To perform the comparisons of interest, we used datasets of a standardized English proficiency measure and five linguistic measures: EI, OP, UGJT, TGJT, and MKT. p y g The same instruments of identical items were used in both Kim (2020) and Godfroid et al. (2018), with the exception of the delivery mode of EI and OP. In Kim (2020), the two oral tasks (i.e., EI and OP) were delivered on the web, and other measures (i.e., UGJT, TGJT, and MKT) in the lab, while all data in Godfroid et al. (2018) were collected from measures administered in a lab setting (see section, Web-based Envi- ronment, for details). Participants In both Kim’s WB study and Godfroid et al.’s LB study, the participants were English L2 speakers attending a large university in the American Midwest. Kim’s study recruited 149 participants, and Godfroid et al. initially recruited 160. Nine participants in Godfroid et al., who missed all five linguistic measures, were excluded from the analysis, resulting in a final sample of 151. For speaker L1, defined here as self-reported dominant language, the most common was reported as Chinese in both Kim (n = 53) and Godfroid et al. (n = 74), followed by any variety of Spanish (Kim, n = 14; Godfroid et al., n = 9), and Korean (Kim, n = 17; Godfroid et al., n = 10). The remaining 65 (Kim) and 58 (Godfroid et al.), reported 24 (Kim) and 30 (Godfroid et al.) other L1s. Table 2 outlines the demographic information of the participants in each project. Current study The current study aims to explore the validity and usefulness of a web-delivered EI task designed to measure English morphosyntactic knowledge. To this end, we examined the comparability, in terms of construct validity, of an EI task delivered in a traditional https://doi.org/10.1017/S0272263124000214 Published online by Cambridge University Press A comparison of lab- and web-based elicited imitation 7 in-person lab setting against an equivalent EI task administered on the web. The following Research Questions (RQs) guided this study: in-person lab setting against an equivalent EI task administered on the web. The following Research Questions (RQs) guided this study: RQ1: To what extent do the EI scores administered in the lab and on the web relate to an English proficiency test? RQ1a: Do grammatical and ungrammatical EI items administered in the lab and on the web exhibit similar relationships to an English proficiency test? RQ2: To what extent do EI tasks administered in the lab and on the web relate to measures of implicit and explicit English morphosyntactic knowledge? With regard to RQ1 and RQ1a, we anticipate EI-based measures of morphosyntactic knowledge to exhibit comparable correlations with English proficiency scores across modalities, for both grammatical and ungrammatical items. With regard to RQ2, we expect to obtain a similar factor structure and loadings for EI scores when situated among several other measures of implicit and explicit morphosyntactic knowledge. Target structures Six grammatical features (Table 3) initially developed by Godfroid et al. (2018) were included in both WB and LB research as target structures across five linguistic tasks: (1) third-person singular -s, (2) mass/count nouns, (3) comparatives, (4) embedded questions, (5) passive, and (6) verb complements. These structures were chosen due to their emergence at various stages of SLA, making them suitable for assessing a broad spectrum of English morphosyntactic knowledge. https://doi.org/10.1017/S0272263124000214 Published online by Cambridge University Press Kathy Minhye Kim et al. Kathy Minhye Kim et al. 8 Table 2. Background information of the L2 speakers in the lab- and web-based environments N Mean SD Min Max Skew Kurtosis Lab–based (n = 151): Godfroid, Kim, Isbell, & Hui (2018) Dataset Residence Length: Months 145 44.33 32.34 2 240 2.72 11.71 Age of Arrival 145 19.96 6.31 0 38 –0.28 2.27 Age of Onset 145 8.09 3.75 0 25 0.92 2.21 Web–based (n = 149): Kim (2020) Dataset Residence Length: Months 137a 34.78 33.11 1 216 2.11 6.89 Age of Arrival 137a 22.92 6.05 3 40 0.12 0.46 Age of Onset 138a 8.16 3.88 2 30 1.87 7.27 aDiscrepancies in the number of participants are due to missing background data. nd information of the L2 speakers in the lab- and web-based environments Table 3. Six target structures Structure Example Third Person Singular –S * The old woman enjoy watching many different famous movies. Mass/Count Noun * The boy had rices in his lunch box. Comparative Adjective * It is more harder to learn Korean than to learn English. Embedded Question * He wanted to know why had he studied for the exam. Passive * The flowers were pick last winter for the festival. Verb Complement * Jim is told his parents want buying a new house. Instruments Lab-based elicited imitation Lab-based elicited imitation The experimental stimuli of the LB EI developed by Godfroid et al. (2018) consisted of 24 critical items (half grammatical and half ungrammatical) and eight filler items. The 24 critical items were counterbalanced by grammaticality yielding two counterbalanced lists (form A and form B). The sentences ranged from 6 to 13 words. Prior to the test phase, four practice sentences (two grammatical and two ungrammatical) were pro- vided. Participants were instructed to listen to each sentence, judge its plausibility, and repeat the sentence in “correct English.” At the beginning of each trial, a fixation cross (+) appeared for 500 milliseconds. The sentence was then played with a speaker icon appearing on the screen. After each sentence, a plausibility question followed (e.g., Do you agree, disagree, or are you unsure about the content of the statement?). Participants had four seconds to decide. A microphone icon then appeared on the screen with a beep sound and the text, “Please repeat now.” They had eight seconds to repeat the sentence. The responses were recorded by a research assistant with a recorder. The task was programmed on Superlab 5.0. An overall accuracy score was calculated based on the correct usage of the target forms in obligatory contexts. Web-based elicited imitation Web-based elicited imitation In the WB EI, two changes were made from the LB EI. First, to enhance test item reliability, only one form of the counterbalanced versions of LB EI (form B) was employed. Second, we added four practice sentences (resulting in eight sentences, four grammatical and four ungrammatical) and model responses to two practice sentences. A comparison of lab- and web-based elicited imitation 9 These changes were made to compensate for the lack of immediate assistance from researchers to address queries. Aside from these adjustments, all other conditions remained identical to the LB EI. The task and the trial sequences of the WB EI were identical to the LB EI. During the repetition trial, an additional text, “Your voice is now being recorded.” was included under the microphone icon. We also designed a progress marker (e.g., 1/32) to inform participants of their progress throughout the experiment. At no point did the task instructions or the model responses include explicit guidance to focus on forms or provide metalinguistic feedback on the linguistic features. Oral production Both Kim’s and Godfroid et al.’s OP tasks instructed participants to read and retell a picture-cued short story containing the target structures (Godfroid et al., 2018; God- froid & Kim, 2021). The pictures provided illustrations of the main context and assisted in the later story retrieval. Each picture was followed by one to four sentences. Participants were asked to read the story twice without time constraints, with no note-taking allowed. Subsequently, they were asked to retell the story within 2.5 minutes, in as much detail as possible, while the pictures remained on the screen. The overall accuracy score was calculated by dividing the number of times a target structure was needed by the number of its correctly used instances. The WB OP tasks were programmed using the same technical setup as the EI tasks. In the current paper, only the WB EI was validated, given its wider use and greater technical sophistication compared to OP (i.e., prompt presentation and timing of responses). Timed and untimed grammaticality judgment tests The LB computerized written grammaticality judgment tests (GJTs) instructed partic- ipants to read a sentence and determine its grammaticality under timed or untimed conditions. The TGJT consisted of 24 critical items (half grammatical and half ungrammatical) and 16 filler items (Godfroid et al., 2018; Godfroid & Kim, 2021). In the UGJT, we only included 12 ungrammatical items, following R. Ellis (2005), and 16 fillers. The TGJT asked participants to make a decision under a time limit, which was set relative to the sentence lengths. The time limit was determined by computing the average audio length of sentences of the same length and adding 50% of its median (Godfroid et al., 2018; Godfroid & Kim, 2021). The UGJT was equivalent to the TGJT, but a time constraint was not imposed. In this way, participants were able to employ explicit knowledge to help with their decisions. Correct responses in both tests were awarded one point. Lab-based elicited imitation The task was programmed as a custom-made Java web application, which consisted of a backend server with Java Servlets doing user authentication and data storage in an SQL database as well as a web front-end accessible from the participants’ browsers. As in the LB EI, correct usage of the target forms in obligatory contexts was used as an outcome measure. English proficiency measure English proficiency measure To obtain participants’ general English proficiency, individuals self-reported their most recent Test of English as a Foreign Language (TOEFL) scores through a background questionnaire. In the interest of inclusivity, the ordinal studies (Godfroid et al., 2018; Kim, 2020) also accepted scores of other standardized tests. In Godfroid et al., the International English Language Testing System (IELTS) was included, and in Kim, IELTS and both the Common European Framework of Reference for Languages and the Test of English for International Communication were accepted. In all cases, the scores were converted into equivalent TOEFL scores using the conversion reference provided by the Educational Testing Service (https://www.ets.org/toefl/institutions/ scores/compare/). Lab-based environment Participants enrolled in the LB research (Godfroid et al., 2018; Godfroid & Kim, 2021) completed the tasks one-on-one with a research assistant in a language research lab. Participants started with two tasks (i.e., OP and EI) that draw attention to the meaning of sentences and proceeded to measures that direct attention to forms (i.e., GJTs and MKT). This order was meant to minimize recognition of the target forms in the meaning-based, implicit knowledge measures. Web-based environment As previously mentioned, participants in the WB experiment (Kim, 2020; Kim Godfroid, 2023) performed two oral tasks (i.e., OP and EI) on the web and the GJ and MKT in the lab setting (Table 4). The decision to conduct the OP and EI tas online was made to enhance research efficiency, especially by reducing the extensi labor involving one-on-one sessions with a research assistant. The GJTs and MKT we purposefully conducted in the lab to prevent learners from accessing external resourc such as metalinguistic explanations of grammar on websites or in grammar textbook Ten days prior to the lab visits, participants received an e-mail with step-by-st directions on accessing and completing the web tasks. This instructional tutorial w recorded using the New Screen Recording function in QuickTime Player (version 10. Seven days prior to the lab visits, participants received another e-mail containing unique web link with a personalized code directed to an interface for the web tasks. O the interface, participants were given general instructions (e.g., find a quiet room f 30 minutes and use Chrome or Firefox). Following the completion of the consent for background questionnaire, and OP, participants proceeded with the EI. The TGJ Table 4. Sequence and settings of web- and lab-based tasks Web–based: Kim (2020) Dataset Lab–based: Godfroid, Kim, Isbell, & Hui (2018) Dataset Measures Setting Measures Setting Background questionnaire Web Background questionnaire Lab Oral production Oral production Elicited imitation Elicited imitation TGJT and UGJT Lab TGJT and UGJT Metalinguistic knowledge test Metalinguistic knowledge test Note: TFJT, timed grammatical judgment test; UGJT, untimed grammatical judgment test. Metalinguistic knowledge tests g g The MKT asked participants to read 12 sentences, each containing one grammatical error. Participants were then asked to identify, correct, and provide detailed explana- tions for each error. Correct responses to each sentence earned one point, allowing a maximum score of 12 points on the MKT. Because explanations required participants to use their most explicit linguistic knowledge, we only considered the explanation part for analysis. https://doi.org/10.1017/S0272263124000214 Published online by Cambridge University Press Kathy Minhye Kim et al. 10 Results We first present the descriptive statistics of the TOEFL scores and the linguistic measures in both WB and LB conditions (Table 5). The average TOEFL score for the participants was 93.03 (SD = 13.11) in the WB condition and 96.77 (SD = 8.86) in the LB condition. The reliability scores of the linguistic tasks ranged from. 51 to. 96, and the skewness and kurtosis values were within the acceptable ranges (±2), indicating the normality of most of the tasks. Web-based environment As previously mentioned, participants in the WB experiment (Kim, 2020; Kim & Godfroid, 2023) performed two oral tasks (i.e., OP and EI) on the web and the GJTs and MKT in the lab setting (Table 4). The decision to conduct the OP and EI tasks online was made to enhance research efficiency, especially by reducing the extensive labor involving one-on-one sessions with a research assistant. The GJTs and MKT were purposefully conducted in the lab to prevent learners from accessing external resources, such as metalinguistic explanations of grammar on websites or in grammar textbooks. Ten days prior to the lab visits, participants received an e-mail with step-by-step directions on accessing and completing the web tasks. This instructional tutorial was recorded using the New Screen Recording function in QuickTime Player (version 10.5). Seven days prior to the lab visits, participants received another e-mail containing a unique web link with a personalized code directed to an interface for the web tasks. On the interface, participants were given general instructions (e.g., find a quiet room for 30 minutes and use Chrome or Firefox). Following the completion of the consent form, background questionnaire, and OP, participants proceeded with the EI. The TGJT, Table 4. Sequence and settings of web- and lab-based tasks Web–based: Kim (2020) Dataset Lab–based: Godfroid, Kim, Isbell, & Hui (2018) Dataset Measures Setting Measures Setting Background questionnaire Web Background questionnaire Lab Oral production Oral production Elicited imitation Elicited imitation TGJT and UGJT Lab TGJT and UGJT Metalinguistic knowledge test Metalinguistic knowledge test Note: TFJT, timed grammatical judgment test; UGJT, untimed grammatical judgment test. Table 4. Sequence and settings of web- and lab-based tasks A comparison of lab- and web-based elicited imitation 11 UGJT, and MKT tasks were conducted in the lab after participants had completed the web tasks in the convenience of their homes. UGJT, and MKT tasks were conducted in the lab after participants had completed the web tasks in the convenience of their homes. Analysis To investigate RQ1 and RQ1a (i.e., the relationship between EIs and standardized English proficiency scores), Pearson correlation coefficients were conducted between the TOEFL scores and two versions of the EI—the WB and LB EIs—as well as their item types: grammatical items (EI_G) and ungrammatical items (EI_UG). We then exam- ined the corresponding 95% CIs and performed a Fisher’s r-to-z transformation (Preacher, 2002) to statistically confirm the relationships (Cumming & Finch, 2005). y p g RQ2 addressed the extent to which both WB EI and LB EI are associated with other measures of implicit and explicit knowledge. To this end, we configured two separate confirmatory factor analysis (CFA) models with Kim’s WB dataset (2020) and God- froid et al.’s LB dataset (2018), respectively. Two latent constructs, implicit and explicit knowledge, were configured with the implicit knowledge factor indicated by EI, OP, and TGJT and the explicit knowledge factor indicated by UGJT and MKT (R. Ellis, 2005; R. Ellis & Loewen, 2007). ) We evaluated the CFA models based on the global goodness of fit, the indices of which provide a (global) summary of the acceptability of the model; that is, whether the model has been properly specified. Model fit indices considered were the χ2 statistic (and corresponding degree of freedom and p value); root mean square error of approximation (RMSEA), corrected for model complexity, taking sample size into account, and its 90% CI; standardized root mean square residual (SRMR); and comparative fit index (CFI), which compares the fitted model to a base model with no parameter restrictions. We followed Hu and Bentler’s (1999) and Kline’s (2016) guidelines for fit interpretation (i.e., RMSEA lower-bound CI ≤0.06, which yields a nonsignificant p value, SRMR ≤0.08, and CFI ≥. 95). Full-information maximum likelihood estimation was used to evaluate CFA models, and robust maximum likeli- hood estimator method was used to accommodate multivariate normality assumption violations. All CFA analyses were carried out in R version 4.2.0, using the lavaan package. https://doi.org/10.1017/S0272263124000214 Published online by Cambridge University Press Skew odfr 0 –0 –1 –0 –0 –0 eb–b –0 –0 –1 0 –0 0 st, on atica wne roid, 0.43 0.02 1.25 0.19 0.48 0.38 base 0.28 0.33 1.93 0.17 0.46 0.62 nly th cal ite ewne roid 0.43 0.02 1.25 0.19 0.48 0.38 base 0.28 0.33 1.93 0.17 0.46 0.62 only t cal it wnes roid, 0.43 0.02 1.25 0.19 0.48 0.38 based 0.28 0.33 1.93 0.17 0.46 0.62 nly th al ite Skew odfr 0 –0 –1 –0 –0 –0 Skew odfro 0 –0 –1 –0 –0 –0 eb–b –0 –0 –1 0 –0 0 st, on atica RQ1 and RQ1a: Relationship with English proficiency In addressing RQ1, we carried out correlation analyses between the TOEFL scores and two versions of the EI tasks separately for the grammatical (EI_G), ungrammatical (EI_UG), and combined EI items. Before stratifying by grammaticality, we first observed the overall EI scores that combined both grammatical and ungrammatical items. Moderate and significant org/10.1017/S0272263124000214 Published online by Cambridge University Press eliab — 70; F 96b 51 65 70 — 67 93b wness Kurtosis k Relia oid, Kim, Isbell, & Hui (2018) 43 –0.14 — 02 –0.83 21c Form A =. 70 25 1.65 250–word story .96 19 0.25 24 .51 48 –0.54 12 .65 38 –0.29 12 .70 ased: Kim (2020) 28 –0.55 — 33 –0.04 23c Form B =. 67 93 4.74 250–word story .93 17 –0.36 21c .68 46 –0.56 12 .68 62 –0.33 12 .79 ly the rule was used; OP = oral production; TOEFL = Test of English As a Fore l items were used. Skew odfr 0 –0 –1 –0 –0 –0 A comparison of lab- and web-based elicited imitation 13 correlations were found between TOEFL and LB EI (r = .60, 95% CI [0.44, 0.72], n = 83) as well as TOEFL and WB EI (r = .52, 95% CI [0.39, 0.63], n = 139). Figure 1 visually represents these relationships. The correlation between LB -EI and TOEFL (r = .60) falls within the 95% CI of the corresponding WB EI and TOEFL pair (95% CI [0.39, 0.63]). Similarly, the WB EI’s relation to the TOEFL score (r = .52) falls within the CI of the LB EI and TOEFL pair (95% CI [0.44, 0.72]). Their comparable associations were sup- ported by the nonsignificant Fisher’s test results (p = .393), failing to reject a null hypothesis (H0 = the strength of EI-TOEFL correlations are comparable between the administrative conditions). When stratified by grammaticality, different patterns were observed. As shown in Figure 1 (left), the grammatical items in both EI versions yielded similar associations to their respective TOEFL scores, returning nonsignificant Fisher’s test results (p = .164). However, we observed differences between the lab and web versions of the ungram- matical EI items (although not reaching statistical significance, p = 0.062), with the web version correlating weaker to TOEFL scores (r = .37, 95% CI [0.22, 0.50]) than those of the lab version (r = .57, 95% CI [0.41, 0.70]). RQ1 and RQ1a: Relationship with English proficiency This trend of ungrammatical items showing less discrimination than the grammatical items is pronounced only in the web condition and not in the lab condition (Figure 1, right), suggesting that the adminis- trative environment of the web might introduce variability in how ungrammatical items are processed and performed by participants. Figure 1. TOEFL and EI correlational values, along with a 95% CI and Fisher’s r-to-z transformation, by conditions (left) and by item types (right). Note: EI = elicited imitation; EI_G = EI with only grammatical items; EI_UG = EI with only ungrammatical items. Figure 1. TOEFL and EI correlational values, along with a 95% CI and Fisher’s r-to-z transformation, by conditions (left) and by item types (right). Note: EI = elicited imitation; EI_G = EI with only grammatical items; EI_UG = EI with only ungrammatical items. Figure 1. TOEFL and EI correlational values, along with a 95% CI and Fisher’s r-to-z transformation, by conditions (left) and by item types (right). Note: EI = elicited imitation; EI_G = EI with only grammatical items; EI_UG = EI with only ungrammatical items. https://doi.org/10.1017/S0272263124000214 Published online by Cambridge University Press Kathy Minhye Kim et al. 14 RQ2: relationship with explicit and implicit measures To address RQ2 (i.e., the relationship between EI and explicit and implicit L2 knowl- edge measures), we first explored the associations between EIs and the five linguistic measures. Figure 2 presents the correlation results for all five linguistic measures separately for the WB (top) and LB (bottom) tasks. Small to moderate relationships Figure 2. Correlations of linguistic tasks performed in the web (top) and in the lab (bottom). Note: p < 0.05, p < 0.01, *p < 0.001. The diagonal cells plot the standardized score distribution of corresponding tests. Figure 2. Correlations of linguistic tasks performed in the web (top) and in the lab (bottom). Note: p < 0.05, p < 0.01, p < 0.001. The diagonal cells plot the standardized score distribution of corresponding tests. Figure 2. Correlations of linguistic tasks performed in the web (top) and in the lab (bottom). Note: p < 0.05, p < 0.01, p < 0.001. The diagonal cells plot the standardized score distribution of corresponding tests. Correlations of linguistic tasks performed in the web (top) and in the lab (bottom). Figure 2. Correlations of linguistic tasks performed in the web (top) and in the lab (bottom). Note: p < 0.05, p < 0.01, p < 0.001. The diagonal cells plot the standardized score distribution o corresponding tests. 05, p < 0.01, **p < 0.001. The diagonal cells plot the standardized score distribution of g tests. A comparison of lab- and web-based elicited imitation 15 were found among most measures (.332 < r <. 430, p < 0.001), except for the correlations between UGJT and OP in the LB versions (r = .168) and the MKT and three implicit knowledge measures, which were either insignificant or weakly correlated (.146 < r <. 226, 0.01 < p < 0.05). Importantly, no statistically significant differences in the strength of correlation were found between LB and WB EI measures with different linguistic tests, with Fisher’s r-to-z transformation with a range of 0.19 < p < 0.72 (see Figure S1, Online Supplementary Materials). To explore the relationships more fully, we submitted all measures to a series of CFAs separately for the WB (n = 149) and the LB (n = 151) tasks. As previously mentioned, for each task version, we configured a model with EI indicated as an implicit knowledge construct along with two implicit knowledge measures (i.e., OP and written TGJT). RQ2: relationship with explicit and implicit measures Table 6 presents fit indices for the WB and LB conditions. Both CFA models of the lab and web versions demonstrated excellent fit to the data, with all fit indices (i.e., CFI, SRMR, and RMSEA lower bound) within the acceptable range and χ2 returning nonsignificant values. The reliability (?) of the implicit factor was .69 (WB) and .67 (LB), and for the explicit factor, .66 (WB) and .66 (LB). Parameter estimates for the models are detailed in Figure 3. All indicators in both models were Table 6. Model fit indices for the web- and lab-based conditions Web-based Lab-based Parameters (n) 16 16 χ2 1.711 5.156 χ2 p (> 0.05) 0.789 0.272 df 4 4 CFI (≥. 95) 1.000 0.986 SRMR (version 0.08) 0.015 0.033 RMSEA 0.000 0.045 RMSEA lower (≤0.06) 0.000 0.000 RMSEA upper 0.079 0.163 Note: χ2 = chi-square; χ2 p = chi-square test p value; CFI = comparative fit index; df = degree of freedom; RMSEA = root mean square error of approximation; SRMR = standardized root mean square residual. Table 6. Model fit indices for the web- and lab-based conditions Web-based Lab-based Parameters (n) 16 16 χ2 1.711 5.156 χ2 p (> 0.05) 0.789 0.272 df 4 4 CFI (≥. 95) 1.000 0.986 SRMR (version 0.08) 0.015 0.033 RMSEA 0.000 0.045 RMSEA lower (≤0.06) 0.000 0.000 RMSEA upper 0.079 0.163 Note: χ2 = chi-square; χ2 p = chi-square test p value; CFI = comparative fit index; df = degree of freedom; RMSEA = root mean square error of approximation; SRMR = standardized root mean square residual. Table 6. Model fit indices for the web- and lab-based conditions able 6. Model fit indices for the web- and lab-based conditions Note: χ2 = chi-square; χ2 p = chi-square test p value; CFI = comparative fit index; df = degree of freedom; RMSEA = root mean square error of approximation; SRMR = standardized root mean square residual. Figure 3. Two-factor CFA model, lab-version (left) and web-version (right). Note: EI = elicited imitation; EI = EI; MKT = metalinguistic knowledge test; OP = oral production; TWGJT = timed grammaticality judgment test; UWGJT = untimed grammaticality judgment test. Figure 3. Two-factor CFA model, lab-version (left) and web-version (right). Figure 3. Two-factor CFA model, lab-version (left) and web-version (right). RQ2: relationship with explicit and implicit measures Note: EI = elicited imitation; EI = EI; MKT = metalinguistic knowledge test; OP = oral production; TWGJT = timed grammaticality judgment test; UWGJT = untimed grammaticality judgment test. Discussion The goal of this study was to verify the validity of a low-fee, web-programmed EI task that measures English morphosyntactic knowledge. Drawing on theoretical and empirical work in L2 assessment and psycholinguistics, we hypothesized that, first, EI scores will yield significant associations with standardized L2 proficiency test scores as L2 grammar knowledge is a central component of L2 proficiency. Second, linguistic knowledge consists of explicit and implicit knowledge, with the EI task operationalizing as either implicit or automatized explicit knowledge; hence, EI is expected to exhibit more meaningful relationships to time-pressured accuracy measures (implicit or automatized explicit knowledge) than untimed linguistic measures (explicit knowledge). To empiri- cally test the validity of web-programmed EI in relation to these assumptions, we compared the extent to which an EI administered on the web yielded a relationship to English proficiency scores and measures of implicit (i.e., OP and TGJT) and explicit (i.e., UGJT and MKT) grammar knowledge similar to that of EI administered in a lab setting. g In this study, we observed a good amount of shared variance between general English proficiency scores and the two versions of the EI scores. We found relationships of medium to large, comparable associative strength between TOEFL and WB EI (r = .52, 95% CI [0.39, 0.63]) and LB EI (r = .60, 95% CI [0.44, 0.72]), providing initial support for the similar importance of grammar competence in overall language proficiency. Previous research has reported strong relationships between standardized English proficiency and EI scores, demonstrating the use of morphosyntactic knowl- edge in language proficiency tests (e.g., Elder & R. Ellis, 2009; Erlam, 2006, 2009; Spada et al., 2015). For instance, in Erlam’s studies (2006, 2009), scores of EI measuring 17 features of English grammar were strongly linked to the overall IELTS scores (r = .76), with the writing component showing the weakest (r = .46) and the listening component yielding the strongest (r = .72) correlations. Including only one target feature in the EI, Spada and colleagues (2015) used a C-test to measure learners’ English proficiency and found one’s knowledge of the English passive voice was moderately related to their general English proficiency (r = .50). In a recent meta-analysis (Kostromitina & Plonsky, 2022), studies using binary scoring, as our study, showed a correlation of . 53 (95% CI [0.43, 0.63]) to proficiency scores, which aligns closer to our findings. https://doi.org/10.1017/S0272263124000214 Published online by Cambridge University Press https://doi.org/10.1017/S0272263124000214 Published online by Cambridge University Press Kathy Minhye Kim et al. 16 significant. Collectively, no significant deviation of the WB EI and LB EI scores was observed in relation to a battery of different explicit and implicit knowledge measures. Such mirroring of the CFA results of the WB and LB measures reaffirms WB EI as a measure sharing a similar underlying construct as the LB EI. significant. Collectively, no significant deviation of the WB EI and LB EI scores was observed in relation to a battery of different explicit and implicit knowledge measures. Such mirroring of the CFA results of the WB and LB measures reaffirms WB EI as a measure sharing a similar underlying construct as the LB EI. https://doi.org/10.1017/S0272263124000214 Published online by Cambridge University Press Discussion On par with these findings, our study, which also used only six features of English, yielded a medium correlation to general English proficiency in both versions of the EIs, thus establishing preliminary evidence for the comparable validity of online EI vis-à-vis in-person EI tasks. p Intriguingly, when the EI items were grouped by grammaticality, we observed some evidence of modality effects. A trend of differences emerged in the correlations between TOEFL scores and the ungrammatical EI items (although not reaching statistical significance, p = 0.062), with a weaker correlation for the web version (r = .37, 95% CI [0.22, 0.50]) compared to that of the lab version (r = .57, 95% CI [0.41, 0.70]). This pattern, where the ungrammatical tasks were less consistent but neither improved nor harmed the overall effectiveness of EI, matches the findings of Yan et al. (2016). In our context, such a trend was specific to the WB format, suggesting that the online A comparison of lab- and web-based elicited imitation 17 administrative context might introduce variability in participant responses to ungram- matical items. The lab setting (more specifically, the affordance of direct researcher interaction) allowed for immediate feedback on participants’ questions on “correct English” responses when task instructions were given. Although the web version attempted to address this by modeling correction of ungrammatical items, the less controlled condition of the web setting might not have achieved the same effectiveness, thereby potentially affecting the results. We note that the observed trend of modality effect does not necessarily diminish the validity of ungrammatical items as a whole (because the observed effects are not statistically meaningful; and the LB version did show a fairly robust correlation to the TOEFL scores, in fact, numerically slightly better than grammatical items; see Figure 1, right). Instead, our findings caution users of WB EI about the potential for misinterpreting instructions, which could reduce the effec- tiveness of ungrammatical sentences in EI tasks. g Second, the effectiveness of the online EI was further corroborated by a series of CFA results. Based on a well-established explicit-implicit knowledge model by R. Ellis (2005) and its extensions (Godfroid & Kim, 2021; Godfroid et al., 2018), another goal was to assess the association between the web-programmed EI with different implicit and explicit knowledge measures. To this end, in both WB and LB versions, we configured CFA models with the same structure, anticipating comparable structural validity. Discussion As expected,wefound excellentmodel fits forboth WB and LBCFA models, with significant loadings for EI scores and other four linguistic measures. Such mirroring of the CFA results adds additional support for both EIs zeroing in on a common construct of L2 knowledge type. g yp To our knowledge, this is one of the first studies to validate the effectiveness of EIs deployed on the web. Our findings show promise for utilizing web-programmed EI as an alternative to its LB counterpart bringing potential to language science and educa- tion. Notably, the capacity to reliably record utterances via the Internet opens great prospects for research that necessitate elicited production data. Research, such as L2 oral proficiency (e.g., Ortega et al., 2002) and explicit-implicit knowledge development (e.g., R. Ellis, 2005; Erlam, 2006; Suzuki & DeKeyser, 2015; Suzuki et al., 2023) that entails an experimental design similar to that of the current study, may benefit from utilizing web-programmed EI in eliciting speech data. Relatedly, our web-programmed EI may serve as an efficient tool for researchers to conduct equitable and statistically powered research. With increased calls for diverse representation of L2 learners in SLA research (Andringa & Godfroid, 2020; Godfroid & Andringa, 2023), this online task, which enables wider and larger recruitment of participant samples, could hold promise for conducting equitable yet reliable research with unconventional learners (e.g., non- WEIRD populations: Western, Educated, Industrialized, Rich, and Democratic) on a larger scale. Beyond EIs used for research purposes, there is potential for their use in educational applications. With EI demonstrating its effectiveness as a language pro- gram placement test (Yan et al., 2016), this WB program, along with the potential for automated scoring of EIs (Isbell et al., 2023), may make the administration and scoring of EIs feasible with limited staff and resources. For researchers and educators interested in web-programming their own EI test, see Appendix S1 (Online Supplementary Material) and Kim et al. (2024.) for the coding guidelines and a tutorial on autoscoring English grammar forms. https://doi.org/10.1017/S0272263124000214 Published online by Cambridge University Press Limitations and future directions As previously mentioned, in the WB condition, the OP task was administered online alongside the EItask.Thisdecision wasmadedeliberatelyto optimize limited resourcesin https://doi.org/10.1017/S0272263124000214 Published online by Cambridge University Press Kathy Minhye Kim et al. 18 Kim’s longitudinal study. However, for the specific focus of this paper (i.e., the validation of online vs. offline EIs), having online OP exclusively in the WB condition and not in the LB condition might introduce variations. To check for potential confounding effects, we looked into how OPs, in both conditions, relate to the three linguistic tests (i.e., TGJT, UGJT, and MKT), all of which were conducted in the lab for both conditions. We then used Fisher’s r-to-z transformation to test for parity between the two conditions. If online modality mediated test performance, the strength of associations between OP and its paired linguistic tasks would yield meaningful differences between conditions. All paired relationships between OP and the linguistic tests were comparable across the conditions, with p values ranging from 0.117 to 0.763 (Figure S2, Online Supplementary Materials). While these results offer some support that the differences in modality between OPs are not meaningful, in future research, it is recommended to eliminate any discrepancies between conditions to facilitate more direct comparisons. p Another important limitation of this study is that participants’ English proficiency scores were self-reported. The secondary data we reanalyzed (Godfroid et al., 2018; Kim, 2020) primarily came from university students, many of whom provided the scores achieved at the commencement of their academic programs. Therefore, their entered language proficiency score may not accurately represent their current profi- ciency levels. To test whether test-taking time changes the associations between EI and standardized proficiency scores, we broke the analysis down by test administration year (Figure S3 and Tables S1–3, Online Supplementary Materials). In essence, there is a gradual decline in correlation strength as the time since taking the test increases. Specifically, among the 139 participants who reported the years of test administration, those who took the English proficiency test within 2 years from the year of experimen- tation showed a correlation score of r = .57 (95% CI [0.33, 0.74], n = 51) to the EI scores. This coefficient is comparable to those who have taken the tests between 3 and 4 years (r = .55, 95% CI [0.33, 0.72], n = 63). https://doi.org/10.1017/S0272263124000214 Published online by Cambridge University Press Limitations and future directions The correlational strength dropped drastically when the years of test-taking dated after 5 years from 2019 (r = .30, 95% CI [–0.11, 0.62], n = 25). Given the strength weakens as the time since test-taking increases, we anticipate a stronger correlation between EI and language proficiency scores when the scores more accurately reflect learners’ current proficiency level. Future studies should incorporate language proficiency tests into the experimental design, if not using secondary data. The current WB EI program used in the study served as a data collection instrument. Nevertheless, its scalability was limited by the reliance on manual procedures for test item development and response scoring. To address this, a more comprehensive system integrating item development, test administration, and automated scoring is envisioned. With the aid of natural language processing technologies, a system could be created to generate orselectsentencesfroma corpus containing target grammaticalconstructs, both with and without errors. The system would store participants’ responses and employ automatic speech recognition and other natural language processing tools for automated scoring (Isbell et al., 2023; Kim et al., 2024). Such a comprehensive WB EI test system has the potential to greatly benefit the SLA and language learning community. p g y g g g y As also suggested by a reviewer, we encourage future studies to utilize a planned within-subjects design, where all participants engage in both offline and online versions of EIs. The reanalytic nature of our study precluded such a design, but the proposed within-subjects approach offers a rigorous way to investigate the impact of modalities (online vs. offline) on test scores while mitigating potential sources of noise, such as sampling error and participant variability. A comparison of lab- and web-based elicited imitation 19 References American Educational Research Association, American Psychological Association, & National Council on Measurement in Education. (2014). Standards for Educational and Psychological Testing. American Educational Research Association. 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The elicited oral imitation test as a measure of implicit knowledge. In R. Ellis, S. Loewen, C. Elder, H. Reinders, R. Erlam & J. Conclusion The aim of this study was to validate the WB EI test by comparing its performance with the traditional LB delivery mode in terms of its correlation with established overall proficiency and linguistic knowledge tests. Using secondary data from Kim (2020) and Godfroid et al. (2018), we found moderate correlations between the EI test results and the TOEFL test for both delivery modes. The factor loadings of the EI test were also similar between the two modes, indicating comparable structural validity in the CFA model. Thus, we show promise for utilizing web-programmed EI as an alternative to its LB counterpart bringing potential to language science and education. The WB delivery mode offers numerous benefits, including logistical convenience, easier participant recruitment, and lower running costs. Supplementary material. 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https://openalex.org/W2990364955	https://www.frontiersin.org/articles/10.3389/fpsyg.2019.02478/pdf	English	null	The Impact of Grammar on Mentalizing: A Training Study Including Children With Autism Spectrum Disorder and Developmental Language Disorder	Frontiers in psychology	2,019	cc-by	13,135	The Impact of Grammar on Mentalizing: A Training Study Including Children With Autism Spectrum Disorder and Developmental Language Disorder Stephanie Durrleman1,2, Morgane Burnel3, Jill Gibson De Villiers4, Evelyne Thommen5, Rachel Yan4 and Hélène Delage1 1 Department of Psycholinguistics, University of Geneva, Geneva, Switzerland, 2 Department of Linguistics, University of Geneva, Geneva, Switzerland, 3 Laboratoire de Psychologie et Neurocognition, Department of Psychology, Université Grenoble Alpes, Saint-Martin-d’Hères, France, 4 Department of Psychology, Smith College, Northampton, MA, United States, 5 EESP, University of Applied Sciences and Arts, Western Switzerland, Lausanne, Switzerland Training on complements in English, German, and Mandarin has been reported to trigger improvements on both complements and Theory of Mind (ToM), with typically developing (TD) pre-schoolers on the verge of developing these skills (Hale and Tager-Flusberg, 2003; Lohmann and Tomasello, 2003; Shuliang et al., 2014). In the current study, we build on the idea that increasing mastery of complementation holds the promise of enhancing ToM, and seek (i) to replicate the positive effects observed in previous work for this effect in French-speaking TD children, and (ii) to pilot extending this to clinical children, more speciﬁcally those with Autism Spectrum Disorder (ASD) and Developmental Language Disorder (DLD), through exploring whether improvement in the latter, clinical groups follows that of the TD group. Sixty children with ToM difﬁculties, 16 with ASD (aged 5;6–11;8), 20 with DLD (aged 4;8–9;0) and 24 typically developing children aged (2;9–5;3 years), participated in a 4-week training program. Half received training targeting sentential complements and half received a control training targeting lexical skills. Complementation training, but not lexical training, led to a signiﬁcant direct increase in complements, and also had the indirect effect of signiﬁcantly boosting belief reasoning. TD and clinical groups followed the same patterns of performance. These results conﬁrm previous ﬁndings in other languages for TD, and further suggest promising new directions for therapeutic programs addressing ToM delays in populations of different aetiologies, namely the incorporation of a motivating training on complementation. ORIGINAL RESEARCH published: 19 November 2019 doi: 10.3389/fpsyg.2019.02478 Citation: Durrleman S, Burnel M, De Villiers JG, Thommen E, Yan R and Delage H (2019) The Impact of Grammar on Mentalizing: A Training Study Including Children With Autism Spectrum Disorder and Developmental Language Disorder. Front. Psychol. 10:2478. doi: 10.3389/fpsyg.2019.02478 Keywords: Autism Spectrum Disorder (ASD), Developmental Language Disorder (DLD), Theory of Mind (ToM), sentential complements, training program Edited by: Ilaria Grazzani, University of Milano-Bicocca, Italy Reviewed by: Francesca Panzeri, University of Milano-Bicocca, Italy Steven Stagg, Anglia Ruskin University, United Kingdom Correspondence: Stephanie Durrleman stephanie.durrleman@unige.ch *Correspondence: Stephanie Durrleman stephanie.durrleman@unige.ch Specialty section: This article was submitted to Developmental Psychology, a section of the journal Frontiers in Psychology Received: 02 May 2019 Accepted: 21 October 2019 Published: 19 November 2019 (3) That doll thinks/ believes / says/ mentions that [her ball is in the basket / some Smarties are in this tube] (1) Sally places a ball in a basket, then leaves. While she is absent, Anne arrives and moves the ball from the basket to a box. Sally returns, and the children are asked: (a) Where Sally will look for her marble? (The critical “belief” question), (b) Where it is really? (The “reality” question), (c) Where it was at the beginning? (The “memory” question). This linguistic tool would serve to eﬃciently represent subjective truths because the content of the complement (in brackets) has an independent truth-value, and consequently can be false while the entire sentence remains true. These semantic and syntactic properties render complements ideal tools for grasping propositional attitudes and thus eﬃciently representing subjective truths (Perner, 1988; de Villiers et al., 2014), albeit with some cross-linguistic variation (Perner et al., 2003; Cheung et al., 2004; Tardif et al., 2007). (2) After being presented with a Smarties tube, the children are asked what they think is inside, to which they typically reply: ‘Smarties.’ It is then revealed that in fact there are pencils inside, at which point the child is asked if s/he can remember the contents of the tube, as well as the critical test question: What would another person would think is inside? In support of the view that complementation assists complex ToM reasoning, authors have reported links between mastery of this structure and success at FB in young TD children (de Villiers, 2000; de Villiers and de Villiers, 2000) as well as in children with ASD (Tager-Flusberg, 2000; Tager-Flusberg and Joseph, 2005; Lind and Bowler, 2009) and language-delayed deaf children (Schick et al., 2007). Interestingly, these links are also found when the complements do not occur with mental state verbs but rather with verbs of communication, which themselves do not refer explicitly to mental states (de Villiers and Pyers, 2002; Durrleman and Franck, 2015). Indeed the latter verbs, being less abstract than mental state verbs, have even been argued to be most crucial for ToM success in children with ASD (Tager-Flusberg and Joseph, 2005). Knowledge of sentential complements, rather than of mental state lexicon, would therefore allow children to bootstrap their meta-representational grasp of beliefs. Accurate responses during tasks such as those above are only attested around the age of 4–5 years in TD (Wellman et al., 2001; Milligan et al., 2007). (3) That doll thinks/ believes / says/ mentions that [her ball is in the basket / some Smarties are in this tube] It is important to emphasize the conceptual diﬃculty involved during FB attribution: the child must reconcile the contradiction between what s/he knows and what the other believes. This is a sophisticated step, preceded by simpler mental states attributions. The attribution of diverse desires and beliefs, for instance, does not require the child to reconcile a perspective in contradiction with what they know to be true, and these FB- precursors emerge earlier in development than the attribution of FB, i.e., before age 4 (Wellman and Liu, 2004). The emergence of successful mentalizing including FB reasoning is important for the development of social cognition on various levels, e.g., ﬂuid conversational skills, conﬂict resolution, popularity amongst peers, etc. (Astington and Jenkins, 1999; Astington, 2003; Astington and Pelletier, 2005; Astington and Edward, 2010; Mazza et al., 2017; Derksen et al., 2018). In certain clinical populations, such as children with Autism Spectrum Disorder, diﬃculties with FB reasoning often persist later in development, aﬀecting performance on FB tasks even at a mental age of 9 years (Baron-Cohen et al., 1985). This marked delay has been interpreted to indicate a core mind- reading deﬁcit (Baron-Cohen, 1990), which would explain weaknesses in communicative and social skills characteristic of the autistic condition (Diagnostic and Statistical Manual of Mental Disorders, 5th edition). However, a subset of children with ASD, from 20 to 50% (Baron-Cohen et al., 1985; Prior et al., 1990), systematically succeeds at FB attribution and thus arguably can surmount their fundamental ToM diﬃculty (Tager-Flusberg and Joseph, 2005). It has been claimed that in order to accomplish this, they apply verbal strategies. Put diﬀerently, children on the spectrum could use language to support their reasoning about others’ beliefs, with some studies suggesting that lexical abilities play a role in ToM (e.g., Happé, 1995), and others pointing rather to the importance of grammatical skills (Fisher et al., 2005; Milligan et al., p g p If language skills, in particular with complementation, serve for belief reasoning, then populations with language diﬃculties that include complementation would also be expected to struggle with this aspect of ToM. This seems to be the case for children with Developmental Language Disorder (DLD). INTRODUCTION The ability to grasp that people’s mental representations of the world may or may not correspond to reality is an important milestone in the development of ‘theory of mind’ (ToM) (Dennett, 1978; Premack and Woodruﬀ, 1978; Flavell, 1999). Typically developing (TD) preschool children struggle at tasks requiring them to predict another’s actions based on their false belief (FB) November 2019 \| Volume 10 \| Article 2478 Frontiers in Psychology \| www.frontiersin.org The Impact of Grammar on Mentalizing Durrleman et al. (Wellman et al., 2001). Such tasks usually involve a protagonist whose mistaken belief about an object has arisen because (1) the object was displaced (Change of Location Task, Wimmer and Perner, 1983; Baron-Cohen et al., 1985) or (2) the object has the appearance that it might contain something else than its actual contents (Unexpected Contents, Gopnik and Astington, 1988). 2007). The linguistic determinism approach (de Villiers, 2007) maintains that a speciﬁc grammatical structure is most crucially solicited during mentalizing, namely complement clauses such as (3), where a proposition is embedded under a verb of mental-state (e.g., think, believe) or communication (e.g., say, mention): (3) That doll thinks/ believes / says/ mentions that [her ball is in the basket / some Smarties are in this tube] (3) That doll thinks/ believes / says/ mentions that [her ball is in the basket / some Smarties are in this tube] Frontiers in Psychology \| www.frontiersin.org (3) That doll thinks/ believes / says/ mentions that [her ball is in the basket / some Smarties are in this tube] The current work is thus concerned with elucidating whether training sentential complements can be beneﬁcial for the remediation of belief reasoning in children with ASD and those with DLD, along the lines of TD. It is also an open question whether enhancing complementation can also be useful for other aspects of ToM beyond false belief reasoning, such as grasping diversity of desires. and ASD (Durrleman et al., 2016a). Taking as a point of departure that language is not only fundamentally related to mentalizing, but also inﬂuences its development rather than vice versa as revealed by longitudinal studies (TD: Astington and Jenkins, 1999; ASD: Tager-Flusberg and Joseph, 2005), researchers have aimed to trigger ToM via the training of complements in preschool TD children. Results have revealed that this training is indeed eﬀective at boosting ToM, even when training involved complements of verbs of communication alone (Hale and Tager-Flusberg, 2003; Shuliang et al., 2014) and when deceptive scenarios (i.e., involving appearance-reality dissociations) were not included to train complements (Lohmann and Tomasello, 2003; Shuliang et al., 2014), although capitalizing on both complements and deceptive scenarios together appears to be especially useful for consolidating ToM. Still, none of these studies on complementation training included participants delayed for either language or ToM, and instead focussed on children on the cusp of developing these skills anyway. It thus remains to be determined whether populations where ToM and/or language is aﬀected would show similar boosts in belief reasoning to that already observed in TD children due to complementation training. The current work is thus concerned with elucidating whether training sentential complements can be beneﬁcial for the remediation of belief reasoning in children with ASD and those with DLD, along the lines of TD. It is also an open question whether enhancing complementation can also be useful for other aspects of ToM beyond false belief reasoning, such as grasping diversity of desires. regressions or ANOVAS, especially when variables are highly intercorrelated, as they are in this study. It models the relationships among multiple independent and dependent variables simultaneously, unlike linear regression, which can only analyze one layer of linkages at a time. Because SEM can test multiple pathways, it allows the investigation of both direct and indirect eﬀects in one hypothesized model (Gefen et al., 2000). This is important in determining the particular role that the complement training plays in advancing false belief understanding. Frontiers in Psychology \| www.frontiersin.org Participants ll f h All of the participants in this study were native French-speakers, recruited in Geneva and Lausanne, Switzerland and Paris, France. The project received approval from the Ethics Committee of the Faculty of Psychology and Educational Sciences of the University of Geneva as well as from the Geneva Cantonal Ethics Commission, and was also declared at ‘La Commission Nationale de l’Informatique et des Libertés (CNIL)’ in France. Children’s parents all provided written, informed consent for their child to participate. Sixty children participated in the study: 16 children with ASD aged 5;6–11;8 (M = 8;3), 20 children with DLD aged 4;8–9;0 (M = 6;9) and 24 TD children aged 2;9–5;3 years (M = 4;3). Diﬀerences in age were due to the fact that diﬃculties on ToM have been attested at diﬀerent phases of development in these three populations. Matching was done on the groups’ linguistic and cognitive characteristics as explained below. Children with ASD were recruited from specialized schools, those with DLD from speech-language centers which they attended after school, and TD children from kindergartens and day-care facilities. We targeted children of the age range when complements and ToM are reportedly not yet mastered, hence for TD children this meant choosing children between the ages of 3 and 6 years (Wellman et al., 2001), for children with DLD the upper cut-oﬀwas age 9 (Andrés-Roqueta et al., 2013; Nilsson and de López, 2016) and for children with ASD this cut-oﬀwas extended to 12 years (Baron-Cohen et al., 1985; Yirmiya et al., 1998). Then, for these children to be included in the study, they had to meet several criteria: (i) TD children had to have no history of language impairment and needed to be included in normal classrooms without support. (ii) In contrast, clinical groups had to have been previously given the relevant diagnosis by a qualiﬁed professional. More speciﬁcally, children with DLD needed to have obtained language scores of at least 2 SDs below age- speciﬁc norms according to standardized tests used by speech and language pathologists in Switzerland and France (CIM 10; De La Santé, 1993), while children with ASD had to have met the criteria for this condition according to the DSM-IV-TR (American Psychiatric Association [APA], 2000), the Autism Diagnostic Observation Schedule, ADOS (Lord et al., 2003) and/or the ADI-R (Rutter et al., 2003). (3) That doll thinks/ believes / says/ mentions that [her ball is in the basket / some Smarties are in this tube] Children with this condition display primary diﬃculties in formal language (Leonard, 2014) including complementation (Tuller et al., 2012; Steel et al., 2016) and are also reportedly delayed in ToM, even if these delays appear to be more subtle than those attested in ASD (Holmes, 2002; Tucker, 2004; Andrés-Roqueta et al., 2013). Moreover, mastery of complements by children with DLD also relates to their success at ToM as measured by false-belief tasks (de Villiers et al., 2003; Miller, 2004). Interestingly, the verbal demands of the ToM tests administered in the studies conducted with this population may impact their performance (Miller, 2001), but these alone do not suﬃce to clearly explain their ToM performance, as even tasks that rely minimally on language pose problems, suggesting that the diﬃculty is at the level of ToM reasoning (Nilsson and de López, 2016). In favor of the view that language inﬂuences ToM reasoning and not only verbal ToM task performance, relations between complements and low verbal ToM tasks have been reported for both DLD (Durrleman et al., 2017a) November 2019 \| Volume 10 \| Article 2478 Frontiers in Psychology \| www.frontiersin.org Frontiers in Psychology \| www.frontiersin.org 2 The Impact of Grammar on Mentalizing Durrleman et al. and ASD (Durrleman et al., 2016a). Taking as a point of departure that language is not only fundamentally related to mentalizing, but also inﬂuences its development rather than vice versa as revealed by longitudinal studies (TD: Astington and Jenkins, 1999; ASD: Tager-Flusberg and Joseph, 2005), researchers have aimed to trigger ToM via the training of complements in preschool TD children. Results have revealed that this training is indeed eﬀective at boosting ToM, even when training involved complements of verbs of communication alone (Hale and Tager-Flusberg, 2003; Shuliang et al., 2014) and when deceptive scenarios (i.e., involving appearance-reality dissociations) were not included to train complements (Lohmann and Tomasello, 2003; Shuliang et al., 2014), although capitalizing on both complements and deceptive scenarios together appears to be especially useful for consolidating ToM. Still, none of these studies on complementation training included participants delayed for either language or ToM, and instead focussed on children on the cusp of developing these skills anyway. It thus remains to be determined whether populations where ToM and/or language is aﬀected would show similar boosts in belief reasoning to that already observed in TD children due to complementation training. 1A total of 11 children were eliminated after the pre-tests from the initial cohort of 71 participants: 3 TD, 4 ASD and 4 DLD. For 10 children, this was because of ceiling performance, and for 1 child with ASD this was because of hypersensitivity to the sounds of the testing material. Participants ll f h (iii) Scores on pre- (training)-tests assessing ToM and complements also had to In the current study, we build on the idea that increasing mastery of complementation holds the promise of enhancing ToM, and seek (i) to replicate the positive eﬀects observed in work on other languages for this eﬀect in French-speaking TD children, and (ii) to pilot extending this to clinical children, more speciﬁcally those with ASD and DLD, through exploring whether improvement in the latter, clinical groups follows that of the TD group. If this proves to be the case, our results would suggest a novel, evidence-based, clinical intervention, addressing both language and ToM in these populations. We explore several other questions as well with our rich data set. We verify that complementation training is more eﬀective for complements and ToM than a more general, lexical training. We test whether the eﬀects of complement training are particular to false beliefs assessed verbally, or encompass low-verbal false beliefs too. We ask whether the contribution of complementation is speciﬁc to false belief reasoning, or whether it can be observed to assist other, earlier-mastered aspects of ToM, like diverse desires and true beliefs. Importantly for clinical purposes, we ask whether the hypothesized ToM gains persist through time by retesting after a delay. Finally, we ask whether the control group, who received lexical training, diﬀerentially improved to the target, complementation training group, on the lexical tasks. In addition to individual analyses on the outcome measures, we undertake a Structural Equation Model (SEM) to look more closely at the pathways of change, for example, asking whether the success of complement training depended on other abilities, such as non-verbal reasoning or language skills at outset. SEM allows several advantages over simple November 2019 \| Volume 10 \| Article 2478 Frontiers in Psychology \| www.frontiersin.org 3 The Impact of Grammar on Mentalizing Durrleman et al. 20 boys and 10 girls. The two groups of 30 were matched on a variety of global cognitive and linguistic standardized measures (all t < 1, see Table 1 for precise p-values), namely non-verbal reasoning (Raven et al., 1998), as well as general morphosyntax and lexicon (Exalang 3–6; Helloin and Thibault, 2006). For more speciﬁc measures, we created tests assessing: (i) complementation understanding (based on de Villiers and Pyers, 2002) and (ii) ToM abilities. Participants ll f h The latter included a verbal measure of false-belief (based on Baron-Cohen et al., 1985), a minimally (low-)verbal measure of false-belief (based on Woolfe et al., 2002) as well as a test assessing skills emerging just before false-belief reasoning (FB precursors), namely diverse desires and diverse beliefs (based on Burnel et al., 2017). We refer to the latter as low-verbal ToM and FB precursors. Table 1 presents the descriptive measures of the children included in each of the training groups. Details on the descriptive characteristics of the cognitive groups (TD, ASD and DLD) are reported in Table 2. While these groups diﬀer for age [F(2,52) = 37.57, p < 0.001], for reasons explained above, they do not diﬀer on standardized measured of non- verbal reasoning (p = 0.09), and morphosyntax (p = 0.23) or lexicon (p = 0.27). leave enough of a margin for progress to be achieved, thus only children performing equivalent to or below 70% were included (equivalent to a maximum of 8 successful items out of 12 on FB and 4 successful items out of 6 on false complements). (iv) In addition, parents had to report that their child’s language comprehension was of the level to understand simple subject- verb-object sentences, which was subsequently conﬁrmed by experimenters upon the ﬁrst meeting during language tasks (Exalang et al., 2006), such that leading them up to complex sentences in a relatively short space of time could be feasible. (v) Finally, only children who could attend to pre-tests could proceed to training.1 Within each population, one half was assigned to the target-training program involving the teaching of sentential complements, while the other half was assigned to an alternative training program focussing on lexical enrichment. The latter group allowed us to conﬁrm that any eﬀects arising with complementation training were not due to general linguistic stimulation. This preliminary study involved small groups of participants for each population of children. Because our hypotheses were identical for all of these populations, we analyzed their data grouped together and focused on the type of training, and then conducted analyses to see whether the overall results were driven by any subgroup/speciﬁc population(s), i.e., whether progress in the TD and clinical groups were similar. November 2019 \| Volume 10 \| Article 2478 Frontiers in Psychology \| www.frontiersin.org Material and Procedure TABLE 2 \| Descriptive characteristics of participants. TD (n = 24) ASD (n = 16) DLD (n = 20) Chronological Age 4.32 (0.67) 8.28 (2.02) 6.92 (1.53) Raven 13.71 (4.72) 15.94 (15.94) 17.90 (4.54) Global morphosyntax 10.29 (2.29) 10.43 (2.06) 11.65 (2.16) Global lexicon 33.75 (3.17) 32.38 (8.31) 34.05 (2.72) TABLE 2 \| Descriptive characteristics of participants. material, so that children had to adjust their predictions depending on the changing epistemic state of the agent (Forgeot d’Arc and Ramus, 2011). We created three sets of tests, meaning that children who participated in the entire study saw a total of 108 diﬀerent items (54 FB items and 54 TB items) over the course of three testing sessions, and never saw the same item twice. The order of the items which made up each test was randomized and counterbalanced across participants. program lasted 4–6 weeks. One to two weeks after training ceased, immediate post-(training)-tests were administered to determine potential gains on abilities targeted by the programs, namely complements, ToM and lexicon. Again 4–6 weeks went by, this time without training, and another set of tests was administered, i.e., ‘follow-up’ or ‘delayed’ tests. These ‘follow-up’ tests were only conducted with children who had made progress of at least 10% between pre-tests and immediate post-tests in order to determine whether gains on complements and ToM could be maintained. Figure 1 outlines the overall experimental design of the study. program lasted 4–6 weeks. One to two weeks after training ceased, immediate post-(training)-tests were administered to determine potential gains on abilities targeted by the programs, namely complements, ToM and lexicon. Again 4–6 weeks went by, this time without training, and another set of tests was administered, i.e., ‘follow-up’ or ‘delayed’ tests. These ‘follow-up’ tests were only conducted with children who had made progress of at least 10% between pre-tests and immediate post-tests in order to determine whether gains on complements and ToM could be maintained. Figure 1 outlines the overall experimental design of the study. Theory of Mind Tests ToM was evaluated via a total of 12 FB items. These were interspersed with 12 TB items. Of the 12 FB items, 6 formed a verbal ToM task and 6 others a low-verbal one (again 6 true and 6 false beliefs). For each task, the child’s response always implied selecting one element amongst three, two involved in the scenario presented (corresponding to a true vs. Material and Procedure Pre-tests assessed a series of relevant measures, namely ToM (via verbal and low-verbal FB tasks as well as a mini-test of FB precursors), complements, lexicon, morphosyntax and non-verbal reasoning (see below for more details on these measures). One to two weeks after being tested for the ﬁrst time, participants were randomly assigned to one of the two training programs, i.e., either that of complements or lexicon. Each Amongst the target-training group, there were 21 boys and 9 girls and amongst the control-training group there were TABLE 1 \| Means (standard deviations) on paired variables at the moment of pre-test for the two groups (syntactic training, lexical training) and the three populations of children (TD, Typically Developing; ASD, Autism Spectrum Disorder; DLD, Developmental Language Disorder). TABLE 1 \| Means (standard deviations) on paired variables at the moment of pre-test for the two groups (syntactic training, lexical training) and the three populations of children (TD, Typically Developing; ASD, Autism Spectrum Disorder; DLD, Developmental Language Disorder). Syntactic training Lexical training T-tests results n = 30 n = 30 TD ASD DLD TD ASD DLD n = 12 n = 8 n = 10 n = 12 n = 8 n = 10 Chronological Age 5.92 (1.97) 6.57 (2.36) t(58) = 1.16, p = 0.25 4.29 7.52 6.58 4.35 9.04 7.25 Raven 15.53 (5.26) 15.87 (4.90) t(58) = 0.25, p = 0.80 12.92 16.25 18.10 14.50 15.63 17.70 Global morphosyntax 10.62 (2.54) 10.97 (1.94) t(56) = 0.58, p = 0.56 10.00 10.57 11.40 10.58 10.29 11.90 Global lexicon 34.2 (2.8) 32.8 (6.4) t(58) = 1.13, p = 0.27 33.8 34.4 34.5 33.7 30.4 33.6 False Complements/6 1.47 (1.50) 1.57 (1.48) t(58) = −0.26, p = 0.79 0.92 1.50 2.10 1.17 2.13 1.60 Verbal FB/6 0.63 (0.76) 0.93 (1.17) t(58) = −1.18, p = 0.25 0.50 0.75 0.70 0.90 1.50 0.70 Low-verbal FB/6 2.60 (1.90) 2.73 (2.18) t(58) = −0.25, p = 0.80 2.58 3.13 2.20 2.50 4.13 1.90 FB Precursors 4.77 (1.48) 4.69 (1.65) t(57) = 0.19, p = 0.85 4.25 5.38 4.90 3.50 5.88 5.22 ) on paired variables at the moment of pre-test for the two groups (syntactic training, lexical training) and the three populations of D, Autism Spectrum Disorder; DLD, Developmental Language Disorder). November 2019 \| Volume 10 \| Article 2478 Frontiers in Psychology \| www.frontiersin.org 4 The Impact of Grammar on Mentalizing Durrleman et al. Material and Procedure a false belief), while the third was unrelated to the story. The verbal ToM task was directly inspired by the Sally- Anne Task (Baron-Cohen et al., 1985). As explained above, in the FB scenarios of this task, the child is confronted with an object being moved from location 1 to location 2 in the main protagonist’s ignorance and has to capitalize on this protagonist’s false belief to predict that s/he will look for the desired object in location 1 (where it is no longer present). For example, one of the proposed scenes in our assessment was: “This is Bob. This is the mother. Bob has a yellow pot. The mother has a green pot. Bob has a ball, he puts the ball in his yellow pot. Bob is going out to play. The mother takes the ball out of the yellow pot and puts it in the green pot. Now Bob comes back, he wants to play with the ball. Where’s Bob going to get his ball?”. The child must then choose from three answers: the initial position (here the basket), the place where the object is actually located (here the box) and the position of the middle representing an object not involved in the story (here a bag). To succeed, children must take into account Bob’s misrepresentation while putting aside their own knowledge of reality. In the true belief, ﬁller scenarios, the displacement occurred in front of Bob (see Figure 2), or while he was absent the object was manipulated and returned to its original position. Pre- and Post-tests All tests, both pre and post, were conducted on laptop computers, to contrast with the materials used for the training itself, which was administered via iPads. This distinction between testing and training modalities allowed us to ascertain that any gains between pre and post-tests could not simply be attributed to increased familiarity with the material used during tests. Tests assessing ToM and sentential complements were speciﬁcally designed for the study, but followed the same basic format of tests which have been used in various previous studies, as explained in more details below. Administering ToM tests on a screen as opposed to having them administered by an experimenter held the promise of being the least penalizing option in particular for children with ASD (Chevallier et al., 2014). Each test of our target variables (i.e., complements and ToM) contained 36 items, which were all animated scenarios. Eighteen of these items involved FB, i.e., they corresponded to the test condition, while the other items involved true beliefs (TB). TB items cannot be taken as unambiguous ToM measures, as accurate responses coincide with reality responses (Dennett, 1978), however, they allowed varying the FIGURE 1 \| Experimental design. Frontiers in Psychology \| www.frontiersin.org 5 November 2019 \| Volume 10 \| Article 2478 FIGURE 1 \| E i t l d i November 2019 \| Volume 10 \| Article 2478 5 The Impact of Grammar on Mentalizing Durrleman et al. The low-verbal ToM evaluation was inspired by Woolfe et al. (2002), who claim that the task “minimize(s) verbal task- performance requirements.” This is because the pictures alone are informative enough for the child to both interpret the scene and to respond. As an illustration, images would appear on the screen clearly depicting someone blindfolded who was trying to obtain an object. This information was then also provided verbally in the form of commentary, which was thus not crucial for task success. For example (see Figure 3), in one scene there was a blindfolded man with a ﬁshing rod and seaweed covering the object at the end of his rod and the commentary went: “Look! The man is ﬁshing! He can’t see anything. Let’s see what is behind the seaweed - Click here!” All children understood and clicked, which made the seaweed move aside. In one scenario there was a ﬁsh, in another test there was a boot. FB Precursors If the participant met the inclusionary criteria deﬁned above after the main tests, we administered a mini-test evaluating the skills emerging before the ability to assign false beliefs, namely the understanding that people diﬀer from each other in their desires and beliefs (Wellman and Liu, 2004). There were a total of 6 items seen in each mini-test, 3 diverse desires and 3 diverse beliefs, such that children participating in the three testing sessions saw a total of 18 items assessing FB precursors. As an illustration of an assessment of diverse desires, the child saw an animated story while hearing the following narration: “What do you prefer: a carrot or a biscuit?” The child would then select one (usually the biscuit) and then see a small scene in which another character chooses the opposite, e.g., “Here is Theo. Theo prefers carrots. Theo is hungry. What do you think Theo will eat?”. For an assessment of diverse beliefs, the child heard: “This is Thomas’ book. Sometimes Thomas’ mother puts his book on the table, sometimes Thomas’ mother puts his book on the shelf. This is Thomas. Thomas is looking for his book. Where do you think the book is? On the shelf or on the table?” The child would then click on one, say the shelf, in which case the story would Pre- and Post-tests Then children were then presented with three objects and asked to click on the object the man was thinking about, in this instance selecting between a ﬁsh, a boot and a wheel. three options to select from, the voice said: “Look here: what is Dad saying that Jean is doing?” (see Figure 4). We pointed back to the picture and maintained the present tense in light of observations that past-tense can be diﬃcult for children with DLD (Rice and Wexler, 1996; Bishop, 2013) and ASD (Tager- Flusberg, 1989; Roberts et al., 2004). In the ﬁllers, the report and the event coincided (true complement) such that it suﬃced for children just to touch the only event evoked (e.g., the father says that Jean is eating ﬁsh and Jean is shown to indeed be eating ﬁsh). Control Training: Lexicon The control, lexical training was based on diﬀerent applications teaching the lexicon, namely Bitsboard, Flashcards, French FEL, Apprends-moi les mots (‘Teach me words”) and Animaux (“Animals”). Several themes are covered during the proposed exercises, such as colors, food, means of transport, animals, etc. At each session, we recorded the words learned and thus in subsequent sessions only checked these again before addressing the novel words. This training involved the same sort of demands as the target training, namely image designation, repetition, truth-value judgment, but also carefully steered away from mental state terms. Non-verbal Reasoning Raven’s matrices (Raven et al., 1998), were also administered during the pre-test to assess the child’s level of non-verbal reasoning. During this task, the child must complete 36 series of increasing diﬃculty. Each series is presented with a piece missing, which the child must select amidst six pieces. FIGURE 4 \| Illustration of the complements test. detail in Supplementary Appendix A. The ﬁrst activity, inspired by Wilson and Fox (2013), dealt with inﬁnitival complements such as: “Sophie sees a baby crying” which are the ﬁrst kind to be mastered (Bloom et al., 1989; Diessel, 2004). All other activities focused on tensed complements of communication verbs, such as: “The little girl screams that there is a spider in the bathtub” which are the kind speciﬁcally hypothesized to support ToM (de Villiers, 2007). Six sessions contained new material, composed of approximately 100 diﬀerent items. All children were presented with the entire material at least once, and some saw it a second time if the experimenters noticed they were still not excelling after 3 weeks. In this case, material from the beginning would start over. Target Training: Complementation For the training of complements, we administered a novel iPad application (Durrleman et al., 2016b), called DIRE, which means ‘to say’ in French. This name indicates that the program focuses mainly on the training of complements of verbs of communication (as well as some complements of verbs of perception or desire), thus abstracting away from verbs of mental state such as ‘think’ or ‘believe.’ DIRE also stands for ‘Diﬀerentiating Ideas from Reality via Exercises,’ since the purpose of the training oﬀered is to assist children with ToM diﬃculties to acquire these complements so that they may in turn apply them during ToM reasoning. We opted for iPad training, as such methods have already proven to be eﬀective with clinical populations (Alzrayer et al., 2014). Our training involved ﬁve types of activities, various using pictorial representations of speech, as previous work has found that visual cues are eﬀective in remediation programs with ASD (e.g., Wellman et al., 2002; Paynter and Peterson, 2013). Frontiers in Psychology \| www.frontiersin.org Language Finally, we evaluated the child’s language level using a test normed for children aged 3–6 years: EXALANG 3-6 tests (Helloin and Thibault, 2006). We opted for this task to assess receptive lexical skills (via the designation of images) and morphosyntactic (via the morphosyntax subtest) because its general format was very similar to our other tests for ToM and complements, namely they contained simple, computerized animations. Also, as mentioned earlier, belief attribution emerges generally around 4–5 years of age, i.e., along with general language skills corresponding to this age range, thus we reasoned that a language task for this age range would be appropriate for our sample, who were still struggling with FB. Training Training programs either focused speciﬁcally on complements (for the target training) or more generally on the lexicon (for the control training). Each involved ﬁve types of activities conducted on iPads, two to three times per week for maximum duration of 6 weeks, and a minimum duration of 3 weeks in the event that children already performed at ceiling at this point of the training program. Complements Test The evaluation of sentential complements was inspired by de Villiers and Pyers (2002). The general format involved one protagonist reporting an event to another, after which the actual event was shown. There were a total of 12 items, 6 test FB items and 6 TB items. In the test items, the complement reported an event inaccurately (false complement). The child had to simply recall the content of the erroneous complement uttered in the ﬁrst scene in order to score a point. An illustration would be: “The mother asks the father what Jean is doing. And the father answers that Jean is eating ﬁsh. Look! Jean is giving ﬁsh to the cat!” Then, pointing back to the picture of the parents now with FIGURE 2 \| Illustration of the verbal ToM task. FIGURE 3 \| Illustration of the low-verbal ToM task. FIGURE 2 \| Illustration of the verbal ToM task. FIGURE 2 \| Illustration of the verbal ToM task. FIGURE 3 \| Illustration of the low-verbal ToM task. FIGURE 3 \| Illustration of the low-verbal ToM task. November 2019 \| Volume 10 \| Article 2478 Frontiers in Psychology \| www.frontiersin.org 6 Durrleman et al. The Impact of Grammar on Mentalizing continue: “For Thomas, the book is on the table. Where will Thomas look for his book?”. continue: “For Thomas, the book is on the table. Where will Thomas look for his book?”. FIGURE 4 \| Illustration of the complements test. Question 1: Did the Target and Control Training Have Differential Effects? Our ﬁrst research question concerns whether the trainings had diﬀerential eﬀects, namely an improvement from pre-test to post-test speciﬁc to the type of intervention (descriptive data for the syntactic and lexical training are provided in Figure 5). Speciﬁcally, it is necessary to show that the syntactic training resulted in improvement on false complements, but the lexical training did not. Next, it is necessary to show that the syntactic training has eﬀects on False Belief performance, and that the lexical training did not. Third, it is necessary to show that the training was not restricted to the verbal false belief tasks, but applied equally to the verbal and low-verbal tasks. The ﬁve activities of DIRE were administered during each training session. The order of appearance of the activities was the same for all children, beginning with activity 1 and ending with activity 5. Each activity addressed a particular aspect of complementation via brief exercises, which are explained in Recall that the children were selected to have poor performance on complements and false belief, with the result that the data were non-normally distributed, as the distributions were truncated. To do ANOVAs, we tried using the Box-Cox transformation but homogenous variance and normal error November 2019 \| Volume 10 \| Article 2478 Frontiers in Psychology \| www.frontiersin.org 7 The Impact of Grammar on Mentalizing Durrleman et al. FIGURE 5 \| Scores on ToM and complements tasks at pre-test and post-test for the syntactic and lexical groups. FIGURE 5 \| Scores on ToM and complements tasks at pre-test and post-test for the syntactic and lexical groups. There was no prediction that these would be aﬀected by syntactic or lexical training, so a second set of analyses looked at the change in these variables compared to the variables targeted in Question 1. distribution could not be achieved due to this truncation at one end of the distributions. Therefore, on the variables that constituted selection criteria, namely false belief (verbal and low-verbal) and false complements, non-parametric tests were necessary. Wilcoxon signed rank tests were used in the analysis of pre- and post-training eﬀects on the false belief and complement measures for the diﬀerent training groups. Eﬀects sizes (using r, Rosenthal, 1994) are reported for the non-parametric test and regular Cohen’s d (Cohen, 1988) are reported for the parametric test. Question 1: Did the Target and Control Training Have Differential Effects? Using Cohen’s guidelines for r, a large eﬀect is 0.5, a medium eﬀect is 0.3, and a small eﬀect is 0.1 (Cohen, 1988). The Box-Cox transformations were applied ﬁrst to variables including precursors to false belief, true belief (verbal and low-verbal) and true complements, after which assumption of homogeneity of variance and normal distribution of residuals were met according to Levene’s tests and Shapiro’s tests. In order to assess if the target training had a speciﬁc eﬀect as compared to the control training on false complements and false belief attribution, both crucial to ToM, we ran factorial ANOVAs with the training group (syntax vs. lexical) and the moment of test (pre-test vs. post-test) as independent variables on the following dependent variables: true complements, verbal TB, low-verbal TB, and precursors to FB. The interaction eﬀect between the training group (syntactic vs. lexical training) and the moment of test (pre-test vs. post-test) was not signiﬁcant eﬀect for true complements [F(1,55) = 0.58, p = 0.45], verbal TB [F(1,58) = 2.47, p = 0.12], low-verbal TB [F(1,58) = 2.03, p = 0.16], and precursors to FB [F(1,57) = 0.18, p = 0.67]. In the syntactic training group, Wilcoxon’s signed rank test showed a statistically signiﬁcant diﬀerence between pre-test and post-test on Verbal False Belief (Pre-test: median = 0.50; post- test : median = 4.0; Z = −4.07, p < 0.001, r = 0.74), low-verbal False Belief (Pre-test: median = 2.0; post-test: median = 5.0; Z = −3.42, p < 0.001, r = 0.63), and False Complements (Pre- test: median = 1.0; post-test: median = 6.0; Z = −4.53, p < 0.001, r = 0.83) with large eﬀect sizes. However, one-way ANOVAs showed that there was no signiﬁcant progression between pre- test and post-test on FB precursors [F(1,58) = 2.89, p = 0.09], Verbal True Belief [F(1,58) = 1.08, p = 0.30], low-verbal true belief [F(1,58) = 2.60, p = 0.11], and true complements [F(1,58) = 3.20, p = 0.08], possibly due to already high scores on these variables (see Supplementary Appendix B). 2One child with ASD in the lexical training group displayed particularly poor performance in lexical designation in the pre-test, which could bias group results in the comparison between pre- and post-test. However, the progression of the “lexical training group” is still signiﬁcant even when removing this child from the analyses (Z = 2.4, p < 0.05). Note also that we proceeded to non-parametrical (Wilcoxon signed-rank) tests for these comparisons, due to ceiling eﬀects on this measure. 3A 10:1 ratio of cases to free parameters is suggested as a minimum sample size for SEM by Bentler and Chou (1987) and echoed by many others as a rule of thumb. Question 3: Are There Population Differences in the Effects of Training? post-test) on verbal false belief, low-verbal false belief and false complements for the three populations of children (TD, Typically Developing; ASD, Autism Spectrum Disorder; DLD, Developmental Language Disorder). and group as the independent variable. The untransformed data met the condition on homogeneity of variance by Levene’s test, and there was no signiﬁcant diﬀerence between the three groups on total post-FB performance after complement training [F(2,27) = 0.546, p = 0.586]. (pre-test vs. follow-up and post-test versus follow-up) for the dependent variables, on 22 children who had showed gains of at least 10% on the post-test in the syntactic training group. The progression between pre-test and follow-up test was statistically signiﬁcant for verbal FB (p = 0.001, r = 0.81), low-verbal FB (p = 0.003, r = 0.64), and false complements (p = 0.001, r = 0.88), with higher scores in follow-up. The mean scores were higher on immediate post-tests compared to follow-up post-tests, but the diﬀerence between these two post-tests was not signiﬁcant for verbal FB (p = 0.265, r = 0.035), and did not reach signiﬁcance for low-verbal FB (p = 0.066, r = 0.39) or false complements (p = 0.096, r = 0.36). Thus there was generally only a small drop between post-test and follow-up 4–6 weeks after the ﬁrst post-test (see Figure 7). Question 4: Did the Lexical Training Group Show Differential Results on the Lexicon? As for results on our standardized test of receptive lexicon, the syntactic training group did not show a diﬀerence between pre- and post-test scores (Z = 1.3, p = 0.2), whereas the lexical training group did (Z = 2.6, p < 0.01).2 For detailed information about participants and their individual results, see Supplementary Appendices C,D. Question 3: Are There Population Differences in the Effects of Training? Differences in the Effects of Training? In order to compare the eﬀects of the two trainings in the three populations, additional factorial ANOVAs were run (see Figure 6). This third ANOVA (time × training × clinical group) is exploratory, given the small sample sizes (approximately 10 children per condition). Results showed no interaction eﬀect between training group (syntax vs. lexical), moment of test (pre-test vs. post-test) and population (TD vs. ASD vs. DLD) for false complement [F(2,105) = 0.54, p = 0.59], Verbal FB [F(2,103) = 0.56, p = 0.57], low-verbal FB [F(2,105) = 0.02, p = 0.98], true complements [F(2,105) = 1.12, p = 0.33], Verbal TB [F(2,108) = 0.60, p = 0.55], non- verbal TB [F(2,108) = 0.77, p = 0.46), and precursors to FB [F(2,106) = 0.68, p = 0.51]. In the lexical group, one-way ANOVAs showed no signiﬁcant progression between pre-test and post-test on precursors of FB [F(1,56) = 1.46, p = 0.23], verbal True Belief [F(1,54) = 0.60, p = 0.44], low-verbal true belief (F < 1), or true complements [F(1,55) = 1.36, p = 0.25]. Non-parametric tests indicate no signiﬁcant changes occurred from pre-test to post-test in false complements (Z = −1.18, p = 0.24, r = 0.21), verbal False Belief (Z = −1.52, p = 0.13, r = 0.28), or low-verbal false belief (Z = −1.31, p = 0.50, r = 0.24). Question 2: Are There Training Effects on the ToM Skills Other Than False Belief? Question 2: Are There Training Effects on the ToM Skills Other Than False Belief? The children were tested also on True Complements, True Beliefs, and Precursors to False belief both pre and post training. The children were tested also on True Complements, True Beliefs, and Precursors to False belief both pre and post training. A one-way ANOVA was conducted on the complement training condition only, with total post FB as the outcome November 2019 \| Volume 10 \| Article 2478 Frontiers in Psychology \| www.frontiersin.org 8 The Impact of Grammar on Mentalizing Durrleman et al. FIGURE 6 \| Interaction between training (syntax vs. lexicon) and moment of test (pre-test vs. post-test) on verbal false belief, low-verbal false belief and false complements for the three populations of children (TD, Typically Developing; ASD, Autism Spectrum Disorder; DLD, Developmental Language Disorder). FIGURE 6 \| Interaction between training (syntax vs. lexicon) and moment of test (pre-test vs. We test six parameters with a sample of 60 participants. Though the 10:1 ratio is often considered safe, there are simulations (e.g., Nevitt and Hancock, 2004; Wolf et al., 2013) that suggest higher numbers are desirable in certain conditions. Latent variables would render the sample size insuﬃcient compared to the simple path model explored here. Missing data also would drastically increase the necessary sample size. An insuﬃcient sample size can result in a failure to converge on a model, or have poor ﬁt estimates. The current model does not show these problems, and is supported by the regressions, so we judge it to be a worthwhile addition despite the small sample. Question 5: Did the Training Result Persist Beyond Immediate Post-test? TABLE 3 \| Regression results predicting children’s total post-false belief (N = 60). A signiﬁcant B-weight indicates the beta-weight and semi-partial correlation are also signiﬁcant. B represents unstandardized regression weights. β indicates the standardized regression weights. sr2 represents the semi-partial correlation squared. r represents the zero-order correlation. LL and UL indicate the lower and upper limits of a conﬁdence interval, respectively. ∗p < 0.05. ∗∗p < 0.01. ∗∗∗p < 0.001. False Belief understanding contribute to the training eﬀect? What about non-verbal intelligence, as measured by Ravens, or the level of general language skill (using EXALANG)? And did the child’s trained mastery of false complements contribute to the false belief post-test score, or did some children succeed on post-test even if they did not improve on complements? groups. For that reason, the groups and populations could be collapsed to explore regressions with the outcome variable of false beliefs (combining low-verbal and verbal tasks into one 12- point score). The ﬁnal regressions contained just the variables that contributed unique variance to this outcome. As shown in Table 3, ﬁrst age was entered and then Raven’s matrices, the total of pre-training score on False belief (non-verbal and verbal combined), then Training condition, then the total post- training score on False complements, since both of these variables contributed to the outcome. groups. For that reason, the groups and populations could be collapsed to explore regressions with the outcome variable of false beliefs (combining low-verbal and verbal tasks into one 12- point score). The ﬁnal regressions contained just the variables that contributed unique variance to this outcome. As shown in Table 3, ﬁrst age was entered and then Raven’s matrices, the total of pre-training score on False belief (non-verbal and verbal combined), then Training condition, then the total post- training score on False complements, since both of these variables contributed to the outcome. The ANOVAs across training and control groups showed no diﬀerence across the TD, ASD and DLD populations in the outcome, nor any interactions between populations and training Having established the signiﬁcant variables in the regression for prediction of the outcome, various SEM models were tried to ﬁnd the model with the best ﬁt. Although the number of subjects is on the low side for a SEM, the ﬁt indices can give an indication of whether the sample has suﬃcient power to justify the model. Question 5: Did the Training Result Persist Beyond Immediate Post-test? More powerful statistics were used to explore the contributing eﬀects of the background variables and training conditions. Although certain criteria had to be met to be included in the study, it would be impossible with such a small sample to match the groups on every variable. Using multiple regressions ﬁrst to discover which variables share variance with the outcome, structural equation modeling (SEM) provided a powerful tool with which to look at the paths of inﬂuence on the outcome of false belief understanding.3 For example, did the initial level of In order to assess if the progression observed at post-test was still present between pre-test and follow-up test, we ran non- parametric comparisons using Wilcoxon matched-pairs signed- ranks tests with the moment of test as the repeated variable 3A 10:1 ratio of cases to free parameters is suggested as a minimum sample size for SEM by Bentler and Chou (1987) and echoed by many others as a rule of thumb. November 2019 \| Volume 10 \| Article 2478 Frontiers in Psychology \| www.frontiersin.org 9 The Impact of Grammar on Mentalizing Durrleman et al. FIGURE 7 \| Scores on verbal FB, low-verbal FB and false complements at the moment of pre-test, post-test, and follow-up. TABLE 3 \| Regression results predicting children’s total post-false belief (N = 60). Predictor B B 95% CI [LL, UL] SE B β sr2 r Fit Difference (Intercept) −0.78 [−3.74, 2.18] 1.48 Age −0.36 [−0.75, 0.04] 0.20 −0.19 0.03 −0.04 Raven’s Total 0.26∗∗ [0.09, 0.42] 0.08 0.31 0.08 0.26∗ Total Pre-False Belief 0.61∗∗ [0.28, 0.93] 0.16 0.35 0.12 0.30∗ Training 4.88∗∗∗ [3.38, 6.39] 0.75 0.60 0.35 0.58∗∗ R2 = 0.547∗∗ (Intercept) −1.08 [−3.65, 1.48] 1.28 Age −0.32 [−0.67, 0.02] 0.17 −0.17 0.02 −0.04 Raven’s Total 0.19∗ [0.04, 0.34] 0.07 0.23 0.04 0.26∗ Total Pre-False Belief 0.51∗∗∗ [0.22, 0.79] 0.14 0.30 0.08 0.30∗ Training 2.50∗∗ [0.81, 4.19] 0.84 0.31 0.05 0.58∗∗ Total Post-False Complements 0.79∗∗∗ [0.43, 1.15] 0.18 0.46 0.12 0.72∗∗ R2 = 0.667∗∗ 1R2 = 0.120∗∗ A signiﬁcant B-weight indicates the beta-weight and semi-partial correlation are also signiﬁcant. B represents unstandardized regression weights. β indicates the standardized regression weights. sr2 represents the semi-partial correlation squared. r represents the zero-order correlation. LL and UL indicate the lower and upper limits of a conﬁdence interval, respectively. ∗p < 0.05. ∗∗p < 0.01. ∗∗∗p < 0.001. TABLE 3 \| Regression results predicting children’s total post-false belief (N = 60). DISCUSSION Theory of Mind plays a fundamental role in social cognition (Harris, 2006), and an important step of ToM development occurs around the age of 4–5 years, when TD children begin to understand that others have beliefs that diﬀer from their own and may be in conﬂict with reality (Wellman et al., 2001). In contrast, marked delays in consolidating this step in mental reasoning can be observed in children with ASD (Yirmiya et al., 1998), and more subtle delays in children with DLD (Nilsson and de López, 2016). The overarching objective of this work was to identify an eﬃcient way to address ToM diﬃculties in ASD and DLD. y Amidst these clinical populations, the subset succeeding at tasks assessing false beliefs has been shown to display a better level of language, and in particular grammatical skills (ASD: Fisher et al., 2005; Paynter and Peterson, 2010; DLD: Farrar et al., 2009; Andrés-Roqueta et al., 2013). Some authors claim that mastery of ‘complement clauses’ such as ‘X thinks/ says that Y,’ would be the grammatical component par excellence facilitating belief reasoning (de Villiers, 2000, 2007), including in ASD and DLD (Tager-Flusberg and Joseph, 2005; Durrleman et al., 2016a, 2017a), because the content of the embedded sentence may refer to a subjective truth. In light of the fact the privileged links have been found between complements and belief reasoning, not only in TD (de Villiers and Pyers, 2002) but also in clinical populations including ASD and DLD (Farrar et al., 2017), our main objective with this work was to see if complementation training could yield similar gains across these populations. As such, our target program aimed to enhance complementation so as to boost ToM performance not only in TD, which has already been found, but also in ASD and DLD, which has never before been investigated. This training was compared to a control training, which promoted lexical enrichment. Our ﬁndings replicate the results for TD children in other languages to French indicating that syntactic training focussing on sentential complements improves both these structures as well as performance on false belief attribution in this population (Hale and Tager-Flusberg, 2003; Lohmann and Tomasello, 2003; Shuliang et al., 2014). Question 5: Did the Training Result Persist Beyond Immediate Post-test? Table 4 shows the results, and Figure 8 shows the optimum model result, with excellent ﬁt indices. November 2019 \| Volume 10 \| Article 2478 Frontiers in Psychology \| www.frontiersin.org 10 The Impact of Grammar on Mentalizing Durrleman et al. TABLE 4 \| Standardized parameter estimates for the hypothesized model (N = 60). B SE β p Path Analysis Raven’s Total →Total Post-FB 0.13∗ 0.06 0.16 0.040 Total Pre-False Belief →Total Post-FB 0.45∗∗∗ 0.13 0.27 0.001 Training →Total Post-FB (c) 2.63∗∗∗ 0.80 0.33 0.001 Total Post-False Complements →Total Post-FB (b) 0.80∗∗∗ 0.17 0.48 0.000 Age →Raven’s Total 1.05∗∗ 0.27 0.46 0.000 Age →Total Pre-False Belief 0.28∗ 0.14 0.26 0.038 Training →Total Post-False Complements (a) 3.00∗∗∗ 0.48 0.62 0.000 Indirect Effect a × b 2.38∗∗∗ 0.63 0.30 0.001 Total Effect c 5.01∗∗∗ 0.74 0.63 0.000 Standardized parameter estimation for the hypothesized model. All reported estimates are the maximum likelihood standardized point estimates. □2(7, N = 60) = 8.934, p = 0.257; comparative ﬁt index = 0.982; Tucker-Lewis index = 0.963, root mean square error of approximation = 0.068, standardized root mean square residual = 0.067. ∗p < 0.05. ∗∗p < 0.01. ∗∗∗p < 0.001. TABLE 4 \| Standardized parameter estimates for the hypothesized model (N = 60). DISCUSSION Moreover, children with DLD and ASD, who can display delays in both syntax and ToM (Yirmiya et al., 1998; Nilsson and de López, 2016), did not show any diﬀerence from the TD group regarding As background variables, the Raven’s score and the pre- test False belief score contributed to the ﬁnal outcome, but age did not have a direct eﬀect. Importantly, the training condition had a signiﬁcant eﬀect on both the False Belief outcome, and also on the children’s skill on the False complement post-task. That skill then contributed signiﬁcantly also to False belief. A further analysis asked whether the Training on False complements was instrumental in the outcome on False Belief via two paths: one direct, and the other with ﬁnal performance on False Complements as the mediating variable. The analysis revealed that there was indeed an additional mediating eﬀect of the false complements, as revealed in Table 4. Training has a highly signiﬁcant eﬀect on complements (a), which then has an eﬀect on False Beliefs (b). So the indirect eﬀect is a x b. In addition, Training has a direct eﬀect (c) on false beliefs, also highly signiﬁcant. FIGURE 8 \| Standardized parameter estimation for the hypothesized model. FIGURE 8 \| Standardized parameter estimation for the hypothesized model. 11 November 2019 \| Volume 10 \| Article 2478 Frontiers in Psychology \| www.frontiersin.org The Impact of Grammar on Mentalizing Durrleman et al. these positive outcomes. This ﬁrst attempt at explicit syntactic training with a group including these clinical populations is thus encouraging regarding the potential direct linguistic gains as well as the indirect cognitive beneﬁts, as measured by ToM tasks. argument structure of cognitive verbs, have children who develop false belief skills earlier (Tompkins et al., 2018). Much debate has arisen over whether the causal eﬀect is on the child’s own language, or directly in providing evidence for the theory the child is building about other minds (de Villiers and de Villiers, 2014). The model in this experimental study hints at a role for each eﬀect. The ToM beneﬁts associated with enhanced complementation skills were observed speciﬁcally for false belief reasoning, and not, e.g., for precursors of this ability such as the comprehension of diverse beliefs and desires (Wellman and Liu, 2004) or true belief items which can be resolved via reality responses. The interest of complementation appears indeed to support a speciﬁc component of ToM. DATA AVAILABILITY STATEMENT The datasets generated for this study are available on request to the corresponding author. DISCUSSION It is important to underline that improvement on FB was observed whether measured via verbal or low-verbal tasks. Performance on both of these measures was indeed correlated to complementation skills, suggesting that language supports not only FB-task performance, but also the reasoning implied in belief attribution (de Villiers, 2007). It is also interesting to note that both general grammatical skills and speciﬁc mastery of complementation relate to mentalizing abilities in typical development on post- tests, while atypical development appears to speciﬁcally capitalize on complementation, in line with previous reports (Farrar et al., 2017). This may suggest diﬀerent pathways to FB understanding, potentially related to diﬀerences between these populations to beneﬁt from social interactions (Farrar et al., 2017). Future work on the eﬀects of complementation training on ToM should seek to include larger cohorts of children with DLD and ASD, as well as other populations such as deaf children, who also show diﬃculties with both embedding (Tuller and Delage, 2014) and ToM (Peterson and Siegal, 2000). Subsequent studies should also seek to determine whether or not the gains are limited in scope (e.g., giving rise merely to verbal strategies for solving ToM tasks, see e.g., Leslie and Roth, 1993; Happé, 1995; Tager-Flusberg etal., 1997; Tager- Flusberg, 2000; Senju et al., 2010) or short-term, as these are important concerns, especially for children on the autism spectrum. It would thus be worthwhile to include a dimension of testing involving more ecological tasks, such as hide and seek, as well as parent questionnaires assessing the quality of the children’s social interactions, testing to be also conducted well after the intervention has ceased. This would allow a deeper understanding of whether children’s enhanced grasp of belief reasoning resulting from complementation training can give rise to more successful social skills, including in the long term. Such results could provide clearer arguments in favor of the beneﬁts of syntactic remediation in ToM programs across aetiologies. Encouragingly, improvements in complements and ToM were observed not only during immediate tests but also in follow-up post-tests, revealing that the training eﬀects were still detectible over time as revealed by higher performance on follow-up post-tests compared to pre-tests. FUNDING The training on complements had two eﬀects, one direct and one indirect via the improvement on children’s own complementation skills. That is, some component of the outcome variance was contributed by being exposed to an enhanced and carefully designed verbal input on complementation, and the other, by the child’s skill in producing correct answers to complementation questions. Evidence for both eﬀects is found in the developmental literature on false beliefs. Research has shown that families that engage in much discourse about mental states, which frequently is coupled with complements given the This work was supported by Swiss National Science Foundation (SNSF), grant number 100014_159606. This work was supported by Swiss National Science Foundation (SNSF), grant number 100014_159606. DISCUSSION Still, despite this durability in improvements between pre-tests and post-tests conducted roughly a month after training ceased, there was nevertheless a very modest drop in performance between immediate post- tests and follow-up post-tests, which could indicate that more training would be required for the results to be sustainable. None of the previous studies examining the eﬀects of complementation training in TD children included follow-up post-tests (Hale and Tager-Flusberg, 2003; Lohmann and Tomasello, 2003; Shuliang et al., 2014), thus more work is needed to determine whether or not this is speciﬁc to our cohort which included clinical children. AUTHOR CONTRIBUTIONS SD designed the study in collaboration with HD as well as JD and ET. SD, HD, and ET orchestrated recruiting and training. MB and RY analyzed data, with RY more speciﬁcally building structural equation models and ﬁne-tuning results. SD wrote the manuscript with the input from HD, JD, ET, and MB. While the children of all populations (TD, DLD, and ASD) who received the control training improved their lexical abilities, they did not improve skills on complements or ToM. The increase in performance observed in the target-training group on both complements and FB is therefore not achievable via just any linguistic stimulation, but rather stems from a speciﬁc training on complementation, here administered via DIRE. Frontiers in Psychology \| www.frontiersin.org REFERENCES The language-cognition interface in ASD: complement sentences and false belief reasoning. Res. Autism Spectrum Disord. 21, 109–120. doi: 10.1016/ j.rasd.2015.10.003 Astington, J. W., and Pelletier, J. (2005). “Theory of mind, language, and learning in the early years: developmental origins of school readiness,” in The development of social cognition and communication, eds B. D. Homer, and C. S. Tamis- Lemonda, (Mahwah, NJ: Erlbaum), 205–230. Durrleman, S., Da Costa, J., and Delage, H. (2016b). 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P., Tompkins, V., and Gage, N. A. (2017). Are there diﬀerent pathways to explicit false belief understanding? General language and complementation in typical and atypical children. Cogn. Dev. 43, 49–66. doi: 10.1016/j.cogdev.2017.02.005 Cheung, H., Hsuan-Chih, C., Creed, N., Ng, L., Wang, S. P., and Mo, L. (2004). Relative roles of general and complementation language in theory-of-mind development: evidence from cantonese and english. Child Dev. 75, 1155–1170. REFERENCES de Villiers, J., Hobbs, K., and Hollebrandse, B. (2014). “Recursive complements and propositional attitudes,” in Recursion: Complexity in Cognition. Heidelberg, eds M. Speas, and T. Roeper, (New York, NY: Springer), 221–242. doi: 10.1007/978- 3-319-05086-7_10 Alzrayer, N., Banda, D. R., and Koul, R. K. (2014). Use of iPad/iPods with individuals with autism and other developmental disabilities: a meta-analysis of communication interventions. Rev. J. Autism Dev. Disord. 1, 179–191. doi: 10.1007/s40489-014-0018-5 de Villiers, J. G., and Pyers, J. (2002). Complements to cognition: a longitudinal study of the relationship between complex syntax and false belief understanding. Cogn. Dev. 17, 1037–1060. doi: 10.1016/s0885-2014(02) 00073-4 American Psychiatric Association [APA], (2000). Diagnostic and Statistical Manual of Mental Disorders, 4th Edn. Washington, DC: American Psychiatric Association. de Villiers, P. A., Burns, F., and Pearson, B. Z. (2003). “The role of language in the theory of mind development of language-impaired children: complementing theories,” in Proceedings of the 27th Annual Boston University Conference on Language Development, eds B. Beachley, A. Brown, and F. Conlin, (Somerville, MA: Cascadilla Press), 232–242. Andrés-Roqueta, C., Adriàn, J. E., Clemente, R. A., and Katsos, N. (2013). Which are the best predictors of Theory of Mind delay in children with speciﬁc language impairment? Int. J. Lang. Commun. Disord. 48, 726–737. doi: 10.1111/ 1460-6984.12045 Dennett, D. (1978). Beliefs about beliefs. Behav. Brain Sci. 4, 568–570. Astington, J. W. (2003). “Sometimes necessary, never suﬃcient: false belief understanding and social competence,” in Individual Diﬀerences in Theory of Mind : Implications for Typical and Atypical Development, eds B. Repachol, and V. Slaughter, (New York, NY: Psychology Press), 13–38. Derksen, D. G., Hunsche, M. C., Giroux, M. E., Connolly, D. A., and Bernstein, D. M. (2018). A systematic review of theory of mind’s precursors and functions. Zeitschrift Psychol. 226, 87–97. doi: 10.1027/2151-2604/a000325 Diessel, H. (2004). The Acquisition of Complex Sentences. Cambridge: Cambridge University Press. Astington, J. W., and Edward, M. J. (2010). Language matters for theory of mind. Int. Soc. Study Behav. Dev. Newsletter 45, 7–9. Durrleman, S., Burnel, M., and Reboul, A. (2017a). Theory of mind in SLI revisited: links with syntax, comparisons with ASD. Int. J. Lang. Commun. Disord. 52, 816–830. doi: 10.1111/1460-6984.12317 Astington, J. W., and Jenkins, J. M. (1999). A longitudinal study of the relation between language and theory-of-mind development. Dev. Psychol. 35, 1311– 1320. doi: 10.1037/0012-1649.35.5.1311 Durrleman, S., Burnel, M., Thommen, E., Foudon, N., Sonié, S., Reboul, A., et al. (2016a). SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpsyg. 2019.02478/full#supplementary-material November 2019 \| Volume 10 \| Article 2478 12 The Impact of Grammar on Mentalizing Durrleman et al. REFERENCES doi: 10.1111/j.1467-8624.2004.00731.x Farrar, M. J., Johnson, B., Tompkins, V., Easters, M., Zilisi-Medus, A., and Benigno, J. P. (2009). Language and theory of mind in preschool children with speciﬁc language impairment. J. Commun. 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Frontiers in Psychology \| www.frontiersin.org November 2019 \| Volume 10 \| Article 2478 REFERENCES doi: 10.1017/s0140525x000 76512 November 2019 \| Volume 10 \| Article 2478 Frontiers in Psychology \| www.frontiersin.org 14 Durrleman et al. The Impact of Grammar on Mentalizing autism, individuals with mental retardation, and normally developing individuals. Psychol. Bull. 124, 283–307. doi: 10.1037/0033-2909.124. 3.283 Wellman, H. M., and Liu, D. (2004). Scaling of theory-of-mind task. Child Dev. 75, 523–541. doi: 10.1111/j.1467-8624.2004.00691.x j Wilson, M. S., and Fox, B. J. (2013). Language for Theory of Mind: Understanding Others’ Perceptions, Wants, and Needs. Winooski, VT: Laureate Learning Systems. Conﬂict of Interest: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Wimmer, H., and Perner, J. (1983). Beliefs about beliefs: representation and constraining function of wrong beliefs in young children’s understanding of deception. Cognition 13, 103–128. doi: 10.1016/0010-0277(83)90004-5 Copyright © 2019 Durrleman, Burnel, De Villiers, Thommen, Yan and Delage. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Wolf, E. J., Harrington, K. M., Clark, S. L., and Miller, M. W. (2013). Sample size requirements for structural equation models: an evaluation of power, bias, and solution propriety. Educ. Psychol. Measure. 73, 913–934. Woolfe, T., Want, S. C., and Siegal, M. (2002). Signposts to development: theory of mind in deaf children. Child Dev. 73, 768–778. doi: 10.1111/1467-8624.00437 g g p p y mind in deaf children. Child Dev. 73, 768–778. doi: 10.1111/1467-8624.00437 Yirmiya, N., Erel, O., Shaked, M., and Solomonica-Levi, D. (1998). Meta-analyses comparing theory of mind abilities of individuals with Yirmiya, N., Erel, O., Shaked, M., and Solomonica-Levi, D. (1998). Meta-analyses comparing theory of mind abilities of individuals with November 2019 \| Volume 10 \| Article 2478 Frontiers in Psychology \| www.frontiersin.org 15
https://openalex.org/W4391025141	https://www.microbiologyresearch.org/deliver/fulltext/acmi/10.1099/acmi.0.000689.v1/acmi.0.000689.v1.1.pdf?itemId=/content/reviewreports/10.1099/acmi.0.000689.v1.1&mimeType=pdf	English	null	Antimicrobial resistance and molecular characteristics of N. gonorrhoea isolates in Ghana	null	2,024	cc-by	3,312	How to cite the SciScore reports for this article: Musah HS, Addy F, Dufailu OA. SciScore reports for: Antimicrobial resistance and molecular characteristics of N. gonorrhoea isolates in Ghana. Access Microbiology. 2023. https://doi.org/10.1099/acmi.0.000689.v1.1 Musah HS, Addy F, Dufailu OA. SciScore reports for: Antimicrobial resistance and molecular characteristics of N. gonorrhoea isolates in Ghana. Access Microbiology. 2023. https://doi.org/10.1099/acmi.0.000689.v1.1 Musah HS, Addy F, Dufailu OA. SciScore reports for: Antimicrobial resistance and molecul isolates in Ghana. 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If SciScore makes any mistakes, please contact us to help us learn and improve. Table 1: Rigor Adherence Table Ethics not detected. Inclusion and Exclusion Criteria not required. Attrition not required. Sex as a biological variable not detected. Subject Demographics Age: not detected. Weight: not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Replication not required. Data Information Availability: Data Availability statement: Molecular data has been deposited on Genbank with accompanying accession numbers as stated earlier. Table 1: Rigor Adherence Table Table 1: Rigor Adherence Table Table 2: Key Resources Table Your Sentences REAGENT or RESOURCE SOURCE IDENTIFIER Experimental Models: Organisms/Strains Briefly , gonococcal DNA sequences were trimmed at consensus regions starting from nucleotide ( nt ) 445 in reference to N . gonorrhoea strain MS11 to yield 490 base pair ( bp ) for porB and for tbpB , from nt 1118 reference to strain UU1008 to yield 390 bp . nt 1118 Software and Algorithms Ltd , South Africa using Sanger sequencing technology on an ABI Genetic Analyser 3500XL ( Thermo Fisher Scientific® , UK) . Thermo Fisher Scientific® Suggestion: (Thermo Fisher Scientific, RRID:SCR_008452)( link) MEGA X MEGA X software was employed to align sequences using ClustalW and to draw the phylogenetic tree using the maximum composite likelihood model while employing 1000 bootstrap replications to test the confidence of the tree. ClustalW Suggestion: (ClustalW, RRID:SCR_017277)( link) Table 2: Key Resources Table SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore is not a substitute for expert review. SciScore also checks for the presence and correctness of several unique identifiers, including RRIDs (research resource identifiers) in the manuscript, detects sentences that appear to be missing RRIDs, and can even suggest RRIDs under certain circumstances. All RRID suggestions should be verified; only the author can know whether the suggestions are correct. SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore is not a substitute for expert review. 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Please refer to the MDAR Elaboration Document for additional context for the MDAR framework. rovided by SciScore \| August 12, 2023 \| Based on DocumentID 2281_64d790b6422720.494476 Materials Materials Antibodies Yes (indicate where provided: page no/section/legend) n/a For commercial reagents, provide supplier name, catalogue number and RRID, if available No antibodes detected. Please add identifiers for all resources where possible Cell Materials Yes (indicate where provided: page no/section/legend) n/a Cell lines: Provide species information, strain. Provide accession number in repository OR supplier name, catalog number, clone number, OR RRID No cell lines detected Please add identifiers for all resources where possible Primary cultures: Provide species, strain, sex of origin, genetic modification status. 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Data availability rovided by SciScore \| August 12, 2023 \| Based on DocumentID 2281_64d790b6422720.494476 Analysis Adherence to community standards Yes (indicate where provided: page no/section/legend) n/a MDAR framework recommends adoption of discipline-specific guidelines, established and endorsed through community initiatives. Journals have their own policy about requiring specific guidelines and recommendations to complement MDAR. State if relevant guidelines (eg., ICMJE, MIBBI, ARRIVE) have been followed, and whether a checklist (eg., CONSORT, PRISMA, ARRIVE) is provided with the manuscript. Not currently checked by SciScore Report provided by SciScore \| August 12, 2023 \| Based on DocumentID 2281_64d790b6422720.49447682 Adherence to community standards rovided by SciScore \| August 12, 2023 \| Based on DocumentID 2281_64d790b6422720.494476 ACMI-S-23-00255.pdf By Haris Musah WORD COUNT 5745 5 5 4 5 14 14 5 14 14 5 12 20 28 1 26 1 1 1 8 8 8 8 1 8 15 1 30 1 1 1 1 2 15 1 1 1 2 9 11 16 9 11 16 9 11 16 10 19 19 19 10 10 9 13 22 9 13 22 9 13 22 9 13 22 27 27 27 3 3 7 7 7 21 29 31 17 18 25 25 23 23 1 1 12 24 24 1 1 6 32 6 32 6 32 ACMI-S-23-00255.pdf ORIGINALITY REPORT PRIMARY SOURCES 24 words — 1% 5 24 words — 1% 5 www.science.gov Internet www.tandfonline.com 4 Iddrisu Baba Yabasin, Mohammed Mohammed Ibrahim, Abass Adam, Sam-Awortwi Wilfred et al. 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https://openalex.org/W4232238438	https://ris.utwente.nl/ws/files/254107226/Cinar_2019_Delving_into_social_entrepreneurship_in_universities_is_it_legitimate_yet.pdf	English	null	Delving into social entrepreneurship in universities: is it legitimate yet?	null	2,021	cc-by	9,669	Regional Studies, Regional Science ISSN: (Print) 2168-1376 (Online) Journal homepage: https://www.tandfonline.com/loi/rsrs20 ISSN: (Print) 2168-1376 (Online) Journal homepage: https://www.tandfonline.com/loi/rsrs20 Delving into social entrepreneurship in universities: is it legitimate yet? Ridvan Cinar ARTICLE HISTORY Received 29 September 2018; Accepted 12 February 2019 social entrepreneurship; universities; new institutionalism; legitimacy; place-based belonging; third missio social entrepreneurship; universities; new institutionalism; legitimacy; place-based belonging; third mission JEL CLASSIFICATIONS I23; L31; D23 JEL CLASSIFICATIONS I23; L31; D23 JEL CLASSIFICATIONS I23; L31; D23 INTRODUCTION There is an increasing awareness regarding the emergence of a class of large-scale problems chal- lenging contemporary society, such as climate change, inefﬁcient use and lack of resources, urban sustainability, and democratic security, that require concerted and coordinated action from a range of partners. Recognizing the limitations of both the public and private sectors to produce mass, coordinated and ﬂexible responses, there is growing interest in new forms of coordinating mechanisms that allow for solutions to these societal challenges to be developed. Social Delving into social entrepreneurship in universities: is it legitimate yet? Ridvan Cinar ABSTRACT Universities have recently been pressurized to go beyond their economic conceptualization of third-mission activities and contribute to solving grand societal challenges in the regions in which they are located. Social entrepreneurship has emerged as one mechanism by which universities can address societal challenges. Despite a growing awareness of universities’ potential and expectations to enhance social entrepreneurship in their geographical vicinities, how these processes become legitimized within a higher education context has received surprisingly little attention. This paper, therefore, explores factors affecting the (de)legitimacy process of social entrepreneurship within universities. Using a single case study design that relies on semi-structured interviews carried out in a Dutch public university, it was found that organizational legitimacy of social entrepreneurship remains unestablished. Furthermore, the legitimacy process is affected by (1) the expectations of stakeholders, the difﬁculty of measuring social impact and third-mission indicators; (2) an overemphasis on high-tech research and application as an organizational identity; (3) the absence of a leader in the ﬁeld and lack of organizational recognition; and (4) stringent regulations of public institutions in the Netherlands. In addition, enhancing social entrepreneurs is hindered by the lack of place-based belonging among the student body. Consequently, this paper argues that a holistic approach that focuses on the speciﬁcities of universities and the increasing competitive environment in which they have come to function, the potential facilitating role of other organizational actors in the ﬁeld, and designing appropriate policy instruments and incentives would beneﬁt universities in their efforts to enhance social entrepreneurship. Ridvan Cinar To cite this article: Ridvan Cinar (2019) Delving into social entrepreneurship in universities: is it legitimate yet?, Regional Studies, Regional Science, 6:1, 217-232, DOI: 10.1080/21681376.2019.1583602 To link to this article: https://doi.org/10.1080/21681376.2019.1583602 © 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group Published online: 08 May 2019. Submit your article to this journal View Crossmark data Citing articles: 1 View citing articles Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=rsrs20 REGIONAL STUDIES, REGIONAL SCIENCE 2019, VOL. 6, NO. 1, 217–232 https://doi.org/10.1080/21681376.2019.1583602 CONTACT (Corresponding author) r Department of Social Political and Territorial Sciences, University of Aveiro, Aveiro, Portugal. © 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group 218 Ridvan Cinar entrepreneurship has been acknowledged as one such potential mechanism where actors create new forms of social organizations, structures and institutions that help solve these challenges (Dees, 1998; Nicholls, 2006). Social entrepreneurship has recently garnered the interest of policy-makers, particularly in Europe. In its Entrepreneurship 2020 Action Plan, the European Commission (EC) stressed the signiﬁcance of social entrepreneurship as follows: ‘We must work on ensuring that being an entrepreneur is an attractive prospect for Europeans. This also includes social entrepreneurs whose potential is often underestimated’ (European Commission, 2013, p. 5). The action plan and successive policy briefs as well as ofﬁcial statements have called for a greater contri- bution of universities to enhance social entrepreneurship. Therefore, it is surprising that whilst universities are expected to make substantive contributions to social entrepreneurship, there is almost no consideration of the way these processes function within the context of higher education. The key issue for social entrepreneurship in universities is the lack of a clear connection to the core tasks of the university. In more traditional technology venturing processes, university knowl- edge resources are converted in terms of exploitable assets that may ultimately generate revenue streams, thereby legitimizing university entrepreneurship activities. However, social entrepre- neurship processes typically involve changes in social processes and organization, not necessarily in monetized settings, beneﬁting the poorer segments of society, unable to provide formal returns to the university knowledge input. This raises the question of how universities legitimize their engagement in social entrepreneurship, particularly in terms of choices regarding which social challenges, societal partners and beneﬁciaries. Suchman (1995, p. 574) posits that ‘legitimacy is a generalized perception or assumption that the actions of an entity are desirable, proper or appropriate within some socially constructed sys- tem of norms, values, beliefs and deﬁnitions’. CONTACT Institutions shape the behaviour of their members by delineating what is expected of them in numerous social circumstances, the foundations of which lie on constructing values and beliefs over time in order to elicit and shape proper actions (Scott, 2008). Having stated that, it is critical to explore whether contributions to social entre- preneurship have manifested themselves as one of the anticipated proper actions in universities, thereby shedding a light onto its (de)legitimacy. Accordingly, this paper will primarily seek to address the overall research question: Which factors affect the legitimacy processes of social entrepreneurship within universities? The paper is structured as follows. First, it reﬂects on the two main strands of social entre- preneurship drawn from the literature and provides a brief account of organizational legiti- macy. Second, it focuses on the universities’ contributions to social entrepreneurship as a third-mission activity and argues that they seek legitimacy in three phases. Third, it outlines in detail the data and methods. Fourth, it presents the ﬁndings, elaborating on factors affect- ing the legitimacy of social entrepreneurship within a public higher education institution: the University of Twente (UT), the Netherlands, in this case. Lastly, it discusses the ﬁndings and concludes with paving the way for new research avenues and implications on academics and policy-makers alike. REGIONAL STUDIES, REGIONAL SCIENCE SOCIAL ENTREPRENEURSHIP: A CONCEPTUAL BACKDROP The term ‘social entrepreneurship’ ﬁrst appeared in the social change literature in the 1960s and 1970s (Gray, 2012), a period when the demand for change was great across the world and reso- nated considerably with individuals. The 1990s witnessed a theory-building process for social entrepreneurship that paved the way for its gaining momentum in scholarly literature, and raised its popularity in the policy sphere (Dees, 1998; Leadbeater, 1997). In spite of the fact that ﬁrms that intended to help solve socioeconomic challenges sowed the ﬁrst seeds of the social REGIONAL STUDIES, REGIONAL SCIENCE Delving into social entrepreneurship in universities: is it legitimate yet? 219 entrepreneurship literature (Shintani, 2011), the concept, as well as the literature, has evolved to encompass civil–society initiatives in the form of non-governmental organizations, not-for-proﬁt organizations, community projects, cooperatives and social enterprises (Cunha, Benneworth, & Oliveira, 2015; Dees, 1998; Seelos & Mair, 2005). Various perspectives from several scholarly disciplines, such as business administration, management, sociology, development studies and economics, have led to social entrepreneurship becoming what Choi and Majumdar (2014, p. 365) characterize as ‘an essentially contested concept’. Whereas some researchers regard social entrepreneurship as the establishment of businesses to beneﬁt the poorer segment of societies (Seelos & Mair, 2005), others view it as a means of harnessing social innovations to generate sol- utions for social problems and thus trigger social change, regardless of whether or not they entail commercial activities (Dees, 1998). Among a variety of descriptions, this paper adheres to Dees’ (1998, p. 4) deﬁnition of a social entrepreneur as it corresponds to the contextual setting (legit- imization of social entrepreneurship in a public university) of this research: Social entrepreneurs play the role of change agents, in the social sector, by; adopting a mission to create and sustain social value (not just private value); recognizing and relentlessly pursuing new opportunities to serve that mission; engaging in a process of continuous innovation, adaptation and learning; acting boldly without being limited by resources currently in hand; and exhibiting heightened accountability to the con- stituencies served and outcomes created. From this perspective, social entrepreneurs can bring about small-scale changes (economic, cultural and/or political) that echo within the dominant system of structures and trigger large-scale changes in the long run. LEGITIMACY IN UNIVERSITIES Institutional theory has taken the academic stage since Selznick (1957) deﬁned institutionaliza- tion as a process where practices are imbued with value, overshadowing the technical require- ments of the tasks involved in an organization. Selznick’s works regarding the institutionalization process laid the foundations for old institutionalism. As a reaction to the great emphasis put upon the internal dynamics within an organization, a new strand in insti- tutional theory was established that directed its focus from the internal dynamics to the external environment. This new strand, traditionally depicted as new institutionalism, underlined the sig- niﬁcance of shifting the focus to ‘examine[ing] how the external environment socially construct[s] organizations, providing them with templates, for their formal structures and policies, and thereby increasing an organization’s legitimacy in the wider world’ (Powell & Bromley, 2015, p. 764). DiMaggio and Powell (1991, p. 8) provided the new strand of institutional theory with the following deﬁnition: The new institutionalism in organization theory and sociology comprises a rejection of rational-actor models, an interest in institution as independent variables, a turn toward cognitive and cultural expla- nations, and an interest in properties of supra-individual units of analysis that cannot be reduced to aggre- gations or direct consequences of individuals’ attributes or motives. A stream of research emerging out of developments in the new institutional theory is legitimacy. It is generally accepted that Max Weber introduced the concept (Johnson, Dowd, & Ridgeway, 2006). Legitimacy is one means by which universities inaugurate congruence between the norms and values of their behaviours and the expected proper actions of the broader social system in which they are embedded (Deephouse & Suchman, 2008). Maurer (1971, p. 361) argues that ‘legitimation is the process whereby an organization justiﬁes to a peer or superordinate system its right to exist, that is, to continue to import, transform and export energy, material or information’. The organizational legitimacy is threatened when there is a prospect of disparity between the two systems of values, and consequently, the organizations either undergo required changes to harmonize their values with the subordinate system of which they are a part (Boxenbaum & Jons- son, 2008), or strive to alter the value of that particular subordinate system itself (Dowling & Pfeffer, 1975). SOCIAL ENTREPRENEURSHIP: A CONCEPTUAL BACKDROP However, they need to be familiar with the broader social system, its spatial characteristics and the actors involved in order to concentrate on the allevia- tion of the roots of social challenges (Alvord, Brown, & Letts, 2004). It can be argued that this process would require some sort of place-based belonging, particularly for those who are new to any social system, such as university students who hail from different cities and countries. Place-based belonging is a process whereby an individual’s attachment to a place is constructed through socio-spatial practices, such as participating in local events, relaxing in a park, etc. (Benson & Jackson, 2012), thus creating memories and meaning surrounding the place (Scan- nell & Gifford, 2010). As such, it is important that potential social entrepreneurs develop place-based belonging if they are to become agents of sustainable, social transformation, especially on a local scale. The road to sustainable social transformation is not unimpeded and linear, regardless of it being oriented locally or globally. Social entrepreneurs face numerous obstacles ranging from pol- itical and economic to cultural and socio-technical. They might overcome the challenges by mobilizing different stakeholders in the process, empowering the people involved and resorting to public, private and non-governmental organizations to use further the expertise required. Uni- versities are regarded as organizations that have made prominent contributions to the regions and societies in which they are embedded (Arbo & Benneworth, 2007), and are understood as pos- sessing the potential to take up the role of being an agent of change in the process of societal transformation, given the knowledge and human resources they contain. Yet, it is surprising that there is almost no consideration of how universities can become an agent of the process of sustainable social transformation, when their members attempt to act accordingly by enhan- cing social entrepreneurship. As such, it is essential to uncover how social entrepreneurship becomes legitimized (or not) within a higher education context, and how universities can foster social entrepreneurs that will seek effective changes in social systems among the immense, highly qualiﬁed, human resources they hold. The new institutional theory, therefore, provides a valuable conceptual lens that helps address the process of organizational legitimacy in a higher education institution. REGIONAL STUDIES, REGIONAL SCIENCE Ridvan Cinar 220 REGIONAL STUDIES, REGIONAL SCIENCE LEGITIMACY IN UNIVERSITIES However, Perrow (1970) notes that inasmuch as changing social values in a super- ordinate system is generally an arduous and risky journey to undertake, organizations are more likely to prefer adapting to the social system for legitimacy. This preference may have several con- sequences such as constraints on organizational behaviour. More speciﬁcally, when organiz- ational practices do not conform to social norms and the values of the broader social system, these practices cease to exist (Dowling & Pfeffer, 1975). Universities have sought legitimacy in numerous areas to be relevant for the social system in which they are embedded and to sustain their right to exist (O’Kane, Mangematin, Geoghegan, & Fitzgerald, 2015). The seeking and establishment of the process of legitimacy is manifest mostly through the practices imbued in third-mission activities, particularly when universities aspire to substantiate that they can collaborate with external stakeholders and contribute to addressing the wicked, global issues (Benneworth & Cunha, 2015). Third-mission activities – occasionally referred to as ‘third stream’ – correspond to links universities establish with actors of external environments such as ﬁrms, government bodies, the public, social enterprises and non-governmental organizations, in addition to their ﬁrst and second missions: teaching and research (Laredo, 2007). Earlier understandings of the third mission centred around commercia- lization activities, which led to a wave of technology transfer ofﬁces in the 1980s and 1990s. REGIONAL STUDIES, REGIONAL SCIENCE Delving into social entrepreneurship in universities: is it legitimate yet? 221 However, recently universities are being expected to go beyond pure commercialization activities, in their understanding of the third mission, and adopt a more developmental role by reaching out to civil society (Goddard, Hazelkorn, Kempton, & Vallance, 2016; Uyarra, 2010). However, recently universities are being expected to go beyond pure commercialization activities, in their understanding of the third mission, and adopt a more developmental role by reaching out to civil society (Goddard, Hazelkorn, Kempton, & Vallance, 2016; Uyarra, 2010). The contribution to social entrepreneurship can be characterized as one of the many third- mission activities through which universities can reach out civil society. Nevertheless, if social entrepreneurship is to be enhanced, the establishment of an organizational legitimacy is necess- ary. These processes require close monitoring of internal behaviours and external values. Scott (2008) has identiﬁed three institutional pillars, namely: regulative, normative and cultural–cog- nitive. LEGITIMACY IN UNIVERSITIES Whereas the regulative pillar entails setting rules, monitoring members’ conformity and, if necessary, a sanction/rewarding of them, the normative pillar elicits proper actions through norms and values. The cultural–cognitive pillar focuses on the cognitive dimension of an individ- ual’s perception of his/her environment, articulated as ‘what a creature does is in large part a func- tion of the creature’s internal representation of its environment’ (D’Andarade, 1984, p. 88). The basis of legitimacy for each pillar is ‘legally sanctioned’ (regulative), ‘morally governed’ (norma- tive) and ‘comprehensible, recognizable, culturally supported’ behaviours (Scott, 2008, p. 60). Based on the three pillar-framework of institutions, it is argued here that the legitimacy in uni- versities occurs in three phases, particularly if the normative or cultural–cognitive pillars are the dominant models inside. Phase 2: Building the legitimacy process Upon the identiﬁcation of nonconformity in the two value systems, and the taking of a decision preferring the implementation of the former option, universities communicate the argument for adaptation to their members. Furthermore, they initiate organizational arrangements, which would allow the members to exercise the proper actions anticipated of them. In other words, organizational change commences. Phase 1: Assessment of organizational values vis-à-vis the norms of a superordinate social system In this phase, universities closely monitor the changing values and external expectations of the broader system in which they participate. They engage with external stakeholders and map out the potential change in the discourse of values and expectations. Further, they try to identify if there is congruence between the two value systems. If not, universities either prefer to adapt and incorporate the changing values (starting the organizational legitimacy process) or attempt to alter the values of the superordinate social system (promulgating their own legitimacy process). When universities prefer the former, the second phase is put in motion. As for the latter option, there is a leap directly towards the third phase. Phase 3: Completion of organizational change and the dissemination of the legitimacy process This phase involves the ﬁnalization of a series of internal arrangements in universities regarding the adaptation to the changing external environment and value systems. Organizational change is completed and the legitimacy process, to a large extent, is established. There is one last step mov- ing forward; the dissemination of this process to external stakeholders. Universities convey the message that they can adapt to changing values and expectations and that they have taken the necessary steps to do so via several means of communication. In case of the latter, universities do not necessarily undergo a process of organizational change; rather, they disseminate the sig- niﬁcance of their values to alter the superordinate social system and its expectations. In this scen- ario, legitimacy is completed when external stakeholders ﬁnally recognize the values of universities and their relevance in the broader social system. REGIONAL STUDIES, REGIONAL SCIENCE REGIONAL STUDIES, REGIONAL SCIENCE Ridvan Cinar 222 DATA AND METHODS The research reported here has adopted a qualitative exploratory case study design. Case studies are necessary when the intention of the research is to explore a certain phenomenon on a deeper level and gain a better understanding of its ramiﬁcations (Stake, 1995). The University of Twente (UT), a public university in the Netherlands, was selected as a case. What makes the UT an inter- esting case is its long commitment to the development of its region since its establishment and efforts to reach out to society. Realizing the prevalence of grand societal challenges, particularly in the last decade, the UT has been searching for ways to mobilize its resources when tackling these challenges. It is the serious commitment and dynamism that has been going on there for some time and its targeted contributions to the socioeconomic development of its geographical vicin- ities that facilitates the UT to be dwelled on as a case study. To gather accurate and insightful data, criterion sampling was administered. In this regard, the research had two prearranged criteria in selecting the samples: . Participants would have experience (academic, managerial or practical level) in either social entrepreneurship or higher education, or both. Academic experience refers to publications on social entrepreneurship and/or higher education studies, while managerial and practical experience corresponds to being part of the university management team and/or being social entrepreneurs/leading social entrepreneurship initiatives, respectively. Moreover, the focus of social entrepreneurship initiatives range from refugee entrepreneurship to cost-effective organic farming and the reduction of the generational divide in citizen engagement. . Participants would have been working or studying in the UT for at least one full academic year. The reason being that this might allow individuals to observe the institutional environment for both semesters, become familiar with organizational culture and thus make better judgments. An interview document, which consisted of 13 semi-structured questions, was sent to two experts (academics) in the ﬁeld. Upon receiving feedback, the interview document was revised, and the potential informants were contacted. Seventeen interviews with individuals such as aca- demics, executive board members and social entrepreneurs (academics/students) were conducted between December 2017 and January 2018. Descriptive information regarding the interviews and interviewee characteristics are outlined in Table 1. Each interview was tailored to the professional background and experience of the participant. Interviews were recorded, transcribed and analyzed thematically (Fereday & Muir-Cochrane, 2006). THE CASE: UNIVERSITY OF TWENTE IN THE TWENTE REGION, THE NETHERLANDS The Twente region is an area in the eastern Netherlands, part of Overijssel province, bordering Germany. It comprises of 14 municipalities with Enschede, Hengelo and Almelo being the Table 1. Interviews and interviewee characteristics. Informant characteristics Academic staff: 9; administrative staff: 6; students: 2 Number of years in the University of Twente 1–4 years: 4; 5–9 years: 6; 10–19 years: 3; 20–29 years: 2; ≥30 years: 2 Relevant experience Academic: 7; managerial: 5; practical: 5 Interview duration Minimum: 37 min; maximum: 73 min Gender Male: 12; female: 5 Table 1. Interviews and interviewee characteristics. Informant characteristics Academic staff: 9; administrative staff: 6; students: 2 Number of years in the University of Twente 1–4 years: 4; 5–9 years: 6; 10–19 years: 3; 20–29 years: 2; ≥30 years: 2 Relevant experience Academic: 7; managerial: 5; practical: 5 Interview duration Minimum: 37 min; maximum: 73 min Gender Male: 12; female: 5 REGIONAL STUDIES, REGIONAL SCIENCE Delving into social entrepreneurship in universities: is it legitimate yet? 223 major cities with a considerable size of rural hinterland. It hosts 3.6% of the Dutch population (approximately 626,500 inhabitants). Since the demise of the textile industry in the 1960s, the unemployment rate in the region has generally been higher than the national average. Consider- ing all the relevant socioeconomic indicators, Twente can be characterized as a peripheral region (Benneworth & Pinheiro, 2017) (Figure 1). Established in the 1961, post the decline of textile industry, the UT is one of the four tech- nical universities in the country. Located in Enschede in Overijssel province, the UT originally set off on its journey with the hope of contributing to its region economically. Starting off as the Technische Hogeschol Twente, primarily a technical applied university, it later gained full uni- versity status around the mid-1980s. Beginning the journey with an entrepreneurial mindset, the UT placed paramount importance on the valorization of knowledge, generating over 1000 spin- off companies since 1984, thereby contributing to the socioeconomic development of the Twente region, mostly, but not limited to, the cities of Enschede and Hengelo. It employs 3074 academic and administrative staff (including doctoral students employed by the university) and enrols 10,435 students as of 2017. Expectation of external stakeholders, difﬁculty in measuring social impact and third-mission indicators Five primary actors constitute the superordinate social system of the UT: the cities of Enschede and Hengelo, the Twente region with its administrative bodies – Regio Twente, Overijssel pro- vince, the Dutch government and the European Commission. Among these, the municipality of Enschede, the Twente region and the Dutch government can be considered as stakeholders, forming the biggest segment of the UT’s superordinate social system. These actors expect the UT to contribute to its region socially and economically; however, they deem these outputs mea- surable. More speciﬁcally, they expect a short-term and immediate outcome of the UT’s actions that is quantiﬁable in socioeconomic terms, as perceived by the UT members. The narrative of an academic staff mirrors the commonly shared perception in the UT: ‘Everybody outside, the municipality (Enschede), the province or the government. … They want something tangible. Something they can see and that can be observed in numbers like jobs, start-ups or money gen- erated’ (Academic staff, 2). In addition, indicators aiming to evaluate third-mission activities of higher education insti- tutions are rather biased towards economic and quantiﬁable outputs such as the number of licences granted, number of patent applications, spin-offs, research contracts and companies in incubators, revenue of external income, etc. The indicators certainly undermine the impact of certain disciplines, particularly in the social sciences and humanities. For instance, indicators value the amount of income a university receives whilst conducting research, but some disciplines within the social sciences and humanities are less costly compared with life and engineering sciences as they may require less equipment. This reinforces the perception that the level of income mirrors impact, which can be highly misleading (Rossi & Rosli, 2014). The indicators not only fail in reﬂecting the full picture of third-mission activities but also lead to the incenti- vization of certain types of behaviours among faculty members. The lack of an efﬁcient measurement of social impact and value further prompts the UT to canalize its members’ efforts towards quantiﬁable third-mission practices, such as the creation of start-ups and spin-offs, patenting and licensing, rather than towards social entrepreneurship. An academic staff expressed it as follows: ‘Of course you are free to contribute to it (social entrepre- neurship) but sooner or later you will realize it does not have an impact on your career. Rather, industry income, establishing start-ups and spinoffs has’ (Academic staff, 4). THE CASE: UNIVERSITY OF TWENTE IN THE TWENTE REGION, THE NETHERLANDS As far as social entrepreneurship is concerned, the UT is involved in Novel-T, formerly known as Kennispark – an incubation and acceleration centre for start- ups – and Design Lab – a creative and interdisciplinary centre that encourages UT members (both students and academic/administrative staff) to work together to develop scientiﬁc ideas, later to be implemented for addressing complex societal challenges. Furthermore, the UT hosts the PC3 (Product Co-Creation Centre for Bottom of the Pyramid) initiative, which facili- tates the interaction of novice social entrepreneurs with specialists in the ﬁeld for the co-creation of products, and VentureLab, which offers business development programmes for technology- based start-ups. These centres and initiatives are platforms through which social entrepreneur- ship, mostly commercial based (start-ups with a social purpose), takes place. Figure 1. Geographical location of the Twente region in relation to the Netherlands and Europe. Source: ITC (2005) (courtesy: Faculty of ITC, University of Twente). Figure 1. Geographical location of the Twente region in relation to the Netherlands and Europe. Source: ITC (2005) (courtesy: Faculty of ITC, University of Twente). REGIONAL STUDIES, REGIONAL SCIENCE Ridvan Cinar 224 Ridvan Cinar 224 RESULTS This section entails a number of factors affecting the legitimacy process of social entrepreneurship within a higher education context, and a preliminary discussion in relation to them. The factors that impact the legitimacy process of social entrepreneurship are as follows: . Expectations of external stakeholders, the difﬁculty of measuring social impact and third- mission indicators. . Overemphasis on high-tech research and application. . Overemphasis on high-tech research and application. . Absence of a leader in the ﬁeld and organizational recognition in the UT. . Stringent regulations of public institutions in the Netherlands. . Lack of place-based belonging among student body, a barrier to fostering social entrepreneurs. . Lack of place-based belonging among student body, a barrier to fostering social entrepreneurs. The factors are further elaborated upon in the following section. REGIONAL STUDIES, REGIONAL SCIENCE Expectation of external stakeholders, difﬁculty in measuring social impact and third-mission indicators Unlike the outcome of social entrepreneurship, in many cases it is possible to quantify the effects of start-ups, spin- offs, patenting and licensing by simply indicating the number of these activities, the revenue they Delving into social entrepreneurship in universities: is it legitimate yet? 225 generate and the jobs they create in a given period. To illustrate, a social entrepreneurship initiat- ive that entails gathering senior citizens and university students to discuss the challenges pertain- ing to the place in which they live, and form a new coordination mechanism to address these challenges, may have considerable positive effects on narrowing the generational gap and age- related social issues in the Netherlands. Nonetheless, as the positive effect is neither quantiﬁable nor fully measurable, this particular initiative is unlikely to take place within the higher education setting of the UT. Recognizing incentivization of certain types of third-mission activities, a senior administrative staff further explains why enhancing social entrepreneurship has not yet been a proper action (Scott, 2008): Well, there are a number of reasons why we [the UT] have not been able to invest more in that issue (social entrepreneurship), particularly when the outside world expects you to deliver immediate, ﬁnancially mea- surable ventures in the city [Enschede]. They just want to have as many start-ups as possible in Enschede. Previously, they were concerned that some successful ones such as Booking.com were moving to the west of the country but now they are not concerned with this anymore. They demand and expect a circulation of ventures. Some will go, the new ones will be created and that is just ﬁne for them, which is another debate in itself. (Administrative staff, 11) Expectation of external stakeholders, difﬁculty in measuring social impact and third-mission indicators are three related factors that negatively affect the legitimacy process of social entrepre- neurship within the higher education context. There exists a path dependency among them. Since there is a lack of an effective measurement for social impact, and third-mission indicators do not reﬂect the full spectrum of activities, external stakeholders have expectations geared towards quantiﬁable outcomes from universities. Similarly, since the superordinate social system expects quantiﬁable outcomes, the UT canalizes its members’ efforts to third-mission activities that focus largely on the commercial value of research, pushing social entrepreneurship to the margin of the organizational agenda. Expectation of external stakeholders, difﬁculty in measuring social impact and third-mission indicators These ﬁndings indicate a need for a framework on how to measure and/or disseminate social impact (Mulgan, 2010). Expectation of external stakeholders, difﬁculty in measuring social impact and third-mission indicators are three related factors that negatively affect the legitimacy process of social entrepre- neurship within the higher education context. There exists a path dependency among them. Since there is a lack of an effective measurement for social impact, and third-mission indicators do not reﬂect the full spectrum of activities, external stakeholders have expectations geared towards quantiﬁable outcomes from universities. Similarly, since the superordinate social system expects quantiﬁable outcomes, the UT canalizes its members’ efforts to third-mission activities that focus largely on the commercial value of research, pushing social entrepreneurship to the margin of the organizational agenda. These ﬁndings indicate a need for a framework on how to measure and/or disseminate social impact (Mulgan, 2010). Overemphasis on high-tech research and application Overemphasis on high-tech research and application Nevertheless, the discontent about the framing of research interests in relation to high-tech among the aca- demic staff has grown, as exempliﬁed by following: To be honest, I am a bit tired and concerned at the same time about the university demanding that we should frame our research in line with high-tech. There is so much emphasis on high-tech. We have sev- eral social challenges in the Netherlands of which solutions do not necessarily entail any kind of high-tech application. What if I come up with such an idea? Nothing much happens because it does not fulﬁl the interest of the UT. (Academic staff, 9) The academic staff is not the only group expected to frame their interests in relation to high-tech. Students also experience it in different ways. We started a project to empower local people working in agriculture sector both in the Netherlands and Colombia. In the beginning, the project was progressing gradually, you know … when you start a project everything becomes clear slowly afterwards. After some weeks UT started to check if we embed high-tech in our project. We did not because we did not need it. After some months when it became clear that this project does not involve any kind of high-tech applications in the process, the support started to fade away gradually. (Student, 16) I am a third-year psychology student and it took me three years to ﬁnally ﬁnd my place in this university. I think it [the emphasis on high tech] is really overwhelming. We [psychology students] constantly question our role, our place in this university. That is so sad because there is so much psychologist can do for the people here [the UT] and outside [the region]. (Student, 17) The above demonstrates that the concentration on the development and application of high-tech has also been incorporated into student projects and initiatives. An overemphasis on this impacts social entrepreneurship in two ways: (1) any social entrepreneurship initiative that does not entail a high-tech dimension receives little to no support, regardless of its beneﬁts for the locals; and (2) the societal challenges the region faces receives little attention inside the UT when the use of high-tech is not part of the solution. Thus, the potential of the academic and student body in contributing to solving grand challenges via social entrepreneurship remains unexplored. Overemphasis on high-tech research and application As mentioned above, the UT is one of the four technical universities in the Netherlands. The ﬁndings suggest that there exists a narrative of rivalry, although not a very robust one, between the UT and the other three technical universities inside the organization. Nevertheless, this nar- rative impels the UT to take actions that will preferably distinguish it from the other three uni- versities by investing in new academic niches, incorporating high-end tech and increasing the quality of existing research areas. Surprisingly, the UT is determined to differentiate itself from the others by increasing the emphasis on high-tech related ﬁelds, as stated by a senior staff: If you are a technical university, you need to do what technical universities do. If you want to survive in this competitive environment [technical universities], you need to differentiate yourself. Investing in high tech is a strategy, differentiating factor for us. With this, we spread the message that if you want to focus on any high-tech related ﬁeld in the Netherlands, University of Twente is the place you should choose. (Administrative staff, 14) This strategy is striking considering that research, development and implementation of high-tech is already an area in which the other three technical universities have heavily invested. This strategy is striking considering that research, development and implementation of high-tech is already an area in which the other three technical universities have heavily invested. This focus is also exacerbated by the will of an external stakeholder, the Enschede municipal- ity in this case, to transform the city into a regional high-tech hub. Accumulation of these factors urged the UT to concentrate on strengthening this particular ﬁeld as a research area and encour- aged its academic staff, including those in the social sciences and humanities, to frame their REGIONAL STUDIES, REGIONAL SCIENCE Ridvan Cinar 226 research accordingly. For instance; a faculty member in the philosophy department is expected to specialize on the philosophy of digital technologies. A social scientist in public administration is expected primarily to specialize in the role of digital technologies in public governance. A psy- chologist is anticipated to conduct research in the ﬁeld of robot–human interaction. Overemphasis on high-tech research and application Absence of a leader in the ﬁeld and organizational recognition Social entrepreneurship as a research ﬁeld does exist in the UT, particularly in the NIKOS aca- demic centre (Netherlands Institute for Knowledge Intensive Entrepreneurship); however, only a handful of researchers afﬁliated with the centre focus on it. Interviewees frequently touched upon the necessity of social scientists who devote a vast portion of their research career to social entre- preneurship. Findings indicate that social scientists with academic expertise, mostly in social entrepreneurship, are fundamental for two reasons: (1) to have a product champion(s) who would lead the development of social entrepreneurship; and (2) consolidate it as a research ﬁeld within the UT. Current efforts of academics focusing on social entrepreneurship are rather fragmented. Among a handful of those researchers, some are afﬁliated with more than one uni- versity, therefore allocating a signiﬁcant period of time to another institution while some others simply reside outside the Twente region or the Netherlands. The following statement of an inter- viewee epitomizes the situation in the UT: REGIONAL STUDIES, REGIONAL SCIENCE Delving into social entrepreneurship in universities: is it legitimate yet? 227 What we are missing here is a leader in social entrepreneurship. There is no product champion at the moment. The ones we have are either very limitedly connected to this university or live elsewhere. That is a pity. This is why I say the ﬁrst thing I think to do here is to attract a professor on social entre- preneurship or somebody who likes social entrepreneurship quite a lot and leads the process. I talked to rectors and also chairman of the board. They are absolutely not against it, but they are not a product cham- pion, and you need a product champion. (Administrative staff, 10) What we are missing here is a leader in social entrepreneurship. There is no product champion at the moment. The ones we have are either very limitedly connected to this university or live elsewhere. That is a pity. This is why I say the ﬁrst thing I think to do here is to attract a professor on social entre- preneurship or somebody who likes social entrepreneurship quite a lot and leads the process. I talked to rectors and also chairman of the board. They are absolutely not against it, but they are not a product cham- pion, and you need a product champion. Overemphasis on high-tech research and application (Administrative staff, 10) Informants also base the need for a product champion for social entrepreneurship on the history of organizational developments in the UT, as articulated by a faculty member: ‘The problem is that we do not have a Van den Kroonenberg (the rector who championed the entrepreneurial university model in 1980s) for social entrepreneurship’ (Academic staff, 5) – pointing to the sig- niﬁcance of endogenous leadership that has manifested itself in organizational developments throughout the history of the UT. Another dimension that has constantly been of focus in terms of this particular theme is the lack of attention devoted to existing social entrepreneurs (academic and administrative staff, and students) in the UT. There is a consensus that the UT has not placed enough emphasis on suc- cessful social entrepreneurs who are, in some way, afﬁliated to the university through various media outlets, such as the independent journalistic medium of U-Today – campus-based, local newspapers, ofﬁcial Facebook and Twitter pages, and YouTube channels: ‘When there is a student starting a company, he or she is all over the place. In the website, Facebook, newspa- pers. When we [students] work in these [social entrepreneurship initiatives], we do not get any attention’ (Student, 16). This can be interpreted as a consequence of social entrepreneurship not yet being legitimized inside the UT, postulated by various media outlets (Deephouse & Such- man, 2008). Lack of place-based belonging One last factor that does not necessarily impact the legitimacy of social entrepreneurship in the UT but makes its manifestation among the student body relatively difﬁcult is the lack of place- based belonging to the region. The UT is located between the cities of Enschede and Hengelo, approximately 5 km from each city, and it has a full campus where almost all faculties, research centres and student facilities are located. Interviewees stated that the interaction of students with these two cities is limited: ‘It is technically possible to live here without having to go to the city throughout your studies. This is not myth. I know people who have ﬁnished their studies and been to city only once or twice’ (Student, 17). Students do not engage much with the region or the local people, leading to a lack of place-based identity formation and a sense of belonging (Benson & Jackson, 2012; Scannell & Gifford, 2010): Our students come from all over Netherlands and the world. You cannot expect them to be enthusiastic in these [social entrepreneurship initiatives] when they do not know much about local people and their chal- lenges. Unfortunately, their interaction is very limited. (Academic staff, 7) This is signiﬁcant in the fostering of social entrepreneurship if the aim is local impact for two reasons: (1) social entrepreneurs need to develop a deep understanding of the region in which a particular social issue is observed, and its socio-political conditions; and (2) social entrepreneurs should sympathize with a particular segment of society that suffers from a social issue to develop initiatives targeting solutions and social change (Dees, 1998). This is signiﬁcant in the fostering of social entrepreneurship if the aim is local impact for two reasons: (1) social entrepreneurs need to develop a deep understanding of the region in which a particular social issue is observed, and its socio-political conditions; and (2) social entrepreneurs should sympathize with a particular segment of society that suffers from a social issue to develop initiatives targeting solutions and social change (Dees, 1998). To sum up, the ﬁndings demonstrate that social entrepreneurship has been pushed to the margins of organizational agenda in the UT due to various reasons. It has only been found to be relevant when the endeavour entails the establishment of a start-up, encompassing a mission of creating social value, somehow incorporating the use of high-tech. Lack of place-based belonging As for fostering social entrepreneurs among the student body, their limited interaction with the region and its actors makes this rather difﬁcult. Stringent regulations of public institutions One of the most frequently highlighted issues in the interviews was the fact that public insti- tutions in the Netherlands have miscellaneous internal regulations and occasionally contrasting organizational interests, making inter-institutional collaborations even more challenging. The working schedule of faculty members in the UT is one such regulation that limits the room for engaging in third-mission activities that do not generate any income. Faculty members are to ﬁll out a weekly timetable stating the areas in which they spent their time working. The time allocated for third-mission activities that do not necessarily generate any income is rather limited. Considering that social entrepreneurship requires a collaboration mechanism of various sectors, agents and institutions, which is a time-consuming process, faculty members can only allocate a limited number of hours to work for social entrepreneurship projects: ‘First of all, how will you arrange your time sheets? This is not something that generates income so how many hours will you be able to dedicate, and more importantly will it be allowed by your depart- ment?’ (Academic staff, 8). Another participant reﬂected on organizational ﬂexibility in public institutions in the Netherlands: The biggest barrier [to enhancing social entrepreneurship] is traditional organizations and professionals who have to look after their responsibility to make sure that everything is organized and absolutely in line with internal regulations. That is the biggest hindrance. And the fact that we have organized every- thing so perfectly in the Netherlands that we think, from working schedules to employee expertise and more, makes the new initiatives extremely hard to realize. (Administrative staff, 15) Relatively restrictive internal regulations alike can be encountered in other public institutions that prevent employees from devoting a signiﬁcant period of time to collaborative, social REGIONAL STUDIES, REGIONAL SCIENCE Ridvan Cinar 228 entrepreneurship projects, as claimed by an UT employee: ‘We worked with them [employees in municipalities]. They also have problems when they spend time in projects with us because their job descriptions can be very strict’ (Academic staff, 3). Although one might infer this to be an issue regarding employee ﬂexibility, the nature of the abovementioned challenges should be con- sidered as a lack of organizational ﬂexibility (Skorstad & Ramsdal, 2016). It is further constrained when public institutions do not see any positive political reciprocity in social entrepreneurship initiatives, particularly when they feel that the continuation of their political mandate is at risk. REGIONAL STUDIES, REGIONAL SCIENCE DISCUSSION AND CONCLUSIONS This paper has sought to shed a light on the factors affecting the legitimacy of social entrepre- neurship in the UT. The ﬁndings suggest that a number of factors hinder this process. Based on these factors, the legitimacy process for social entrepreneurship remains unestablished. As a matter of choice, the UT has attempted to garner legitimacy for a rather limited form of social entrepreneurship, which entails encouraging the establishment of start-ups that have a social mis- sion, indicating isomorphism and decoupling (Boxenbaum & Jonsson, 2008) surrounding the practice of subject matter. This decision is in line with the expectation of external stakeholders, therefore suggesting congruence between its values and the superordinate system to which it belongs (Dowling & Pfeffer, 1975). Although the municipality (Enschede), the provincial government (Overijsel), the Dutch government and the European Commission expect the UT to contribute to the social and econ- omic development of its region (as perceived by the UT), the demand for quantiﬁable and REGIONAL STUDIES, REGIONAL SCIENCE Delving into social entrepreneurship in universities: is it legitimate yet? 229 economic outcomes and assistance in transforming the region into a high-tech hub outstrips the social expectation. In other words, while economic expectations from the UT have long been clear (i.e., generating start-ups and spin-offs, creation of jobs via these, etc.), the social expec- tations are rather fuzzy, leaving room for dozens of interpretations. How does a university have a social impact on its region? Is it by enhancing social entrepreneurship, community engage- ment and industry collaboration or preparing the students for future jobs? In the midst of all these possibilities, the UT has decided to prioritize economic expectations in a quantiﬁable manner and argue its social impact through positive externalities of economic outcomes (i.e., jobs created in the region via start-ups), thereby seeking congruence between its own and the stakeholders’ values to survive (Dowling & Pfeffer, 1975; Maurer, 1971). Consequently, social entrepreneur- ship has not been promoted as one of the ‘proper actions’ in the university (Scott, 2008). Actors perceive this template through the cultural–cognitive pillar of the UT whereby they realize that enhancing social entrepreneurship is not a ‘comprehensible, recognizable, culturally supported’ behaviour (Scott, 2008, p. 60). p With regard to the three phases of the legitimacy framework provided above, the ﬁrst phase has taken place, though not necessarily for social entrepreneurship. DISCUSSION AND CONCLUSIONS More speciﬁcally, the expec- tation from external stakeholders lies mainly in three areas: to contribute to its region socioeco- nomically, to transform the region into a high-tech hub and to address grand social challenges. The ﬁrst two were found to have signiﬁcantly higher inﬂuence. In addition, the UT argues that by focusing on the ﬁrst and second expectations, it is already addressing grand challenges. As such, there was no value conﬂict between the superordinate social system and the organizational values of the UT, particularly reinforced by those who are in the steering (university governance) positions, since they also share similar views. Thus, the ﬁrst phase of the legitimacy process is completed, without much value conﬂict and a further leap to the second and third phases. The implications drawn from this particular case are manifold. . Pattern of triggering institutional change in response to the expectations of the external environment can be historically contingent (Thornton & Ocasio, 2008). More speciﬁcally, signiﬁcant institutional change processes were realized through the great efforts of strong, key individuals throughout the history of the UT, as is the case with former rector Harry Van den Kroonenberg who championed the entrepreneurial university model. It appears that this has become a tradition in the university. As such, it can be argued that if similar institutions are to contribute to social entrepreneurship, a prominent ﬁgure who can make the case and lead the processes becomes essential (Bolman & Deal, 2017). . If social entrepreneurship is expected to be enhanced by universities and other public insti- tutions, the importance of organizational legitimacy should not be underestimated. To assist in constructing it, there are several roles external stakeholders (institutions and pol- icy-makers) must assume. Upon clarifying expectations with respect to the social impact of universities, policy-makers should also design appropriate strategies, such as incentive sys- tems, funding mechanisms, embracing the often underestimated, unmeasurable or hard- to-gauge impacts, work ﬂexibility of the employees of a region and focus on regionally rel- evant science that would help universities fulﬁl these expectations. In this regard, univer- sities should not be left alone. . Universities’ contributions to social entrepreneurship should be addressed holistically. This requires a focus and analysis of the increasingly competitive environment within which they function (Hazelkorn, 2011). FUNDING The author acknowledge that the paper was supported by funding from the European Commis- sion’s Horizon 2020 Research and Innovation Programme (Marie Sklodowska-Curie Actions) grant agreement No: 722295. DISCUSSION AND CONCLUSIONS This environment has urged the UT to construct an organiz- ational identity based on competitiveness, rivalry and differentiation due to the metriﬁca- tion of academia, evaluation systems and research funding regimes. Without it, perhaps the construction of an organizational identity that is also based on regional alignment would be possible. REGIONAL STUDIES, REGIONAL SCIENCE Ridvan Cinar 230 . Whenever a university’s enhancement of social entrepreneurship is discussed, students usually receive the attention and are seen as the potential agents of change (Pache & Chowdhury, 2012). However, challenges emerge for campus universities located relatively far from cities, where students have little or no interaction with the region. Such universities should aim at designing an organizational environment that will foster university-region interactions at all levels. Designing courses regarding the characteristics of the regions, planning some of the academic and social events with local residents of the cities, and aligning student projects with regional needs are some instances. Initiatives should go beyond enabling only a small segment of students to undertake internships and should incorporate university–region interaction into teaching and research (Goddard et al., 2016). In addition to ﬁlling the empirical gap that exists in legitimacy studies, with most scholarship being highly theoretical (Deephouse & Suchman, 2008), this study also points to new research avenues. A study exploring institutional logics (with respect to social entrepreneurship) occupy- ing the organizational ﬁeld, in a given region composed of several actors, is highly recommended. Such a study will likely provide academics and policy-makers with a holistic glimpse into the state of social entrepreneurship. Ridvan Cinar http://orcid.org/0000-0002-5640-224X Ridvan Cinar http://orcid.org/0000-0002-5640-224X ACKNOWLEDGEMENTS The author thanks Dr. David Charles (Northumbria University), Dr. Anna Kosmutzky (Leibniz University Hannover) Dr. Paul Benneworth (Western Norway University of Applied Sciences & University of Twente), Dr. Helena Aittola (University of Jyvaskyla) and two anonymous reviewers for their constructive feedback on earlier version of this paper. DISCLOSURE STATEMENT No potential conﬂict of interest was reported by the author. REFERENCES Alvord, S. H., Brown, L. D., & Letts, C. W. (2004). Social entrepreneurship and societal transformation. Journal of Applied Behavioral Science, 40(3), 260–282. doi:10.1177/0021886304266847 Alvord, S. H., Brown, L. D., & Letts, C. W. (2004). Social entrepreneurship and societal transformation. 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https://openalex.org/W2774924390	https://www.scielo.br/j/spmj/a/SLBcWrVSThKJLKKpwDfSbpt/?lang=en&format=pdf	English	null	What do Cochrane systematic reviews say about probiotics as preventive interventions?	São Paulo medical journal	2,017	cc-by	7,006	KEY WORDS: Review [publication type]. Probiotics. Preventive medicine. Evidence-based medicine. Evidence-based practice. ABSTRACT IUndergraduate Medical Student, Escola Paulista de Medicina (EPM), Universidade Federal de São Paulo (Unifesp), São Paulo (SP), Brazil. orcid.org/0000-0001-6736-2928 IIUndergraduate Medical Students, Escola Paulista de Medicina (EPM), Universidade Federal de São Paulo (Unifesp), São Paulo (SP), Brazil. IIIMD, MSc. Psychologist, Postgraduate Student, Evidence-Based Health Program, Universidade Federal de São Paulo (Unifesp), and Assistant Researcher, Cochrane Brazil, São Paulo (SP), Brazil. orcid.org/0000-0001-9146-4684 IVMD, PhD. Rheumatologist, Adjunct Professor, Discipline of Evidence-Based Medicine, Escola Paulista de Medicina (EPM), Universidade Federal de São Paulo (Unifesp), and Assistant Coordinator, Cochrane Brazil, São Paulo (SP), Brazil. orcid.org/0000-0002-9522-1871 IUndergraduate Medical Student, Escola Paulista de Medicina (EPM), Universidade Federal de São Paulo (Unifesp), São Paulo (SP), Brazil. orcid.org/0000-0001-6736-2928 IIUndergraduate Medical Students, Escola Paulista de Medicina (EPM), Universidade Federal de São Paulo (Unifesp), São Paulo (SP), Brazil. IIIMD, MSc. Psychologist, Postgraduate Student, Evidence-Based Health Program, Universidade Federal de São Paulo (Unifesp), and Assistant Researcher, Cochrane Brazil, São Paulo (SP), Brazil. orcid.org/0000-0001-9146-4684 IVMD, PhD. Rheumatologist, Adjunct Professor, Discipline of Evidence-Based Medicine, Escola Paulista de Medicina (EPM), Universidade Federal de São Paulo (Unifesp), and Assistant Coordinator, Cochrane Brazil, São Paulo (SP), Brazil. orcid.org/0000-0002-9522-1871 BACKGROUND: Probiotics have been used for a range of clinical situations and their use is strongly en- couraged by the media worldwide. This study identified and summarized all Cochrane systematic reviews about the preventive effects of probiotics in clinical practice. f DESIGN AND SETTING: Review of systematic reviews, conducted in the Discipline of Evidence-Based Medicine, Escola Paulista de Medicina (EPM), Universidade Federal de São Paulo (Unifesp). METHODS: We included all Cochrane reviews on any probiotics when they were used as preventive in- terventions and compared with no intervention, placebo or any other pharmacological or non-pharma- cological intervention. RESULTS: 17 Cochrane systematic reviews fulfilled our inclusion criteria and were summarized in this re- port. None of the reviews included in the present study provided high-quality evidence for any outcome. The benefits from use of probiotics included decreased incidence of antibiotic-associated diarrhea and Clostridium difficile-associated diarrhea; decreased incidence of upper respiratory tract infections and du- ration of episodes; decreased need for antibiotics and absences from school due to colds; and decreased incidence of ventilator-associated pneumonia. Probiotics seem to decrease the incidence of gestational diabetes mellitus, birthweight, risk of vaginal infection and incidence of eczema. CONCLUSION: Despite the marketing and the benefits associated with probiotics, there is little scientific evidence supporting the use of probiotics. COCHRANE HIGHLIGHT COCHRANE HIGHLIGHT DOI: 10.1590/1516-3180.2017.0310241017 ABSTRACT None of the reviews provided any high-quality evidence for prevention of illnesses through use of probiotics. More trials are needed to gain better knowledge of pro- biotics and to confirm when their use is beneficial and cost-effective. Setting Discipline of Evidence-Based Medicine of Escola Paulista de Medicina (EPM), Universidade Federal de São Paulo (UNIFESP). Selection of systematic reviews Two of the three researchers (VLB, LPDSR and DDB) indepen- dently and randomly selected and evaluated all references that were retrieved through the systematic search, to confirm their eligibility in accordance with the inclusion criteria. Any dis- agreements were resolved by consulting a more experienced author (RR). INTRODUCTION More than 400 species of microorganisms dwell in the human gastrointestinal tract.1,2 Balance between them is vital for the host’s health. Present-day high usage of antibiotics, together with environmental and physiological factors, can alter this ecosystem. This imbalance can cause illnesses such as diarrhea, which was responsible for 1.31 million deaths in 2015, including 499,000 among children under five.3,4 Some research has shown that use of probiotics can con- fer some health benefits, such as treatment for diarrheal disease, prevention of systemic infec- tions and other effects.2,5 The 2001 definition from the World Health Organization (WHO) states that probiotics are “live microorganisms which, when administered in adequate amounts, confer a health benefit on the host”.6 They are currently presented by media sources as an attractive health promotion method that prevents or cures a range of clinical situations.7 Indeed, many trials assessing the effects of probiotics (including using different species of microorganisms) as preventive or thera- peutic options for a range of diseases have been conducted and published.2,5 Consequently, a con- siderable amount of published data is currently available through MEDLINE. Corroborating this, a search in this database carried out on July 26, 2017, using the MeSH (medical subheading) term probiotics, retrieved 12,370 records, which corresponded to an increase of 278% in the number of records over the last ten years (from December 2007 to July 2017). The questions that therefore arise are: Should probiotics be indicated for preventive pur- poses? And if so, for which patients? Which types of probiotics should be used, and at what dose and for how long? In this review, we identified and summarized all Cochrane systematic reviews about the pre- ventive effects of probiotics in clinical practice. 578 Sao Paulo Med J. 2017; 135(6):578-86 What do Cochrane systematic reviews say about probiotics as preventive interventions? \| COCHRANE HIGHLIGHT What do Cochrane systematic reviews say about probiotics as preventive interventions? \| COCHRANE HIGHLIGHT OBJECTIVE To summarize the evidence from Cochrane systematic reviews focusing on probiotics for prevention of any disease or condition. Results from systematic reviews y Among the 16 systematic reviews included, a range of probi- otic strains was used. Four systematic reviews tested their use only among adults,8-11 three only among children12-14 and five among both adults and children,15-19 while another four stud- ies did not specify the age range of the population evaluated.20-23 Two systematic reviews addressed prevention of respiratory dis- eases,11,15 nine addressed prevention of gastroenterological dis- eases,9,10,13,14,16-18,20,21 three addressed gynecological and obstet- ric diseases,8,22,23 one addressed urological diseases19 and one addressed immunological/allergic diseases.12 A summary of the reviews included is presented below. The main findings for each comparison and the quality of the evidence (based on the GRADE approach) are presented in Table 2.8-23 Among the 16 systematic reviews included, a range of probi- otic strains was used. Four systematic reviews tested their use only among adults,8-11 three only among children12-14 and five among both adults and children,15-19 while another four stud- ies did not specify the age range of the population evaluated.20-23 Criteria for including reviews • Types of studies We only included the latest version of completed Cochrane systematic reviews (SR). We excluded any published protocols or any SR marked as “withdrawn” in the Cochrane Database of Systematic Reviews (CDSR). Two systematic reviews addressed prevention of respiratory dis- eases,11,15 nine addressed prevention of gastroenterological dis- eases,9,10,13,14,16-18,20,21 three addressed gynecological and obstet- ric diseases,8,22,23 one addressed urological diseases19 and one addressed immunological/allergic diseases.12 A summary of the reviews included is presented below. The main findings for each comparison and the quality of the evidence (based on the GRADE approach) are presented in Table 2.8-23 • Types of participants We included healthy participants or those diagnosed with any clinical condition or disease. • Types of intervention This review included any probiotics that were used as preven- tive interventions and compared with no intervention, pla- cebo or any other pharmacological or non-pharmacological intervention. #1 “Probiotics” OR “Probiotic” OR “lactobacillus” OR “lactobacilus” OR “lactobacilli” OR “lactobacili” OR “betabacterium” OR “lactobacileae” OR “lactobacilleae” OR “lactobacteria” OR “Lactobacillus acidophilus” OR “Lactobacilus acidophilus” OR “Lactobacillus casei” OR “Lactobacilus casei” OR “Lactobacillus delbrueckii” OR “Lactobacilus delbrueckii” OR “Lactobacillus fermentum” OR “Lactobacilus fermentum” OR “Lactobacillus helveticus” OR “Lactobacilus helveticus” OR “Lactobacillus leichmannii” OR “Lactobacilus leichmannii” OR “Lactobacillus plantarum” OR “Lactobacilus plantarum” OR “Lactobacillus reuteri” OR “Lactobacilus reuteri” OR “Lactobacillus rhamnosus” OR “Lactobacilus rhamnosus” OR “bifidobacterium” OR “lactococcus” OR “streptococcus thermophilus” OR “saccharomyces” OR “bifidobacterium” OR “bacillus subtilis” OR “bacillus licheniformis” OR “bugarian bacillus” OR “enterococcus faecalis” (Probiotics) OR (Probiotic) OR (lactobacillus) OR (lactobacilus) OR (lactobacilli) OR (lactobacili) OR (betabacterium) OR (lactobacileae) OR (lactobacilleae) OR (lactobacteria) OR (Lactobacillus acidophilus) OR (Lactobacilus acidophilus) OR (Lactobacillus casei) OR (Lactobacilus casei) OR (Lactobacillus delbrueckii) OR (Lactobacilus delbrueckii) OR (Lactobacillus fermentum) OR (Lactobacilus fermentum) OR (Lactobacillus helveticus) OR (Lactobacilus helveticus) OR (Lactobacillus leichmannii) OR (Lactobacilus leichmannii) OR (Lactobacillus plantarum) OR (Lactobacilus plantarum) OR (Lactobacillus reuteri) OR (Lactobacilus reuteri) OR (Lactobacillus rhamnosus) OR (Lactobacilus rhamnosus) OR (bifidobacterium) OR (lactococcus) OR (streptococcus thermophilus) OR (saccharomyces) OR (bifidobacterium) OR (bacillus subtilis) OR (bacillus licheniformis) OR (bugarian bacillus) OR (enterococcus faecalis) in Title, Abstract, Keywords C h R ( l ) Table 1. Search strategy (July 1, 2017) • Type of outcomes We considered any clinical, social and laboratory outcomes, as evaluated in the systematic reviews that were included. Search for reviews We carried out a sensitive systematic search in the Cochrane Database of Systematic Reviews (via Wiley) on July 1, 2017. The search strategy is presented in Table 1. Design Review of Cochrane systematic reviews. Search resultsh The initial search resulted in 39 reviews and 13 protocols. First, we excluded all protocols. After full-text assessment, we excluded 23 reviews since they either considered use of probi- otics to be therapeutic interventions rather than preventive inter- ventions or did not analyze probiotics alone. Thus, 16 Cochrane systematic reviews fulfilled our inclusion criteria and were sum- marized in this report. Antibiotic-associated diarrhea among children The review13 assessed the efficacy and safety of probiotics for pre- vention of antibiotic-associated diarrhea (AAD) among chil- dren and included 23 RCTs (938 children, aged 0 to 18 years) that compared different types of probiotics versus active treat- ment, placebo or no treatment. The incidence of antibiotic-asso- ciated diarrhea was lower in the probiotic group (relative risk, RR 0.46; 95% CI 0.35 to 0.61; number needed to treat [NNT] 10; 22 RCTs; 3,898 participants; I2 = 55%; moderate quality of evidence). No adverse events attributable to the treatment were found (2455 participants; 16 RCTs) and there was no difference between probiotics and controls regarding the risk of adverse events for the overall population (risk difference [RD] 0.00; 95% CI -0.01 to 0.01; 16 RCTs; 2455 participants). The authors con- cluded that there was moderate quality of evidence that probiot- ics seemed to prevent AAD. However, in some groups of patients such as immunocompromised individuals and others, serious events have been observed, and therefore probiotics should be avoided in these groups. For further details, and to check the pro- biotics used in each study, refer to the original abstract, avail- able at: http://onlinelibrary.wiley.com/doi/10.1002/14651858. CD004827.pub4/full. • the number of people who had one or more URTIs (odds ratio, OR 0.53; 95% confidence interval, CI 0.37 to 0.76; 7 RCTs; 1,927 participants; low quality of evidence); • the number of people who had three or more URTIs (OR 0.53; 95% CI 0.36 to 0.80; three RCTs; 650 participants; low quality of evidence); • the duration of the event (mean difference [MD] -1.89; 95% CI -2.03 to -1.75; 3 RCTs; 831 participants; low quality of evidence); • the need for antibiotics (OR 0.65; 95% CI 0.45 to 0.94; 4 RCTs; 1,184 participants; moderate quality of evidence); and • missing school due to colds (OR 0.10; 95% CI 0.02 to 0.47; 1 RCT; 80 children; very low quality of evidence). No difference between the groups was found regarding adverse events (OR 0.88; 95% CI 0.65 to 1.19; 4 RCTs; 1,234 participants; low quality of evidence). The authors concluded that, based on very low to low quality of evidence due to the heterogeneity between studies, use of probiotics may be associated with reductions in the numbers of URTIs, duration of the event, need for antibiotics and missing school due to URTIs. Clostridium difficile-associated diarrhea fi The review17 evaluated the efficacy and safety of probiotics for prevention of Clostridium difficile-associated diarrhea (CDAD) and included 31 RCTs (4,492 adults and children), with compari- sons between probiotics and placebo or no treatment. Probiotics presented benefits for reducing the incidence of CDAD (risk ratio [RR] 0.36; 95% CI 0.26 to 0.51; 23 RCTs; 4,213 participants, mod- erate quality of evidence) and of adverse events (RR 0.80; 95% CI 0.68 to 0.95; 26 RCTs; 3,964 participants; moderate quality of evidence). There was no statistical difference between the groups regarding the incidence of C. difficile infection (RR 0.89; 95% CI 0.64 to 1.24; 13 RCTs; 961 participants; moderate quality of evi- dence). The authors concluded that, based on the moderate qual- ity of evidence, use of probiotics seemed to be associated with reduction of CDAD and its adverse events. For further details, and to check the form of probiotics used in each study, refer to the original abstract, available at: http://onlinelibrary.wiley.com/ doi/10.1002/14651858.CD006095.pub3/full. Presentation of the results We presented all the reviews included in this synthesis in a nar- rative manner (qualitative synthesis). The key points considered were their relevance, methods, results, quality of the body of the evidence for each outcome, and applicability. #2 #1 in Cochrane Reviews (complete) Sao Paulo Med J. 2017; 135(6):578-86 579 COCHRANE HIGHLIGHT \| Braga VL, Rocha LPS, Bernardo DD, Cruz COC, Riera R Antibiotic-associated diarrhea among children For further details, and to check the form of probiotics used in each study, refer to the original abstract, available at: http://onlinelibrary.wiley.com/doi/10.1002/14651858. CD006895.pub3/full. Bacterial sepsis and wound complications after liver transplantation The review16 assessed the effects of different interventions for prevention of bacterial sepsis and wound complications in patients undergoing liver transplantation. Seven RCTs were included in this review, but only two (161 participants) assessed preventive effects of probiotics. There was no difference between probiotics plus prebiotics and selective bowel decontamination regarding the risk of needing retransplantation (OR 2.91; 95% CI 0.12 to 68.81; 1 RCT; 63 participants) or the risk of graft rejection requiring medical treatment (OR 1.94; 95% CI 0.38 to 9.83; 1 RCT; 63 participants). In comparing probiotics plus prebiotics with prebiotics, there was no difference regarding the risk of retransplantation (OR 0.33; 95% CI 0.01 to 7.90; 2 RCTs; 129 participants) or the risk of graft rejection requiring medi- cal treatment (OR 0.69; 95% CI 0.12 to 3.84; 1 RCT; 63 partici- pants). The authors’ conclusion was that there was no evidence to support use of probiotics for reducing wound complications and bacterial sepsis in patients with previous liver transplan- tation. For further details, and to check the probiotics used in each study, refer to the original abstract, available at: http:// Moderate quality of evidence (URTIs) and included 13 RCTs (3,780 participants) that com- pared probiotics with placebo. Probiotics presented benefits through reducing the following outcomes: Acute upper respiratory tract infections Acute upper respiratory tract infections The review15 evaluated the effectiveness and safety of probiot- ics for prevention of acute upper respiratory tract infections 580 Sao Paulo Med J. 2017; 135(6):578-86 What do Cochrane systematic reviews say about probiotics as preventive interventions? \| COCHRANE HIGHLIGHT What do Cochrane systematic reviews say about probiotics as preventive interventions? \| COCHRANE HIGHLIGHT Table 2. Characteristics, main findings and quality of evidence from systematic reviews focusing on patient-directed interventions with probiotics Population and aim Comparison Benefits and harm of probiotics Quality of evidence (GRADE approach) Pediatric patients receiving antibiotics13 Probiotics versus placebo Benefits: decreased incidence of antibiotic-associated diarrhea Moderate Risk of some adverse effects in immunocompromised, severely debilitated and other patients Adults and children receiving antibiotics17 Probiotics versus placebo or no treatment Benefit: decreased incidence of adverse events relating to Clostridium difficile-associated diarrhea Moderate No benefit regarding incidence of Clostridium difficile infection Adults, children and the elderly15 Probiotics versus placebo Benefit: decreased incidence of upper respiratory tract infections, duration of episodes, need for antibiotics and missing school due to colds Very low to low No difference in adverse events Patients undergoing liver transplantation16 Prebiotics alone or plus probiotics versus selective bowel decontamination; Prebiotics plus probiotics versus prebiotics Benefits: prebiotics plus probiotics decreased the proportion of participants with infections and the number of infectious episodes Very low No benefit regarding mortality, need for retransplantation, graft rejection, intensive care unit stay or hospital stay Patients after liver resection9 Probiotics alone or plus prebiotics versus placebo or prebiotics plus probiotics postoperatively No benefit regarding mortality Low Patients with quiescent ulcerative colitis20 Probiotics versus placebo or mesalazine No benefit regarding prevention of relapses and adverse events Low Patients undergoing ileal pouch-anal anastomosis for chronic ulcerative colitis10 Probiotics versus placebo or other treatment No benefit regarding prevention of pouchitis. Acute upper respiratory tract infections Very low Patients susceptible to urinary tract infection and healthy people19 Probiotic versus placebo; probiotics versus antibiotics; probiotic versus no treatment No benefit regarding symptomatic bacterial urinary tract infection Low Patients receiving mechanical ventilation11 Probiotics versus placebo Benefit: decreased incidence of ventilator-associated pneumonia Very low to low No effects regarding intensive care unit mortality, in-hospital mortality, incidence of diarrhea, length of intensive care unit stay, duration of mechanical ventilation or antibiotic use Women with HIV-infection for prevention of vulvovaginal candidiasis23 Probiotics versus placebo or clotrimazole No benefit regarding prevention of vulvovaginal candidiasis infection Low Infants with family history of allergy or food hypersensitivity and healthy infants12 Probiotics alone or plus prebiotics versus placebo Benefit: decreased incidence of infant eczema Not assessed No benefit regarding food hypersensitivity, asthma, atopic eczema, allergic rhinitis, food allergy or urticaria Pregnant women without metabolic or chronic diseases8 Probiotics versus placebo or diet Benefit: probiotics decreased both the rate of gestational diabetes mellitus and the birthweight Not assessed No benefit regarding death (abortion, intrauterine fetal death, stillbirth or neonatal death), risk of premature birth or cesarean delivery rate Patients with Crohn’s disease18 Probiotics versus placebo or other treatment No benefit regarding reduction of the risk of relapse after surgically-induced remission, compared with use of aminosalicylates or azathioprine. Not assessed Risk of some adverse effects from Lactobacillus GG Infants born at gestational age of less than 37 weeks or weighing less than 2500 g at birth, or both14 Probiotics versus placebo or no treatment Benefit: decreased incidence of severe necrotizing enterocolitis and mortality among preterm infants Not assessed No effect regarding nosocomial sepsis Patients undergoing surgery related to Crohn’s disease21 Probiotics versus placebo No benefit regarding clinical recurrence, severe endoscopic recurrence or any endoscopic recurrence Not assessed Pregnant women22 Probiotics versus placebo or acetic acid Benefit: decreased risk of vaginal infection Not assessed No effect regarding prevention of preterm birthi g q y y g p with probiotics Sao Paulo Med J. 2017; 135(6):578-86 581 COCHRANE HIGHLIGHT \| Braga VL, Rocha LPS, Bernardo DD, Cruz COC, Riera R placebo (RR 1.43; 95% CI 0.66 to 3.11; 1 RCT; 12 participants; very low quality of evidence). onlinelibrary.wiley.com/doi/10.1002/14651858.CD006660. pub3/full. Another outcome assessed related to treatment of acute pouchi- tis. Acute upper respiratory tract infections One RCT with 20 participants compared the use of Lactobacillus GG with placebo and there was no difference in clinical improve- ment (RR 3.95%; CI 0.14 to 65.9), with very low quality of evidence. Maintenance of remission in ulcerative colitis The review20 assessed the efficacy and safety of probiotics for prevention of relapses in cases of ulcerative colitis. Four RCTs (587 participants) were included and these showed the follow- ing: probiotics versus mesalazine: no difference between the groups regarding the risk of relapse (OR 1.33; 95% CI 0.94 to 1.90; 3 RCTs; 555 participants; low quality of evidence) or the incidence of adverse events (OR 1.21; 95% CI 0.80 to 1.84; 2 RCTs; 430 participants; moderate quality of evidence); probi- otics versus placebo: no difference between the groups regard- ing the risk of relapse (OR 0.27; 95% CI 0.03 to 2.68; 1 RCT; 32 participants; moderate quality of evidence). The authors con- cluded that there was insufficient evidence to support use of probiotics for preventing relapses in cases of ulcerative colitis. For further details, and to check the form of probiotics used in each study, refer to the original abstract, available at: http:// onlinelibrary.wiley.com/doi/10.1002/14651858.CD007443. pub2/full. The authors concluded that, based on the few studies avail- able, which were of small size (low quality of evidence), there was no benefit from using probiotics, in comparison with pla- cebo or no treatment. For further details, and to check the pro- biotics used in each study, refer to the original abstract, avail- able at: http://onlinelibrary.wiley.com/doi/10.1002/14651858. CD008772.pub2/full. Infections after liver resection The review9 assessed the benefits and harm of different interven- tions for prevention of infectious complications and improving the outcomes after liver resection. Seven RCTs were included, but only two RCTs (125 participants) evaluated probiotics as pre- ventive interventions. One of these compared use of prebiotics and probiotics versus placebo and found that there was no dif- ference in mortality (RR 0.36; 95% CI 0.10 to 1.35; 44 partici- pants). The other RCT, with 81 participants, compared use of pre- operative and postoperative prebiotics and probiotics versus use of postoperative prebiotics and probiotics and found that there was no significant difference in mortality (RR 0.39; 95% CI 0.15 to 1.00). Both of these studies presented low quality of evidence. The authors’ conclusions, based on the very low quality of evidence, was that probiotics did not seem to prevent pouchitis after ileal pouch-anal anastomosis in cases of chronic ulcerative colitis or to have any effect in treatments for patients with acute pouchitis. For more information about the other types of treat- ment in this study, refer to this link: http://onlinelibrary.wiley.com/ doi/10.1002/14651858.CD001176.pub3/full. Urinary tract infections h The review19 evaluated the effects of probiotics for prevention of urinary tract infections in susceptible or healthy adults and children. Nine RCTs (735 participants) were included and these showed that there was no difference in the risk of symptomatic bacterial urinary tract infection, in comparisons of use of pro- biotics versus placebo (OR 0.82; 95% CI 0.60 to 1.12; 6 RCTs; 352 participants) or use of probiotics versus antibiotics (OR 1.12; 95% CI 0.95 to 1.33; 1 RCT; 158 women). The authors’ conclusion was that there was no evidence to support or refute the use of any type of treatment to decrease the frequency of infectious complications after liver resection. For further information, refer to: http://onlinelibrary.wiley.com/ doi/10.1002/14651858.CD006933.pub2/full. Maintenance of remission of Crohn’s disease The review18 assessed the effectiveness of probiotics for mainte- nance of remission in cases of Crohn’s disease. Seven RCTs, com- paring use of probiotics versus placebo, were included. In evalu- ating the risk of relapse among adults, there were no differences between the following: Vulvovaginal candidiasis in HIV-infected women • cesarean delivery (RR 1.23; 95% CI 0.65 to 2.32; 218 women). g The review23 aimed to assess the effects of many antifungals that are administered vaginally or orally, including probiotics, for treatment and prevention of vulvovaginal candidiasis (VVC) in HIV-infected women. Two trials (431 participants) on use of probiotics as preventive interventions were included. No differ- ence regarding this outcome was found in comparisons of probi- otics versus clotrimazole (RR 1.11; 95% CI 0.45 to 2.76; low qual- ity of evidence) or versus placebo (RR 0.54; 95% CI 0.26 to 1.13; low quality of evidence). The authors’ conclusion was that no implications for practice could be determined. For other results from this review, refer to this link: http://onlinelibrary.wiley.com/ doi/10.1002/14651858.CD008739.pub2/full. All the infants included in this study were within the normal range for birthweight. The authors concluded that use of probi- otics seemed to be associated with reduction in GDM. However, they considered that further studies would be required to confirm these results. For more details, and to check the form of probiot- ics used in each study, refer to the original abstract, available at: http://onlinelibrary.wiley.com/doi/10.1002/14651858.CD009951. pub2/full. Gestational diabetes in women without metabolic or chronic diseases Gestational diabetes in women without metabolic or chronic diseases • duration of mechanical ventilation (MD -6.15; 95% CI -18.77 to 6.47; 2 RCTs; 203 participants; very low quality of evidence); and The review8 evaluated the effects of probiotics for prevention of gestational diabetes mellitus (GDM). One RCT (256 pregnant women) was included and it found that there were benefits from use of probiotics (compared with placebo or diet) for reducing the rate of GDM (RR 0.38; 95% CI 0.20 to 0.70; 225 women) and for reducing the birthweight (MD -127.71 g; 95% CI -251.37 to -4.06; 256 women). No difference between the groups was found for the following outcomes: • antibiotic use (OR 1.23; 95% CI 0.51 to 2.96; 1 RCT; 259 par- ticipants; low quality of evidence). The authors concluded that, based on the low quality of evidence, use of probiotics may be associated with reduction in VAP. For further details, and to check the probiotics used in each study, refer to the original abstract, available at: http:// onlinelibrary.wiley.com/doi/10.1002/14651858.CD011513. pub2/abstract. • death (OR 2.00; 95% CI 0.35 to 11.35; 256 women); • risk of premature birth (RR 3.27; 95% CI 0.44 to 24.43; 238 women); Ventilator-associated pneumonia The review11 assessed the effects of probiotics for prevention of ventilator-associated pneumonia (VAP). Eight randomized clin- ical trials (RCTs) (1,083 adults) that compared use of probiot- ics with placebo, usual care and multiple treatment arms were included. Probiotics were shown to present some benefit regard- ing reduction of the incidence of VAP (odds ratio, OR 0.70; 95% CI 0.52 to 0.95; 8 RCTs; 1,018 participants; low quality evidence). No difference was found between the probiotics and control groups in relation to the following outcomes,: No difference was found between the probiotics and control groups in relation to the following outcomes,: Pouchitis after ileal pouch-anal anastomosis for ulcerative colitis The review10 assessed the effectiveness of different interventions for prevention of pouchitis after ileal pouch-anal anastomosis in cases of chronic ulcerative colitis. Thirteen RCTs were included in this review, but only two studies were about the use of pro- biotics. One RCT assessed prevention of pouchitis in patients with ileal pouch-anal anastomosis and showed that there were no benefits from using Bifidobacterium longum, in comparison with • intensive care unit (ICU) mortality (OR 0.84; 95% CI 0.58 to 1.22; 5 RCTs; 703 participants; very low quality of evidence); • in-hospital mortality (OR 0.78; 95% CI 0.54 to 1.14; 4 RCTs; 524 participants; very low quality of evidence); • incidence of diarrhea (OR 0.72; 95% CI 0.47 to 1.09; 4 RCTs; 618 participants; low quality of evidence); • length of ICU stay (mean difference, MD -1.60; 95% CI -6.53 to 3.33; 4 RCTs; 396 participants; very low quality of evidence); 582 Sao Paulo Med J. 2017; 135(6):578-86 What do Cochrane systematic reviews say about probiotics as preventive interventions? \| COCHRANE HIGHLIGHT What do Cochrane systematic reviews say about probiotics as preventive interventions? \| COCHRANE HIGHLIGHT Quality of evidence not assessed Allergic disease and food hypersensitivity among children Allergic disease and food hypersensitivity among children The review12 assessed the effect of probiotics for prevention of allergic disease relating to food hypersensitivity among infants. It included 12 RCTs that compared use of probiot- ics with placebo or use of probiotics plus prebiotics with pla- cebo. The overall results from the pooled data showed that the incidence of infant eczema was reduced in the probiot- ics group (RR 0.82; 95% CI 0.70 to 0.95; 5 RCTs; 1,477 par- ticipants; I2 = 63.6%). However, these studies were hetero- geneous and there was no statistical difference when the analysis was limited to atopic eczema (RR 0.80; 95% CI 0.62 to 1.02). There was also no significant difference regarding the other outcomes evaluated. The authors concluded that there was insufficient evidence to support use of probiot- ics for preventing allergies or food hypersensitivity among infants, given that the findings were inconsistent and there were many follow-up losses. For further details, and to check the form of probiotics used in each study, refer to the orig- inal abstract, available at: http://onlinelibrary.wiley.com/ doi/10.1002/14651858.CD006475.pub2/full. • E. coli Nissle and placebo (RR 0.43; 95% CI 0.15 to 1.20; 1 RCT; 20 participants); • Lactobacillus GG and placebo after surgically-induced remis- sion (RR 1.58; 95% CI 0.30 to 8.4; 1 RCT; 37 participants) or medically-induced remission (RR 0.83; 95% CI 0.25 to 2.80; 1 RCT; 9 participants); • Lactobacillus GG and maintenance therapy with aminosalic- ylates or azathioprine (RR 0.67; 95% CI 0.13 to 3.30; 1 RCT; 24 participants). Regarding the risk of relapse among children, there was no difference between Lactobacillus GG and placebo (RR 1.85; 95% CI 0.77 to 4.40; 1 RCT; 75 participants). Regarding the risk of adverse events, Lactobacillus GG was associated with a risk of adverse events in comparison with maintenance therapy using aminosalicylates or azathioprine (no numerical data provided). A small RCT found that, at the end of the study, there was no difference in use of Saccharomyces boulardii plus conventional maintenance therapy versus placebo Sao Paulo Med J. 2017; 135(6):578-86 583 COCHRANE HIGHLIGHT \| Braga VL, Rocha LPS, Bernardo DD, Cruz COC, Riera R plus conventional maintenance therapy for relapses, according to the clinical disease activity index (CDAI) (RR 0.17; 95% CI 0.02 to 1.23). DISCUSSIONh This overview found that despite increasing marketing of probi- otics, there are still few systematic reviews on the preventive use of probiotics and there is a scarcity of high-quality randomized trials. None of the reviews included in the present study provided high-quality evidence for any outcome. Many clinical trials assessed in this study showed very low or low quality of evidence. Another point that needs to be noted is the huge variety of probiotics that have been considered in RCTs. This made it difficult to identify the individual effect of each pro- biotic agent, and also precluded meta-analyses. Postoperative recurrence of Crohn’s disease Most of the studies focused on gastrointestinal diseases. We found that there were some benefits from use of probiot- ics, with moderate quality of evidence, regarding their use for decreasing the incidence of antibiotic-associated diarrhea among children13 and the incidence of Clostridium difficile-associated diarrhea among adults and children.17 Other benefits that were observed with very low or low quality of evidence were that use of probiotics decreased the incidence of infections and the num- ber of infectious episodes in patients undergoing liver trans- plantation.16 The benefit of decreased incidence of severe nec- rotizing enterocolitis and mortality among preterm infants was noted in another review, but the quality of its evidence could not be assessed.14 The objective of the review21 was to evaluate the medical thera- pies for prevention of postoperative recurrence of Crohn’s dis- ease. It included 23 RCTs, but only 4 RCTs were about probi- otics. These RCTs found that there was no difference between probiotics and placebo regarding the risk of clinical recur- rence (RR 1.41; 95% CI 0.59 to 3.36; 3 RCTs; 213 adults), risk of severe endoscopic recurrence (RR 0.96; 95% CI 0.58 to 1.59; four RCTs; 333 adults) or risk of any endoscopic recurrence (RR 0.98; 95% CI 0.74 to 1.29; 3 RCTs; 213 adults). The authors con- cluded that there was no difference in effect between use of pro- biotics and placebo. For information on the other medical thera- pies included in this review, refer to this link: http://onlinelibrary. wiley.com/doi/10.1002/14651858.CD006873.pub2/full. In relation to respiratory diseases, probiotics showed some benefits regarding decreased incidence of upper respiratory tract infections and duration of episodes, the need for antibi- otics and missing school due to colds,15 and regarding the inci- dence of ventilator-associated pneumonia in patients receiv- ing mechanical ventilation.11 These studies were classified as presenting very low or low quality of evidence, using the GRADE approach. Quality of evidence not assessed in comparison with the controls (acetic acid or placebo) (RR 0.19; 95% CI 0.08 to 0.48; 88 pregnant women); comparison of use of probiotics plus dietary counselling versus control (pla- cebo plus dietary counselling or placebo only) showed that there was no difference between the groups regarding birth at gesta- tional ages of less than 32 weeks (RR 0.65; 95% CI 0.03 to 15.88; 1 RCT; 238 pregnant women) or birth between the gestational ages of 32 and 37 weeks (RR 3.95; 95% CI 0.36 to 42.91; 1 RCT; 238 pregnant women). The authors concluded that there was no evidence that probiot- ics were beneficial for maintenance of remission in cases of Crohn’s disease. For further details, and to check the probiotics used in each study, refer to the original abstract, available at: http://onlinelibrary. wiley.com/doi/10.1002/14651858.CD004826.pub2/full. Necrotizing enterocolitis in premature newborns The review14 assessed the efficacy and safety of probiotics for prevention of severe necrotizing enterocolitis (NEC) or sep- sis in premature infants. Twenty-four RCTs, comparing use of probiotics with placebo or no treatment, were included. Probiotics showed some benefit regarding reduction of the inci- dence of severe NEC (RR 0.43; 95% CI 0.33 to 0.56; 20 RCTs; 5,529 infants) and all-cause mortality (RR 0.65; 95% CI 0.52 to 0.81; 17 RCTs; 5,112 infants). Comparison between use of pro- biotics and placebo or no treatment showed that there was no statistical difference in the incidence of nosocomial sepsis (RR 0.91; 95% CI 0.80 to 1.03; 19 RCTs; 5,338 infants). The authors concluded that use of enteral probiotics seemed to be associated with reduction of severe NEC and mortality among premature infants. For further details, and to check the form of probiot- ics used in each study, refer to the original abstract, available at: http://onlinelibrary.wiley.com/doi/10.1002/14651858. CD005496.pub4/full. The authors concluded that use of probiotics seemed to be associated with lower risk of vaginal infection during pregnancy and that there was insufficient evidence to support their use for prevention of premature birth. For further details, and to check the form of probiotics used in each study, refer to the original abstract, available at: http://onlinelibrary.wiley.com/doi/10.1002/14651858. CD005941.pub2/full. REFERENCES Our systematic review has the advantage of the number of studies included, given that the topic of probiotics is currently a matter of debate and that there are uncertainties regarding their effectiveness. Another advantage is that it summarizes the evidence relating to probiotics and their use that has been gathered in the Cochrane Library, which is recognized as the largest database of systematic reviews, given that the information about probiotics is distributed among many studies. 1. Shokryazdan P, Faseleh Jahromi M, Liang JB, Ho YW. Probiotics: From Isolation to Application. J Am Coll Nutr. 2017;1-11. 1. Shokryazdan P, Faseleh Jahromi M, Liang JB, Ho YW. Probiotics: From Isolation to Application. J Am Coll Nutr. 2017;1-11. 2. Markowiak P, Śliżewska K. Effects of Probiotics, Prebiotics, and Synbiotics on Human Health. Nutrients. 2017;9(9):pii. E1021. 3. International Vaccine Access Center (IVAC), Johns Hopkins Bloomberg School of Public Health. (2015). Pneumonia and Diarrhea Progress Report 2015: Sustainable Progress in the Post-2015 Era. Available from www. jhsph.edu/research/centers-and-institutes/ivac/resources/IVAC-2015- Pneumonia-Diarrhea-Progress-Report.pdf. Accessed in 2017 (Nov 14). 4. GBD Diarrhoeal Diseases Collaborators. Estimates of global, regional, and national morbidity, mortality, and aetiologies of diarrhoeal diseases: a systematic analysis for the Global Burden of Disease Study 2015. Lancet Infect Dis. 2017;17(9):909-48. This overview has some limitations. Our search was con- ducted in a single database, even though the Cochrane Library is recognized as the most important database of systematic reviews. The limited data available is a consequence of the small number of papers, and the low quality of evidence is related to the small sample sizes and bias of the RCTs. Another point that should be noted is the huge variety of prebiotics that have been considered in RCTs, which led to difficulty in identifying the individual effect of each probiotic agent, and also precluded meta-analyses. The NNT was not determined in some reviews, which made it more challenging to analyze cost-effectiveness. 5. Sun J, Marwah G, Westgarth M, et al. Effects of Probiotics on Necrotizing Enterocolitis, Sepsis, Intraventricular Hemorrhage, Mortality, Length of Hospital Stay, and Weight Gain in Very Preterm Infants: A Meta-Analysis. Adv Nutr. 2017;8(5):749-63. 6. 6. Joint FAO/WHO Working Group Report on Drafting Guidelines for the Evaluation of Probiotics in Food London, Ontario, Canada, April 30 and May 1, 2002. Guidelines for the Evaluation of probiotics in food. Available from: http://www.who.int/foodsafety/fs_management/en/ probiotic_guidelines.pdf. Accessed in 2017 (Nov 14). Preterm laborh The review22 assessed the effectiveness and safety of probiotics for prevention of premature labor and delivery and included three RCTs (344 pregnant women) that compared use of pro- biotics with acid acetic and placebo. The following results were found: use of probiotics reduced the risk of vaginal infection 584 Sao Paulo Med J. 2017; 135(6):578-86 What do Cochrane systematic reviews say about probiotics as preventive interventions? \| COCHRANE HIGHLIGHT In three systematic reviews about gynecological and obstetric diseases, we found that there were some bene- fits in relation to decreasing the rate of gestational diabetes mellitus, decreasing both the birthweight8 and the risk of vaginal infection,22 although the quality of evidence could not be assessed. One RCT found that there was a benefit in relation to reducing the incidence of eczema among infants with a family history of allergy or food hypersensitivity and among healthy infants.12 be recommended when there is uncertainty about their bene- fits and harm. Here, we make it clear that much needs to be done in rela- tion to studying probiotics. Firstly, basic research is needed in order to elucidate the pathophysiological links between different diseases and use of probiotics. Secondly, RCTs with high-quality evidence are needed, with larger sample sizes and better con- trol over variables. Thirdly, research on the cost-effectiveness of use of probiotics needs to be stimulated, because their use must be analyzed in terms of their consequences for health and economic repercussions. Despite the potential benefits of probiotics, we did not find any high-quality evidence that could change clinical practice or recom- mendations for their use. Furthermore, some probiotics may be harmful in groups of patients such as those presenting immuno- suppression, severe debilitation and other such conditions. On the other hand, it is important to examine the number needed to treat (NNT) and to analyze the cost-effectiveness of use of probiotics. Goldenberg et al. concluded that the NNT to prevent one case of diarrhea was ten. Thus, in this example, probiotics reduced the number of cases of diarrhea even with only a few patients treated.13 To prevent Clostridium difficile-associated diarrhea, 29 patients would need to be treated.17 CONCLUSION This overview included 16 Cochrane systematic reviews about the use of probiotics as preventive measures within clin- ical practice. There was little scientific evidence to support the use of probiotics. None of the reviews provided high-qual- ity evidence for preventive action achieved through use of probiotics and each review analyzed only a few randomized controlled trials. REFERENCES Regarding the implications for practice, our study summa- rizes the use of probiotics as a preventive intervention for some clinical settings and shows the situations in which there is a real benefit. From this, healthcare professionals can decide when to indicate probiotics for patients and can improve outcomes in their hospitals. For example, probiotics can be used to reduce the incidence of vaginal infection during pregnancy and to decrease the incidence of VAP. On the other hand, probiotics should not 7. Stanton C, Gardiner G, Meehan H, et al. Market potential for probiotics. Am J Clin Nutr. 2001;73(2 Suppl):476S-483S. 8. Barrett HL, Dekker Nitert M, Conwell LS, Callaway LK. Probiotics for preventing gestational diabetes. Cochrane Database Syst Rev. 2014;(2):CD009951. Sao Paulo Med J. 2017; 135(6):578-86 585 COCHRANE HIGHLIGHT \| Braga VL, Rocha LPS, Bernardo DD, Cruz COC, Riera R 9. Gurusamy KS, Naik P, Davidson BR. Methods of decreasing infection to improve outcomes after liver resections. Cochrane Database Syst Rev. 2011;(11):CD006933. Date of first submission: September 29, 2017 Last received: October 23, 2017 Accepted: October 24, 2017 10. Singh S, Stroud AM, Holubar SD, Sandborn WJ, Pardi DS. Treatment and prevention of pouchitis after ileal pouch-anal anastomosis for chronic ulcerative colitis. Cochrane Database Syst Rev. 2015;(11):CD001176. 11. Bo L, Li J, Tao T, et al. Probiotics for preventing ventilator-associated pneumonia. Cochrane Database Syst Rev. 2014;(10):CD009066. 11. Bo L, Li J, Tao T, et al. Probiotics for preventing ventilator-associated pneumonia. Cochrane Database Syst Rev. 2014;(10):CD009066. Address for correspondence: Daniel Damasceno Bernardo Liga de Medicina Baseada em Evidências, Escola Paulista de Medicina (EPM), Universidade Federal de São Paulo (Unifesp) Rua Botucatu, 740 – 3o andar Vila Clementino – São Paulo (SP) – Brasil CEP 04023-900 Tel. (11) 5576-4203 E-mail: danieldamascenobernardo1@gmail.com 12. Sinn J. Mechanisms of asthma and allergic disease-1075. Probiotics in infants for prevention of allergic disease and food hypersensitivity. World Allergy Organization Journal. 2013;6(Suppl 1):P72. Available from: https://www.researchgate.net/publication/274954663_Mechanisims_ of_asthma_and_allergic_disease_-_1075_Probiotics_in_infants_for_ prevention_of_allergic_disease_and_food_hypersensitivity. Accessed in 2017 (Oct 31). 13. Johnston BC, Goldenberg JZ, Vandvik PO, Sun X, Guyatt GH. Probiotics for the prevention of pediatric antibiotic-associate diarrhea. Cochrane Database Syst Rev. 2011;(11):CD004827. 13. Johnston BC, Goldenberg JZ, Vandvik PO, Sun X, Guyatt GH. Probiotics for the prevention of pediatric antibiotic-associate diarrhea. Cochrane Database Syst Rev. 2011;(11):CD004827. 14. Alfaleh K, Bassler D. Probiotics for prevention of necrotizing enterocolitis in preterm infants. Cochrane Database Syst Rev. 2008;(1):CD005496. 14. Alfaleh K, Bassler D. REFERENCES Probiotics for prevention of necrotizing enterocolitis in preterm infants. Cochrane Database Syst Rev. 2008;(1):CD005496. 15. Hao Q, Dong BR, Wu T. Probiotics for preventing acute upper respiratory tract infections. Cochrane Database Syst Rev. 2015;(2):CD006895. 16. Gurusamy KS, Nagendran M, Davidson BR. Methods of preventing bacterial sepsis and wound complications after liver transplantation. Cochrane Database Syst Rev. 2014;(3):CD006660. 17. Goldenberg JZ, Ma SS, Saxton JD, et al. Probiotics for the prevention of Clostridium difficile-associated diarrhea in adults and children. Cochrane Database Syst Rev. 2013;(5):CD006095. Cochrane Database Syst Rev. 2013;(5):CD006095. 18. Rolfe VE, Fortun PJ, Hawkey CJ, Bath-Hextall F. Probiotics for maintenance of remission in Crohn’s disease. Cochrane Database Syst Rev. 2006;(4):CD004826. 18. Rolfe VE, Fortun PJ, Hawkey CJ, Bath-Hextall F. Probiotics for maintenance of remission in Crohn’s disease. Cochrane Database Syst Rev. 2006;(4):CD004826. 19. Schwenger EM, Tejani AM, Loewen PS. Probiotics for preventing urinary tract infections in adults and children. Cochrane Database Syst Rev. 2015;(12):CD008772. 20. Naidoo K, Gordon M, Fagbemi AO, Thomas AG, Akobeng AK. Probiotics for maintenance of remission in ulcerative colitis. Cochrane Database Syst Rev. 2011;(12):CD007443. 21. Doherty G, Bennett G, Patil S, Cheifetz A, Moss AC. Interventions for prevention of post-operative recurrence of Crohn’s disease. Cochrane Database Syst Rev. 2009;(4):CD006873. 21. Doherty G, Bennett G, Patil S, Cheifetz A, Moss AC. Interventions for prevention of post-operative recurrence of Crohn’s disease. Cochrane Database Syst Rev. 2009;(4):CD006873. 22. Othman M, Neilson JP, Alfirevic Z. Probiotics for preventing preterm labour. Cochrane Database Syst Rev. 2007;(1):CD005941. 22. Othman M, Neilson JP, Alfirevic Z. Probiotics for preventing preterm labour. Cochrane Database Syst Rev. 2007;(1):CD005941. 23. Ray A, Ray S, George AT, Swaminathan N. Interventions for prevention and treatment of vulvovaginal candidiasis in women with HIV infection. Cochrane Database Syst Rev. 2011;(8):CD008739. 586 Sao Paulo Med J. 2017; 135(6):578-86
https://openalex.org/W2732107752	https://openresearch.lsbu.ac.uk/download/b379ada6b4acc3236da7472278e9d5c608b4399843768b92fef4caba9674e04c/1045074/Hobbins%20et%20al.%2C%202017.pdf	English	null	Normobaric hypoxic conditioning to maximize weight loss and ameliorate cardio-metabolic health in obese populations: a systematic review	American journal of physiology. Regulatory, integrative and comparative physiology/American journal of physiology. Regulatory, integrative, and comparative physiology	2,017	cc-by	24,472	Abstract Abstract 37 Background: Normobaric hypoxic conditioning (HC) is defined as exposure to 38 systemic and/or local hypoxia at rest (passive) or combined with exercise training (active). 39 HC has been previously used by healthy and athletic populations to enhance their physical 40 capacity, and improve performance in the lead up to competition. Recently, HC has also been 41 applied acutely (single exposure) and chronically (repeated exposure over several weeks) to 42 overweight and obese populations with the intention of managing and potentially increasing 43 cardio-metabolic health and weight loss. At present, it is unclear what the cardio-metabolic 44 health and weight loss responses of obese populations are in response to passive and active 45 HC. Exploration of potential benefits of exposure to both passive and active HC may 46 provide pivotal findings for improving health and well being in these individuals. 47 Methodology: A systematic literature search for articles published between 2000 and 48 2017 was carried out. Studies investigating the effects of normobaric HC as a novel 49 therapeutic approach to elicit improvements in the cardio-metabolic health and weight loss of 50 obese populations were included. 51 Results: Studies investigated passive (n = 7; 5 animal, 2 human), active (n = 4; all 52 human) and a combination of passive and active (n = 4; 3 animal, 1 human) HC to an 53 inspired oxygen fraction (FiO2) between 4.8–15.0%, ranging between a single session and 54 daily sessions per week, lasting from 5 days up to 8 months. Passive HC led to reduced 55 insulin concentrations (-37 – +22%) in obese animals and increased energy expenditure 56 (+12 – +16%) in obese humans, while active HC lead to reductions in body weight (-4 – - 57 2%) in obese animals and humans, and blood pressure (-8 – -3%) in obese humans, 58 compared to a matched workload in normoxic conditions. Inconclusive findings, 59 however, exist in determining the impact of acute and chronic HC on markers such as 60 triglycerides, cholesterol levels and fitness capacity. Importantly, most of the studies that 61 included animal models involved exposure to severe levels of hypoxia (FiO2 = 5.0%; 62 simulated altitude >10,000 m) that are not suitable for human populations. Abstract 63 Conclusions: Overall, normobaric HC demonstrated observable positive findings in 64 relation to insulin and energy expenditure (passive), and body weight and blood pressure 65 (active), which may improve the cardio-metabolic health and body weight management of 66 obese populations. However, further evidence on responses of circulating biomarkers to both 67 passive and active HC in humans is warranted. 68 69 Word count: 391 70 Background: Normobaric hypoxic conditioning (HC) is defined as exposure to 38 systemic and/or local hypoxia at rest (passive) or combined with exercise training (active). 39 HC has been previously used by healthy and athletic populations to enhance their physical 40 capacity, and improve performance in the lead up to competition. Recently, HC has also been 41 applied acutely (single exposure) and chronically (repeated exposure over several weeks) to 42 overweight and obese populations with the intention of managing and potentially increasing 43 cardio-metabolic health and weight loss. At present, it is unclear what the cardio-metabolic 44 health and weight loss responses of obese populations are in response to passive and active 45 Title 1 Normobaric hypoxic conditioning to maximise weight-loss and ameliorate cardio-me 2 health in obese populations: A systematic review 3 4 Authors 5 L Hobbins 1, S Hunter 1, N Gaoua 1, O Girard 2,3 6 7 Institutions 8 1 Sport and Exercise Science Research Centre, London South Bank University, L 9 United Kingdom 10 2 Athlete Health and Performance Research Centre, Aspetar Orthopaedic and 11 Medicine Hospital, Doha, Qatar 12 3 ISSUL, Institute of Sport Sciences, University of Lausanne, Switzerland 13 14 Address for correspondence 15 Mr. Liam Hobbins 16 hobbinsl@lsbu.ac.uk 17 Sport and Exercise Science Research Centre 18 School of Applied Sciences 19 London South Bank University 20 London, United Kingdom 21 SE1 0AA 22 23 Running head 24 Therapeutic use of hypoxia in obese individuals. 25 26 Key words 27 Obesity, hypoxia, altitude training, weight loss, cardio-metabolic health. 28 29 Word count: 7411 30 31 Number of tables: 4 32 33 Number of figures: 1 34 35 Number of references: 84 36 Title 1 Normobaric hypoxic conditioning to maximise weight-loss and ameliorate cardio-me 2 health in obese populations: A systematic review 3 4 Authors 5 L Hobbins 1, S Hunter 1, N Gaoua 1, O Girard 2,3 6 7 Institutions 8 1 Sport and Exercise Science Research Centre, London South Bank University, L 9 United Kingdom 10 2 Athlete Health and Performance Research Centre, Aspetar Orthopaedic and 11 Medicine Hospital, Doha, Qatar 12 3 ISSUL, Institute of Sport Sciences, University of Lausanne, Switzerland 13 14 Address for correspondence 15 Mr. Liam Hobbins 16 hobbinsl@lsbu.ac.uk 17 Sport and Exercise Science Research Centre 18 School of Applied Sciences 19 London South Bank University 20 London, United Kingdom 21 SE1 0AA 22 23 Running head 24 Therapeutic use of hypoxia in obese individuals. 25 26 Key words 27 Obesity, hypoxia, altitude training, weight loss, cardio-metabolic health. 28 29 Word count: 7411 30 31 Number of tables: 4 32 33 Number of figures: 1 34 35 Number of references: 84 36 Title 1 Normobaric hypoxic conditioning to maximise weight-loss and ameliorate cardio-metabolic 2 health in obese populations: A systematic review 3 4 Authors 5 L Hobbins 1, S Hunter 1, N Gaoua 1, O Girard 2,3 6 7 Institutions 8 1 Sport and Exercise Science Research Centre, London South Bank University, London, 9 United Kingdom 10 2 Athlete Health and Performance Research Centre, Aspetar Orthopaedic and Sports 11 Medicine Hospital, Doha, Qatar 12 3 ISSUL, Institute of Sport Sciences, University of Lausanne, Switzerland 13 14 Address for correspondence 15 Mr. Liam Hobbins 16 hobbinsl@lsbu.ac.uk 17 Sport and Exercise Science Research Centre 18 School of Applied Sciences 19 London South Bank University 20 London, United Kingdom 21 SE1 0AA 22 23 Running head 24 Therapeutic use of hypoxia in obese individuals. 25 26 Key words 27 Obesity, hypoxia, altitude training, weight loss, cardio-metabolic health. 28 29 Word count: 7411 30 31 Number of tables: 4 32 33 Number of figures: 1 34 35 Number of references: 84 36 1 1 Abstract 37 Background: Normobaric hypoxic conditioning (HC) is defined as exposure to 38 systemic and/or local hypoxia at rest (passive) or combined with exercise training (active). 39 HC has been previously used by healthy and athletic populations to enhance their physical 40 capacity, and improve performance in the lead up to competition. Title 1 Normobaric hypoxic conditioning to maximise weight-loss and ameliorate cardio-me 2 health in obese populations: A systematic review 3 4 Authors 5 L Hobbins 1, S Hunter 1, N Gaoua 1, O Girard 2,3 6 7 Institutions 8 1 Sport and Exercise Science Research Centre, London South Bank University, L 9 United Kingdom 10 2 Athlete Health and Performance Research Centre, Aspetar Orthopaedic and 11 Medicine Hospital, Doha, Qatar 12 3 ISSUL, Institute of Sport Sciences, University of Lausanne, Switzerland 13 14 Address for correspondence 15 Mr. Liam Hobbins 16 hobbinsl@lsbu.ac.uk 17 Sport and Exercise Science Research Centre 18 School of Applied Sciences 19 London South Bank University 20 London, United Kingdom 21 SE1 0AA 22 23 Running head 24 Therapeutic use of hypoxia in obese individuals. 25 26 Key words 27 Obesity, hypoxia, altitude training, weight loss, cardio-metabolic health. 28 29 Word count: 7411 30 31 Number of tables: 4 32 33 Number of figures: 1 34 35 Number of references: 84 36 Recently, HC has also been 41 applied acutely (single exposure) and chronically (repeated exposure over several weeks) to 42 overweight and obese populations with the intention of managing and potentially increasing 43 cardio-metabolic health and weight loss. At present, it is unclear what the cardio-metabolic 44 health and weight loss responses of obese populations are in response to passive and active 45 HC. Exploration of potential benefits of exposure to both passive and active HC may 46 provide pivotal findings for improving health and well being in these individuals. 47 Methodology: A systematic literature search for articles published between 2000 and 48 2017 was carried out. Studies investigating the effects of normobaric HC as a novel 49 therapeutic approach to elicit improvements in the cardio-metabolic health and weight loss of 50 obese populations were included. 51 Results: Studies investigated passive (n = 7; 5 animal, 2 human), active (n = 4; all 52 human) and a combination of passive and active (n = 4; 3 animal, 1 human) HC to an 53 inspired oxygen fraction (FiO2) between 4.8–15.0%, ranging between a single session and 54 daily sessions per week, lasting from 5 days up to 8 months. Passive HC led to reduced 55 insulin concentrations (-37 – +22%) in obese animals and increased energy expenditure 56 (+12 – +16%) in obese humans, while active HC lead to reductions in body weight (-4 – - 57 2%) in obese animals and humans, and blood pressure (-8 – -3%) in obese humans, 58 compared to a matched workload in normoxic conditions. Inconclusive findings, 59 however, exist in determining the impact of acute and chronic HC on markers such as 60 triglycerides, cholesterol levels and fitness capacity. Importantly, most of the studies that 61 included animal models involved exposure to severe levels of hypoxia (FiO2 = 5.0%; 62 simulated altitude >10,000 m) that are not suitable for human populations. 63 Conclusions: Overall, normobaric HC demonstrated observable positive findings in 64 relation to insulin and energy expenditure (passive), and body weight and blood pressure 65 (active), which may improve the cardio-metabolic health and body weight management of 66 obese populations. However, further evidence on responses of circulating biomarkers to both 67 passive and active HC in humans is warranted. 68 69 Word count: 391 70 g p p p p p HC. Exploration of potential benefits of exposure to both passive and active HC may 46 provide pivotal findings for improving health and well being in these individuals. 47 M h d l A i li h f i l bli h d b 2000 d 8 Methodology: A systematic literature search for articles published between 2000 and 48 2017 was carried out. Studies investigating the effects of normobaric HC as a novel 49 therapeutic approach to elicit improvements in the cardio-metabolic health and weight loss of 50 obese populations were included. 51 Conclusions: Overall, normobaric HC demonstrated observable positive findings in 4 relation to insulin and energy expenditure (passive), and body weight and blood pressure 5 (active), which may improve the cardio-metabolic health and body weight management of 6 obese populations. However, further evidence on responses of circulating biomarkers to both 7 passive and active HC in humans is warranted. 8 2 2 1. Introduction 71 Obesity has been labeled as the global epidemic of the 21st century (78). In the United 72 Kingdom alone, 58% of women and 65% of men are considered to be overweight or obese, 73 i.e., defined as having a body mass index (BMI) of 25–29.9 or ≥30 kg.m2, respectively (49). 74 Compared to the early 1990s, whereby obesity prevalence was estimated to be ~15%, those 75 living in today’s society have a 1 in 4 chance of becoming obese (49). Further, co-morbidities 76 such as cardiovascular disease, type II diabetes and cancer are at greater risk of development 77 in obese popuations resulting in the possibility of higher mortality rates (21). 78 Obesity is typically caused by a consistently positive energy balance, i.e., greater 79 calories consumed versus those expended, which eventually leads to excess fat accumulation 80 (28) – the negative impact of which is profound in terms of health consequences. Carrying 81 additional weight can result in elevated blood pressure (7), metabolic deficiencies (28) and 82 mechanical complications (11) amongst other factors – all of which create an increased 83 functional demand on the body of obese individuals. Further, the increased mechanical 84 demand during weight-bearing activities of obese populations may be deleterious on lower 85 limb joints (i.e., knee and ankle) and limit the functional capabilities compared to healthy and 86 normal weight populations (70). Aside from bariatric surgery, that is primarily available 87 for the most severe cases (BMI ≥40 kg.m2 [3]), various interventions including diet 88 manipulation, caloric restriction, and increased physical activity and exercise (12), are 89 proposed to counteract these problems. g p p p p p HC. Exploration of potential benefits of exposure to both passive and active HC may 46 provide pivotal findings for improving health and well being in these individuals. 47 M h d l A i li h f i l bli h d b 2000 d 8 90 For weight loss to be considered clinically significant, a change of ≥3% in body 91 weight is required (12) – and then ≤3% change to be deemed as weight maintenance over the 92 1. Introduction 71 Obesity has been labeled as the global epidemic of the 21st century (78). In the United 72 Kingdom alone, 58% of women and 65% of men are considered to be overweight or obese, 73 i.e., defined as having a body mass index (BMI) of 25–29.9 or ≥30 kg.m2, respectively (49). 74 Compared to the early 1990s, whereby obesity prevalence was estimated to be ~15%, those 75 living in today’s society have a 1 in 4 chance of becoming obese (49). Further, co-morbidities 76 such as cardiovascular disease, type II diabetes and cancer are at greater risk of development 77 in obese popuations resulting in the possibility of higher mortality rates (21). 78 Obesity is typically caused by a consistently positive energy balance, i.e., greater 79 calories consumed versus those expended, which eventually leads to excess fat accumulation 80 (28) – the negative impact of which is profound in terms of health consequences. Carrying 81 additional weight can result in elevated blood pressure (7), metabolic deficiencies (28) and 82 mechanical complications (11) amongst other factors – all of which create an increased 83 functional demand on the body of obese individuals. Further, the increased mechanical 84 demand during weight-bearing activities of obese populations may be deleterious on lower 85 limb joints (i.e., knee and ankle) and limit the functional capabilities compared to healthy and 86 normal weight populations (70). Aside from bariatric surgery, that is primarily available 87 for the most severe cases (BMI ≥40 kg.m2 [3]), various interventions including diet 88 manipulation, caloric restriction, and increased physical activity and exercise (12), are 89 proposed to counteract these problems. 90 For weight loss to be considered clinically significant, a change of ≥3% in body 91 weight is required (12) – and then ≤3% change to be deemed as weight maintenance over the 92 duration of several months (65). Typically, weight loss is achieved in the first six months of 93 commencing a new diet and/or exercise programme, but a plateau is then reached and often 94 the weight lost is subsequently regained (66). Given the inadequacy of current weight 95 3 3 management strategies, innovative approaches are warranted for clinically-relevant weight 96 loss treatment and significant improvements in the health and general well-being of those 97 who are overweight and obese beyond what is achieved to date. 1. Introduction 71 For a range of exercise 124 intensities (55–65% of maximal O2 uptake [VO2max] / 60–70% of maximum heart rate 125 [HRmax]) and similar levels of simulated altitude (~2600 m), other studies (18, 32, 46, 51, 76) 126 have suggested that active HC induces specific molecular adaptations that do not occur when 127 training in a normoxic environment (66). These positive adaptations, in particular, include 128 increased basal noradrenaline levels (4), arteriole diameter and peripheral vasodilation (45), 129 mitochondria number (66), glycolytic enzyme activity (16), insulin sensitivity (40), as well as 130 reduced diastolic blood pressure (63) and leptin levels (29). Such physiological adaptations 131 would in turn improve the metabolic phenotype of obese individuals. 132 sedentary overweight males, for instance, passive acute (single 3-hour exposure session) 121 normobaric HC increased energy expenditure and altered fuel utilisation (reduced glucose 122 and increased lipid oxydation), while further passive HC (multiple 3-h exposure sessions on 7 123 consecutive days) magnified these metabolic adjustments (77). For a range of exercise 124 intensities (55–65% of maximal O2 uptake [VO2max] / 60–70% of maximum heart rate 125 [HRmax]) and similar levels of simulated altitude (~2600 m), other studies (18, 32, 46, 51, 76) 126 have suggested that active HC induces specific molecular adaptations that do not occur when 127 training in a normoxic environment (66). These positive adaptations, in particular, include 128 increased basal noradrenaline levels (4), arteriole diameter and peripheral vasodilation (45), 129 mitochondria number (66), glycolytic enzyme activity (16), insulin sensitivity (40), as well as 130 reduced diastolic blood pressure (63) and leptin levels (29). Such physiological adaptations 131 would in turn improve the metabolic phenotype of obese individuals. 132 Recent reviews have investigated the impact of O2 availability as a therapeutic 133 intervention for body weight management (56), intermittent hypoxia for fat loss and 134 enhancement of cardiovascular health (66), the role of hypoxia in energy balance (28), 135 hypoxic conditioning for several pathological diseases (67) and the effectiveness of hypoxic 136 training on cardio-metabolic risk factors (71). Overall, these reviews tend to agree that 137 both hypoxia and hyperoxia (i.e., environmental conditions posing a challenge to O2 138 homeostasis) may play a significant role in the processes associated with obesity and 139 weight loss paradigm. 1. Introduction 71 98 management strategies, innovative approaches are warranted for clinically-relevant weight 96 loss treatment and significant improvements in the health and general well-being of those 97 who are overweight and obese beyond what is achieved to date. 98 Hypoxia is defined as a reduced (or insufficient) oxygen (O2) supply to tissues caused 99 by decreases in O2 saturation of arterial blood (24). Hypoxic conditioning (HC) relates to 100 passive (i.e., during rest) or active (i.e., during exercise) exposure to systemic (whole body) 101 and/or local (tissue) hypoxia, resulting in a decrease in arterial O2 availability (38). HC can 102 be implemented acutely (single exposure) or chronically (multiple exposures over prolonged 103 periods of time). Permenant residence in a hypobaric hypoxic (terrestrial altitude due to 104 lower-than-sea level barometric pressure) environment has shown to reduce the likleyhood of 105 becoming obese (68). Several studies have reported weight loss (1, 58, 81), reduced blood 106 pressure (35, 61) and improved metabolic function (35, 61, 64, 72, 73) after a 1–3 week 107 residential stay (e.g. hotel and food provided, light entertainment acitivites throughout the 108 day, no structured exercise program) at terrestrial altitude (1500–8800 m). However, 109 permanent living or travelling regularly to terrestrial altitude may not be feasible to all (i.e., 110 re-location, elevated cost, lack of time). In obese populations, this practice could also lead to 111 side effects such as physiological and metabolic deficiencies (44), including obstructive sleep 112 apnea (30) or the development of acute mountain sickness (81). 113 Alternatively, exposure to normobaric hypoxia (or simulated altitude via a reduced 114 inspired O2 fraction [FiO2]), is increasingly popular as the number of commercially-available 115 devices permitting simulated hypoxic exposure is growing. Primarily, this intervention allows 116 living at or near sea level and then exposing, periodically, individuals to hypoxic conditions 117 at rest or whilst exercising. This is typically accomplished by breathing through a mask 118 or staying in an environmentally controlled chamber/room/tent whereby the FiO2 is 119 typically reduced to 15–12% (equivalent to simulated altitudes of ~2600–4300 m). In 120 4 sedentary overweight males, for instance, passive acute (single 3-hour exposure session) 121 normobaric HC increased energy expenditure and altered fuel utilisation (reduced glucose 122 and increased lipid oxydation), while further passive HC (multiple 3-h exposure sessions on 7 123 consecutive days) magnified these metabolic adjustments (77). 1. Introduction 71 However, the aforementioned reviews are limited in terms of 140 systematically examining the potential impact of passive and active HC on markers of cardio- 141 metabolic health and well-being (71), while some focus solely on human research with no 142 consideration of findings from animal models (66, 71). Further, combining the literature on 143 HC of populations with a multitude of diseases (e.g., cardiovascular and pulmonary) does not 144 provide conclusive evidence in relation to the specific treatment of obese populations (67). 145 5 Due to the mechanical restrictions and weight loading implications on lower limbs (i.e., on 146 the knee and ankle joints) when completing exercise in at-risk (obese, overweight and 147 sedentary) populations (70), the exploration of potential benefits of exposure to both passive 148 and active HC may provide pivotal findings for weight loss and maintenance strategies. 149 Therefore, the aim of this systematic review is to a) summarise the current literature 150 surrounding passive and active normobaric HC as a therapeutic method for improving cardio- 151 metabolic health and managing weight-loss in obese animals and humans, and b) offer 152 perspectives for future research within this area of literature. 153 154 2. Materials and methods 155 2.1. Literature search 156 A literature search was carried out in the Pubmed, ScienceDirect, Scopus, Web of 157 Science and SportsDiscus databases. The terms (intermittent hypoxia OR passive hypoxic 158 exposure OR hypoxic training OR altitude training OR live-low train-high) AND 159 (obesity OR overweight OR weight loss OR physiological response OR metabolic 160 response OR cardiovascular response) were combined to search the full text of 161 experimental articles published after 2000 and before January 2017. Each title, abstract 162 and full text were assessed for relevance to the topic and selected if they met the inclusion 163 criteria as follows: an original research article; randomised and controlled design; human or 164 animal experimentation; overweight (BMI: 25–30 kg.m2), obese (BMI: 30–38 kg.m2) and/or 165 sedentary participants; normobaric hypoxic intervention; assessment of at least one of the 166 following parameters: blood pressure, glucose concentrations, insulin levels or cholesterol; 167 English language; and published in a peer-reviewed journal. 1. Introduction 71 Exlcusion criteria were: 168 athletic/sport population/performance focus; involved obstructive sleep apnoea; clinical 169 studies; implemented hypobaric/no hypoxia; or included a physically active or 170 Due to the mechanical restrictions and weight loading implications on lower limbs (i.e., on 146 the knee and ankle joints) when completing exercise in at-risk (obese, overweight and 147 sedentary) populations (70), the exploration of potential benefits of exposure to both passive 148 and active HC may provide pivotal findings for weight loss and maintenance strategies. 149 6 ado esce t popu at o . O y u te t a t c es we e ev ewed. add t search, references were scanned for further relevant articles and were incl 172 inclusion criteria. 173 174 2.2. Assessment of Methodological Quality 175 A modified scale to assess the methodological quality of the stud 176 review was carried out following selection of full text articles. The m 177 applied due to the greater representation for experiments employing a t 178 compared to the Delphi, PEDro and Cochrane scales (53). A 10 item 179 included the criteria listed below and guided the assessment scoring of ea 180 0 = clearly no; 1 = maybe; 2 = clearly yes; range = 0 (poor)–20 (excellent 181 1. Inclusion criteria were clearly stated; 182 2. Subjects were randomly allocated to groups; 183 3. Intervention was clearly defined; 184 4. Groups were tested for similarity at baseline; 185 5. A control group was used; 186 6. Outcome variables were clearly defined; 187 7. Assessments were practically useful; 188 8. Duration of intervention was practically useful; 189 9. Between-group statistical analysis was appropriate; 190 10. Point measures of variability; 191 192 3. Results 193 3.1. Search results 194 7 Fig. 1 illustrates a flow chart of the search results. The search yielded a total of 212 195 publications. After removal of irrelevant titles, 23 items remained in relation to the focus of 196 the review, reduced to eight following abstract assessment, and subsequently four full texts 197 that met the inclusion criteria. Additionally, a further eleven full text items were added via 198 reference list searching. 199 Fig. 1 near here 200 201 3.2. Methodological quality assessment 202 The average quality of the 15 studies included in this review was 16/20 according to 203 Paul et al. (53). One study scored 20/20, and the lowest score was 12/20. 204 205 3.3. 1. Introduction 71 Intermittent protocols adopted a pattern of 30 s of 235 exposure to hypoxia followed by 30 s of exposure to normoxia, lasting for 8 h (2) and 12–16 236 h per day (55). There were modifications to this approach in two of the investigations as 237 follows: 40 and 80 s of exposure to hypoxia and normoxia, respectively (52), and 2 x 15-min 238 periods of exposure to hypoxia interspersed with 5 and 10 min of exposure to normoxia (34). 239 Only Rodriguez et al. (59) implemented a sustained exposure period of 24 h per day. The 240 hypoxic level ranged between FiO2 = 4.8% (2, 52, 55, 59) and 14.3% (34), while most studies 241 used a FiO2 of ~5.0% (2, 52, 55, 59) All interventions involved daily exposure. Most studies 242 examined responses over a prolonged period of time (2–6 weeks [2, 34, 52, 59]), with only 243 remaining three including males only (46, 69, 77). Further, four studies used obese 220 (BMI = 30–37.1 kg.m2 [18, 32, 51, 76]), one overweight (BMI = 27 kg.m2 [77]) and one 221 sedentary (normal weight with a BMI = 22.2 kg.m2 [69]) participants. The body 222 composition of one participant cohort was not reported (46). Participants were aged 223 between 21–51 years. Where mentioned, participants were free from hypertension (18, 224 77), diabetes (76), stroke (18), acute and chronic cardio-vascular, pulmonary and 225 respiratory diseases/infections (18, 69, 76, 77), barriers to physical activity (32), 226 altitude/hypoxic exposure (32, 76), medication to control weight or metabolism (32, 69, 227 77), alcohol/drug abuse and smoking (33, 69, 76, 77), and exercise (32, 46, 69, 77) within 228 ≥3 months of enrolling. 229 Table 1 near here 230 231 3.4. Animal studies 232 3.4.1. Passive hypoxic exposure 233 The five investigations reviewed implemented two modes of passive HC, namely 234 intermittent and sustained hypoxia. Intermittent protocols adopted a pattern of 30 s of 235 exposure to hypoxia followed by 30 s of exposure to normoxia, lasting for 8 h (2) and 12–16 236 h per day (55). There were modifications to this approach in two of the investigations as 237 follows: 40 and 80 s of exposure to hypoxia and normoxia, respectively (52), and 2 x 15-min 238 periods of exposure to hypoxia interspersed with 5 and 10 min of exposure to normoxia (34). 239 Only Rodriguez et al. 1. Introduction 71 Study characteristics 206 Table 1 illustrates the details of the studies included in this review. Eight studies 207 used animal models (2, 6, 34, 37, 52, 55, 59, 79). Five of these implemented a protocol of 208 passive HC only (2, 34, 52, 55, 59), two active normoxic periods followed by passive HC 209 (6, 79), and one used passive and active HC combined (37). All animal studies included 210 obese rodents (mice or rats) aged between 3 and 24 weeks, seven used male (2, 6, 37, 52, 211 55, 59, 79) and one involved female (34) models. Five of the animal model groups were 212 genetically obese (2, 6, 55, 59, 79), while three were fed a high-fat diet (34, 37, 52). Other 213 than one study stating leptin deficiency in their animal models (34), no other difference 214 in the health of animals across studies was mentioned. 215 Seven of the eligible studies investigated human participants (18, 32, 46, 51, 69, 216 76, 77). Two of these employed passive HC only (69, 77), four active HC only (32, 46, 51, 217 76), and one investigated both passive and active HC (18). Four of the human 218 investigations were composed of both males and females (18, 32, 51, 76), with the 219 195 8 remaining three including males only (46, 69, 77). Further, four studies used obese 220 (BMI = 30–37.1 kg.m2 [18, 32, 51, 76]), one overweight (BMI = 27 kg.m2 [77]) and one 221 sedentary (normal weight with a BMI = 22.2 kg.m2 [69]) participants. The body 222 composition of one participant cohort was not reported (46). Participants were aged 223 between 21–51 years. Where mentioned, participants were free from hypertension (18, 224 77), diabetes (76), stroke (18), acute and chronic cardio-vascular, pulmonary and 225 respiratory diseases/infections (18, 69, 76, 77), barriers to physical activity (32), 226 altitude/hypoxic exposure (32, 76), medication to control weight or metabolism (32, 69, 227 77), alcohol/drug abuse and smoking (33, 69, 76, 77), and exercise (32, 46, 69, 77) within 228 ≥3 months of enrolling. 229 Table 1 near here 230 231 3.4. Animal studies 232 3.4.1. Passive hypoxic exposure 233 The five investigations reviewed implemented two modes of passive HC, namely 234 intermittent and sustained hypoxia. 1. Introduction 71 (59) implemented a sustained exposure period of 24 h per day. The 240 hypoxic level ranged between FiO2 = 4.8% (2, 52, 55, 59) and 14.3% (34), while most studies 241 used a FiO2 of ~5.0% (2, 52, 55, 59) All interventions involved daily exposure. Most studies 242 examined responses over a prolonged period of time (2–6 weeks [2, 34, 52, 59]), with only 243 9 9 Polotsky et al. (55) investigating both short-term (5 days) and long-term (12 weeks) 244 responses. 245 246 3.4.2. Combined passive and active hypoxic exposure 247 Chen et al. (6) and Wu et al. (79) implemented a live high-train low (LHTL) 248 intervention, with 90-min exercise sessions (moderate-intensity swimming) carried out in 249 normoxia, followed by sustained passive HC periods (8 h per day, FiO2 = 14.0%). Lu et al. 250 (37) employed a live high-train high (LHTH) intervention, with implementation of 60-min 251 active HC (moderate-intensity running), and the remaining hours of the day living in the 252 same hypoxic environment (FiO2 = 13.6%). These interventions ranged between 4–6 weeks. 253 254 3.5. Human studies 255 3.5.1. Passive hypoxic exposure 256 Wang et al. (69) and Workman & Basset (77) both implemented sustained passive HC 257 periods corresponding to a period of 60 min and 3 h, respectively. The hypoxic level during 258 these sessions was controlled via two methods: FiO2 clamped at 12–15% (69), and 259 manipulation of FiO2 to clamp the arterial O2 saturation (SpO2) at ~80% (77). 260 Whereas Wang et al. (69) implemented a 4-week intervention (5 days of exposure per 261 week, 60-min sessions), Workman & Basset (77) investigated responses to both a single 3-h 262 session as well as the same period of exposure and hypoxic level on an additional 6 263 consecutive days. 264 265 3.5.2. Active hypoxic exposure 266 Active investigations have used a live low-train high (LLTH) approach and 267 implemented exercise of a moderate intensity (55–65% VO2max / 60–70% HRmax). Exercise 268 Polotsky et al. (55) investigating both short-term (5 days) and long-term (12 weeks) 244 responses. 245 246 3.4.2. Combined passive and active hypoxic exposure 247 Chen et al. (6) and Wu et al. (79) implemented a live high-train low (LHTL) 248 intervention, with 90-min exercise sessions (moderate-intensity swimming) carried out in 249 normoxia, followed by sustained passive HC periods (8 h per day, FiO2 = 14.0%). Lu et al. 1. Introduction 71 250 (37) employed a live high-train high (LHTH) intervention, with implementation of 60-min 251 active HC (moderate-intensity running), and the remaining hours of the day living in the 252 same hypoxic environment (FiO2 = 13.6%). These interventions ranged between 4–6 weeks. 253 254 3.5. Human studies 255 3.5.1. Passive hypoxic exposure 256 Wang et al. (69) and Workman & Basset (77) both implemented sustained passive HC 257 periods corresponding to a period of 60 min and 3 h, respectively. The hypoxic level during 258 these sessions was controlled via two methods: FiO2 clamped at 12–15% (69), and 259 manipulation of FiO2 to clamp the arterial O2 saturation (SpO2) at ~80% (77). 260 10 programmes were typically cardiovascular-based (running, cycling, stepping [32, 46, 51, 269 76]), with one study adding strength training (40–50% of 1 repetition maximum, 3 sets of 15 270 repetitions, interspersed with 2–3-min rest periods [32]). 271 The FiO2 in all studies was 15.0%. Typical exercise prescription included sessions of 272 60–90 min in duration, performed three times per week, over a 4-week period (32, 46, 76), 273 with one study implementing a longer training period of eight weeks (51). Kong et al. (32) 274 took their participants to a sea-level residential camp for 4 weeks, which permitted a greater 275 amount of time for exercise per week (22 h) and dietary control. Although, the hypoxic group 276 spent only 6 h in hypoxia per week (exercise modality unknown) with the remainder of the 277 sessions (16 h) carried out in normoxic conditions. 278 279 3.5.3. Combined passive and active exposure 280 Gatterer et al. (18) utilised a combination of passive and active HC via a LLTH 281 approach over a period of 8 months. Participants completed 90-min moderate intensity (65– 282 70% of HRmax) exercise sessions on an exercise ergometer of their choice (cycle, treadmill, 283 cross-trainer), immediately followed by 90 mins rest, all in hypoxic (FiO2 = 12–14%) 284 conditions, twice weekly. 285 286 4. Discussion 287 4.1. Animal studies 288 4.1.1. Passive hypoxic exposure 289 Table 2 presents the overall findings of the animal studies included in this 290 review. 1. Introduction 71 Glucose concentrations are commonly measured in obese animals following passive 291 programmes were typically cardiovascular-based (running, cycling, stepping [32, 46, 51, 269 76]), with one study adding strength training (40–50% of 1 repetition maximum, 3 sets of 15 270 repetitions, interspersed with 2–3-min rest periods [32]). 271 programmes were typically cardiovascular-based (running, cycling, stepping [32, 46, 51, 269 76]), with one study adding strength training (40–50% of 1 repetition maximum, 3 sets of 15 270 repetitions, interspersed with 2–3-min rest periods [32]). 271 programmes were typically cardiovascular-based (running, cycling, stepping [32, 46, 51, 269 76]), with one study adding strength training (40–50% of 1 repetition maximum, 3 sets of 15 270 repetitions, interspersed with 2–3-min rest periods [32]). 271 The FiO2 in all studies was 15.0%. Typical exercise prescription included sessions of 272 60–90 min in duration, performed three times per week, over a 4-week period (32, 46, 76), 273 with one study implementing a longer training period of eight weeks (51). Kong et al. (32) 274 took their participants to a sea-level residential camp for 4 weeks, which permitted a greater 275 amount of time for exercise per week (22 h) and dietary control. Although, the hypoxic group 276 spent only 6 h in hypoxia per week (exercise modality unknown) with the remainder of the 277 sessions (16 h) carried out in normoxic conditions. 278 ble 2 presents the overall findings of the animal studies included in this In contrast, Briancon-Marjollet et al. 296 (2) reported significant glucose concentration increases in obese rats after 8 h of intermittent 297 (30 s :30 s) HC to an extreme hypoxic level (FiO2 = 5.0%) per day over 2 weeks. Other 298 investigations have shown unchanged values when animals were exposed intermittently to 299 similar hypoxic levels using protocols of 40 s :80 s for 8 h (52) and 30 s :30 s for 12 h (55) 300 per day. It seems that the common response of glucose concentrations in obese animals, 301 passively exposed to varying levels of hypoxia, is yet to be verified. This variation in the 302 present findings may be partly explained through the differences in pre-analytical 303 conditions of sampled tissue, which was subsequently utilised for glucose concentration 304 assessment (84). 305 Insulin is receiving a great deal of attention due to its dominance in Type II Diabetes 306 control and development (26). In obese rats, insulin concentrations were unchanged 307 following intermittent HC for 8 h per day (40 s :80 s [52]; and 30 s :30 s [2]). This is perhaps 308 due to the severity of the hypoxic stimulus (FiO2 = ~5.0%) blunting improvements in this 309 health marker (50). Only one study has reported significant increases in insulin levels, which 310 occurred following both a 5 day (+356%) and 12 week (+185%) hypoxic intervention in 311 obese mice (55). The highly significant increase in insulin concentrations shown here may 312 not actually be of benefit. Perhaps, exacerbation of insulin resistance occurred, leading to 313 hyperinsulinemia (79). It is interesting to note that the hypoxic level employed in these 314 studies was similar (FiO2 = ~5.0%), and animals were intermittently exposed to hypoxia over 315 1 :1 (30 s :30 s) and 1 :2 (40 s :80 s) sequences. Reducing the severity of hypoxia during 316 exposure periods may prevent dramatic increases, as reported here by Polotsky et al. (55), 317 concentrations following intermittent HC, despite exposure time/cycle (30 s :30 s versus 15 294 min :5–10 min, respectively) and severity of hypoxic exposure (FiO2 = ~5.0% versus 14.3%, 295 respectively) being largely different between protocols. In contrast, Briancon-Marjollet et al. ble 2 presents the overall findings of the animal studies included in this 11 concentrations following intermittent HC, despite exposure time/cycle (30 s :30 s versus 15 294 min :5–10 min, respectively) and severity of hypoxic exposure (FiO2 = ~5.0% versus 14.3%, 295 respectively) being largely different between protocols. In contrast, Briancon-Marjollet et al. 296 (2) reported significant glucose concentration increases in obese rats after 8 h of intermittent 297 (30 s :30 s) HC to an extreme hypoxic level (FiO2 = 5.0%) per day over 2 weeks. Other 298 investigations have shown unchanged values when animals were exposed intermittently to 299 similar hypoxic levels using protocols of 40 s :80 s for 8 h (52) and 30 s :30 s for 12 h (55) 300 per day. It seems that the common response of glucose concentrations in obese animals, 301 passively exposed to varying levels of hypoxia, is yet to be verified. This variation in the 302 present findings may be partly explained through the differences in pre-analytical 303 conditions of sampled tissue, which was subsequently utilised for glucose concentration 304 assessment (84). 305 Insulin is receiving a great deal of attention due to its dominance in Type II Diabetes 306 control and development (26). In obese rats, insulin concentrations were unchanged 307 following intermittent HC for 8 h per day (40 s :80 s [52]; and 30 s :30 s [2]). This is perhaps 308 due to the severity of the hypoxic stimulus (FiO2 = ~5.0%) blunting improvements in this 309 health marker (50). Only one study has reported significant increases in insulin levels, which 310 occurred following both a 5 day (+356%) and 12 week (+185%) hypoxic intervention in 311 obese mice (55). The highly significant increase in insulin concentrations shown here may 312 not actually be of benefit. Perhaps, exacerbation of insulin resistance occurred, leading to 313 hyperinsulinemia (79). It is interesting to note that the hypoxic level employed in these 314 studies was similar (FiO2 = ~5.0%), and animals were intermittently exposed to hypoxia over 315 1 :1 (30 s :30 s) and 1 :2 (40 s :80 s) sequences. Reducing the severity of hypoxia during 316 exposure periods may prevent dramatic increases, as reported here by Polotsky et al. (55), 317 concentrations following intermittent HC, despite exposure time/cycle (30 s :30 s versus 15 294 min :5–10 min, respectively) and severity of hypoxic exposure (FiO2 = ~5.0% versus 14.3%, 295 respectively) being largely different between protocols. ble 2 presents the overall findings of the animal studies included in this 296 (2) reported significant glucose concentration increases in obese rats after 8 h of intermittent 297 (30 s :30 s) HC to an extreme hypoxic level (FiO2 = 5.0%) per day over 2 weeks. Other 298 investigations have shown unchanged values when animals were exposed intermittently to 299 similar hypoxic levels using protocols of 40 s :80 s for 8 h (52) and 30 s :30 s for 12 h (55) 300 per day. It seems that the common response of glucose concentrations in obese animals, 301 passively exposed to varying levels of hypoxia, is yet to be verified. This variation in the 302 present findings may be partly explained through the differences in pre-analytical 303 conditions of sampled tissue, which was subsequently utilised for glucose concentration 304 assessment (84). 305 Insulin is receiving a great deal of attention due to its dominance in Type II Diabetes 306 control and development (26). In obese rats, insulin concentrations were unchanged 307 following intermittent HC for 8 h per day (40 s :80 s [52]; and 30 s :30 s [2]). This is perhaps 308 due to the severity of the hypoxic stimulus (FiO2 = ~5.0%) blunting improvements in this 309 health marker (50). Only one study has reported significant increases in insulin levels, which 310 occurred following both a 5 day (+356%) and 12 week (+185%) hypoxic intervention in 311 obese mice (55). The highly significant increase in insulin concentrations shown here may 312 not actually be of benefit. Perhaps, exacerbation of insulin resistance occurred, leading to 313 hyperinsulinemia (79). It is interesting to note that the hypoxic level employed in these 314 studies was similar (FiO2 = ~5.0%), and animals were intermittently exposed to hypoxia over 315 1 :1 (30 s :30 s) and 1 :2 (40 s :80 s) sequences. Reducing the severity of hypoxia during 316 exposure periods may prevent dramatic increases, as reported here by Polotsky et al. (55), 317 12 12 and protect against subsequent exacerbation and development of hyperinsulinemia. This 318 assumption, however, needs to be verified in an obese human population. 319 Varying findings of cholesterol following HC have been reported. Reductions in total 320 cholesterol were found following hypoxic exposure (15 min :5–10 min, 8 times per day for 321 40 days, FiO2 = 14.3%) in both lean and obese mice (34). ble 2 presents the overall findings of the animal studies included in this 347 Notwithstanding, equal changes occurred in the control group (+30%), whom didn’t received 348 any hypoxic treatment. In addition to this, mean arterial blood pressure increased similarly in 349 both groups. This may have been a result of the investigators feeding animals a high-fat diet 350 alongside the hypoxic intervention, and subsequently blunting the potentially beneficial 351 effects. These findings further highlight the co-morbidity relationship between obesity and 352 hypertension whilst consuming a high-fat diet, which will not be reduced with severe hypoxic 353 levels as shown in other studies (2, 52). 354 In summary, a small amount of evidence suggests that leptin may be a marker 343 associated with weight loss, due to the findings of slower weight gain following passive 344 associated with weight loss, due to the findings of slower weight gain following passive 344 HC. 345 Triglycerides, an important part of fat storage (13), have been found to increase 346 following intermittent (40 s :80 s) HC for 8 h per day over 2 weeks in obese rats (+30% [52]). 347 Notwithstanding, equal changes occurred in the control group (+30%), whom didn’t received 348 any hypoxic treatment. In addition to this, mean arterial blood pressure increased similarly in 349 both groups. This may have been a result of the investigators feeding animals a high-fat diet 350 alongside the hypoxic intervention, and subsequently blunting the potentially beneficial 351 effects. These findings further highlight the co-morbidity relationship between obesity and 352 hypertension whilst consuming a high-fat diet, which will not be reduced with severe hypoxic 353 levels as shown in other studies (2, 52). 354 Finally, only three studies have measured body weight before and after a chronic 355 passive hypoxic intervention in obese animals. Ling et al. (34) found weight to increase 356 equally in the hypoxic group (+79%) and the control group (+78%). Rodriguez et al. (59) 357 reported weight to increase in the hypoxia group (+9%) but with slightly greater increases in 358 the control group (+13%). Previously, weight loss has generally been observed in the first 359 days of exposure (41, 42). However, this has not been the case in the present review. 360 Finally, Polotsky et al. (55) reported no change in any group. The discrepancy in findings of 361 body weight following passive HC presented here may be due to a lack of dietary control. ble 2 presents the overall findings of the animal studies included in this Contrastingly, an increase in total 322 cholesterol values occured following HC (40 s :80 s for 8 h per day over 14 days, FiO2 = 323 5.0%) in obese rats, and in control (no hypoxic exposure) lean and obese rats (52). In another 324 study, no difference was reported in both lean and obese animals exposed to hypoxia (30 s 325 :30 s for 8 h per day over 14 days, FiO2 = 5.0%) or those who received no hypoxic exposure 326 (2). Although the variance is apparent, it is difficult to interpret findings due to there being a 327 lack of individual evaluation of levels of high-density (HDL) and low-density (LPL) 328 lipoprotein. Increases in total cholesterol in response to HC may in fact be a result of an 329 increase in the HDL/LDL ratio, which would actually be beneficial but is not yet clear in the 330 current literature. 331 Leptin, a satiety hormone, is suggested to be associated with weight loss due to its 332 action on hypothalamic metabolism and appetite suppression, potentiating a reduced energy 333 intake (47). It is also considered a growing marker of weight loss during and following HC 334 (50). Studies have reported increases in leptin in both hypoxic and normoxic groups (2, 52), 335 but there was no assessment of body weight changes. Increases in serum leptin have been 336 found following intermittent, moderate hypoxic and normoxic exposure of 15 min :5–10 min, 337 respectively, compared to those who received no exposure to hypoxia (34), which was also 338 aligned with slower rates of weight gain (+79% versus +100%, respectively). Notably, the 339 animal models were fed a high-fat diet during the course of the intervention, which therefore 340 may explain the reports of weight gain in this study. It could be that the weight gain was a 341 result of increases in muscle mass of the animal models, but this measure was not assessed. 342 13 13 In summary, a small amount of evidence suggests that leptin may be a marker 343 associated with weight loss, due to the findings of slower weight gain following passive 344 HC. 345 Triglycerides, an important part of fat storage (13), have been found to increase 346 following intermittent (40 s :80 s) HC for 8 h per day over 2 weeks in obese rats (+30% [52]). ble 2 presents the overall findings of the animal studies included in this (37) concluded that, compared to a control group, who received no exposure to 384 hypoxia or exercise completion, obese rats lost significant amounts of weight, fat mass, LDL 385 and total cholesterol after a combination of 60-min running sessions in hypoxic conditions 386 (FiO2 = 13.6%) and permenant residence in the same hypoxic environment, conducted over 4 387 weeks. Therefore, perhaps the increased physical workload, regardless of the conditions the 388 animal models were in, led to improvements in cardio-metabolic health and reductions in 389 weight. Notably, HDL cholesterol was reduced in the hypoxic group (37), presenting a 390 negative effect of active hypoxic exposure, as HDL cholesterol is deemed as ‘good’ 391 actually report weight gain after repeated passive HC over 4–12 weeks. To summarise, 367 passive HC in obese animals, fed a high-fat diet, does not lead to conclusive weight loss. 368 Table 2 near here 369 370 4.1.2. Combined passive and active hypoxic exposure 371 Unlike passive HC alone, reductions in fasting glucose and insulin responses have 372 typically been found following a combination of passive HC and normoxic active periods (6, 373 79). Interestingly, the hypoxic level (FiO2 = ~14.0%) as well as the duration and mode of 374 exercise employed (1.5 h of swimming) was similar across studies. Notably, Wu et al. (79) 375 also found reductions in fasting glucose concentrations within the group whom carried out 376 normoxic exercise without passive HC. This raises questions as to whether exercise alone is 377 more effective than a combination of exposure modes. Passive HC and normoxic active 378 periods, when combined, could potentially improve metabolic and hormonal responses 379 of obese animals. Pending confirmatory research, this could at least in part be ascribed to 380 improved insulin sensitivity and cellular glucose uptake. 381 The primary question regarding the use of passive and active HC is whether it leads to 382 more benefical health outcomes than a similar workload completed in normoxic conditions. 383 Lu et al. (37) concluded that, compared to a control group, who received no exposure to 384 hypoxia or exercise completion, obese rats lost significant amounts of weight, fat mass, LDL 385 and total cholesterol after a combination of 60-min running sessions in hypoxic conditions 386 (FiO2 = 13.6%) and permenant residence in the same hypoxic environment, conducted over 4 387 weeks. ble 2 presents the overall findings of the animal studies included in this 362 From an experimental perspective, controlling caloric intake may be difficult in animal 363 models and subsequently leads to disparate changes in weight (i.e., weight gain). Only 364 Polotsky et al. (55) stated what the animal models were fed throughout the intervention and 365 reported no change in body weight. The majority of available studies presented here 366 Finally, only three studies have measured body weight before and after a chronic 355 passive hypoxic intervention in obese animals. Ling et al. (34) found weight to increase 356 equally in the hypoxic group (+79%) and the control group (+78%). Rodriguez et al. (59) 357 reported weight to increase in the hypoxia group (+9%) but with slightly greater increases in 358 the control group (+13%). Previously, weight loss has generally been observed in the first 359 days of exposure (41, 42). However, this has not been the case in the present review. 360 14 14 actually report weight gain after repeated passive HC over 4–12 weeks. To summarise, 367 passive HC in obese animals, fed a high-fat diet, does not lead to conclusive weight loss. 368 Table 2 near here 369 370 4.1.2. Combined passive and active hypoxic exposure 371 Unlike passive HC alone, reductions in fasting glucose and insulin responses have 372 typically been found following a combination of passive HC and normoxic active periods (6, 373 79). Interestingly, the hypoxic level (FiO2 = ~14.0%) as well as the duration and mode of 374 exercise employed (1.5 h of swimming) was similar across studies. Notably, Wu et al. (79) 375 also found reductions in fasting glucose concentrations within the group whom carried out 376 normoxic exercise without passive HC. This raises questions as to whether exercise alone is 377 more effective than a combination of exposure modes. Passive HC and normoxic active 378 periods, when combined, could potentially improve metabolic and hormonal responses 379 of obese animals. Pending confirmatory research, this could at least in part be ascribed to 380 improved insulin sensitivity and cellular glucose uptake. 381 The primary question regarding the use of passive and active HC is whether it leads to 382 more benefical health outcomes than a similar workload completed in normoxic conditions. 383 Lu et al. ble 2 presents the overall findings of the animal studies included in this Therefore, perhaps the increased physical workload, regardless of the conditions the 388 animal models were in, led to improvements in cardio-metabolic health and reductions in 389 weight. Notably, HDL cholesterol was reduced in the hypoxic group (37), presenting a 390 negative effect of active hypoxic exposure, as HDL cholesterol is deemed as ‘good’ 391 The primary question regarding the use of passive and active HC is whether it leads to 382 more benefical health outcomes than a similar workload completed in normoxic conditions. 383 Lu et al. (37) concluded that, compared to a control group, who received no exposure to 384 hypoxia or exercise completion, obese rats lost significant amounts of weight, fat mass, LDL 385 and total cholesterol after a combination of 60-min running sessions in hypoxic conditions 386 (FiO2 = 13.6%) and permenant residence in the same hypoxic environment, conducted over 4 387 weeks. Therefore, perhaps the increased physical workload, regardless of the conditions the 388 animal models were in, led to improvements in cardio-metabolic health and reductions in 389 weight. Notably, HDL cholesterol was reduced in the hypoxic group (37), presenting a 390 negative effect of active hypoxic exposure, as HDL cholesterol is deemed as ‘good’ 391 15 cholesterol (17). The change in HDL levels may be a reflection in the overall reduction in 392 total cholesterol. Therefore, this may have led to a reduction in HDL and LDL, but with the 393 maintenance of relative concentrations and HDL:LDL ratio. 394 cholesterol (17). The change in HDL levels may be a reflection in the overall reduction in 392 total cholesterol. Therefore, this may have led to a reduction in HDL and LDL, but with the 393 maintenance of relative concentrations and HDL:LDL ratio. 394 The remaining two combined normoxic active periods and passive HC studies 395 included in this review, which measured weight pre- and post-intervention, reported similar 396 findings. Both Chen et al. (6) and Wu et al. (79) implemented identical protocols consisting 397 of daily 90-min swimming sessions in normoxic conditions followed by passive HC (FiO2 = 398 14.0%, sustained for 8-h per day). Both studies found greater body weight attenuation of the 399 obese animal models, in comparison to the increase in the control group (no passive and 400 active exposure). Further, weight did not change in the group who completed exercise in 401 normoxic conditions without passive HC (6, 79). ble 2 presents the overall findings of the animal studies included in this These findings suggest that a combination 402 of passive and active HC is possibly more beneficial for weight control than a matched 403 workload in normoxia. To date, however, the mechanisms that induce this response remain 404 unclear (28). Possible increases in daily metabolic rate of only those in the hypoxic groups, 405 causing a negative energy balance, may have occurred. Or perhaps appetite was surpressed 406 through increased leptin concentrations, resulting in a reduced calorie intake. However, 407 neither of these reponses were assessed in these investigations. 408 ble 3 presents the overall findings of the human studies included in this Therefore, previously employed passive HC protocols in 423 humans may not be beneficial to improve cardio-metabolic health (reduce blood pressure) or 424 lose weight. 425 moderate (FiO2 = 15.0%) hypoxia (69). However, the participants included in these studies 417 had a healthy BMI (22–27 kg.m2), yet deemed as sedentary, which may explain the 418 ineffective treatment on blood pressure and body weight. Moreover, it could be suggested 419 that the participant cohort in these studies (69, 77) required a more severe level of hypoxia to 420 elicit positive responses. In support of this, recent reviews (50, 67) have indicated a linear 421 continuum between no additive effect and a deleterious effect with HC that is dependent on 422 the severity of the hypoxic stimulus. Therefore, previously employed passive HC protocols in 423 humans may not be beneficial to improve cardio-metabolic health (reduce blood pressure) or 424 lose weight. 425 lose weight. 425 In their study, Workman & Basset (77) assessed metabolic responses, via a 30-min 426 metabolic rate determination test pre- and post-intervention. They found increases in energy 427 expenditure following acute (+16%) and short-term (+12%) HC, as did lipid metabolism 428 (+44% and +29%, respectively); whereas, glycogen metabolism decreased (-31% and -49%, 429 respectively). Collectively, these findings suggest that passive HC may be an effective 430 modality to induce a shift in fuel utilisation and expend a greater quantity of lipid-based 431 energy stores. Over a longer duration, this may lead to a substantially consistent negative 432 energy balance which may promote measurable weight loss. To date, such a protocol has not 433 been employed in an obese human population. 434 Table 3 near here 435 436 4.2.2. Active hypoxic exposure 437 Metabolic responses have been assessed following active HC (60–90-min moderate- 438 intensity cardiovascular activity, 3 sessions per week, 4–6 weeks, FiO2 = 15.0%) in obese 439 humans. Netzer et al. (51) reported greater enhancements in triglycerides, total cholesterol 440 and HDL in those whom completed 8 weeks training for 90-min at 60% of HRmax in hypoxic 441 In their study, Workman & Basset (77) assessed metabolic responses, via a 30-min 426 metabolic rate determination test pre- and post-intervention. They found increases in energy 427 expenditure following acute (+16%) and short-term (+12%) HC, as did lipid metabolism 428 (+44% and +29%, respectively); whereas, glycogen metabolism decreased (-31% and -49%, 429 respectively). ble 3 presents the overall findings of the human studies included in this Collectively, these findings suggest that passive HC may be an effective 430 modality to induce a shift in fuel utilisation and expend a greater quantity of lipid-based 431 energy stores. Over a longer duration, this may lead to a substantially consistent negative 432 energy balance which may promote measurable weight loss. To date, such a protocol has not 433 been employed in an obese human population. 434 ble 3 presents the overall findings of the human studies included in this 16 moderate (FiO2 = 15.0%) hypoxia (69). However, the participants included in these studies 417 had a healthy BMI (22–27 kg.m2), yet deemed as sedentary, which may explain the 418 ineffective treatment on blood pressure and body weight. Moreover, it could be suggested 419 that the participant cohort in these studies (69, 77) required a more severe level of hypoxia to 420 elicit positive responses. In support of this, recent reviews (50, 67) have indicated a linear 421 continuum between no additive effect and a deleterious effect with HC that is dependent on 422 the severity of the hypoxic stimulus. Therefore, previously employed passive HC protocols in 423 humans may not be beneficial to improve cardio-metabolic health (reduce blood pressure) or 424 lose weight. 425 In their study, Workman & Basset (77) assessed metabolic responses, via a 30-min 426 metabolic rate determination test pre- and post-intervention. They found increases in energy 427 expenditure following acute (+16%) and short-term (+12%) HC, as did lipid metabolism 428 (+44% and +29%, respectively); whereas, glycogen metabolism decreased (-31% and -49%, 429 respectively). Collectively, these findings suggest that passive HC may be an effective 430 modality to induce a shift in fuel utilisation and expend a greater quantity of lipid-based 431 energy stores. Over a longer duration, this may lead to a substantially consistent negative 432 energy balance which may promote measurable weight loss. To date, such a protocol has not 433 been employed in an obese human population. 434 Table 3 near here 435 436 4.2.2. Active hypoxic exposure 437 Metabolic responses have been assessed following active HC (60–90-min moderate- 438 intensity cardiovascular activity, 3 sessions per week, 4–6 weeks, FiO2 = 15.0%) in obese 439 moderate (FiO2 = 15.0%) hypoxia (69). However, the participants included in these studies 417 had a healthy BMI (22–27 kg.m2), yet deemed as sedentary, which may explain the 418 ineffective treatment on blood pressure and body weight. Moreover, it could be suggested 419 that the participant cohort in these studies (69, 77) required a more severe level of hypoxia to 420 elicit positive responses. In support of this, recent reviews (50, 67) have indicated a linear 421 continuum between no additive effect and a deleterious effect with HC that is dependent on 422 the severity of the hypoxic stimulus. 4.2.2. Active hypoxic exposure Metabolic responses have been assessed following active HC (60–90-min moderate- 438 intensity cardiovascular activity, 3 sessions per week, 4–6 weeks, FiO2 = 15.0%) in obese 439 humans. Netzer et al. (51) reported greater enhancements in triglycerides, total cholesterol 440 and HDL in those whom completed 8 weeks training for 90-min at 60% of HRmax in hypoxic 441 17 versus normoxic conditions. In other studies, no change has been found in both the hypoxic 442 and normoxic groups for triglycerides, total cholesterol and HDL following a similar exercise 443 intensity range and duration over 4 weeks (46, 76). Morishima et al. (46) also reported that 444 glucose concentrations decreased in both the hypoxic (-8%) and normoxic (-7%) group over 445 the course of the intervention. These findings are interesting as all intervention groups 446 exercised under the same hypoxic level and completed the same type of exercise at an 447 ‘absolute’ intensity, i.e. an intensity regardless of the environmental condition. Consequently, 448 differences in findings may have been related primarily to the total duration of the studies (8 449 [51] versus 4 weeks [46, 76]). Therefore, it appears that further improvements in metabolic 450 markers such as triglycerides, total cholesterol and HDL with HC would require an 451 intervention of more than 4 weeks in duration for positive effects. 452 versus normoxic conditions. In other studies, no change has been found in both the hypoxic 442 and normoxic groups for triglycerides, total cholesterol and HDL following a similar exercise 443 intensity range and duration over 4 weeks (46, 76). Morishima et al. (46) also reported that 444 glucose concentrations decreased in both the hypoxic (-8%) and normoxic (-7%) group over 445 the course of the intervention. These findings are interesting as all intervention groups 446 exercised under the same hypoxic level and completed the same type of exercise at an 447 ‘absolute’ intensity, i.e. an intensity regardless of the environmental condition. Consequently, 448 differences in findings may have been related primarily to the total duration of the studies (8 449 [51] versus 4 weeks [46, 76]). Therefore, it appears that further improvements in metabolic 450 markers such as triglycerides, total cholesterol and HDL with HC would require an 451 intervention of more than 4 weeks in duration for positive effects. 4.2.2. Active hypoxic exposure 452 In two studies, fasting insulin reductions have been found in both hypoxic (FiO2 = 453 15.0%) and normoxic exercise (60 mins, moderate intensity, 3 times per week, for 4 weeks) 454 groups over the course of an intervention (hypoxia: -37%, normoxia: -33% [76]; hypoxia: - 455 22%, normoxia: -36% [46]). Although not significant, baseline assessment in both studies of 456 insulin concentrations were ~2 arbitary units larger in the hypoxic compared to normoxic 457 group. Therefore, this may explain the insignificant effect of the hypoxic stimulus as those in 458 the control group started the intervention at a lower concentration. Additional consideration 459 of other hormonal markers, such as growth horomone and insulin-like growth factor, that 460 may further lead to enhancements of potential weight loss through promotion of mechanistic 461 responses (60) warrant further investigation. 462 Hypertension is extremely prevelant in obese populations, causing an increased strain 463 on an already laboured cardiovascular system (33). Kong et al. (32) implemented 464 cardiovascular- and strength-based exercise in an obese population and found significant 465 improvements of systolic (-8%) and diastolic (-7%) blood pressure after 4 weeks of 22 h of 466 18 exercise per week in the hypoxic group. Notably, their hypoxic group participants completed 467 6 h of the weekly training schedule (type of exercise session unknown) in a hypoxic 468 environment, with the remainder carried out in normoxic conditions. Whereas, those who 469 carried out all of the 22 h training load in normoxic conditions had less improvement in 470 systolic (-3%) and diastolic (-1%) blood pressures. Compared to the normoxic group, 471 Wiesner et al. (76) also reported a similar reduction in systolic (-2% versus -2%) but greater 472 reduction in diastolic (-4% versus -1%) blood pressures in the hypoxic group over a similar 473 duration of 4 weeks, yet with a reduced volume of exercise (180 min per week). All in all, 474 active HC demonstrates more supportive evidence for improved blood pressure responses 475 compared to active normoxic periods. That said, a previous review (62) concluded significant 476 benefits to blood pressure values following active HC compared to normoxic conditions in 477 those with various cardiovascular diseases, including normalisation and 3 month maintenance 478 of stage 1 hypertensive patients (39). 4.2.2. Active hypoxic exposure It could also be suggested that multiple combinations of 479 exercise (cardiovascular and strength) carried out in hypoxic conditions are more beneficial 480 than cardiovascular exercise alone to reduce blood pressure in obese populations. This is 481 supported by the findings of Kong et al. (32), perhaps through enhanced vascular endothelial 482 growth factor transcription leading to improved human vasculature control and capillary 483 action (82). 484 467 Reductions in heart rate, for a given exercise workload, have been observed for both 485 hypoxic (-18%) and normoxic (-20%) groups post-intervention (32), yet only statistically 486 significant in the normoxic group. In other studies, no change in heart rate during an exercise 487 test before and after the intervention period was found in the hypoxic or normoxic group (46, 488 76) – although lactate accumulation was reduced in both intervention groups (hypoxic: -11%, 489 normoxic: -13% [76]). It could be suggested that due to obese humans having a lower 490 baseline fitness level compared to athletic and healthy populations, it is likely that any form 491 19 of training will lead to an improved recovery response, via assessment of heart rate. 492 Arguably, adding in an additional stimulus such as hypoxia likely reduces the potential of an 493 increased recovery, and therefore, be less beneficial than the same workload in normoxic 494 conditions. 495 of training will lead to an improved recovery response, via assessment of heart rate. 492 Arguably, adding in an additional stimulus such as hypoxia likely reduces the potential of an 493 increased recovery, and therefore, be less beneficial than the same workload in normoxic 494 conditions. 495 Kong et al. (32) showed non-significant reductions in BMI (-6%) and weight (-7%) of 496 the hypoxic group, however, obese humans in the normoxic group also showed non- 497 significant weight loss post-intervention (-4%). Netzer et al. (51) reported non-significant 498 reductions in weight and BMI in the hypoxic group, however, this did not occur in the 499 normoxic group. In another study, no change was found in BMI and fat mass following both 500 the hypoxic (FiO2 = 15.0%) and normoxic intervention (moderate-intensity cycling, 3 times 501 per week, 4 weeks), but the normoxic group did lose slightly more weight after the 502 intervention compared to those in the hypoxic group (-1% versus -0.5%, respectively [46]). 4.2.2. Active hypoxic exposure 503 Overall, reductions in weight, BMI and individual tissue mass are found following active HC 504 (moderate-intensity cardio-based exercise, 3 sessions per week, 4–8 week duration). This also 505 occurs without hypoxia but to a lesser extent. Non-significant improvements in these studies 506 may be strengthed if the small participant cohorts (~10 individuals per group) were increased 507 to permit a greater effect size. Alternatively, it could be considered that participants became 508 acclimatised to the hypoxic level (FiO2 = 15.0%), which was consistently maintained 509 throughout the whole intervention period (4–8 weeks). This could have lead to a rapid plateau 510 of adaptations in body composition as the absence of periodisation may not perpetuate 511 beneficial gains. 512 20 12.0%) twice per week, for 8 months in obese males and females. After 5 weeks, similar 517 changes in both hypoxic and normoxic groups were reported for body weight (-2% and -1%) 518 and fat mass (+1% and -1%). After 3 months, these responses were futher improved in 519 comparison to the baseline assessment in the hypoxic (body weight: -4%, fat mass: -1%) as 520 well as normoxic (body weight: -3%, fat mass: -2%) group. Additionally, similar reductions 521 were found in both hypoxic and normoxic groups for values of systolic (-3% and -2%) and 522 diastolic blood pressure (-3% and -3%). Following completion of the 8 month intervention 523 period, those in the hypoxic group displayed reductions in fat mass (-1%) and blood pressure 524 (systolic: -4%, diastolic: -2%). However, similar responses were found in the normoxic group 525 (fat mass: -2%; systolic blood pressure: -6%; diastolic blood pressure: -5%). Interestingly, 526 body weight was equally reduced in both groups (-3%) post-intervention. In the only 527 available study, it seems that a combination of both passive and active HC has no added 528 benefit compared to a matched workload in normoxic conditions on weight loss and cardio- 529 metabolic responses assessed here. The main explantion would be that unaltered stimuli (i.e., 530 hypoxic level, exercise intensity/duration) throughout the intervention lead to a near plateau 531 in most measures assessed over this 8 month period. 532 5. Additional considerations At present, it is difficult to affirm that overall fitness is improved following active HC 535 versus similar exercise training in normoxia of obese populations. Exercise performance in an 536 obese population, assessed via total running distance over the course of a 4-week 537 intervention, showed a tendency of being higher in the hypoxic compared to the normoxic 538 group (+18% [32]). In contrast, workload during hypoxic in reference to normoxic sessions in 539 other studies was typically lower (-17.5% [76], -20% [46]). When exercising in hypoxia, 540 exercise may be perceived as ‘harder’ (majored internal load as evidenced by higher heart 541 21 rate, rating of perceived exertion or blood lactate values) versus a matched workload in 542 normoxia, leading to a reduced total workload. Therefore, it may be that obese humans 543 require multiple exercise modalities to continue exercising at a clamped intensity and 544 complete a greater total workload. 545 rate, rating of perceived exertion or blood lactate values) versus a matched workload in 542 normoxia, leading to a reduced total workload. Therefore, it may be that obese humans 543 require multiple exercise modalities to continue exercising at a clamped intensity and 544 complete a greater total workload. 545 Cardiorespiratory fitness (VO2max) is a key determinant of morbidity and mortality 546 (74). Following active HC (60-mins cardiovascular-based exercise, 55–65% VO2max, 3 times 547 per week, for 4 weeks) non-significant increases in this determinant have been reported (46, 548 76). However, these enhancements were visible in both the hypoxic and normoxic exercise 549 groups (+5.6% versus +3.1% [76], +12.6% versus +8.7% [46]; hypoxia versus normoxia, 550 respectively). Taken as a whole, this could indicate that the mode of exercise is primarily 551 responsible for gains (i.e., not the addition of the hypoxic stimulus). Undoubtedly, detection 552 of adaptations to the intervention is paramount to select training intensity, modality and 553 duration for successful interventions in obese populations. One may argue that the studies 554 included in the present review have primarily implemented exercise performance tests that 555 are overly challenging for obese populations, due to the requirement of exercising to 556 volitional exhaustion (46, 76). Other sea-level training studies of obese populations have 557 incorporated a 10-m walk test (23), a 6-minute step test (5) and a 6-minute walk test (27) to 558 assess post-intervention changes in aerobic exercise performance. 5. Additional considerations Further research of this area is required to validate this 604 claim and differentiate the effect of adding hypoxia in comparison to the effect of exercising 605 at different intensities. It could be that, clamping the metabolic demand (i.e., working at a 606 given relative exercise intensity in hypoxia versus normoxia) may be beneficial for obese 607 populations. Arguably, the muscoskeletal system load is likely reduced in O2-deprived 608 environments and thereby could prevent further damage to joints, tendons and ligamets 609 during locomotor activities (e.g., outdoor or treadmill walking). 610 In line with current American College of Sports Medicine (12) and UK National 611 Health Service recommendations (49), the reviewed literature here suggests that a moderate- 612 intensity, continuous exercise training programme (60–75% HRmax for 60–90-min, 3 times 613 per week) is the recommended method to achieve weight loss. However, a growing body of 614 literature is indicating that implementation of high-intensity intermittent exercise (3–5 sets of 615 A number of studies in this review mention a reduced workload of participants 592 carrying out moderate-intensity, continuous exercise in hypoxia compared to those in 593 normoxia (46, 76), which has also been proposed elsewhere when clamping the metabolic 594 demand (20). It would be interesting to investigate whether the cardio-metabolic responses of 595 obese populations are significantly different between relative and absolute exercise intensities 596 using direct comparisons (i.e., same participants), which may inform which exercise intensity 597 is more suitable for setting training goals in this population. For example, cycling at 100 598 watts in hypoxic conditions will create a greater physiological strain (increased heart rate, 599 cardiac output) on the human body compared to the same absolute intensity in normoxic 600 conditions; thus inducing a higher internal (physiological) load for a matched external (power 601 output) load. When cycling at a similar relative intensity, the internal load most likely will be 602 reduced during hypoxia to match the external load of exercising in normoxia, as 603 demonstrated by Wiesner et al. (76). Further research of this area is required to validate this 604 claim and differentiate the effect of adding hypoxia in comparison to the effect of exercising 605 at different intensities. It could be that, clamping the metabolic demand (i.e., working at a 606 given relative exercise intensity in hypoxia versus normoxia) may be beneficial for obese 607 populations. 5. Additional considerations To date, the inclusion of 559 such performance tests is lacking in the field of HC. 560 Other than one study which utilised a fixed SpO2 (77), all studies presented in this 561 review have implemented a fixed FiO2 during exposure to hypoxia. One potential issue, 562 however, is that the variance in individual response to a given simulated altitude is 563 significant. In support of this, Hamlin et al. (22) concluded that for exposure to the same 564 hypoxic level (FiO2 = 10.0%), there is a greater inter-individual variability in the extent of 565 arterial desaturation compared to a clamped SpO2 of 75%. Additionally, obese humans are 566 22 considered as having a higher ‘resistance to hypoxia’ in comparison to healthy humans, and 567 thereby a delayed/minimal desaturation (or SpO2 decrease) when exposed to low hypoxic 568 doses (FiO2 12.0% [54]). To negate this, implementing fixed SpO2 values may minimise the 569 number of ‘non-responding’ participants to a given hypoxic stimulus. Costalat et al. (8) 570 recently investigated individualised intermittent passive exposure to hypoxia (SpO2 ~80%), 571 including normoxia phases (re-oxygenation to ~95%), in overweight and obese individuals. 572 However, this investigation was not included in this review due to a lack of a 573 control/normoxic condition. 574 23 A number of studies in this review mention a reduced workload of participants 592 carrying out moderate-intensity, continuous exercise in hypoxia compared to those in 593 normoxia (46, 76), which has also been proposed elsewhere when clamping the metabolic 594 demand (20). It would be interesting to investigate whether the cardio-metabolic responses of 595 obese populations are significantly different between relative and absolute exercise intensities 596 using direct comparisons (i.e., same participants), which may inform which exercise intensity 597 is more suitable for setting training goals in this population. For example, cycling at 100 598 watts in hypoxic conditions will create a greater physiological strain (increased heart rate, 599 cardiac output) on the human body compared to the same absolute intensity in normoxic 600 conditions; thus inducing a higher internal (physiological) load for a matched external (power 601 output) load. When cycling at a similar relative intensity, the internal load most likely will be 602 reduced during hypoxia to match the external load of exercising in normoxia, as 603 demonstrated by Wiesner et al. (76). 7.2. Psychological aspect of weight loss 7.2. Psychological aspect of weight loss 623 A large, and often underestimated, factor in achieving weight loss is related to 624 psychological behaviours. Exercising regularly requires motivation and enjoyment to 625 maintain adherence (31). At present, pleasure-displeasure responses of healthy populations 626 exercising at a high-intensity in normoxic conditions are varied with both positive affects 627 (43) and negative moods (48) reported. To our knowledge, this type of investigation does not 628 exist during and following HC of obese humans. Implementing such affect-perceptual 629 measurements would significantly aid levels of adherence to achieve weight loss through 630 long-term interventions. Interestingly, Ekkekakis & Linds (14) concluded that enjoyment 631 was reduced when obese populations had an imposed exercise intensity 10% greater 632 than a self-selected speed. It remains to be verified whether implementation of self- 633 selected speeds during shorter work periods in hypoxia would be more applicable in an 634 obese population, as previously reported (14, 25). 635 636 7.3. Differences within obese populations 637 Although this review is focused on the treatment of obese (BMI: 30–38 kg.m2) 638 populations, some studies have been included with participant groups of overweight and 639 sedentary animals and humans, with a large majority of evidence derived from obese animal 640 findings. Further comparative research is warranted to investigate the responses of different 641 A large, and often underestimated, factor in achieving weight loss is related to 624 psychological behaviours. Exercising regularly requires motivation and enjoyment to 625 maintain adherence (31). At present, pleasure-displeasure responses of healthy populations 626 exercising at a high-intensity in normoxic conditions are varied with both positive affects 627 (43) and negative moods (48) reported. To our knowledge, this type of investigation does not 628 exist during and following HC of obese humans. Implementing such affect-perceptual 629 measurements would significantly aid levels of adherence to achieve weight loss through 630 long-term interventions. Interestingly, Ekkekakis & Linds (14) concluded that enjoyment 631 was reduced when obese populations had an imposed exercise intensity 10% greater 632 than a self-selected speed. It remains to be verified whether implementation of self- 633 selected speeds during shorter work periods in hypoxia would be more applicable in an 634 obese population, as previously reported (14, 25). 635 636 5. Additional considerations Not only is this 617 form of exercise more time- and metabolically-efficient (36), but also would be more 618 beneficial for weight loss compared to moderate-intensity during normoxia (9, 60, 83). In 619 prescribing such exercise, a careful manipulation of work :rest ratios depending on the aim 620 of the session (aerobically versus anaerobically-based responses) is needed. 621 622 5. Additional considerations Arguably, the muscoskeletal system load is likely reduced in O2-deprived 608 environments and thereby could prevent further damage to joints, tendons and ligamets 609 during locomotor activities (e.g., outdoor or treadmill walking). 610 In line with current American College of Sports Medicine (12) and UK National 611 592 24 recovery periods of 2–3 min) in obese populations is beneficial (19, 54, 75). Not only is this 617 form of exercise more time- and metabolically-efficient (36), but also would be more 618 beneficial for weight loss compared to moderate-intensity during normoxia (9, 60, 83). In 619 prescribing such exercise, a careful manipulation of work :rest ratios depending on the aim 620 of the session (aerobically versus anaerobically-based responses) is needed. 621 622 7.2. Psychological aspect of weight loss 623 A large, and often underestimated, factor in achieving weight loss is related to 624 psychological behaviours. Exercising regularly requires motivation and enjoyment to 625 maintain adherence (31). At present, pleasure-displeasure responses of healthy populations 626 exercising at a high-intensity in normoxic conditions are varied with both positive affects 627 (43) and negative moods (48) reported. To our knowledge, this type of investigation does not 628 exist during and following HC of obese humans. Implementing such affect-perceptual 629 measurements would significantly aid levels of adherence to achieve weight loss through 630 long-term interventions. Interestingly, Ekkekakis & Linds (14) concluded that enjoyment 631 was reduced when obese populations had an imposed exercise intensity 10% greater 632 than a self-selected speed. It remains to be verified whether implementation of self- 633 selected speeds during shorter work periods in hypoxia would be more applicable in an 634 obese population, as previously reported (14, 25). 635 636 7.3. Differences within obese populations 637 Although this review is focused on the treatment of obese (BMI: 30–38 kg m2) 638 recovery periods of 2–3 min) in obese populations is beneficial (19, 54, 75). Not only is this 617 form of exercise more time- and metabolically-efficient (36), but also would be more 618 beneficial for weight loss compared to moderate-intensity during normoxia (9, 60, 83). In 619 prescribing such exercise, a careful manipulation of work :rest ratios depending on the aim 620 of the session (aerobically versus anaerobically-based responses) is needed. 621 recovery periods of 2–3 min) in obese populations is beneficial (19, 54, 75). 7.3. Differences within obese populations Although this review is focused on the treatment of obese (BMI: 30–38 kg.m2) 638 populations, some studies have been included with participant groups of overweight and 639 sedentary animals and humans, with a large majority of evidence derived from obese animal 640 findings. Further comparative research is warranted to investigate the responses of different 641 25 stages of obese populations (e.g., I, II and III [10]), males versus females, and young versus 642 older populations with or without associated complications (i.e., pre-diabetes). 643 stages of obese populations (e.g., I, II and III [10]), males versus females, and young versus 642 older populations with or without associated complications (i.e., pre-diabetes). 643 644 7.4. Experimental considerations 645 Finally, determining the extent of metabolic stress associated with HC for inducing 646 clinically relevant (>3%) weight losses (66) should be a key focus area. Arguably, many 647 confounding variables likely affect determination of the optimal dose-response during HC, 648 such as food consumption, in the lead up to and following the completion of sessions. If these 649 were to be controlled, and short-term (single session) cardio-metabolic responses were to be 650 assessed in obese populations, it will be possible to implement the ‘optimal’ exposure 651 protocol (i.e., most beneficial dose, duration and intensity) for long-term improvements in 652 cardio-metabolic health and weight loss, as proposed recently by Serebrovskaya et al. (62). 653 Additional consideration of potential drawbacks associated with HC, such as onset of 654 obstructive sleep apnoea and acute mountain sickness, should be made to increase the 655 possibility of developing optimal passive and active HC protocols. 656 657 8. A summary of passive and active HC protocols 658 Table 4 states a summary of passive and active HC protocols in relation to the 659 literature presented in this review for improving cardio-metabolic health and 660 promoting weight loss in obese humans. HC-induced physiological, metabolic, 661 cardiovascular and hormonal responses are undoubtedly highly individual. 662 Importantly, all of the animal models and human participant cohorts included here 663 were free from associated cardio-metabolic complications. In reality, this may not 664 always be the case. 7.3. Differences within obese populations Therefore, we recommend full general practitioner clearance to be 665 obtained from prior to undertaking any HC, similar to the process of beginning any 666 Finally, determining the extent of metabolic stress associated with HC for inducing 646 clinically relevant (>3%) weight losses (66) should be a key focus area. Arguably, many 647 confounding variables likely affect determination of the optimal dose-response during HC, 648 such as food consumption, in the lead up to and following the completion of sessions. If these 649 were to be controlled, and short-term (single session) cardio-metabolic responses were to be 650 assessed in obese populations, it will be possible to implement the ‘optimal’ exposure 651 protocol (i.e., most beneficial dose, duration and intensity) for long-term improvements in 652 cardio-metabolic health and weight loss, as proposed recently by Serebrovskaya et al. (62). 653 Additional consideration of potential drawbacks associated with HC, such as onset of 654 obstructive sleep apnoea and acute mountain sickness, should be made to increase the 655 possibility of developing optimal passive and active HC protocols. 656 26 physical activity programme/dietary intervention. Positive outcomes would also likely 667 depend on the level of hypoxia employed and careful manipulation of key variables 668 structuring the HC routine (e.g., number of cycles, duration, intensity, mode of exercise 669 and/or periodisation). Importantly, this summary should be interpreted with caution 670 and seenas a starting point only, as it is based upon the findings of a small amount of 671 evidence (passive: 7 studies; active: 8 studies). We therefore encourage clinicians and 672 researchers to refine them to reach a consensus. 673 Table 4 near here 674 675 9. Conclusions 676 The findings of this review in obese populations suggest that a) passive HC could 677 lead to reduced insulin concentrations (-37 – -22%) in animals and increased energy 678 expenditure (+12 – +16%) in humans, while active HC may reduce body weight (-4 – - 679 2%) in both animals and humans as well as blood pressure (-8 – -3%) in humans; b) 680 inconsistent findings and limited understanding still exist for determining the impact of acute 681 and chronic HC on markers such as triglycerides, cholesterol levels and fitness capacity; and 682 c) a large majority of studies include animal models exposed to severe levels of hypoxia 683 (FiO2 = ~5.0%) that are not suitable for obese humans. 7.3. Differences within obese populations Also, published findings, at present, 684 do not clearly show changes in responses of heart rate, fat and muscle mass following HC 685 being significantly larger than a matched exposure and/or exercise period in normoxic 686 conditions. Nevertheless, the promising findings need larger cohorts, more mechanistic 687 measures and real-world applications of findings to improve the potential clinical impact of 688 this novel intervention. Finally, the industrial and technological advancement, including 689 miniaturised equipment for home use and accessibility to environmental chambers, will 690 certainly contribute to the expansion in the use of these methods. 691 physical activity programme/dietary intervention. Positive outcomes would also likely 667 depend on the level of hypoxia employed and careful manipulation of key variables 668 structuring the HC routine (e.g., number of cycles, duration, intensity, mode of exercise 669 and/or periodisation). Importantly, this summary should be interpreted with caution 670 and seenas a starting point only, as it is based upon the findings of a small amount of 671 evidence (passive: 7 studies; active: 8 studies). We therefore encourage clinicians and 672 researchers to refine them to reach a consensus. 673 Table 4 near here 674 675 9. Conclusions 676 The findings of this review in obese populations suggest that a) passive HC could 677 lead to reduced insulin concentrations (-37 – -22%) in animals and increased energy 678 expenditure (+12 – +16%) in humans, while active HC may reduce body weight (-4 – - 679 2%) in both animals and humans as well as blood pressure (-8 – -3%) in humans; b) 680 inconsistent findings and limited understanding still exist for determining the impact of acute 681 and chronic HC on markers such as triglycerides, cholesterol levels and fitness capacity; and 682 c) a large majority of studies include animal models exposed to severe levels of hypoxia 683 (FiO2 = ~5.0%) that are not suitable for obese humans. Also, published findings, at present, 684 do not clearly show changes in responses of heart rate, fat and muscle mass following HC 685 being significantly larger than a matched exposure and/or exercise period in normoxic 686 conditions. Nevertheless, the promising findings need larger cohorts, more mechanistic 687 measures and real-world applications of findings to improve the potential clinical impact of 688 this novel intervention. 7.3. Differences within obese populations Finally, the industrial and technological advancement, including 689 miniaturised equipment for home use and accessibility to environmental chambers, will 690 certainly contribute to the expansion in the use of these methods. 691 692 692 27 27 References References 693 Obese Surgery, 15(9), 1238–1242. 729 1. Boyer, S. J. & Blume, F. D. (1984). Weight loss and changes in body composition at high altitude. Journal of Applied Physiology, 57, 1580–1585. 1. Boyer, S. J. & Blume, F. D. (1984). 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Normobaric Intermittent Hypoxia over 8 Months Does Not 747 Reduce Body Weight and Metabolic Risk Factors-a Randomized, Single Blind, 748 Placebo-Controlled Study in Normobaric Hypoxia and Normobaric Sham 749 Hypoxia. Obesity Facts, 8(3), 200-209. 750 19. Gibala, M. J., Little, J. P., MacDonald, M. J., & Hawley, J. A. (2012). Physiological 751 adaptations to low‐volume, high‐intensity interval training in health and disease. The 752 Journal of Physiology, 590(5), 1077-1084. 753 20. Girard, O., Malatesta, D., & Millet, G. P. (2017). Walking in hypoxia: an efficient 754 treatment to lessen mechanical constraints and improve health in obese 755 individuals? Frontiers in Physiology, 8. 756 21. Guh, D. P., Zhang, W., Bansback, N., Amarsi, Z., Birmingham, C. L. & Anis, A. H. 757 (2009). The incidence of co-morbidities related to obesity and overweight: a 758 systematic review and meta-analysis. BMC Public Health, 9,88. 759 22. Hamlin, M. J., Marshall, H. C., Hellemans, J., & Ainslie, P. N. (2010). Effect of 760 intermittent hypoxia on muscle and cerebral oxygenation during a 20-km time trial in 761 elite athletes: a preliminary report. Applied Physiology, Nutrition, and 762 Metabolism, 35(4), 548-559. 763 23. Hayes, H. B., Jayaraman, A., Herrmann, M., Mitchell, G. S., Rymer, W. Z., & 764 Trumbower, R. D. (2014). Daily intermittent hypoxia enhances walking after chronic 765 spinal cord injury A randomized trial. Neurology, 82(2), 104-113. 766 12. Donnelly, J. E., Blair, S. N., Jakicic, J. M., Manore, M. M., Rankin, J. W. & Smith, B. 730 K. (2009). American College of Sports Medicine Position Stand. Appropriate physical 731 activity intervention strategies for weight loss and prevention of weight regain for 732 adults. Medicine and Science in Sport and Exercise, 41(2), 459-471. 733 13. Ebbert, J. O., & Jensen, M. D. (2013). Fat depots, free fatty acids, and dyslipidemia. 4. Calbet, J. A. (2003). 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The Journal of Physiology, 551(1), 379-386. 826 Affective and enjoyment responses to high-intensity interval training in overweight- 827 to-obese and insufficiently active adults. Journal of Sport and Exercise 828 Psychology, 37(2), 138-149. 829 44. Miele, C. H., Schwartz, A. R., Gilman, R. H., Pham, L., Wise, R. A., Davila-Roman, 830 V. G. & Checkley, W. (2016). Increased cardiometabolic risk and worsening 831 hypoxemia at high altitude. High Altitude Medicine & Biology, 17(2), 93-100. 832 45. Montero, D., & Lundby, C. (2016). Effects of exercise training in hypoxia versus 833 normoxia on vascular health. Sports Medicine, 46(11), 1725-1736. 834 46. Morishima, T., Kurihara, T., Hamaoka, T., & Goto, K. (2014). Whole body, regional 835 fat accumulation, and appetite‐related hormonal response after hypoxic 836 training. Clinical Physiology and Functional Imaging, 34(2), 90-97. 837 47. Morton, G. J., Cummings, D. E., Baskin, D. G., Barsh, G. S., & Schwartz, M. W. 838 (2006). Central nervous system control of food intake and body 839 weight. Nature, 443(7109), 289-295. 840 36. Little, J. P., Jung, M. E., Wright, A. E., Wright, W., & Manders, R. J. (2014). Effects of high-intensity interval exercise versus continuous moderate-intensity exercise on postprandial glycemic control assessed by continuous glucose monitoring in obese adults. Applied Physiology, Nutrition, and Metabolism, 39(7), 835-841. 37. Lu, Y. L., Jing, W., Feng, L. S., Zhang, L., Xu, J. F., You, T. J., & Zhao, J. (2014). Effects of hypoxic exercise training on microRNA expression and lipid metabolism in obese rat livers. Journal of Zhejiang University Science, 15(9), 820-829. 38. Lundby, C., Calbet, J. A. L. & Robach, P. (2009). The response of human skeletal muscle tissue hypoxia. Cellular and Molecular Life Sciences, 66, 3615-3623. 39. Lyamina, N. P., Lyamina, S. V., Senchiknin, V. N., Mallet, R. T., Downey, H. F., & Manukhina, E. B. (2011). 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(2010). Brown adipose tissue: is it affected by intermittent 821 hypoxia?. Lipids in Health and Disease, 9(1), 121. 822 42. Martinez, D., Vasconcellos, L. F., de Oliveira, P. G., & Konrad, S. P. (2008). 823 Weight loss and brown adipose tissue reduction in rat model of sleep 824 apnea. Lipids in Health and Disease, 7(1), 26. 825 43. Martinez, N., Kilpatrick, M. W., Salomon, K., Jung, M. E., & Little, J. P. (2015). 4. Calbet, J. A. (2003). Chronic hypoxia increases blood pressure and noradrenaline spillover in healthy humans. The Journal of Physiology, 551(1), 379-386. Acute altitude-induced hypoxia suppresses plasma glucose and leptin in healthy humans. Metabolism, 59(2), 200-205. 30. Kendzerska, T., Leung, R. S., Gershon, A. S., Tomlinson, G., & Ayas, N. (2016). The interaction of obesity and nocturnal hypoxemia on cardiovascular consequences in adults with suspected obstructive sleep apnea. A historical observational study. Annals of the American Thoracic Society, 13(12), 2234-2241. 31. Kong, Z., Fan, X., Sun, S., Song, L., Shi, Q., & Nie, J. (2016). Comparison of high- intensity interval training and moderate-to-vigorous continuous training for cardiometabolic health and exercise enjoyment in obese young women: a randomized controlled trial. PLoS One, 11(7), e0158589. 32. Kong, Z., Zang, Y., & Hu, Y. (2013). Normobaric hypoxia training causes more weight loss than normoxia training after a 4-week residential camp for obese young adults. Sleep and Breathing, 18(3), 591-597. 33. Kotchen, T. A. (2010). Obesity-related hypertension: epidemiology, pathophysiology, and clinical management. American Journal of Hypertension, 23(11), 1170-1178. 34. Ling, Q., Sailan, W., Ran, J., Zhi, S., Cen, L., Yang, X., & Xiaoqun, Q. (2008). The effect of intermittent hypoxia on bodyweight, serum glucose and cholesterol in obesity mice. Pakistan Journal of Biological Sciences, 11(6), 869-875. 35. Lippl, F. J., Neubauer, S., Schipfer, S., Lichter, N., Tufman, A., Otto, B. & Fischer, R. 801 (2010). Hypobaric hypoxia causes body weight reduction in obese subjects.Obesity, 802 18, 675-681. 803 30 3 36. Little, J. P., Jung, M. E., Wright, A. E., 804 of high-intensity interval exercise versu 805 postprandial glycemic control assessed 806 adults. Applied Physiology, Nutrition, a 807 37. Lu, Y. L., Jing, W., Feng, L. S., Zhang 808 Effects of hypoxic exercise training on 809 obese rat livers. Journal of Zhejiang Un 810 38. Lundby, C., Calbet, J. A. L. & Robach 811 muscle tissue hypoxia. Cellular and M 812 39. Lyamina, N. P., Lyamina, S. V., Sench 813 Manukhina, E. B. (2011). Normobaric 814 and normalizes nitric oxide synthesis in 815 Hypertension, 29(11), 2265-2272. 816 40. Mackenzie, R., Maxwell, N., Castle, P. 817 hypoxia and exercise improve insulin s 818 diabetes. Diabetes, Metabolism Resear 819 41. Martinez, D., Fiori, C. Z., Baronio, D 820 & Bos, Â. (2010). Brown adipose tiss 821 hypoxia?. 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Intermittent hypoxia and diet-induced obesity: effects on 853 oxidative status, sympathetic tone, plasma glucose and insulin levels, and arterial 854 pressure. Journal of Applied Physiology, 117(7), 706-719. 855 53. Paul, D. J., Gabbett, T. J., & Nassis, G. P. (2016). Agility in team sports: Testing, 856 training and factors affecting performance. Sports Medicine, 46(3), 421-442. 857 54. Piper, A. J., & Grunstein, R. R. (2010). Big breathing: the complex interaction of 858 obesity, hypoventilation, weight loss, and respiratory function. Journal of Applied 859 Physiology, 108(1), 199-205. 860 55. Polotsky, V. Y., Li, J., Punjabi, N. M., Rubin, A. E., Smith, P. L., Schwartz, A. R., & 861 O'Donnell, C. P. (2003). Intermittent hypoxia increases insulin resistance in 862 genetically obese mice. The Journal of Physiology, 552(1), 253-264. 863 56. Quintero, P., Milagro, F. I., Campion, J., & Martinez, J. A. (2010). 4. Calbet, J. A. (2003). 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The 908 biomechanics of restricted movement in adult obesity. Obesity Reviews, 7(1), 13-24. 909 71. Wee, J., & Climstein, M. (2015). Hypoxic training: Clinical benefits on 910 cardiometabolic risk factors. Journal of Science and Medicine in Sport, 18(1), 56-61. 911 33 34 72. Westerterp, K. R., Kayser, B., Wouters, L., Le Trong, J. L., & Richalet, J. P. (1994). 912 Energy balance at high altitude of 6,542 m. Journal of Applied Physiology, 77(2), 913 862-866. 914 73. Westerterp-Plantenga, M. S., Westerterp, K. R., Rubbens, M., Verwegen, C. R., 915 Richelet, J. P., & Gardette, B. (1999). Appetite at “high altitude”[Operation Everest 916 III (Comex-’97)]: a simulated ascent of Mount Everest. Journal of Applied 917 Physiology, 87(1), 391-399. 918 74. Weston, K. S., Wisløff, U., & Coombes, J. S. (2014). High-intensity interval training 919 in patients with lifestyle-induced cardiometabolic disease: a systematic review and 920 meta-analysis. 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Chronic hypoxia increases blood pressure and noradrenaline spillover in healthy humans. The Journal of Physiology, 551(1), 379-386. Influences of normobaric hypoxia training on physical fitness and metabolic 926 risk markers in overweight to obese subjects. Obesity, 18(1), 116-120. 927 77. Workman, C. & Basset, F. A. (2012). Post-metabolic response to passive normobaric 928 hypoxic exposure in sedentary overweight males: A pilot study. Nutrition and 929 Metabolism, 9(103). 930 78. World Health Organisation (2003). Obesity and Overweight. Global Strategy on Diet, 931 Physical Activity and Health. 932 79. Wu, M. C., Tsai, Y. L., Huang, C. Y., Kao, C. L., Hou, C. W., Chen, C. Y. & Kuo, C. 933 H. (2013). Hyperinsulinemia and overweight in obese Zucker rats effectively 934 suppressed by exercise training with hypoxia recovery. European Journal of Sport 935 Science, 13(2), 221-230. 936 80. Wu, T. Y., Ding, S. Q., Liu, J. L., Yu, M. T., Jia, J. H., Chai, Z. C. & Liang, B. Z. 7 (2007). Who should not go high: chronic disease and work at altitude during 8 construction of the Qinghai-Tibet railroad. High Altitude Medicine and Biology, 8(2), 9 88-107. 0 81. Yang, B., Sun, Z. J., Cao, F., Zhao, H., Li, C. W. & Zhang, J. (2015). Obesity is a risk factor for acute mountain sickness: A prospective study in Tibet railway construction workers on Tibetan plateau. European Review for Medical and Pharmalogical Sciences, 19, 119-122. 82. You, T., Arsenis, N. C., Disanzo, B. L. & LaMonte, M. J. (2013). Effects of exercise 945 training on chronic inflammation in obesity. Sports Medicine, 43, 243-256. 946 83. Zhang, H., Tong, T. K., Qiu, W., Zhang, X., Zhou, S., Liu, Y., & He, Y. (2017). Comparable Effects of High-Intensity Interval Training and Prolonged Continuous 83. Zhang, H., Tong, T. K., Qiu, W., Zhang, X., Zhou, S., Liu, Y., & He, Y. (2017). 83. Zhang, H., Tong, T. K., Qiu, W., Zhang, X., Zhou, S., Liu, Y., & He, Y. (2017). Comparable Effects of High-Intensity Interval Training and Prolonged Continuous 34 Exercise Training on Abdominal Visceral Fat Reduction in Obese Young 949 Women. Journal of Diabetes Research. 950 84. Zhang, X., Rong, C., Li, H., Qin, X., Li, S., Lai, Z., & Chen, X. (2015). Glucose 951 Stability Study: NaF/Citrate Plasma Versus Serum. Clinical Laboratory, 62(3), 952 389-393. 953 Exercise Training on Abdominal Visceral Fat Reduction in Obese Young 949 Women. Journal of Diabetes Research. 950 84. Zhang, X., Rong, C., Li, H., Qin, X., Li, S., Lai, Z., & Chen, X. (2015). Glucose 951 Stability Study: NaF/Citrate Plasma Versus Serum. Clinical Laboratory, 62(3), 952 389-393. 953 954 Exercise Training on Abdominal Visceral Fat Reduction in Obese Young 949 Women. Journal of Diabetes Research. 950 84. Zhang, X., Rong, C., Li, H., Qin, X., Li, S., Lai, Z., & Chen, X. (2015). Glucose 951 Stability Study: NaF/Citrate Plasma Versus Serum. Clinical Laboratory, 62(3), 952 389-393. 953 954 35 Disclosures 955 The authors declare no conflict of interest is in relation to the production of this manuscript 956 and no funding was received. 957 958 Figure caption 959 Fig. 1: Flow chart of literature search results; OSA = obstructive sleep apnoea. 960 Fig. 1: Flow chart of literature search results; OSA = obstructive sleep apnoea. 960 36 Table 1: Experimental details of studies included in this review that have investigated passive and active hypoxic conditioning. Participants Intervention Study Type Age Gender BMI (kg.m2) Groups Exposure type Approach Protocol Mode Duration Level of hypoxia (FiO2 %) Briancon- Marjollet et al. (2016) Zucker rats 9 w 48 M NM Obese hypoxia (n = 12) Passive N/a Intermittent (30 s :30 s) 8 h.d N/a 2 w 5.0 Lean hypoxia (n = 12) Obese control (n = 12) N/a N/a Lean control (n = 12) Chen et al. (2011) Zucker rats 14 w 56 M NM Obese exercise (n = 7) Active LHTL 90 mins daily exercise Swimming 6 w N/a Lean exercise (n = 7) Obese hypoxia (n = 7) Passive Sustained 8 h.d N/a 14 Lean hypoxia (n = 7) Obese exercise and hypoxia (n = 7) Active 90 mins daily exercise sustained 8 h.d Swimming 20.9, 14.0 Lean exercise and hypoxia (n = 7) Obese controls (n = 7) N/a N/a N/a N/a Lean controls (n = 7) Gatterer et al. (2015) Humans 51.4 y 22 F, 10 M 37.1 Hypoxia (n = 16) Combined LLTH 90 mins moderate (65- 70% HRmax) intensity exercise, 90 mins rest Cycling, running, cross trainer 2x w, 8 m 14.0, 12.0 Control (n = 16) N/a Kong et al. Exercise Training on Abdominal Visceral Fat Reduction in Obese Young 949 Women. Journal of Diabetes Research. 950 84. Zhang, X., Rong, C., Li, H., Qin, X., Li, S., Lai, Z., & Chen, X. (2015). Glucose 951 Stability Study: NaF/Citrate Plasma Versus Serum. Clinical Laboratory, 62(3), 952 389-393. 953 (2013) Humans 21.1 y 8 F, 10 M 34.3 Hypoxia (n = 10) Active LLTH Moderate (60-70% HRmax) intensity exercise, strength (40-50% 1 rep max, 3 sets of 15 reps, 2 – 3 min rest periods) training Running, stepping, cycling, strength exercising 22 h w, 4 w 15.0 Control (n = 8) N/a Ling et al. (2008) Kunming mice NM 80 F NM Hypoxia-normal diet (n = 20) Passive N/a Intermittent (15 min :5 – 10 min) N/a 8x d, 40 d 14.3 Hypoxia-fatty diet (n = 20) Control-normal diet (n = 20) N/a N/a 961 ed in this review that have investigated passive and active hypoxic conditioning. I i Intervention 37 Control-fatty diet (n = 20) Lu et al. (2014) Sprague Dawley rats 3 w 20 M NM Hypoxia (n = 10) Combined LHTH 1 h exercise 6 d.w, lived in hypoxia Running 4 w 13.6 Control (n = 10) N/a N/a N/a N/a Morishima et al. (2014) Humans 31y 20M NM Hypoxia (n = 9) Active LLTH 60 mins moderate (55 % VO2max) intensity cycling Cycling 3x w, 4 w 15.0 Control (n = 11) N/a Netzer et al. (2008) Humans 47.8 y 10 F, 22 M 33.1 Hypoxia (n = 10) Active LLTH 90 mins moderate (60% HRmax) exercise Stepping, running, cycling 3x w, 8 w 15.0 Control (n = 10) N/a Olea et al. (2014) Wister rats 24 w 160 M NM Hypoxia obese (n = 40) Passive N/a Intermittent (40s :80 s) 8 h.d N/a 2 w 5.0 Hypoxia control (n = 40) Obese (n = 40) N/a N/a N/a Control (n = 40) Polotsky et al. (2003) Mice NM 74 M NM Obese short-term hypoxia (n = 15) Passive N/a Intermittent (30 s :30 s) 16 h.d N/a 5 d 4.8-5.0 Lean short-term hypoxia (n = 16) Obese long-term hypoxia (n = 7) Intermittent (30 s:30 s) 12 h.d 12 w Lean short-term controls (n = 15) N/a N/a 5 d N/a Obese short-term controls (n = 14) Controls (n = 7) Rodriguez et al. Exercise Training on Abdominal Visceral Fat Reduction in Obese Young 949 Women. Journal of Diabetes Research. 950 84. Zhang, X., Rong, C., Li, H., Qin, X., Li, S., Lai, Z., & Chen, X. (2015). Glucose 951 Stability Study: NaF/Citrate Plasma Versus Serum. Clinical Laboratory, 62(3), 952 389-393. 953 (2014) Mice 10 w 82 M NM Obese hypoxia (n = 10) Passive N/a Intermittent (30 s:30 s) 12 h.d N/a 4 w 5.0 Lean hypoxia (n = 11) Obese controls (n = 10) N/a N/a N/a Lean controls (n = 11) Obese hypoxia (n = 9) Passive Sustained 24 h.d 10 Lean hypoxia (n = 10) Control-fatty diet (n = 20) Sustained 24 h.d 38 Obese controls (n = 10) N/a N/a N/a Lean controls (n = 11) Wang et al. (2007) Humans 24 y 30 M 22.2 Severe hypoxia (n = 10) Passive N/a Sustained 1 h.d N/a 4 w 12.0 Moderate hypoxia (n = 10) 15.0 Control (n = 10) N/a N/a N/a Wiesner et al. (2009) Humans 42.2 y 27 F, 18 M 30 Hypoxia (n = 24) Active LLTH 60 mins moderate (65% VO2max) intensity running Running 3x w, 4 w 15.0 Control (n = 21) N/a Workman & Basset (2012) Humans 28 y 15 M 27 Acute hypoxia (n = 11) Passive N/a Sustained 3 h N/a 1d Target SpO2: 80% Short-term hypoxia (n = 6) 1x d, 1 w Control (n = 4) N/a N/a N/a N/a Wu et al. (2013) Zucker rats 14 w 56 M NM Obese exercise (n = 7) Active LHTL 90 mins daily swimming Swimming 6 w N/a Lean exercise (n = 7) Obese hypoxia (n = 7) Passive Sustained 8 h.d N/a 14 Lean hypoxia (n = 7) Obese exercise and hypoxia (n = 7) Active 90 mins daily swimming, sustained 8 h.d Swimming 20.9, 14.0 Lean exercise and hypoxia (n = 7) Obese controls (n = 7) N/a N/a N/a N/a Lean controls (n = 7) BMI = body mass index; d = day(s); F = female(s); FiO2 = fraction of inspired oxygen; h = hour(s); HRmax = heart rate maximum; LHTH = live-high train-high; LHTL = live-high train-low; LLTH = live-low train-high; M = male(s); mins = minutes; m = months; n = number; N/a = not applicable; NM = not measured; rep max = repetition maximum; s = seconds; VO2max = maximal oxygen uptake; w = week(s); y = years. Exercise Training on Abdominal Visceral Fat Reduction in Obese Young 949 Women. Journal of Diabetes Research. 950 84. Zhang, X., Rong, C., Li, H., Qin, X., Li, S., Lai, Z., & Chen, X. (2015). Glucose 951 Stability Study: NaF/Citrate Plasma Versus Serum. Clinical Laboratory, 62(3), 952 389-393. 953 N/a BMI = body mass index; d = day(s); F = female(s); FiO2 = fraction of inspired oxygen; h = hour(s); HRmax = heart rate maximum; LHTH = live-high train-high; LHTL = live-high train-low; LLTH = live-low train-high; M = male(s); mins = minutes; m = months; n = number; N/a = not applicable; NM = not measured; rep max = repetition maximum; s = seconds; VO2max = maximal oxygen uptake; w = week(s); y = years. BMI = body mass index; d = day(s); F = female(s); FiO2 = fraction of inspired oxygen; h = hour(s); HRmax = heart rate maximum; LHTH = live-high train-high; LHTL = live-high train-low; LLTH = live-low train-high; M = male(s); mins = minutes; m = months; n = number; N/a = not applicable; NM = not measured; rep max = repetition maximum; s = seconds; VO2max = maximal oxygen uptake; w = week(s); y = years. 39 Table 2: A summary of the findings for animal studies included in this review. Measures Study Condition Glucose Insulin Cholesterol HDL LDL Triglycerides Leptin BP Body weight Briancon- Marjollet et al. (2016) Obese hypoxia     Lean hypoxia   Obese control   Lean control   Chen et al. (2011) Obese exercise  Obese exercise and hypoxia    Obese controls  Ling et al. (2008) Hypoxia-normal diet   Hypoxia-fatty diet   Control-normal diet  Control-fatty diet  Lu et al. (2014) Hypoxia     Control  Olea et al. (2014) Hypoxia obese       Hypoxia control   Obese    Control  Polotsky et al. (2003) Obese short-term hypoxia    Lean short-term hypoxia  Obese long-term hypoxia    Lean short-term control  Obese short-term control  Control  Rodriguez et al. (2014) Obese hypoxia  Lean hypoxia  Wu et al. (2013) Obese exercise   Obese exercise and hypoxia    Obese control  BP = blood pressure; HDL = high-density lipoprotein cholesterol; LDL = low-density lipoprotein cholesterol;  = increase;  = decrease;  = maintenance. 962 BP = blood pressure; HDL = high-density lipoprotein cholesterol; LDL = low-density lipoprotein cholesterol;  = increase;  = decrease;  = maintenance. 963 40 40 Table 3: A summary of the findings for human studies included in this review. Exercise Training on Abdominal Visceral Fat Reduction in Obese Young 949 Women. Journal of Diabetes Research. 950 84. Zhang, X., Rong, C., Li, H., Qin, X., Li, S., Lai, Z., & Chen, X. (2015). Glucose 951 Stability Study: NaF/Citrate Plasma Versus Serum. Clinical Laboratory, 62(3), 952 389-393. 953 4 Measures Study Condition Glucose Insulin Cholesterol HDL Triglycerides EE Lipid metabolism Glycogen metabolism HR BP La+ BMI Body weight Gatterer et al. (2015) Hypoxia    Control    Kong et al. (2013) Hypoxia     Control     Morishima et al. (2014) Hypoxia        Control         Netzer et al. (2008) Hypoxia      Wang et al. (2007) Severe hypoxa  Wiesner et al. (2009) Hypoxia         Control        Workman & Basset (2012) Acute hypoxia      Short-term hypoxia    BMI = body mass index; BP = blood pressure; EE = energy expenditure; HDL = high-density lipoprotein cholesterol; HR = heart rate; La+ = lactate accumulation;  = increase;  = decrease;  = maintenance. 5 3: A summary of the findings for human studies included in this review. 964 yp BMI = body mass index; BP = blood pressure; EE = energy expenditure; HDL = high-density lipoprotein cholesterol; HR = heart rate; La+ = lactate accumulation;  = increase;  = decrease;  = maintenance. 965 41 Table 4: A summary of the passive and active hypox 966 improving cardio-metabolic health and promoting weig 967 humans, based on the evidence presented in this review. 968 Type of exposure Variable Passive Active Level of hypoxia (FiO2 %) 10.0–12.0 13.0–14.0 Number of cycles 5–15 N/a Intensity N/a 55–65% VO2max / 60–70% HRmax Duration (hours) 1–1.5 1–1.5 Frequency Daily 2–3 times per week Periodisation (weeks) 2–4 4–6 FiO2 = fraction of inspired oxygen; HR = heart rate; N/a = not applicable; VO2max = maximal oxygen uptake. 969 Table 4: A summary of the passive and active hypoxic conditioning protocols for 966 improving cardio-metabolic health and promoting weight loss of overweight or obese 967 humans, based on the evidence presented in this review. 968 Type of exposure Variable Passive Active Level of hypoxia (FiO2 %) 10.0–12.0 13.0–14.0 Number of cycles 5–15 N/a Intensity N/a 55–65% VO2max / 60–70% HRmax Duration (hours) 1–1.5 1–1.5 Frequency Daily 2–3 times per week Periodisation (weeks) 2–4 4–6 FiO2 = fraction of inspired oxygen; HR = heart rate; N/a = not applicable; VO2max = maximal oxygen uptake. 969 Table 4: A summary of the passive and active hypoxic conditioning protocols for 966 improving cardio-metabolic health and promoting weight loss of overweight or obese 967 humans, based on the evidence presented in this review. 968 FiO2 = fraction of inspired oxygen; HR = heart rate; N/a = not applicable; VO2max = maximal oxygen uptake. 969 42
https://openalex.org/W2755934374	https://europepmc.org/articles/pmc5605617?pdf=render	English	null	Role of Creativity in the Effectiveness of Cognitive Reappraisal	Frontiers in psychology	2,017	cc-by	8,395	ORIGINAL RESEARCH published: 15 September 2017 doi: 10.3389/fpsyg.2017.01598 Specialty section: Specialty section: This article was submitted to Cognition, a section of the journal Frontiers in Psychology This article was submitted to Cognition, a section of the journal Frontiers in Psychology Keywords: cognitive reappraisal, creativity, negative emotions, regulation, effectiveness Role of Creativity in the Effectiveness of Cognitive Reappraisal Xiaofei Wu1†, Tingting Guo2†, Tengteng Tang1, Baoguo Shi1* and Jing Luo1,2* 1 Beijing Key Laboratory of Learning and Cognition, The Collaborative Innovation Center for Capital Education Development, Department of Psychology, Capital Normal University, Beijing, China, 2 Key Laboratory of Mental Health, Institute of Psychology, Chinese Academy of Sciences, Beijing, China As a well-recognized and widely adopted emotional regulation strategy, cognitive reappraisal has generally been proven to be efﬁcient. However, the cognitive mechanism underlying regulatory efﬁciency, particularly the role of creativity, in cognitive reappraisal is unclear. Although previous studies have evaluated the relationship between creativity and reappraisal from the perspectives of generation (i.e., generating cognitive reappraisals and generating creative ideas involve similar cognitive neural networks) and individual differences (i.e., the ability to generate different cognitive reappraisals can be predicted by scores on creativity-related tests), how cognitive reappraisal’s efﬁciency can be related to creativity is still unknown. In this research, we assessed the relationship between cognitive reappraisal’s creativity and its effectiveness in regulating negative emotion. In Study 1, participants were asked to generate reappraisals of negative stimuli and then evaluate the creativity and regulatory effectiveness of these reappraisals. The results indicated positive correlation between creativity rating and regulatory effectiveness, but we found that it was difﬁcult for the participants to generate highly creative reappraisals on their own. Therefore, in Study 2, we showed participants well-prepared reappraisal materials that varied in their creativity and asked them to evaluate their regulatory effectiveness and creativity. The results suggested that creativity and appropriateness were signiﬁcant predictors of the regulating effects of the reappraisal and that creativity was the most dominant predictor. In summary, both experiments found a positive correlation between reappraisal’s creativity and effectiveness, thus implying that creativity plays an important role in reappraisal. INTRODUCTION The ability to control eﬀective response and to initiate more adaptive behavior has important consequences for our physical and mental well-being (Gross and John, 2003). Cognitive reappraisal, a linguistic strategy that alters the trajectory of emotional responses by reformulating the meaning of a situation (Gross, 1998), has been regarded as an eﬀective method for regulating negative emotion (Aldao et al., 2010; Webb et al., 2012). However, the cognitive mechanisms mediating the generation of cognitive reappraisal and its eﬀectiveness in regulating emotion remain to be speciﬁed. A recent meta-analysis of neuroimaging studies of cognitive reappraisal found that prefrontal and parietal control regions exert their eﬀects through changes in the lateral temporal areas associated with semantic and perceptual representations (Buhle et al., 2014), thus y gy Received: 14 June 2017 Accepted: 31 August 2017 Published: 15 September 2017 Citation: Wu X, Guo T, Tang T, Shi B and Luo J (2017) Role of Creativity in the Effectiveness of Cognitive Reappraisal. Front. Psychol. 8:1598. doi: 10.3389/fpsyg.2017.01598 Received: 14 June 2017 Accepted: 31 August 2017 Published: 15 September 2017 Edited by: Massimiliano Palmiero, University of L’Aquila, Italy Reviewed by: Zhao Xin, Northwest Normal University, China Saskia Jaarsveld, Kaiserslautern University of Technology, Germany Correspondence: Jing Luo luoj@psych.ac.cn Baoguo Shi baoguoshi@126.com †These authors have contributed equally to this work. Edited by: Massimiliano Palmiero, University of L’Aquila, Italy Massimiliano Palmiero, University of L’Aquila, Italy Reviewed by: Zhao Xin, Northwest Normal University, China Saskia Jaarsveld, Kaiserslautern University of Technology, Germany Correspondence: Jing Luo luoj@psych.ac.cn Baoguo Shi baoguoshi@126.com †These authors have contributed equally to this work. Specialty section: This article was submitted to Cognition, a section of the journal Frontiers in Psychology Received: 14 June 2017 Accepted: 31 August 2017 Published: 15 September 2017 Citation: Wu X, Guo T, Tang T, Shi B and Luo J (2017) Role of Creativity in the Effectiveness of Cognitive Reappraisal. Front. Psychol. 8:1598. doi: 10.3389/fpsyg.2017.01598 Citation: The ill-deﬁned problem has been found to be facilitated to a certain extent by inhibition or dysfunction in the executive neural network (Reverberi et al., 2005; Radel et al., 2015) as well as by the participation of the default mode network (DMN) and medial temporal lobe (MTL) association and memory systems (Takeuchi et al., 2012; Duﬀet al., 2013). Regarding cognitive reappraisal, studies have indicated that the tactics of reappraisal can be categorized into limited types, such as change current circumstances, reality challenge, change future consequences, and distancing (McRae et al., 2012), thus implying the possibility of ordinary change in reappraisal. However, it is far more plausible that the change will be a creative or insightful one, given that it essentially requires an individual to search for and ﬁnd new of individual diﬀerences (Weber et al., 2014) and generation processes (Fink et al., 2016). While adopting concepts from the studies on creative divergent thinking, Weber et al. (2014) developed the Reappraisal Inventiveness Test (RIT) to investigate individual diﬀerences in the ability to generate diﬀerent reappraisals for an anger-eliciting situation, ﬁnding that the RIT can be positively associated with openness to experience and tests that measure divergent thinking. Fink et al. (2016) ﬁnd that the generation of cognitive reappraisal is associated with a brain activation pattern (the pattern of alpha power) similar to that observed in verbal creative ideation but with higher cognitive control and fewer spontaneous imaginative thought processes relative to the latter; this ﬁnding thus implies that the representational change in cognitive reappraisal, in terms of generation processes, can also be creative. However, as an emotional regulation strategy, the goal of an individual in generating cognitive reappraisal is to change his/her emotions, particularly negative emotions. Therefore, in addition to the perspective of individual diﬀerences and generation processes, the eﬀect of emotional regulation should also be considered. Only if we ﬁnd a correlation between reappraisal’s creativity and its eﬀectiveness in regulating emotion can we conclude that the representational change in cognitive reappraisal is creative. The roles of insight and creativity in psychological therapy have been widely recognized by therapists of various theoretical orientations (Hill, 2007; Novotney, 2013), particularly those engaged in cognitive behavioral therapy (CBT) (Elliott et al., 1994) from which the strategy of cognitive reappraisal originates and to which it ultimately applies. Citation: September 2017 \| Volume 8 \| Article 1598 Frontiers in Psychology \| www.frontiersin.org 1 Role of Creativity in Reappraisal Wu et al. supporting a mechanism of representational change for cognitive reappraisal to regulate emotion (Ochsner and Gross, 2005, 2007; Bebko et al., 2011). In other words, the process of cognitive reappraisal, by its very nature, means the process of changing or altering the mental set or the information-processing bias one uses to represent the situation. As an abstract representation of the encountered problem that problem solvers retain in their mind, the problem space could be classiﬁed as well-deﬁned or ill-deﬁned, according to the constraints that are formulated for the solution (Simon and Newell, 1971; Simon, 1973; Jaarsveld and Leeuwen, 2005; Jaarsveld et al., 2010, 2012, 2015; Jaarsveld and Lachmann, 2017). Accordingly, a change in the mental representation of a problem space can occur in two diﬀerent ways (Simon and Newell, 1971). In the well-deﬁned problem situation, in which the rules and mental operators are clear and limited, a change in strategy is explicit and directly available. For example, in the Wisconsin Card Sorting Test (WCST), a change in sorting strategies is always made among several limited selections (e.g., color, number, or shape), and this type of change is found to be mainly mediated by the neural network for cognitive control and executive function, particularly the prefrontal cortex (PFC) (e.g., Monchi et al., 2001). Another type of mental set or representation change, however, is made under an ill-deﬁned problem situation, such as the one encountered in divergent thinking, in which the rules and mental operators are highly uncertain and the stepwise application of mental operators based on ordinary reasoning (e.g., to derive the possible applications of an empty can based on its major function) typically cannot result in truly original solutions. Unlike the ordinary representational change in the well-deﬁned problem situation, which largely depends on the executive function, the representational change in the ill-deﬁned problem situation typically occurs in a creative or insightful manner. More, in open or ill-deﬁned problem space, cognitive productions tend to be more original, hence, more creative, than in well-deﬁned or closed problem space. Therefore, changes in mental representation made under conditions of an ill-deﬁned problem space, can result in more original solutions than changes made in a well-deﬁned space (Jaarsveld and Lachmann, 2017). Citation: However, there is still no experiment that directly assesses the relationship between cognitive reappraisal’s creativity and its eﬀectiveness in regulating negative emotion. The aim of this study was to assess this relationship. In Study 1, participants were asked to generate reappraisal with the aim of alleviating and improving the unpleasant feelings that arose from looking at a picture and to then evaluate the creativity and regulatory eﬀectiveness of these reappraisals. We found that it was diﬃcult for the participants to generate truly, highly creative reappraisals by themselves; thus, in Study 2, we showed the participants well-prepared reappraisal materials that varied in their creativity and asked them to evaluate the materials’ regulatory eﬀectiveness and creativity. Both experiments demonstrated a positive correlation between reappraisal’s creativity and eﬀectiveness, thus proving that creativity plays an important role in reappraisal. Frontiers in Psychology \| www.frontiersin.org Materials and Methods Participants A total of 30 Chinese participants (16 women) with a mean age of 22.2 years (SD = 1.6, range = 19–25) took part in this study. They were all undergraduate or graduate students at Capital Normal University. The participants were paid 40 RMBs, and written consent was obtained prior to the experiment. This study was approved by the Ethical Committee of Capital Norman University. The transcribed text of the participants’ cognitive reappraisals and the recalled reappraisals were then rated by 8 graduate students (experts) who majored in related psychological ﬁelds (such as psychological counseling) and had knowledge and experiences associated with cognitive reappraisal and emotional regulation on 2 dimensions: (1) the degree of creativity (on a 9-point scale ranging from 1 = not at all to 9 = completely); and (2) the degree of consistency between a given participant’s initial reappraisal of a given picture and their subsequent memories of it (also performed on a 9-point scale). Before making their evaluation, these graduate students were instructed to reach a full understanding of the meaning of “creativity” and “consistency.” Through these evaluation procedures, we wanted to learn (1) how well the participants could successfully recall the initial cognitive reappraisal they had made for the given picture; and (2) the consistency between self-rated creativity and other-rated creativity. Procedure The participants were tested individually and viewed in 25 negative pictures twice each. The pictures were selected from the International Aﬀective Picture System (IAPS) (Lang et al., 2008) and covered a variety of negative stimuli and situations (e.g., threat/attack scenes, horrible animals, disgusting things, etc.). These stimuli were selected to have a mildly negative valence, with an average score of 2.56 (SD = 0.52, range = 1.51–3.55; 9-point scale), and to be arousing, with an average score of 5.43 (SD = 0.85, range = 3.93–7.35; 9-point scale). Participants were asked to view all 25 negative pictures one by one and to describe their reappraisals aloud for each picture during its presentation (30,000 ms) on a screen with the goal of alleviating and improving the unpleasant feelings that arose from looking at the pictures (Figure 1). After generating the reappraisal solution, participants were asked to press the button “1,” and the evaluated picture would be presented on the screen. If participants could not generate the reappraisal solution in 30,000 ms, the next picture was presented, and participants were asked to answer the next reappraisal solution immediately. This procedure has typically been used to study cognitive reappraisal (Ochsner et al., 2004; Goldin et al., 2008; Kanske and Kotz, 2011). Immediately after pressing the button, a 9-point scale for eﬀectiveness appeared, based on which participants were to rate the degree of eﬀectiveness in regulating their negative emotion associated with the unpleasant pictures (1 = not at all, 9 = totally). Eﬀectiveness was deﬁned as “the degree or extent to which the reappraisal could improve (which means to decrease the negative emotion or even promote the positive emotion) your feelings toward the picture.” Before the formal procedure of cognitive reappraisal, the participants were instructed and trained on how to perform reappraisals of the unpleasant pictures, which also followed the standard procedure reported in similar studies (Ochsner et al., 2004; Goldin et al., 2008; Kanske and Kotz, 2011). The participants also received 6 training trials prior to the formal experimental procedure to familiarize them with the procedure Frontiers in Psychology \| www.frontiersin.org EXPERIMENT 1 The aim of this study was to test the hypothesis that creativity plays an important role in cognitive reappraisal’s regulation of unwanted emotions. We proposed that all types of reappraisals, by their very nature, could be viewed as a process of cognitive restructuring and contained, to some extent, the components of creativity. The level of creativity of a given cognitive reappraisal could signiﬁcantly predict its eﬀectiveness in regulating emotion. More importantly, recent studies have found connections between cognitive reappraisal and creativity from the perspective September 2017 \| Volume 8 \| Article 1598 Frontiers in Psychology \| www.frontiersin.org 2 Role of Creativity in Reappraisal Wu et al. and to practice the reappraisal strategies. The oral reappraisals were recorded and transcribed for further analysis. We instructed a group of participants on how to generate cognitive reappraisal in response to unpleasant pictures and then asked them to do so by themselves. The participants were then required to evaluate the eﬀectiveness and creativity of their reappraisals. The creativity of these reappraisals was also evaluated by another group of people who had expertise in emotional regulation strategies, including cognitive reappraisal. We predicted that a cognitive reappraisal’s eﬀectiveness in regulating emotion would be signiﬁcantly correlated with its creativity. After the participants had generated their cognitive reappraisals for all 25 pictures, they were required to view the same 25 pictures again and to write down their answers to the following 2 questions: (1) What was the reappraisal you made for the picture the last time that you saw it (the memory recall task)? (2) Do you think your reappraisal is a creative one (the self-rating of creativeness)? The participants rated the degree of creativity using a 9-point scale (1 = not at all, 9 = completely). We deﬁned creativity as “diﬃcult to obtain from other people.” Each picture was presented in random order in the ﬁrst presentation, and the second time, the picture was presented in the same order as the ﬁrst time for each individual. After the experiment, they were showed a comedy for 10 min with the aim of regulating the uncomfortable feelings that arose from looking at the negative pictures. None of our participants reported feeling uncomfortable during or after the experiment. Results and Discussion Effectiveness was deﬁned as “the degree or extent to which the reappraisal could improve (which means to decrease the negative emotion or even promote the positive emotion) your feelings toward the picture.” Results and Discussion A signiﬁcant trial-by-trial positive correlation was observed between the participants’ self-rating on the eﬀectiveness of the reappraisal and their self-rating on its creativity (r = 0.44, p < 0.001, N = 748; Mself-rating of creativeness = 5.41, SD = 2.18; Mself-rating of eﬀectiveness = 6.72, SD = 1.91). This result demonstrated that the creative factor might play an important role in a given cognitive reappraisal’s regulating eﬀect on the unwanted emotion. The degree of consistency between the participants’ initial reappraisal and their later memories of it was 7.69 (SD = 3.82) on the 9-point scale. This ﬁnding implies that the participants’ memories of their initial reappraisal were very reliable. There was a signiﬁcant, but lower, trial-by-trial positive correlation between the experts’ ratings of creativity and the participants’ self-ratings of creativity (r = 0.16, p < 0.001, N = 748). However, the experts’ ratings of creativity were signiﬁcantly lower than the self-ratings of creativity [Mexperts-rating of creativeness = 4.68 (SD = 0.18) vs. Mself-rating of creativeness = 5.41 (SD = 0.48); t(36) = 2.80, p < 0.01, Cohen’s d = 0.47], and the experts’ ratings of creativity were not correlated with the self-ratings of eﬀectiveness (r = 0.07, September 2017 \| Volume 8 \| Article 1598 Frontiers in Psychology \| www.frontiersin.org 3 Wu et al. Role of Creativity in Reappraisal FIGURE 1 \| The procedure of experiment 1. The participants were asked to view negative pictures and describe their reappraisals aloud during a 30,000 ms presentation; then, a 9-point scale for effectiveness appeared, and participants were asked to rate the degree of effectiveness in regulating their negative emotion associated with the unpleasant pictures (1 = not at all, 9 = completely). Effectiveness was deﬁned as “the degree or extent to which the reappraisal could improve (which means to decrease the negative emotion or even promote the positive emotion) your feelings toward the picture.” FIGURE 1 \| The procedure of experiment 1. The participants were asked to view negative pictures and describe their reappraisals aloud during a 30,000 ms presentation; then, a 9-point scale for effectiveness appeared, and participants were asked to rate the degree of effectiveness in regulating their negative emotion associated with the unpleasant pictures (1 = not at all, 9 = completely). Materials and Methods Participants p = 0.07, N = 748). These results indicated that (1) the creativity of a given reappraisal could to some extent be objective and could be recognized by both themselves and others and (2) there were fairly large discrepancies between one’s self-rating and the evaluation of others, relative to people who have expertise on the strategies of emotional regulation. The participants might have a tendency to exaggerate the creativity of their own reappraisal. Importantly, we found that the chances of obtaining highly creative reappraisals were very low if we asked people to generate reappraisals on their own. Only 31.9% of the reappraisals were rated as highly creative (≥7 on a 9-point scale) by the participants themselves, and only 0.5% of them were rated as highly creative by the graduate students with relevant expertise. Therefore, to obtain suﬃcient cases of high-quality creative reappraisal, we should develop a database. Two groups of people participated in this study. One group generated the creative reappraisal materials pertaining to unpleasant IAPS pictures, whereas the other evaluated these materials. Seventy-ﬁve participants [36 females, with a mean age of 24.5 years (SD = 2.31, range = 20–36)], recruited for college students in Beijing, participated in the task of generating creative reappraisal materials. Given that everyone enjoys viewing negative pictures and generating reappraisals of those pictures (indeed, there were some individuals who could not stand viewing the negative pictures or appeared to be averse to generating reappraisals for negative scenes). Therefore, each of the people we recruited were interested in participating in this task and considered themselves highly creative people who could generate creative cognitive reappraisals for these negative pictures. The participants were paid 1–5 RMBs for each reappraisal according to the quality of their work, i.e., the creativity and appropriateness of the reappraisal that they produced for a given picture. Another group of 45 participants [27 females, with a mean age of 24.2 years (SD = 1.60, range = 21–28)] took part in the evaluation task. They were paid 150 RMBs for their participation. A written consent was obtained from all participants prior to the task. This study was approved by the Ethical Committee of Capital Normal University. EXPERIMENT 2 Experiment 2 was conducted because we found that it was diﬃcult, if not impossible, for the participants to generate highly creative reappraisals on their own (see Study 1). Therefore, we invited one group participants (75) to generate the creative reappraisal sentences pertaining to unpleasant IAPS pictures and the other group participants (45) to evaluate these materials. It meant that the second group participants were just showed the well-prepared reappraisal sentences that varied in their creativity and asked them to evaluate their regulatory eﬀectiveness and creativity. We collected more than 900 creative reappraisals for the standard IAPS negative pictures from a large group of participants and asked another group of participants to evaluate their creativity, eﬀectiveness and appropriateness. Procedure We ﬁrst selected 75 negative pictures from the International Aﬀective Picture System (IAPS; Lang et al., 2008) based on their normative valence and arousal levels. The mean valence rating was 2.50 (SD = 0.45; max: 3.55, min: 1.51), and the mean arousal rating was 5.53 (SD = 0.84). We then created a manual September 2017 \| Volume 8 \| Article 1598 Frontiers in Psychology \| www.frontiersin.org 4 Role of Creativity in Reappraisal Wu et al. that contained detailed instructions on how to approach the 75 selected pictures and sent the manual to the participants through e-mail. In the instructions, we asked the participants to try their best to generate new interpretations (creative reappraisals) that were both novel and appropriate for each unpleasant picture, with the aim of alleviating and improving the unpleasant feelings that arose from looking at the pictures. We also provided examples of highly creative reappraisals for the target pictures in the manual, and the participants were informed that for each reappraisal, they made would be paid 1–2 RMBs but for highly creative reappraisals, they would be paid as much as 4–5 RMBs. Our pilot study found that these procedures could be very helpful for the participants in understanding the requirements of the task and to eﬀectively generate their own creative reappraisals. Only the participants who (a) did not have extremely uncomfortable feelings while viewing the negative pictures and (b) considered themselves able and motivated to generate the creative materials were recruited for the study. The participants were given 2 weeks to think about and write down their reappraisals. After all of the participants returned the creative reappraisal materials, the experimenters, together with students majoring in Chinese language and who were particularly good at wording and phrasing, inspected each item, deleted unqualiﬁed descriptions (e.g., those that had no intention to regulate negative emotion, those that exhibited aggressive intentions, or those that were apparently inappropriate for the target picture), combined descriptions with similar meanings and polished all of the satisfactory descriptions. The ﬁnal database included 946 sentences, the lengths of which were 30–40 Chinese characters. The number of reappraisals/descriptions for each picture ranged from 8 to 23 descriptions. Figure 2 shows examples of reappraisal descriptions for a given picture. (1) A woman who wanted to have a baby was surprised and found herself to be pregnant when she started vomiting. Frontiers in Psychology \| www.frontiersin.org Procedure (2) After a Halloween party, a mother poured leftover pumpkin into the toilet. (3) A girl deliberately poured leftovers into the toilet and had the opportunity to call the boy she liked to help her ﬁx the problem. reappraisal descriptions for a given target picture were presented at the same time when the target picture was shown on the screen. This feature was designed for two reasons: (1) If only one part of a reappraisal description for a given target picture was presented, it would take too long to evaluate all pictures, as each target picture would be presented 8–23 times and 25 target pictures would be presented a total of 948 times; (2) participants could directly compare diﬀerent reappraisal descriptions to which they were exposed at the same time. To avoid confusion and interactions, the evaluations of creativity, appropriateness, and eﬀectiveness were not conducted in the same trial but were evaluated in separate sessions, and the sequence of these three types of evaluations was pseudo-randomly assigned and counter- balanced across participants. After the experiment, they were shown a comedy for 10 min with the aim of regulating the uncomfortable feelings that arose from looking the negative pictures. None of our participants reported feeling uncomfortable during or after the experiment. On the contrary, some of our participants even expressed their willingness to take part in our study again because they felt they were greatly inspired by the examples of highly creative reappraisals and “learn something” from this study. Results and Discussion The average creativity rating of all participants for each piece of creative description ranged from 2.95 to 7.80 (M = 5.59, SD = 0.86). The eﬀectiveness rating of all parts of reappraisals ranged from 2.48 to 7.33 (M = 5.69, SD = 0.61), and the appropriateness rating of those parts ranged from 4.05 to 7.68 (M = 6.21, SD = 0.58). For all creative reappraisals, there was a signiﬁcant positive correlation (r = 0.56, p < 0.001) between creativity and eﬀectiveness, which was consistent with the results obtained in part 1. We also found a signiﬁcant negative correlation (r = −0.58, p < 0.001) between appropriateness and eﬀectiveness and a signiﬁcant negative correlation (r = −0.60, p < 0.001) between creativity and appropriateness, which shows that an increase in creativity was inevitably accompanied by a sacriﬁce of appropriateness. These creative reappraisal descriptions were then evaluated by the other 45 participants on 3 dimensions: (1) creativity (the extent to which one feels that these are novel and unexpected); (2) appropriateness (the extent to which the description is appropriate for or ﬁts with the scene depicted in the picture); and (3) eﬀectiveness (the extent to which the description can make one’s feelings and emotion in looking at the picture better) using a 9-point scale ranging from 1 (not at all) to 9 (completely). We predicted that both creativity and appropriateness were signiﬁcant predictors of eﬀectiveness in regulating negative emotion. Stepwise regression demonstrated that creativity and appropriateness could together explain 44% of the total variance of eﬀectiveness [F(2,943) = 370.30, p < 0.001), βcreativeness = 0.83 (t = 27.15, p < 0.001), βappropriateness = 0.45 (t = 14.81, p < 0.001)], which shows that both creativity and appropriateness were signiﬁcant predictors of the regulating eﬀects of the reappraisal and that creativity was the most dominant predictor. DISCUSSION The participants were ﬁrst instructed to reach a full understanding of the meaning of “creativity,” “appropriateness” and “eﬀectiveness” before beginning the formal procedure. They were then required to input their evaluations of the descriptions, which were presented together with the target pictures one by one. The entire evaluation process was divided into 3 sessions, and each session included 25 pictures and the corresponding descriptions (each session took approximately 2–3 h). All In this study, we assessed the relationship between creativity and cognitive reappraisal by investigating how the regulatory eﬀectiveness of a given reappraisal could be related to its creativity. In Study 1, participants were asked to generate cognitive reappraisals of negative IAPS pictures, and we found that there was a signiﬁcant positive correlation between the eﬀectiveness of the cognitive reappraisal and its creativity rating, In this study, we assessed the relationship between creativity and cognitive reappraisal by investigating how the regulatory eﬀectiveness of a given reappraisal could be related to its creativity. In Study 1, participants were asked to generate cognitive reappraisals of negative IAPS pictures, and we found that there was a signiﬁcant positive correlation between the eﬀectiveness of the cognitive reappraisal and its creativity rating, September 2017 \| Volume 8 \| Article 1598 5 Wu et al. Role of Creativity in Reappraisal FIGURE 2 \| An example of stimuli used in the experiment: Vomitus. FIGURE 2 \| An example of stimuli used in the experiment: Vomitus. thus proving the role of creativity in cognitive reappraisal’s regulatory eﬀectiveness. However, given that the participants could rarely generate high-quality creative reappraisals by themselves, we prepared, in advance, novel and appropriate reappraisal materials that varied in their creativity and presented these well-prepared materials to the participants together with negative IAPS pictures. Our result again proved the relationship between reappraisal’s creativity and its eﬀectiveness in regulating negative emotion in an emotional regulatory procedure involving passively reading (rather than positively generating) the high- quality creative reappraisals. Both studies demonstrated the critical role of creativity in cognitive reappraisals. of evidence by proving that a given cognitive reappraisal’s eﬀectiveness in regulating negative emotion could be predicted by its perceived creativity. Our results, together with those obtained from the perspective of individual diﬀerences and the mental generation process, proved the essential association between creativity and cognitive reappraisal from a relatively comprehensive person-process-eﬀectiveness perspective. Frontiers in Psychology \| www.frontiersin.org DISCUSSION That is, only when an individual generates a novel and appropriate interpretation of the scene that he/she has perceived as negative can he/she eﬃciently alter the initial negative tendency. The representational change account of cognitive reappraisal emphasizes the role of creativity and insightfulness in this emotional regulation strategy. y g g gy A potential diﬃculty in applying the creative principle of cognitive reappraisal to practice is that it will be diﬃcult, if not impossible, for individuals to generate reappraisals that are truly creative. Study 1 clearly demonstrated that only one-third of the self-generated reappraisals were rated as highly creative by the participants who generated the reappraisals themselves, and according to the rating of the graduate students with relevant expertise, only 0.5% of these reappraisals were rated highly “creative” (≥7). From an evolutionary perspective, negative stimuli are closely related to survival, and it can be easier for such stimuli to automatically and preferentially induce negative emotions. Negative emotions make people narrow their focus of attention (Frolli et al., 2016), focusing all their mental energy on the negative emotion. At that moment, however, spontaneously generating a highly creative cognitive reappraisal requires one not only to break from the constraints of dominant negative emotions but also to develop a creative interpretation that is both novel and appropriate. This process requires a considerable amount of initiative and consumes more psychological resources. Under these circumstances, individuals typically tend to adopt a ﬁxed mode of thinking to generate their reappraisal, such as “this is not true,” “things will be better” and “this picture content has nothing to do with myself,” because this strategy can help them save their depleted cognitive resources. The results of this ordinary thinking strategy, however, can eventually lead to a mediocre reappraisal and can cause it to lose its insightful nature and remarkable eﬃciency. g Another issue that may have theoretical implications is the appropriateness rating. Although appropriateness and novelty have been widely recognized as the essential features of creativity (Barron, 1955; Runco and Jaeger, 2012; Huang et al., 2015), the present study indicated an inverse relationship between the creativity and appropriateness ratings. Because the scene that an IAPS picture depicts and its implications are typically clear and unambiguous, the most appropriate manner in which to interpret the picture is to objectively describe it. DISCUSSION As demonstrated in Study 2, one method of solving thi “ ti diﬃ lt ” i t t hi h lit ti not be easy for an individual who is experiencing psychological problems to be clearly aware of what exactly his/her problems are, develop a creative reappraisal of them. Yu et al. (2015) attempted to use low- and high-restructuring (creative) solutions for coping with real psychological distress (“problem”). By pooling the psychological issues that college students frequently encounter in everyday lives, Yu et al. (2015) identiﬁed a list of typical problems (for example, “I feel extremely frustrated because of beginning a career I dislike”) and created corresponding reappraisal-like counseling dialogs for these problems. The authors found that the relatively high-restructuring (creative) dialogs (for example, “Success in life is not holding good cards but playing bad cards well” for the above mentioned problem) can be more “insightful” (which refers to the extent to which there is a cognitive “click” or new enlightenment that would be helpful in understanding life’s puzzles) than the non-creative, ordinary dialogs (“Success mainly depends on eﬀort; it is important to do a good job now”). However, these restructuring dialogs are, by nature, metaphorical expressions of vaguely deﬁned real-life issues; thus, the creative components of these dialogs still cannot be clearly identiﬁed, as in the creative reappraisals that we used in this study, because the IAPS pictures typically depict relatively simple and clear problem situations for which creative solutions can reasonably be formulated. How we can obtain psychotherapeutic problems and dialogs whose creativity or, more speciﬁcally, whose key components of representational change can be more precisely deﬁned remains to be investigated? be altered. The application of representational change theory from creative insight problem solving to cognitive reappraisal includes three major points: (1) the individual’s initial mental representations of the situation can lead to negative emotional arousal and cognitive consequences; (2) these negative mental representations function as a type of “mental set” or stubborn cognitive-emotional response bias that is diﬃcult to eﬃciently change through one’s ordinary mode of rational thinking or stepwise reasoning; and (3) an individual must have insightful creative ideas that are particularly appropriate for reinterpreting the situation in a new manner that can lead to apparently less negative cognitive-emotional consequences. Frontiers in Psychology \| www.frontiersin.org DISCUSSION Accordingly, we proposed a “representational change account” for cognitive reappraisal speciﬁc to a given unpleasant situation (such as viewing a negative IAPS picture). Ohlsson and Knoblich established representational change theory for creative insight problem solving, which suggests that problem solvers initially have a low probability of success because they retrieve inappropriate knowledge and set unnecessary constraints on the problem and that only when they break these inappropriate mental sets and establish a new but suitable representation of the problem can they have success in a seemingly unsolvable insight problem (Ohlsson, 1984, 1992; Knoblich et al., 1999, 2001; Luo et al., 2006; Wu et al., 2009, 2013; Huang et al., 2015; Tang et al., 2016). It is reasonable for us to propose that similar processes can also occur in cognitive reappraisal’s regulation of emotion: when encountering an apparently unpleasant stimulus, such as a picture of vomitus, people’s initial feeling or intuitive emotional response can be disgust; only when they have restructured the situation and given the scene a new meaning (for instance, reinterpreting the vomitus as a sign of pregnancy for a woman who wants to have a baby) can these negative feelings y g As previously noted, there were already two categories of evidence that indicated the relationship between cognitive reappraisal and creativity: (a) the perspective of individual diﬀerences, which indicated that one’s ability to generate diﬀerent cognitive reappraisals could be predicted by one’s score on openness to experience, a feature that is believed to be strongly related to creativity, as well as one’s performance in divergent thinking (Weber et al., 2014); and (b) the generation process perspective, which indicated that the generation of cognitive reappraisal could be accompanied by a brain activation pattern (and, thus, psychological process) similar to that observed in verbal creative ideation (Fink et al., 2016). However, neither of these perspectives covers regulatory eﬀectiveness, which is undoubtedly the core concern of the cognitive reappraisal strategy. The present study provided the appropriate chain September 2017 \| Volume 8 \| Article 1598 Frontiers in Psychology \| www.frontiersin.org 6 Role of Creativity in Reappraisal Wu et al. be altered. DISCUSSION The application of representational change theory from creative insight problem solving to cognitive reappraisal includes three major points: (1) the individual’s initial mental representations of the situation can lead to negative emotional arousal and cognitive consequences; (2) these negative mental representations function as a type of “mental set” or stubborn cognitive-emotional response bias that is diﬃcult to eﬃciently change through one’s ordinary mode of rational thinking or stepwise reasoning; and (3) an individual must have insightful creative ideas that are particularly appropriate for reinterpreting the situation in a new manner that can lead to apparently less negative cognitive-emotional consequences. That is, only when an individual generates a novel and appropriate interpretation of the scene that he/she has perceived as negative can he/she eﬃciently alter the initial negative tendency. The representational change account of cognitive reappraisal emphasizes the role of creativity and insightfulness in this emotional regulation strategy. A potential diﬃculty in applying the creative principle of cognitive reappraisal to practice is that it will be diﬃcult, if not impossible, for individuals to generate reappraisals that are truly creative. Study 1 clearly demonstrated that only one-third of the self-generated reappraisals were rated as highly creative by the participants who generated the reappraisals themselves, and according to the rating of the graduate students with relevant expertise, only 0.5% of these reappraisals were rated highly “creative” (≥7). From an evolutionary perspective, negative stimuli are closely related to survival, and it can be easier for such stimuli to automatically and preferentially induce negative emotions. Negative emotions make people narrow their focus of attention (Frolli et al., 2016), focusing all their mental energy on the negative emotion. At that moment, however, spontaneously generating a highly creative cognitive reappraisal requires one not only to break from the constraints of dominant negative emotions but also to develop a creative interpretation that is both novel and appropriate. This process requires a considerable amount of initiative and consumes more psychological resources. Under these circumstances, individuals typically tend to adopt a ﬁxed mode of thinking to generate their reappraisal, such as “this is not true,” “things will be better” and “this picture content has nothing to do with myself,” because this strategy can help them save their depleted cognitive resources. The results of this ordinary thinking strategy, however, can eventually lead to a mediocre reappraisal and can cause it to lose its insightful nature and remarkable eﬃciency. September 2017 \| Volume 8 \| Article 1598 REFERENCES Huang, F., Fan, J., and Luo, J. (2015). The neural basis of novelty and appropriateness in processing of creative chunk decomposition. Neuroimage 113, 122–132. doi: 10.1016/j.neuroimage.2015.03.030 Aldao, A., Nolenhoeksema, S., and Schweizer, S. (2010). Emotion-regulation strategies across psychopathology: a meta-analytic review. Clin. Psychol. Rev. 30, 217–237. doi: 10.1016/j.cpr.2009.11.004 Jaarsveld, S., Fink, A., Rinner, M., Schwab, D., Benedek, M., and Lachmann, T. (2015). Intelligence in creative processes: an EEG study. Intelligence 49, 171–178. doi: 10.1016/j.ijpsycho.2012.02.012 Barron, F. (1955). The disposition toward originality. J. Abnorm. Psychol. 51, 478–485. doi: 10.1037/h0048073 Jaarsveld, S., and Lachmann, T. (2017). Intelligence and creativity in problem solving: the importance of test features in cognition research. Front. Psychol. 8:134. doi: 10.3389/fpsyg.2017.00134 Bebko, G. M., Franconeri, S. L., Ochsner, K. 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Hippocampus 23, 1143–1149. doi: 10.1002/hipo.22208 Jaarsveld, S., and Leeuwen, C. (2005). Sketches from a design process: creative cognition inferred from intermediate products. Cogn. Sci. 29, 79–101. doi: 10.1207/s15516709cog2901_4 Elliott, R., Shapiro, D. A., Firthcozens, J., Stiles, W. B., Hardy, G. E., Llewelyn, S. P., et al. (1994). Comprehensive process analysis of insight events in cognitive- behavioral and psychodynamic-interpersonal psychotherapies. J. Counsel. Psychol. 41, 449–463. doi: 10.1037/0022-0167.41.4.449 Kanske, P., and Kotz, S. A. (2011). Emotion triggers executive attention: anterior cingulate cortex and amygdala responses to emotional words in a conﬂict task. Hum. Brain Mapp. 32, 198–208. doi: 10.1002/hbm.21012 Fink, A., Weiss, E. M., Schwarzl, U., Weber, H., Assunção, V. L. D., Rominger, C., et al. (2016). Creative ways to well-being: reappraisal inventiveness in the context of anger-evoking situations. Cogn. Aﬀect. Behav. Neurosci. 17, 94–105. doi: 10.3758/s13415-016-0465-9 Knoblich, G., Ohlsson, S., Haider, H., and Rhenius, D. (1999). DISCUSSION For example, for the picture of vomitus, the solution of the pregnancy of a woman who wants to have a baby is a positive regulative intention solution; however, if we reappraise the picture as vomitus after knowing that she ate something that was left over after being eaten by a dog, although both are unexpected or creative reappraisals, the eﬃciency of emotional regulation may be diﬀerent. The emotional regulative intention may be an important factor and must be investigated in future studies. Second, the materials that we have established could be adapted to examine the brain mechanism underlying creative cognitive reappraisal, which is important and valuable for real- life emotional regulation. Third, in our study, all stimuli were selected to have a mildly negative picture, and extremely negative scenes (such as died bodies) were excluded. The applicability of the creative reappraisal strategy should be considered in conjunction with the severity of the negative situation. In our future work, we want to determine whether extremely negative scenes could be regulated using the cognitive reappraisal as mildly negative pictures. Finally, we have considered the contribution of reappraisal’s creativeness and appropriateness to its eﬃciency; however, we should also keep in mind that other situational and emotional factors could alter the regulatory eﬀects of reappraisal, such as stress. The study performed by Raio et al. (2013) suggested that although the cognitive reappraisal could reduce the subjective angry feeling evoked, this regulatory eﬀect was no longer observed when participants were in a stress state. For creative cognitive reappraisal, we want to determine whether there is a similar regulatory eﬀect in a stress state. AUTHOR CONTRIBUTIONS TG and JL designed and supervised the study. TG collected the data. TT analyzed the data. BS, JL, and XW wrote the manuscript. ACKNOWLEDGMENTS This work was supported by funding programs from the National Natural Science Foundation of China (31271079, 31671124), the Beijing Municipal Commission of Education (PXM2016_014203_000027, KZ201410028034), and the Beijing Advanced Innovation Center for Imaging Technology (BAICIT-2016018). DISCUSSION Therefore, any reappraisal eﬀorts that alter the original meaning of the picture can lead to a decrease in appropriateness. Thus, an increase in creativity may inevitably lead to a decrease in appropriateness. Fortunately, the present study found that creative reappraisals could still regulate emotion with high eﬃciency if their appropriateness reached a certain level. Thus, an individual may have a certain degree of tolerance in accepting reappraisals with imperfect appropriateness. It could be interesting to investigate why and the extent to which individuals can accept a reappraisal that is less perfect in its appropriateness and the role that other factors, such as internal motivation and the reappraisal’s perceived creativity, play in this procedural tolerance. As demonstrated in Study 2, one method of solving this “generation diﬃculty” is to present high-quality creative cognitive reappraisal materials that are elaborately prepared by others. The advantage of this approach is that it does not require the individual to generate the creative reappraisal him/herself. Moreover, because the reappraisal made by others can be more unexpected and novel to individuals than the reappraisal made by their own self, it has a greater chance of helping individuals establish a new, favorable mental representation. The restriction of this approach, however, is that it requires a relatively clear deﬁnition of the problem situation and a universally recognized creative reinterpretation; meeting these two requirements can be diﬃcult under real psychological conditions. For example, it may Thus, in this study, we proved our assumption that creativeness is an important factor for a cognitive reappraisal to regulate negative emotion by illustrating the signiﬁcant correlation between the self- or other-rated creativity and the eﬀectiveness of cognitive reappraisal. Nevertheless, some issues must be more speciﬁcally addressed in future research. First, our study only investigated the role of the positive regulative intention of creativity in emotional regulation. However, highly creative reappraisal includes not only positive regulative September 2017 \| Volume 8 \| Article 1598 7 Role of Creativity in Reappraisal Wu et al. intention but also negative regulative intention solutions. 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https://openalex.org/W4372290863	https://zenodo.org/records/7902561/files/IJISRT23APR1181.pdf	English	null	Implants in Esthetic Zone	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	6,159	Implants in Esthetic Zone Dr. Nandini Manjunath2 Head of the Department Department of Periodontology A.J Institute of Dental Sciences Dr. Abraham Davis1 Post Graduate Department of Periodontology A.J Institute of Dental Sciences Dr. Anju K S3 Post Graduate Department of Periodontology Aj Institute of Dental Sciences Dr. Saurav Das4 Post Graduate Department of Periodontology A.J Institute of Dental Sciences Abstract:- Oral rehabilitation has undergone a revolutionary change thanks to the development of osseointegration by Branemark and colleagues, as well as the use of dental implants to replace missing teeth.In edentulous or partially edentulous individuals, implant- supported restorations are fairly predictable, according to numerous studies. Early on in the development of contemporary implantology, tissue health and implant survival were the main priorities. In the past ten years, there has been a growing understanding of how important esthetics along with health contributes to the success of the final restoration. It does, after all, stand for a particular facet of health. As well as being functional, esthetic restorations are becoming more and more in demand from patients. Contrary to implants in the early stages of osseointegration, numerous implants are currently being placed in the maxillary region and other esthetically delicate places. The placement of implants to replace one or more teeth in the esthetic zone can be quite challenging for a dentist, especially in regions where there are several missing teeth as well as soft tissue or bone insufficiency. Preserving or creating the soft tissue scaffold necessary to mimic a natural tooth can be quite difficult. The placement of dental implants in the esthetic region is a sensitive technique with limited margin for an error. A small error in the implant's location or incorrect soft- or hard-tissue care could lead to esthetic failure and patient dissatisfaction. esthetically challenging condition, it's important to remember that the prosthetic tooth's shape, size, placement, and colour must completely match the opposing tooth. It's also important to build healthy peri-implant soft tissue. The soft tissue profile is a common esthetic concern in today's implant dentistry. For many years following the introduction of the Branemark system, the esthetic criteria was ignored. Palacci was the first medical professional to take into account the peri-implant zone's esthetic issues5. Volume 8, Issue 4, April – 2023 Volume 8, Issue 4, April – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Keywords:- Esthetic Zone, Implants Implants in Esthetic Zone  From a Surgical Perspective, the Primary Esthetic Goals of Implant Therapy are:  From a Surgical Perspective, the Primary Esthetic Goals of Implant Therapy are:  Creating a Harmonious Gingival Border without Significantly Altering the Tissue's Height  Preserving the Papillae's Integrity  Achieving a Convex Alveolar Crest form6. The cornerstone of implant treatment in the esthetic zone is proper interdisciplinary treatment planning. It necessitates careful consideration of the technical, surgical, and prosthetic components of the care to be given The Systematic and Interdisciplinary Approach is the first of several elements to be taken into account during the treatment planning process. The patient's medical, dental, and psychological assessments are included in this. Restorative factors for implants in the esthetic zone are then discussed. Functional, biological, and esthetic issues are the three main considerations in restorative care7 It is essential to have adequate bone and soft tissue in the apico-coronal, mesio-distal, and buccolingual dimensions, as well as the correct implant angulations, to achieve overall aesthetic and functional success surrounding implants.  Soft and Hard Tissue Esthetics Determinants  Soft and Hard Tissue Esthetics Determinants Periodontal soft tissue esthetics are influenced by many variables including The accomplishment of four goals—placing dental implants in ideal positions with adequate bone and soft tissue support, correction of any discrepancies in soft tissue contour and form, and ensuring adequate tissue support for facial aspect and embrasure areas—is crucial to the success of restorative-driven implant placement in the anterior maxilla. The placement of a permanent implant restoration that blends in with the surrounding soft tissue and neighbouring teeth without significantly changing colour or contour are the final two elements. Other components include the use of temporary restorations to shape soft tissue around implants before a permanent implant restoration8.  Tooth size, position, and proximity to neighbouring teeth.  Periodontal biotype.  Supporting bone.  Periodontal health.  Periodontal health. All of these factors will also influence peri-implant soft tissue esthetics9  Periodontal Examination If periodontal disease is present, determining the attachment level (probing depths and gingival recession) as well as any contributory elements, such as mucogingival problems (lack of keratinized connected tissue) and furcation involvement, will have an impact on the prognosis of the remaining teeth. A bone sounding is the most reliable clinical sign for identifying the attachment level. The interproximal attachment level on the teeth close to a possible implant site will determine the future gingival architecture. Fig 1 (a) -(a)patient with low smile line. There is limited or no gingival display while smiling. (b)patient with average smile line. There is partial gingival display while smiling. (c) patient with high sm ile line. There is significant gingival display upon smiling9 Fig 2 In a normal dentition without restorations or missing teeth, ideal periodontal esthetics exist.The shapes of soft tissues are bilaterally symmetrical. Up to the point of contact, papilla fill the interproximal voids. Each tooth's axial inclination affects the gingival border, which follows the cementoenamel junction and has its zenith coincident with or just distal from the midline, as shown by the dotted line through the tooth's long axis9. II. After acquiring information such as a thorough medical and dental history, clinical findings, pictures, mounted diagnostic casts, and radiographs, the restorative dentist will design and oversee the plan. It will be necessary to consult with additional specialists (such as an endodontist, an orthodontist, a periodontist, and an oral surgeon) in order to address any occlusal, skeletal, and space issues as well as periodontal and endodontic health concerns. A correct diagnosis and knowledge of the long- term prognosis are necessary to develop an integrated treatment plan.  Optimal Soft Tissue Esthetics The degree of a person's concern with soft tissue esthetics will likely depend on how much gingival display that person has. However, regardless of gingival presentation, individual personal preference and esthetic requirements may range from frank indifference to an obsessive attention to detail and a need for perfection, even when soft tissues are not apparent in their regular smile9. Fig 1 (a) -(a)patient with low smile line. There is limited or no gingival display while smiling. (b)patient with average smile line. There is partial gingival display while smiling. (c) patient with high sm ile line. There is significant gingival display upon smiling9 Fig 2 In a normal dentition without restorations or missing teeth, ideal periodontal esthetics exist.The shapes of soft tissues are bilaterally symmetrical. Up to the point of contact, papilla fill the interproximal voids. Each tooth's axial inclination affects the gingival border, which follows the cementoenamel junction and has its zenith coincident with or just distal from the midline, as shown by the dotted line through the tooth's long axis9. Fig 1 (a) -(a)patient with low smile line. There is limited or no gingival display while smiling. (b)patient with average smile line. There is partial gingival display while smiling. (c) patient with high sm ile line. There is significant gingival display upon smiling9 The team's treating specialists must work together to analyse their findings, which may includes the following tests:  Occlusal Analysis It is important to identify patients who have occlusal wear or abfraction type problems as a result of clenching or bruxism.A thorough evaluation covers the occlusal arrangement and recommendations for lateral and protrusive motions.Mounted diagnostic casts are used to assess occlusion.It is crucial to consider the degree of accessible restorative space, as well as the amount of vertical and horizontal overlap. Fig 2 In a normal dentition without restorations or missing teeth, ideal periodontal esthetics exist.The shapes of soft tissues are bilaterally symmetrical. Up to the point of contact, papilla fill the interproximal voids. Each tooth's axial inclination affects the gingival border, which follows the cementoenamel junction and has its zenith coincident with or just distal from the midline, as shown by the dotted line through the tooth's long axis9. I. The dentoalveolar segment, which includes the teeth, gingiva, and lips, is an essential esthetic zone. Together, these factors influence the patient's smile and facial beauty. Therefore, esthetic implant therapy not only aids in the patient's quest for the perfect smile but also tackles the need for impairment and handicap brought on by the esthetic deficiency1.Through continual research and development, the evolution of implant dentistry has proceeded with notable improvements in biomaterials and clinical methods2. The aim of surgical therapy is to set implants in such a way that the anterior maxilla's dental implant restorations can effectively blend in with the surrounding natural dentition8. Surgical placement of dental implants in locations that will lead to the best possible implant restoration is advised as part of a restorative-driven strategy. In order to do this, dental implants must be inserted in locations where there is sufficient bone volume and soft tissue contouring. Typically, this entails the use of bone grafts and soft tissue In the current situation, esthetic implant therapy attempts to produce an exceptional likeness and functional treatment outcome inside the given edentulous area. In an www.ijisrt.com IJISRT23APR1181 1566 Volume 8, Issue 4, April – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 augmentation to correct the shortage in bone and soft tissue and produce the best locations for implant insertion.  Soft and Hard Tissue Esthetics Determinants Periodontal soft tissue esthetics are influenced by many variables including evaluation in order to prevent compromising future implant sites. evaluation in order to prevent compromising future implant sites. be used during treatment to offer information.It is important to pay close attention to the root’s position and angulation within the alveolar bone. The tipping and drifting of the remaining teeth can occasionally result in space problems, especially mesially-distally. Clear directions must be given to the orthodontist when opening or closing spaces with relation to root positions. be used during treatment to offer information.It is important to pay close attention to the root’s position and angulation within the alveolar bone. The tipping and drifting of the remaining teeth can occasionally result in space problems, especially mesially-distally. Clear directions must be given to the orthodontist when opening or closing spaces with relation to root positions.  Restorative Predicting the future gingival contours of the teeth next to the implant site is crucial in the esthetic zone. A bad or hopeless prognosis will be given for teeth that are fractured, broken down beyond expected restoration, or have compromised support. Prior to removal, each tooth's strategic value must be assessed. Even teeth with a poor prognosis can frequently be saved in complex instances in the initial stages to support a fixed interim prosthesis while implant healing develops. The complicated case will be managed with this stepwise strategy of progressive extraction, sometimes over extended stretches of time (two years), as the implant sites mature and heal.  Radiographic Examination In the pre- and postoperative examination of implants in the esthetic zone, radiography is a crucial diagnostic tool. Information on the morphology, volume, and orientation of the alveolar ridge must be gathered before the doctor moves further with implant placement.The vicinity of important structures, the presence of anatomical variations, and the absence of pathology must all be considered while evaluating the area. The clinician can choose from a variety of imaging modalities to achieve this. These alternatives include cross-sectional imaging modalities like computed tomography (CT) and cone-beam computed tomography (CBCT), as well as conventional plain-film radiography like periapical and panoramic radiographs10.  Endodontic It is necessary to determine the vitality and endodontic integrity of the remaining dentition. It is necessary to assess any pathological alterations, such as periapical lesions and incomplete root canals already present. Any tooth with a poor endodontic prognosis should undergo a thorough risk 1567 IJISRT23APR1181 www.ijisrt.com Volume 8, Issue 4, April – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 evaluation in order to prevent compromising future implant sites. Fig 4 Radiographic imaging of implants: A comparison of methods11  Buccolingual Position Excessive implant countersinking can result in saucerization, which is an unfavourable loss of crestal bone around the implant's perimeter, and subsequent gingival recession following loading. On the other hand, shallow implant placement might result in a damaged restoration without a progressive, appealing emerging profile, obvious metal edge, or optical reflection22. In the absence of gingival recession, it is frequently appropriate to locate the cemento- enamel junction (CEJ) of nearby teeth as a point of reference to determine the apicocoronal position of the implant platform. The sink depth of the implant shoulder, whether it is a one-stage implant or a two-stage implant, should be between 1 and 2 millimetres (mm) apically to the imaginary line connecting the mid-buccal of the neighbouring teeth's CEJs without the implant. An implant that is positioned too far buccally frequently causes buccal cortical plate dehiscence and high risk of gingival recession. The healing of the implant is also made significantly more difficult by this placement14. A suitable emergence profile is produced, the restorative process is simplified, and oral hygiene is made easier with an implant that is properly buccolingually positioned15. The desired crown placement, as well as the features of the implant and abutment, influence the optimal buccal- lingual position. The crown should naturally arise from the soft tissue scaffold during implant placement in order to mimic a natural tooth16. To achieve this, the implant's centre line must frequently be situated at or close to the centre of the tooth it replaces17. The CEJs of the nearby teeth must be taken into account. For instance, the CEJ of the canine and central incisor next to it are typically situated 1 mm further coronally than the CEJ of the maxillary lateral incisor. In the presence of gingival regression, the mid-buccal gingival margin can be used as a reference rather than the CEJ23. Fig 5- Implant positioned buccolingually. An imagined line that reaches the incisal borders of the surrounding teeth is touched by the buccal portion of the implant platform17. International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 ISSN No:-2456-2165  Apicocoronal Position or Countersink the emergence profile  Select the right abutment and final restoration design. To hide the metal of the implant and abutment, the implant must be positioned apically. The osteotomy site may need to be countersunk to achieve this placement. This will be done to varying degrees, and how it is done will be determined in part by the implant head's design. The implant diameter20 has some bearing on the amount of countersinking necessary. The emerging profile is often better when implant is placed more apically21.The repair is more esthetically pleasing (and the surrounding tissue is less healthy) the more apical the implant placement. The effectiveness of the restoration depends on four positional factors, all of which must be carefully taken into account during implant placement13. These include the angulation of the implant as well as the buccolingual, mesiodistal, and apicocoronal placements in relation to the implant platform. Design considerations for prosthetics, such as whether they are screw- or cement-retained, are crucial as well.  Mesiodistal Position The available mesiodistal space must be precisely measured in order to choose the right implant and arrange for the right implant spacing in order to prevent an unpleasant esthetic result. A minimum gap of 1.5 to 2 mm should be kept between implants and neighbouring teeth when there are multiple implants, with a spacing of 3 to 4 mm between each implant at the implant abutment level19. The available mesiodistal space must be precisely measured in order to choose the right implant and arrange for the right implant spacing in order to prevent an unpleasant esthetic result. A minimum gap of 1.5 to 2 mm should be kept between implants and neighbouring teeth when there are multiple implants, with a spacing of 3 to 4 mm between each implant at the implant abutment level19. The horizontal distance of the implant fixture from the tooth or implant is highly inversely linked with crestal bone loss at neighbouring teeth or between implants. It could be better to position the implant slightly distally in order to match the natural symmetry of the maxillary central incisor. The surgical strategy needs to be carefully planned and carried out. For implant insertion and rehabilitation in the esthetic zone, Tischler has established guidelines12. The surgeon should, in accordance with these recommendations:  Employ a conservative flap design., The horizontal distance of the implant fixture from the tooth or implant is highly inversely linked with crestal bone loss at neighbouring teeth or between implants. It could be better to position the implant slightly distally in order to match the natural symmetry of the maxillary central incisor.  Evaluate the preexisting bone and soft tissue,  Precisely time the placement,  Visualise the implant's three-dimensional position,  Take into account the healing period before implant loading,  Take into consideration about the factors that influence th fil ake into consideration about the factors that influenc  Apically positioned flaps (APFs).  APF/Vestibuloplasty (APF/V) combined with autogenous tissue (subepithelial connective tissue graft [SCTG]/free gingival graft [FGG]).  Orthodontic Analysis It is important to pay close attention to the roots' position and angulation within the alveolar bone. The tipping and drifting of the remaining teeth can occasionally result in space problems, especially mesially-distally. When opening or closing spaces in relation to root placements, the orthodontist must have clear instructions. Radiographs will Fig 3 Using a medium field of view, cone-beam computed tomography images were acquired in the multiplanar view (MPR). Axial image of the mandibular arch in (a). Sagittal view of the central incisors in (b). (c) A coronal image showing the molar tee11 Fig 3 Using a medium field of view, cone-beam computed tomography images were acquired in the multiplanar view (MPR). Axial image of the mandibular arch in (a). Sagittal view of the central incisors in (b). (c) A coronal image showing the molar tee11 Fig 4 Radiographic imaging of implants: A comparison of methods11 1568 www.ijisrt.com Volume 8, Issue 4, April – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165  Apically positioned flaps (APFs). V. SOFT TISSUE AUGMENTATION FOR IMPLANT DENTISTRY24 Surgical techniques to increase the width of inadequate mucosa are typically used before implant implantation or when submerged implants are accidentally exposed.  APF/Vestibuloplasty (APF/V) combined with autogenous tissue (subepithelial connective tissue graft [SCTG]/free gingival graft [FGG]). Fig 5- Implant positioned buccolingually. An imagined line that reaches the incisal borders of the surrounding teeth is touched by the buccal portion of the implant platform17. 1569 www.ijisrt.com IJISRT23APR1181 l Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Fig 6 (c)- 2 months postoperative after buccal tissue augmentation with prosthetic abutment placed International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Volume 8, Issue 4, April – 2023  FGG  The epithelialized palatal graft method  SCGT technique or Acellular dermal matrix graft (ADMG)/ collagen matrix (CM) Fig 6 -connective tissue graft for augmentation of buccal keratinized tissue (purse string technique): (a) connective tissue graft donor site (palatal) in relation to 21 (b) CTG graft threaded on the eptfe suture (c) CTG graft sutured 24 Fig 6 (c)- 2 months postoperative after buccal tissue augmentation with prosthetic abutment placed  Allografts  Allografts FGGs and CTGs are associated with considerable patient morbidity because of the wound at the palatal donor site and the likelihood of insufficient donor tissue. To make up for these shortcomings, soft tissue replacements have been used. Acellular dermal matrix graft28 and human fibroblast-derived dermal replacement are two types of dermal allografts that have been used for palatal donor tissue. Allograft materials led to an increase in keratinized tissue width29.  Allografts FGGs and CTGs are associated with considerable patient morbidity because of the wound at the palatal donor site and the likelihood of insufficient donor tissue. To make up for these shortcomings, soft tissue replacements have been used. Acellular dermal matrix graft28 and human fibroblast-derived dermal replacement are two types of dermal allografts that have been used for palatal donor tissue. Allograft materials led to an increase in keratinized tissue width29. g f FGGs and CTGs are associated with considerable patient morbidity because of the wound at the palatal donor site and the likelihood of insufficient donor tissue. To make up for these shortcomings, soft tissue replacements have been used. Acellular dermal matrix graft28 and human fibroblast-derived dermal replacement are two types of dermal allografts that have been used for palatal donor tissue. Allograft materials led to an increase in keratinized tissue width29.  Pink Esthetic Score /White Esthetic Score Achieving clinical esthetic success depends critically on the soft tissue esthetic outcome. The pink aesthetic score /white aesthetic score (PES/WES) can be used to assess the esthetic outcome of peri implant soft tissue25. In order to achieve continuous improvement in PES score, connective tissue grafts may be required in around one-third of patients after immediate implant placement after tooth extraction26. When followed for at least three years following crown installation, CTG augmentation during Stage 2 implant surgery or after final restoration indicated spontaneous improvements of PES score in the short-term follow-up27. Fig 6 -connective tissue graft for augmentation of buccal keratinized tissue (purse string technique): (a) connective tissue graft donor site (palatal) in relation to 21 (b) CTG graft threaded on the eptfe suture (c) CTG graft sutured 24 Fig 7- PES and WES 25  Allografts FGGs and CTGs are associated with considerable patient morbidity because of the wound at the palatal donor site and the likelihood of insufficient donor tissue. To make up for these shortcomings, soft tissue replacements have been used. Acellular dermal matrix graft28 and human fibroblast-derived dermal replacement are two types of dermal allografts that have been used for palatal donor tissue. Allograft materials led to an increase in keratinized tissue width29. Fig 7- PES and WES 25  All ft Fig 7- PES and WES 25 Fig 7- PES and WES 25  Anterior Jaw Regions A surgery to increase the sagittal and/or vertical bone dimensions will be necessary if there is less vertical bone volume. Studies using autografts, allografts, xenografts, and alloplasts for vertical and sagittal bone augmentation at single tooth implants have all demonstrated similarly successful and comparable outcomes. No augmentation method appears to be better than another32. Fig 8 (a)- Radiograph of buccal onlay block bone fixed with two mini-screws(b)Mini screws used as tent-poles to keep bone particles in position32 Fig 8 (a)- Radiograph of buccal onlay block bone fixed with two mini-screws(b)Mini screws used as tent-poles to keep bone particles in position32  Xenografts Allografts come from human cadavers, which raises potential ethical issues as well as the danger of disease transmission. Collagen membrane (CM) of porcine origin, which has been employed with oral wound-healing/around natural teeth, has been tried to overcome the aforementioned drawbacks30. In comparison to autogenous CTGs, Mucograft , a collagen matrix of porcine origin, successfully enhanced the width of keratinized gingiva while also improving the esthetic results31. 1570 IJISRT23APR1181 www.ijisrt.com Volume 8, Issue 4, April – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 ISSN No:-2456-2165  Timing of Implant Placement Following tooth extraction, implant can be placed  Timing of Implant Placement Following tooth extraction, implant can be placed  Immediately (Type 1)  Early after soft tissue healing (Type 2)  Partial bone healing (Type 3)  After complete socket healing (Type 4)33.  Garber Class IV In areas with negligible vertical bone loss but considerable horizontal loss, it is crucial to use a staged strategy in which the ridge is extended with directed bone regeneration. After a few months of required healing time38, implants are then placed using block bone grafts or GBR methods.  Infected sites can be loaded immediately post- extraction.  The fixture should be positioned according to generally accepted standards, however the vestibular/palatal position of the implant depends on the abutment morphology. VIII.  Due to the need to produce the best possible aesthetic outcome, implantological rehabilitation of the esthetic zone is one of the most demanding and complicated treatments. The following conclusions can be reached from the extensive clinic experience and relevant literature:  Garber Class I When both the horizontal and vertical levels of soft tissue and bone are favourable, selecting the optimum placement for an implant is an easy task. It is ideal to perform a soft tissue augmentation at the same time as implant placement in order to lower the risk of soft tissue recession and buccal bone resorption in patients with a thin gingival biotype. Fig 12 The surgical guide for implant placement.CBCT and CAD/CAM are used to produce a surgical Guide for implant placement 42 VII. Fig 11- Immediate implant placement. Implant positioned in the fresh extraction socket35  Custom Implant using Three-Dimensional Printing The use of CAD/CAM as a supportive tool to enhance the outcomes of implant therapy has increased with the development of implant dentistry. For challenging circumstances where standard abutments might not offer an acceptable choice for a future prosthesis, customised implant abutments have been successfully manufactured utilising CAD/CAM. In order to give a more correct impression in such circumstances, customised coping was also produced in addition to customised abutments 40. Clinicians have proposed ideas for applying 3DP and CAD/CAM at the planning stage of implantation, in addition to their use in the production of prosthesis-related components. Cone beam computed tomography (CBCT) along with CAD/CAM have been recommended in order to provide a surgical guide for implant placement 41. Fig 11- Immediate implant placement. Implant positioned in the fresh extraction socket35 When an edentulous site in the esthetic zone is planned for implant placement, the site must be thoroughly evaluated. Garber has proposed a classification for such sites36 . This classification depends on the type of reconstruction needed to get good positioning of the implant. Fig 12 The surgical guide for implant placement.CBCT and CAD/CAM are used to produce a surgical Guide for implant placement 42  Garber Class III If an initial stability is attained, implant placement can be tried in sites with no vertical bone loss and horizontal bone loss greater than Class II. Bone regeneration that is guided is required.  Immediate placement is less traumatic for the patient as there are fewer surgical procedures involved, and patients tend to prefer this clinical approach regarding quality of life. Immediate placement can be a successful procedure in terms of esthetics, but it is technique sensitive and requires an experienced team.The diagnostic step is crucial for treating bone and soft- tissue injuries as well.  Garber Class II According to the method proposed by Summers37, sites with no vertical bone loss and a small horizontal bone shortage measuring 1 to 2 mm narrower than usual can be extended by employing successive osteotomes rather than drilling.By compressing the maxillary cancellous bone while slightly expanding the bony ridge horizontally, this procedure will enhance the bone's quality38  After complete socket healing (Type 4)33. 4 to 8 weeks after tooth extraction, Type 2 placement is the recommended technique for the esthetic zone. At that point, the buccal wall has somewhat flattened due to a bundle of bone resorption, and the soft tissue has healed. The fundamental goal of this protocol is to repair the soft tissue in a way that gives it enough volume and creates a large zone of keratinized mucosa that enables tension-free primary closure after guided bone regeneration surgery. The possibility of esthetic issues is reduced in this approach. It is required to deviate from this technique when there are significant apical bone abnormalities that affect the stability of the initial implant.In such a situation, early implant placement with partial bone healing following 12 to 16 weeks (Type 3) is indicated.33 Fig 9 Type 2 placement.(a) Following GBR, the soft tissue recovered entirely and allowed for the initial tension-free closure.32 (b) Bundle bone resorption has caused a little flattening of the buccal wall34. Fig 9 Type 2 placement.(a) Following GBR, the soft tissue recovered entirely and allowed for the initial tension-free closure.32 (b) Bundle bone resorption has caused a little flattening of the buccal wall34. 1571 IJISRT23APR1181 www.ijisrt.com International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Volume 8, Issue 4, April – 2023 In the esthetic zone, immediate implant insertion is extremely difficult. Only a small percentage of patients who are low risk for esthetic issues, have healthy bone walls, thick facial bone walls (at least 1 mm), no infections at the extraction site, and enough bone volume to support the primary implant are candidates for this approach33. Even if the treatment period is shorter and there is ideal bone volume for implant insertion, rapid procedure is linked to a higher risk of gingival recession. Immediate implant insertion protocol under unfavourable gingival biotype conditions, a patient's absence of bone or soft tissue, or a high smile line can all result in esthetic failure, which is crucial in the esthetic region33,34 1572 IJISRT23APR1181 www.ijisrt.com Volume 8, Issue 4, April – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165  Implant Placement in Edentulous Sites  Implant Placement in Edentulous Sites  Implant Placement in Edentulous Sites REFERENCES [17]. Sadan A, Blatz MB, Bellerino M, Block M. Prosthetic design considerations for anterior single‐ implant restorations. J. Esthet. Dent.. 2004 ;16(3):165-75. [1]. Dash S, Srivastava G, Choudhury GK, Samal M. Implant Positioning in Esthetic Zone. Indian J Public Health Res Dev. 2018;9(12). [18]. Jones AR, Martin W, Adam R, William. Comparing pink and white esthetic scores to layperson perception in the single-tooth implant patient. Int J Oral Maxillofac Implants. 2014 ;29(6):1348-1353. [2]. Paolantonio M, Dolci M, Scarano A, d'Archivio D, Di Placido G, Tumini V, Piattelli A. Immediate implantation in fresh extraction sockets. A controlled clinical and histological study in man. J. Periodontol.. 2001;72(11):1560-71. [19]. Esposito M, Ekestubbe A, Gröndahl K. Radiological evaluation of marginal bone loss at tooth surfaces facing single Brånemark implants. Clin. Oral Implants Res.. 1993 ;4(3):151-7 [3]. Magne P, Magne M, Belser U. Natural and restorative oral esthetics part I: rationale and basic strategies for successful esthetic rehabilitations J. Esthet. Dent.. 1993;5(4):161-73. [20]. Davarpanah M, Martinez H, Celletti R, Tecucianu JF. Three-stage approach to esthetic implant restoration: emergence profile concept. Pract Periodontics Aesthet Dent. 2001 ;13(9):761. [4]. Magne P, Magne M, Belser U. Natural and restorative oral esthetics. PartII: esthetic treatment modalities. J Esthet Dent 1993;5(6):239–46. [21]. Cochran DL, Hermann JS, Schenk RK, Higginbottom FL, Buser D. Biologic width around titanium implants. A histometric analysis of the implanto‐ gingival junction around 118 unloaded and loaded nonsubmerged implants in the canine mandible. J. Periodontol.. 1997 ;68(2):186-97 [5]. Palacci P. Peri-implant soft tissue management: Papilla regeneration technique. Optimal implant positioning and soft tissue management for the Brånemark system. Chicago: Quintessence. 1995:59 70. [6]. Belser U. Implant placement in the esthetic zone. J Periodontal Implant Sci. 2003. [22]. Marković A, Calvo‐Guirado JL, Lazić Z, Gómez‐ Moreno G, Ćalasan D, Guardia J, Čolic S, et al. Evaluation of primary stability of self‐tapping and non‐self‐tapping dental implants. A 12‐week clinical study. Clin Implant Dent Relat Res . 2013 ;15(3):341- 9 [7]. Schoenbaum TR, editor. Implants in the esthetic Zone: A Guide for Treatment of the Partially Edentulous Patient. Springer; 2018 [8]. Buser D, Daniel ,William, Martin W, Belser, Urs C. Optimizing esthetics for implant restorations in the anterior maxilla: anatomic and surgical considerations. Int J Oral Maxillofac Implants 2004 ;19(7):43-61 [23]. Oesterle LJ, Cronin Jr RJ, Ranly DM. Maxillary implants and the growing patient. Int J Oral Maxillofac Implants. 1993 ;8(4):377-387. [24].  Garber Class V Sites with considerable apicocoronal bone loss pose a surgical challenge. As was already said, there are no reliable surgical techniques that can consistently increase bone ridge height. According to some case reports, GBR utilising a non-resorbable membrane and delayed implant implantation are surgical approaches39.  When a shoulderless abutment is used, the long axis of the implant, aimed at the incisal edge of the future restorations, is the most suitable implant position since it permits a restorative crown morphology with a cervical contour similar to a natural tooth. 1573 IJISRT23APR1181 www.ijisrt.com Volume 8, Issue 4, April – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 REFERENCES Caneva M, Botticelli D, Viganò P, Morelli F, Rea M, Lang NP. Connective tissue grafts in conjunction with implants installed immediately into extraction sockets. An experimental study in dogs. Clin. Oral Implants Res.. 2013 ;24(1):50-6. [9]. 9.Todd R. Schoenbaum Implants in the esthetic Zone.2019 [10]. Handelsman M. Surgical guidelines for dental implant placement. Br. Dent. J.. 2006 ;201(3):139-52. [11]. Mallya SM. Evidence and Professional Guidelines for Appropriate Use of Cone Beam Computed Tomography. J Calif Dent Assoc .. 2015 ;43(9):512- 20 [25]. Cosyn J, Raes M, Packet M, Cleymaet R, De Bruyn H. Disparity in embrasure fill and papilla height between tooth‐and implant‐borne fixed restorations in the anterior maxilla: a cross‐sectional study. J. Clin. Periodontol.. 2013 ;40(7):728-33 [12]. Garber DA, Belser UC. Restoration-driven implant placement with restoration-generated site development. Compend Contin Educ Dent (Jamesburg, NJ: 1995). 1995;16(8):796-8. [26]. Gu YX, Shi JY, Zhuang LF, Qiao SC, Xu YY, Lai HC. Esthetic outcome and alterations of soft tissue around single implant crowns: a 2‐year prospective study. Clin. Oral Implants Res.. 2015 ;26(8):909-14. [13]. Garber DA. The esthetic dental implant: letting restoration be the guide. J Am Dent Assoc .. 1995 ;126(3):319-25. y p [27]. Dorfman HS, Kennedy JE, Bird WC. Longitudinal evaluation of free autogenous gingival grafts. J. Clin. Periodontol.. 1980 ;7(4):316-24 [14]. Belser UC, Bernard JP, Buser D. Implant-supported restorations in the anterior region: prosthetic considerations. Pract Periodontics Aesthet Dent:. 1996 ;8(9):875-3 [28]. McGuire MK, Scheyer ET, Nunn ME, Lavin PT. A pilot study to evaluate a tissueengineered bilayered cell therapy as an alternative to tissue from the palate. J. Periodontol. 2008;79:1847-56 [15]. Spray JR, Black CG, Morris HF, Ochi S. The influence of bone thickness on facial marginal bone response: stage 1 placement through stage 2 uncovering. Ann. Periodontol.. 2000 Dec;5(1):119-28 [29]. Canullo L, Fedele GR, Iannello G, Jepsen S. Platform switching and marginal bone‐ level alterations: the results of a randomized‐controlled trial. Clin. Oral Implants Res.. 2010 ;21(1):115-21 [16]. Lazzara RJ. Immediate implant placement into extraction sites: surgical and restorative advantages. Int J Periodontics Restorative Dent. 1989;9:332-43 1574 IJISRT23APR1181 www.ijisrt.com Volume 8, Issue 4, April – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 [30]. Sanz M, Lorenzo R, Aranda JJ, Martin C, Orsini M. REFERENCES Clinical evaluation of a new collagen matrix (Mucograft® prototype) to enhance the width of keratinized tissue in patients with fixed prosthetic restorations: a randomized prospective clinical trial. J. Clin. Periodontol.. 2009 ;36(10):868-76. [31]. Jung RE, Hürzeler MB, Thoma DS, Khraisat A, Hämmerle CH. Local tolerance and efficiency of two prototype collagen matrices to increase the width of keratinized tissue. J. Clin. Periodontol.. 2011 ;38(2):173-9. [32]. Friberg B. Bone augmentation for single tooth implants: A review of the literature. Eur J Oral Implantol. 2016 ;9(Suppl 1):123-34. [33]. Chen B, Buser D. 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https://openalex.org/W2982028539	https://europepmc.org/articles/pmc6857621?pdf=render	English	null	Exploring Actinobacteria Associated With Rhizosphere and Endosphere of the Native Alpine Medicinal Plant Leontopodium nivale Subspecies alpinum	Frontiers in microbiology	2,019	cc-by	10,258	Exploring Actinobacteria Associated With Rhizosphere and Endosphere of the Native Alpine Medicinal Plant Leontopodium nivale Subspecies alpinum Martina Oberhofer1, Jaqueline Hess2, Marlene Leutgeb1, Florian Gössnitzer1†, Thomas Rattei3, Christoph Wawrosch1 and Sergey B. Zotchev1 Citation: Oberhofer M, Hess J, Leutgeb M, Gössnitzer F, Rattei T, Wawrosch C and Zotchev SB (2019) Exploring Actinobacteria Associated With Rhizosphere and Endosphere of the Native Alpine Medicinal Plant Leontopodium nivale Subspecies alpinum. Front. Microbiol. 10:2531. doi: 10.3389/fmicb.2019.02531 Keywords: endophytes, rhizosphere, Edelweiss, Actinobacteria, alpine medicinal plant Specialty section: Specialty section: This article was submitted to Terrestrial Microbiology, a section of the journal Frontiers in Microbiology Received: 08 May 2019 Accepted: 21 October 2019 Published: 08 November 2019 ORIGINAL RESEARCH published: 08 November 2019 doi: 10.3389/fmicb.2019.02531 Edited by: Paolina Garbeva, Netherlands Institute of Ecology (NIOO-KNAW), Netherlands The rhizosphere of plants is enriched in nutrients facilitating growth of microorganisms, some of which are recruited as endophytes. Endophytes, especially Actinobacteria, are known to produce a plethora of bioactive compounds. We hypothesized that Leontopodium nivale subsp. alpinum (Edelweiss), a rare alpine medicinal plant, may serve as yet untapped source for uncommon Actinobacteria associated with this plant. Rhizosphere soil of native Alpine plants was used, after physical and chemical pre- treatments, for isolating Actinobacteria. Isolates were selected based on morphology and identiﬁed by 16S rRNA gene-based barcoding. Resulting 77 Actinobacteria isolates represented the genera Actinokineospora, Kitasatospora, Asanoa, Microbacterium, Micromonospora, Micrococcus, Mycobacterium, Nocardia, and Streptomyces. In parallel, Edelweiss plants from the same location were surface-sterilized, separated into leaves, roots, rhizomes, and inﬂorescence and pooled within tissues before genomic DNA extraction. Metagenomic 16S rRNA gene amplicons conﬁrmed large numbers of actinobacterial operational taxonomic units (OTUs) descending in diversity from roots to rhizomes, leaves and inﬂorescences. These metagenomic data, when queried with isolate sequences, revealed an overlap between the two datasets, suggesting recruitment of soil bacteria by the plant. Moreover, this study uncovered a profound diversity of uncultured Actinobacteria from Rubrobacteridae, Thermoleophilales, Acidimicrobiales and unclassiﬁed Actinobacteria speciﬁcally in belowground tissues, which may be exploited by a targeted isolation approach in the future. Reviewed by: Muhammad Saleem, Alabama State University, United States Daniel Rozen, Leiden University, Netherlands Correspondence: Martina Oberhofer martina.oberhofer@univie.ac.at †††Present address: Florian Gössnitzer, Plant Breeding and Genetics Laboratory, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Seibersdorf, Austria Reviewed by: Muhammad Saleem, Alabama State University, United States Daniel Rozen, Leiden University, Netherlands Reviewed by: Muhammad Saleem, Alabama State University, United States Daniel Rozen, Leiden University, Netherlands Correspondence: Martina Oberhofer martina.oberhofer@univie.ac.at Correspondence: Martina Oberhofer martina.oberhofer@univie.ac.at †††Present address: Florian Gössnitzer, Plant Breeding and Genetics Laboratory, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Seibersdorf, Austria INTRODUCTION The endosphere entails the entire microbial community that inhabits the interior of the host plant. Generally, host plants provide an environment with high nutrient content to their microbial inhabitants and shelter them from adverse environmental factors (Compant et al., 2010; Wang M. et al., 2016). Thus, endophytes experience high selective pressure competing for their beneﬁcial, but spatially limited habitat (Caraballo-Rodríguez et al., 2017). Within the endosphere, diﬀerent plant tissues are characterized by their speciﬁc chemoproﬁles and tissue architecture, which can act as “ﬁlters” and attract diﬀerent subsets of the endophyte community. Both the rhizosphere and the endosphere impose strong selective forces on microbes, which promote highly interactive communities. This suggests involvement of secondary metabolites in interactive signaling aﬀecting antibiosis (Yim et al., 2007), quorum sensing and bioﬁlm formation (Abisado et al., 2018). Hence, these two symbiotic habitats bear prime potential for the discovery of novel natural products (Strobel et al., 2004; Tiwari and Gupta, 2013). Recently, medicinal plants have come to the forefront of bioprospecting in search for new or unusual sources for rare Actinobacteria (Golinska et al., 2015), whereby the term rare in this context speciﬁes Actinobacteria phylogenetically distant to Streptomyces and that are not commonly isolated (all genera except Streptomyces) (Kurtböke, 2003; Janso and Carter, 2010; Khanna et al., 2011; Jose and Jebakumar, 2013). Actinobacteria are Gram-positive bacteria, which have a characteristically high GC-content in their DNA and are ubiquitous in nature. Their habitats range from diverse terrestrial to aquatic ecosystems, but they are also often found associated with other organisms and live in extremophile environments (Quintana et al., 2013). Actinobacteria are recognized as highly proliﬁc producers of secondary metabolites. Bioactive molecules produced by Actinobacteria include antitumor, antiviral, antiparasitic, insecticidal, antibacterial, and antifungal and immunomodulatory agents and herbicides (Barka et al., 2016). Most notably, they are the origin of the majority of all antibiotic compounds currently known (Baltz, 2008). Numerous studies on Actinobacteria in medicinal plants were conducted in Asia, where the use of herbal remedies is deeply anchored in culture and medicine since millennia, e.g., Ayurveda, Traditional Chinese Medicine (Qin et al., 2009; Zhao et al., 2012). Few studies are available on medicinal plants native to South America, Australia, and the Paleotropics (Golinska et al., 2015; Caraballo-Rodríguez et al., 2017), however, investigations on temperate medicinal herbs are yet underrepresented. Among these, studies on alpine medicinal plants are especially rare. Hence, we hypothesized that L. nivale subsp. INTRODUCTION Microbial habitats in plant and in soil encompass the bulk soil, the rhizosphere, and the endosphere (Compant et al., 2010; Wang M. et al., 2016). The bulk soil is a meso- to oligotrophic habitat characterized by pronounced heterogeneity in physical properties, nutrient availability, and other abiotic factors (Lemanceau et al., 2017) and therefore, may both enhance microbial diversity, and November 2019 \| Volume 10 \| Article 2531 Frontiers in Microbiology \| www.frontiersin.org 1 Actinobacteria Associated to Edelweiss Oberhofer et al. support proliferation of particular species (Gottel et al., 2011; Wang M. et al., 2016). In contrast, the rhizosphere is in immediate contact with the plant roots and is actively enriched by a complex mixture of carbon/nutrient sources, such as amino acids, sugars, and other nutrients provided by the plant, in a process known as rhizodeposition (Hütsch et al., 2002; Bais et al., 2006). This environment attracts a plant species-speciﬁc microbial community (Minz et al., 2013; Essarioui et al., 2017) and is then modiﬁed by both the plant and microorganisms. The assemblies of microorganisms present in the rhizosphere are dependent on the soil type, the host species, the host plant genotype, and root system architecture (Ofek-Lalzar et al., 2014; Pérez-Jaramillo et al., 2017; Saleem et al., 2018). The endosphere is adjacent to the rhizosphere and is intimately linked to it by acquiring its microbial inhabitants, the endophytes, not exclusively, but to great extent through horizontal transmission (Frank et al., 2017). Some root endophytes may also result from vertical transmission through seeds and subsequently migrate into root tissues (Johnston-Monje and Raizada, 2011; Mitter et al., 2017). In contrast to pathogens, endophytes live in plant tissues without causing harm to their host. Particularly bacterial endophytes often originate from the rhizosphere of their host plant (Compant et al., 2010; Hardoim et al., 2015). The endosphere entails the entire microbial community that inhabits the interior of the host plant. Generally, host plants provide an environment with high nutrient content to their microbial inhabitants and shelter them from adverse environmental factors (Compant et al., 2010; Wang M. et al., 2016). Thus, endophytes experience high selective pressure competing for their beneﬁcial, but spatially limited habitat (Caraballo-Rodríguez et al., 2017). Within the endosphere, diﬀerent plant tissues are characterized by their speciﬁc chemoproﬁles and tissue architecture, which can act as “ﬁlters” and attract diﬀerent subsets of the endophyte community. Frontiers in Microbiology \| www.frontiersin.org INTRODUCTION Both the rhizosphere and the endosphere impose strong selective forces on microbes, which promote highly interactive communities. This suggests involvement of secondary metabolites in interactive signaling aﬀecting antibiosis (Yim et al., 2007), quorum sensing and bioﬁlm formation (Abisado et al., 2018). Hence, these two symbiotic habitats bear prime potential for the discovery of novel natural products (Strobel et al., 2004; Tiwari and Gupta, 2013). Moreover, leoligin is a plant lignan compound that has been shown to inhibit intimal hyperplasia in venous bypass grafts by reverse cholesterol transport without showing signs of cytotoxicity (Reisinger et al., 2009; Wang L. et al., 2016). However, because of overharvesting, Edelweiss became threatened and subsequently its protection status prevents its collection and use as herbal remedy in Europe. In a yet increasing number of medicinal plants, endophytes are being discovered producing either precursor molecules or the actual bioactive compounds previously attributed to their host plant (Köberl et al., 2013; Golinska et al., 2015), which may unlock possibilities for their biotechnological production and relief the focus on the threatened plant. support proliferation of particular species (Gottel et al., 2011; Wang M. et al., 2016). In contrast, the rhizosphere is in immediate contact with the plant roots and is actively enriched by a complex mixture of carbon/nutrient sources, such as amino acids, sugars, and other nutrients provided by the plant, in a process known as rhizodeposition (Hütsch et al., 2002; Bais et al., 2006). This environment attracts a plant species-speciﬁc microbial community (Minz et al., 2013; Essarioui et al., 2017) and is then modiﬁed by both the plant and microorganisms. The assemblies of microorganisms present in the rhizosphere are dependent on the soil type, the host species, the host plant genotype, and root system architecture (Ofek-Lalzar et al., 2014; Pérez-Jaramillo et al., 2017; Saleem et al., 2018). The endosphere is adjacent to the rhizosphere and is intimately linked to it by acquiring its microbial inhabitants, the endophytes, not exclusively, but to great extent through horizontal transmission (Frank et al., 2017). Some root endophytes may also result from vertical transmission through seeds and subsequently migrate into root tissues (Johnston-Monje and Raizada, 2011; Mitter et al., 2017). In contrast to pathogens, endophytes live in plant tissues without causing harm to their host. Particularly bacterial endophytes often originate from the rhizosphere of their host plant (Compant et al., 2010; Hardoim et al., 2015). November 2019 \| Volume 10 \| Article 2531 INTRODUCTION alpinum and its rhizosphere may serve as untapped habitats for rare Actinobacteria that could be of major interest for bioprospecting and drug discovery. Leontopodium nivale subsp. alpinum (Cass.) Greuter, syn. is commonly known as Edelweiss, a hemicryptophytic herbaceous plant species of family Asteraceae. The focal area of diversity in the genus Leontopodium is the Sino-Himalayan region, where several species are used as medicinal plants (Safer et al., 2011). Edelweiss occurs in subalpine to alpine grassland communities (Hörandl et al., 2011) and is currently believed to be monotypic for the genus in Europe. The plant has been used historically as a medicinal herb against numerous ailments in traditional medicine. It was used to treat dysenteria in humans and animals, bronchitis, diarrhea, fever, and was generally used for its anti-inﬂammatory and antibacterial properties (Dobner et al., 2003). Recent studies show that the root extract exhibits analgesic eﬀects in the rat paw edema test (Speroni et al., 2006). Leontopodic acid, one of the active compounds of the plant, is a potent antioxidant (Schwaiger et al., 2005). In this study, Actinobacteria associated with three Edelweiss plants were explored with a dual concept consisting of targeted isolation from rhizosphere soil suspension and a culture- independent approach exploring the endophytic communities of diﬀerent plant tissues. Phylogenetic analysis revealed an overlap between the rhizosphere isolates and plant endophytic Actinobacteria identiﬁed using 16S rRNA gene amplicon sequencing. Endophytic actinobacterial community of Edelweiss appears to be rich in rare and probably underexplored species, which can be targeted for isolation in the future bioprospecting eﬀorts aimed at drug discovery. November 2019 \| Volume 10 \| Article 2531 Frontiers in Microbiology \| www.frontiersin.org Frontiers in Microbiology \| www.frontiersin.org 2 Actinobacteria Associated to Edelweiss Oberhofer et al. Sample Collection and Treatment Special permission for collecting L. nivale subsp. alpinum and rhizosphere soil samples was issued by the Environmental Agency Austria in 2015. Three plant individuals and their adjacent rhizosphere soils were collected at the Mount Rax in the Austrian Alps on September 26, 2015 at 1624 m above sea level close to Praterstern at the coordinates N474258.34 and E154542.63. To avoid accidentally harvesting clones originating from laterally arising leaf rosettes on rhizomes of the same plant individual, plants were chosen to be a minimum of 20 m apart from each other. Plants and soils were transported under cooled conditions and upon arrival stored at 4◦C until processing. Plants were dissected in leaves, inﬂorescences, rhizomes, and roots and pooled within tissues. Plant tissues were ﬁrst carefully rinsed with tap water and then surface sterilized using an aqueous solution of sodium hypochlorite (2% active chlorine) for 10 min. Tissues were then washed twice with autoclaved distilled water and treated with 70% ethanol for 1 min and rinsed with sterile water. Surface sterilized plant tissues were ground in a mortar and 20% glycerol added before storing at −80◦C until further processing. Soil Suspensions and Pre-treatments p Soil stock suspensions were produced using 250 mg air dried rhizosphere soil and 2.25 ml autoclaved distilled water for each treatment. Pre-treatments were applied to diminish fast growing and abundant soil bacteria that would hinder slow growing Actinobacteria isolation by overgrowth (Kurtböke, 2003). Dry heat pre-treatment was achieved with dry rhizosphere soil, which was exposed to 100◦C for 1 h. Then, soil was re-suspended in 2.25 ml of sterile water after cooling to room temperature. The mixture was vortexed and immediately used for serial dilutions. The phenol pre-treatment was accomplished by adding 4.5 ml of a 5 mM phosphate buﬀer (pH 7) containing 1.5% phenol to one of the four soil stock suspensions described above. This mixture was kept at 30◦C in a water bath for 30 min and was then used for producing serial dilutions. Inoculated plates from this pre-treatment were stored in the fume hood covered with aluminum foil at room temperature for 3 days to allow the safe evaporation of the phenol. Microwave pre-treatment was performed by exposing the soil stock suspension to microwaves for 30 sec (2460 MHz frequency, 100 W power). No pre- treatment as a control was achieved by untreated soil stock suspension as origin of further dilutions. For all isolation plates and dilutions applied that 100 µl of pre-treated soil suspension was spread on the nutrient agar surface. All nutrient media plates were incubated at 28◦C for microbe isolation, phenol treated plates joined at day four. 1https://www.dsmz.de/microorganisms/medium/pdf/DSMZ_Medium12.pdf TABLE 1 \| Carbon and nitrogen sources, B-vitamins, and soil extract in selective media targeting Actinobacteria (Kurtböke, 2003). Media C-source N-source B-vitamins1 AV Glucose Arginine + Glycerol MC Glucose NaNO3 MGA-SE2 Glucose L-Asperagine Soil extract GAC Glucose L-Asperagine HV Humic acid Humic acid + LSV-SE2 Lignin Soy bean ﬂour + Soil extract 1Composed of 0.5 mg each of thiamine-HCl, riboﬂavin, niacin, pyridoxin-HCl, inositol, Ca-pantothenate, p-aminobenzoic acid and 0.25 mg of biotin. 2SE = soil extract. 1https://www.dsmz.de/microorganisms/medium/pdf/DSMZ_Medium12.pdf TABLE 1 \| Carbon and nitrogen sources, B-vitamins, and soil extract in selective media targeting Actinobacteria (Kurtböke, 2003). Antibiotic Treatment Media were supplemented with either no antibiotics, a dual combination of nystatin and cycloheximide (NC) or a triple combination of nystatin, cycloheximide, and novobiocine (NCN). Nystatin (20 µg/ml) and cycloheximide (20 µg/ml) suppress fungi, which can rapidly colonize entire isolation plates. Novobiocine (100 µg/ml) acts against certain Gram-negative and most Gram-positive bacteria targeting abundant unicellular bacteria in soil suspensions. MATERIALS AND METHODS of soil extract (Table 1), which was prepared from common garden soil at the medicinal plant garden of the Department of Pharmacognosy at the University of Vienna. Soil was dried in a beaker at 120◦C for 90 min and ground with mortar and pestle. 400 g of ground soil was used following the protocol for soil extract medium without the addition of agar1. Resulting extract was stored at −20◦C until use. Experimental Design The experiment included a total of 864 isolation plates resulting from factorially combining four pre-treatments of soil suspension, six serial dilutions thereof, six media selective for rare Actinobacteria and three antibiotic treatments. Each combination was replicated twice. After inoculation, plates were investigated twice per week manually and isolates matching morphological criteria of Actinobacteria were picked selectively with tooth picks and streaked out to new plates with the same media and antibiotic regime as present on the original isolation plates. Puriﬁed strains were then inoculated onto solid media facilitating spore formation. Spores were harvested from each sporulating strain and re-suspended in 20% glycerol and stored at −80◦C (Kieser et al., 2000). Actinobacteria with no spore formation were maintained as scrapings as described above. Simultaneously, liquid cultures were inoculated with the strains using Tryptic Soy Broth (TSB) media as default and 2x Yeast Extract/Tryptone (2xYT) media, in case no suﬃcient growth Illumina HiSeq2500 Sequencing q q g Plant samples were separated into leaves, roots, rhizomes and inﬂorescences at harvest and were pooled among tissue from three plant individuals. Total genomic DNA was extracted using the FastDNATM SPIN Kit for Soil DNA Extraction (MP Biomedicals, Santa Ana, CA, United States) according to manufacturer‘s recommendations. Plant tissues were thawed and rinsed once with sterile water to remove excess glycerol. Resulting material was lyophilized for 48 h and 100 mg were weighed in for DNA extraction. Genomic DNA quantity and quality was estimated by gel electrophoresis and Nanodrop lite (Thermo Fisher Scientiﬁc, Waltham, MA, United States), adjusted to company’s requirements (>1 mg DNA per sample, >25 ng/ml) and submitted for Illumina HiSeq 2500 Sequencing to CD Genomics (Shirley, New York, NY, United States). Amplicons were acquired with the primers 357F and 806R and entailed the V3 and V4 region of 16S rDNA as paired end reads of 250 bases. This yielded a total of 30,000–50,000 sequences per tissue. Sequences were processed according to the Mothur MiSeq SOP by using mothur 1.39.5 (Schloss et al., 2009; Kozich et al., 2013). Barcodes, primers, adapters, and chloroplast sequences were removed and sequences assembled, whereby all sequences unlike a ﬁnal length of 380–430 bp were excluded from downstream analyses. Chimeras were identiﬁed and removed using chimera.vsearch (Rognes et al., 2016). Resulting sequences were clustered into operational taxonomic units (OTUs) with an identity cutoﬀof 97%. Taxonomy was assigned using the RDB classiﬁer (Wang et al., 2007) and Silva databases4 with a bootstrap cutoﬀof 80% (Supplementary Figure 1). To mitigate the impact of potential sequencing artifacts, we removed singleton OTUs from further analyses. Original metagenomic sequence data have been deposited at the European Nucleotide Archive [PRJEB32100 (ERP114738)]. The full analysis script is available on GitHub5. The trimmed sequence alignment and the phylogenetic analysis of Supplementary Figure 1 is available at Dryad3. y g y Pellets of puriﬁed microbial strains were extracted using the Wizard SV Genomic DNA puriﬁcation system following the producer’s instructions (Promega Corporation, Madison, WI, United States). Genomic DNA quality and concentration was evaluated by gel electrophoresis in a 0.8% agarose gel supplemented with GelRed (Biotium, Inc., Fremont, CA, United States). Isolation Media Selective nutrient media were employed as means of targeting rare Actinobacteria genera. Media presented speciﬁc carbon (C) and nitrogen (N) sources, micronutrients and in some media addition of B-vitamins (Table 1; Kurtböke, 2003; Magnúsdóttir et al., 2015). Two media (MGA, LSV) required the addition 1Composed of 0.5 mg each of thiamine-HCl, riboﬂavin, niacin, pyridoxin-HCl, inositol, Ca-pantothenate, p-aminobenzoic acid and 0.25 mg of biotin. 2SE = soil extract. 1Composed of 0.5 mg each of thiamine-HCl, riboﬂavin, niacin, pyridoxin-HCl, inositol, Ca-pantothenate, p-aminobenzoic acid and 0.25 mg of biotin. 2SE = soil extract. Frontiers in Microbiology \| www.frontiersin.org November 2019 \| Volume 10 \| Article 2531 3 Actinobacteria Associated to Edelweiss Oberhofer et al. was observed in TSB. Cell suspensions were centrifuged and the pellets stored at −20◦C until genomic DNA extraction. (Haas et al., 2011). GenBank accession numbers are listed in Figures 1, 2 (SUB4176280). Phylogenies were made available at Dryad3. Illumina HiSeq2500 Sequencing Polymerase chain reaction (PCR) of the 16S rDNA barcoding region for bacteria was performed in a ﬁnal volume of 40 µl using the standard primers 27F and 1492R (Hongoh et al., 2003) each 1 µl, 0.5 µl Taq polymerase and 4 µl of commercial 10xTaq reaction buﬀer, 0.5 µl dNTPs and 5% DMSO. Genomic DNA was added depending on concentrations as inferred from gel electrophoresis and was modiﬁed accordingly with ddH2O to reach the ﬁnal volume. PCR products were veriﬁed with gel electrophoresis and products of expected length and speciﬁcity were puriﬁed with DNA Clean & ConcentratorTM-5 (Zymo Research, Irvine, CA, United States) and submitted to sequencing by Euroﬁns Scientiﬁc (Brussels, Belgium). Sequences were then assembled and manually edited and multiple alignments were performed in MEGA version 7.0.18 using ClustalW algorithms (Kumar et al., 2016). Most likely evolutionary models of base exchange were estimated using the same software. Phylogenetic analyses were achieved with maximum composite likelihood analysis (MCL) heuristic method of Neighbor- Join and BioNJ algorithms and were tested for robustness using 1000 bootstrap replicates. Gaps were treated as missing values (Figures 1, 2). Isolates from the rhizosphere of L. nivale subsp. alpinum are abbreviated with the preﬁx RLA- followed by an individual isolate number and GenBank accession numbers in brackets. Isolates were supplemented with their closest matches in GenBank BLASTN2.8.1 (Zhang et al., 2000; Morgulis et al., 2008) and their closest related type strains (T)2. Single strand sequences and amplicons shorter than 850 bp were excluded from phylogenetic analyses. For better resolution, the alignment of Streptomyces (Figure 2) was analyzed separately from other Actinobacteria genera (Figure 1). 2http://www.straininfo.net 3https://doi.org/10.5061/dryad.mkkwh70vg 4https://www.arb-silva.de 5https://github.com/JackyHess/Leontopodium_metagen Metabarcoding of Plant Tissues Isolates From Rhizosphere Cultures Rhizosphere isolates were obtained by applying three pre-treatments (microwave, dry heat, phenol) and no pre- treatment, two diﬀerent antibiotic combinations (NCN, NC) and no antibiotics and media selective for Actinobacteria combined with serial dilutions. Serial dilutions of soil suspensions were performed from 10−1 to 10−6. While the absolute numbers of chosen isolates were randomly distributed, the percentage of rare Actinobacteria was highest in the lowest dilution. Besides Actinobacteria, 184 other bacterial and two fungal strains were isolated, but not further investigated. Tissue speciﬁc Actinobacteria endophyte communities were investigated with metabarcoding. Based on data from V3 and V4 regions of 16S rDNA, we were able to identify a total of 336 unique Actinobacterial OTUs across the four tissues examined. Of these, 292 were found in the root, 213 in the rhizome, 29 in the leaves, and 11 in the inﬂorescence (Figures 3A,B). Among these OTUs we were able to identify 127 to the genus level, which yielded a representation of 45 diﬀerent genera, of which ﬁve were shared with the culture isolates, namely Actinokineospora, Nocardia, Mycobaterium, Micrococcus, and Streptomyces. We also identiﬁed 6 OTUs in the Microbacteriaceae and 24 OTUs in the Micromonosporacea that the RDB classiﬁer was unable to resolve to the genus level, suggesting that incomplete recovery of genera also discovered in the culture-based approach (5 of the 10 genera) is at least partially due to the inability to accurately classify to genus level based on short sequencing reads. Phylogenetic analyses included a total of 77 strains isolated from L. nivale subsp. alpinum rhizosphere soil that were identiﬁed by 16S rDNA barcoding as either belonging to rare Actinobacteria or the genus Streptomyces. Both groups were separately analyzed to retain proper resolution of branches. MCL analysis was used to infer phylogenies for 34 Actinobacteria isolates (Figure 1) and for 43 Streptomyces isolates including the closely related genus Kitasatospora (Figure 2). The maximum likelihood analysis of 16S rDNA sequences of Actinobacteria isolates (Figure 1) inferred eight distinct clades and two subclades in agreement with genera delimitations of Micromonospora (20 isolates), Asanoa (1), Actinokineospora (3), Mycobacterium (1), Nocardia (2), Leifsonia (1), Microbacterium (1), and Micrococcus (5). Modes of isolation are speciﬁed by color-coded geometric ﬁgures in Figures 1, 2. Relating Rhizosphere Isolates to the Endophyte Microbiota Composition The Actinobacteria alignment comprised 794 informative sites from a total of 53 sequences including 36 isolate strains. The best ﬁtting nucleotide substitution model was TN93 plus invariate sites and Gamma distribution with – lnL = −3535 (Tamura and Nei, 1993). The Streptomyces alignment included 1124 informative sites from 42 isolate strains. The total alignment consisted of 67 sequences and Mega7 Modeltest identiﬁed T92 with invariate sites and Gamma distribution as best ﬁtting evolutionary model with a likelihood value of – lnL = −3444,35 (Tamura, 1992). All sequences were checked for possible chimera formation twice at Genbank Submission using chimera.slayer within Mothur version 1.35.1 (Schloss et al., 2009) and Uchime Sanger sequences obtained from each isolate were aligned to Actinobacteria OTUs using PAGAN v.0.61 to analyze rhizosphere isolates together with the endophyte microbiota composition (Löytynoja et al., 2012). Poorly aligning regions were removed from the alignments using trimAl 1.2 (Capella- Gutiérrez et al., 2009) with the -automated1 setting. RAxML v.8.2.10 (Stamatakis, 2014) was used to infer a Maximum Likelihood phylogenetic tree using the option for rapid bootstrap and search for best-scoring ML tree (-f a) with the GTRGAMMA model of evolution. The optimal number of November 2019 \| Volume 10 \| Article 2531 Frontiers in Microbiology \| www.frontiersin.org 4 Actinobacteria Associated to Edelweiss Oberhofer et al. FIGURE 1 \| Continued November 2019 \| Volume 10 \| Article 2531 Frontiers in Microbiology \| www.frontiersin.org 5 Actinobacteria Associated to Edelweiss Oberhofer et al. FIGURE 1 \| Maximum composite likelihood analysis (MCL) of Actinobacteria isolates from the rhizosphere of Leontopodium nivale subsp. alpinum based on Tamura and Nei (1993) model with Gamma distribution and invariate sites using Mega software version 7.0.18 (Kumar et al., 2016). The phylogenetic tree contains experimental sequences (RLA) with their GenBank accession numbers, their closest matches in GenBank Blast search and, respectively, closest type strains (T). The tree is drawn to scale with branch length representing the substitutions per site. Bootstrap support values were calculated with 1000 replicates. Isolates identify by the preﬁx -RLA followed by an individual isolate number and GenBank accession numbers in brackets. Conditions for each isolation are depicted as symbols behind accessions. Relating Rhizosphere Isolates to the Endophyte Microbiota Composition Hexagonal symbols encode selective media (light blue = MGA-SE; dark blue = MC; turquois = AV; dark green = GAC; light green = LSV-SE), square symbols describe pre-treatments (dark red = microwave; blue = dry heat; pink = phenol; lilac = control) and triangles symbolize antibiotic treatments (orange = NCN; yellow = NC; white = control). B-vitamin addition as speciﬁed in Table 1 is indicated by a + behind the symbols of isolates. bootstraps was determined using the extended majority rule consensus criterion (-autoMRE) and the search converged after 650 bootstrap replicates. (1), Streptomyces sp. NEAU-PCY-2 (2, type strain unknown). The rare genus Kitasatospora is typically embedded within the Streptomyces phylogeny (Zhang et al., 1997) and housed two isolates. One more clade grouped with S. erringtonii (1). The clade most divergent to all others is clade 17, whose isolates RLA042a and -042c are related to the plant pathogen S. scabrisporus. RESULTS Isolates From Rhizosphere Cultures Rhizosphere isolates were obtained by applying three pre-treatments (microwave, dry heat, phenol) and no pre- treatment, two diﬀerent antibiotic combinations (NCN, NC) and no antibiotics and media selective for Actinobacteria combined with serial dilutions. Serial dilutions of soil suspensions were performed from 10−1 to 10−6. While the absolute numbers of chosen isolates were randomly distributed, the percentage of rare Actinobacteria was highest in the lowest dilution. Besides Actinobacteria, 184 other bacterial and two fungal strains were isolated, but not further investigated. Phylogenetic analyses included a total of 77 strains isolated from L. nivale subsp. alpinum rhizosphere soil that were identiﬁed by 16S rDNA barcoding as either belonging to rare Actinobacteria or the genus Streptomyces. Both groups were separately analyzed to retain proper resolution of branches. MCL analysis was used to infer phylogenies for 34 Actinobacteria isolates (Figure 1) and for 43 Streptomyces isolates including the closely related genus Kitasatospora (Figure 2). The maximum likelihood analysis of 16S rDNA sequences of Actinobacteria isolates (Figure 1) inferred eight distinct clades and two subclades in agreement with genera delimitations of Micromonospora (20 isolates), Asanoa (1), Actinokineospora (3), Mycobacterium (1), Nocardia (2), Leifsonia (1), Microbacterium (1), and Micrococcus (5). Modes of isolation are speciﬁed by color-coded geometric ﬁgures in Figures 1, 2. Frontiers in Microbiology \| www.frontiersin.org Metabarcoding of Plant Tissues In order to infer taxonomic representation among culture isolates and NGS samples independent of automatically assigned classiﬁcations, we built a phylogenetic tree including both, cultured isolates and one sequence representing each Actinobacteria OTU (Figure 3A). Phylogenetic analysis revealed four deep clades harboring Rubrobacteridae and Thermoleophilales (clade A; bootstrap 100), Acidimicrobiales (clade B; bootstrap 65), Actinomycetales (clade C; bootstrap 79), and clade D, which houses unclassiﬁed Actinobacteria (bootstrap 99). All culture isolates were found to belong to clade C suggesting either a pervasive culture bias in favor of Actinomycetales or enrichment of clades A and B in endophytic tissues compared to the rhizosphere. Clade C also houses 39 of the 45 identiﬁed genera, while the remaining 6 are found in clades A and B (3 genera in each). Based on phylogenetic relationships, we found NGS OTUs branching with all culture isolates suggesting near complete recovery of the taxonomic diversity represented among the cultures using NGS data, despite the inability to accurately classify OTUs to genus level in all cases (Figure 3A). Nevertheless, we found a relative overrepresentation of culture-based isolates compared to NGS OTUs, especially in the Streptomyces and Micromonospora clades (Figure 3A). This is likely due to over-clustering of closely related species The Streptomyces phylogeny comprised 16 clades, and one representing the rare actinobacterial genus Kitasatospora (Figure 2). Clades represent isolates grouping to Streptomyces subrutilus (2 isolates), Streptomyces avidinii (1), S. chinensis (4), a clade not grouping with any type strain closely (2), S. mirabilis (3), and S. olivochromogenes (3). One isolate grouped closely to the type strains of S. brevispora and S. spororaveus, whose 16S rDNA diﬀer marginally from each other. Moreover, another clade formed with S. anulatus (3). Three isolates of the next clade were closest related to Streptomyces sp. WW4-2c, but lacked any closely related type strain. Furthermore, another clade grouped to S. atratus (6), S. venezuelae (1), S. camponoticapitis November 2019 \| Volume 10 \| Article 2531 Frontiers in Microbiology \| www.frontiersin.org 6 Oberhofer et al. Actinobacteria Associated to Edelweiss We also investigated abundance of diﬀerent taxa across diﬀerent tissues based on presence/absence variation of OTUs (Figure 4). Family-level relative abundances showed signiﬁcant diﬀerences between aboveground and belowground tissues (P = 8.5e-06; two-sided Fisher exact test with simulated P-values based on 1e+07 replicates). DISCUSSION The endosphere of a plant often receives a signiﬁcant part of its bacterial inhabitants from the rhizosphere through horizontal transmission (Compant et al., 2010; Wang M. et al., 2016). To our knowledge, this is the ﬁrst study providing initial insights in the presence of rare Actinobacteria from both habitats associated with the ancient and threatened alpine medicinal plant, L. nivale subsp. alpinum, which have been obtained with a combinatory approach of selective isolation and tissue-speciﬁc metagenomic barcoding. Medicinal plants are a promising source of rare Actinobacteria for bioprospecting and have been recently reviewed (Köberl et al., 2013; Golinska et al., 2015), yet temperate and especially alpine plant species remain understudied in this respect. p A range of known selective techniques were employed aiming to target isolation of rare Actinobacteria from the rhizosphere of L. nivale subsp. alpinum. Pre-treatments of soil such as dry heat, microwave irradiation, and phenol reduce the numbers of abundant co-occurring bacteria (Hayakawa, 2003). Nutrient poor media with speciﬁc C- and N-sources (Table 1) promote actinobacterial growth because of their versatile metabolic capabilities compared to other bacteria and may also promote their sporulation. B-vitamins, known as growth factors for certain Actinobacteria, were also utilized (Figures 1, 2 and Table 1; Kurtböke, 2003). Antibiotics act as additional selectors favoring Actinobacteria as resourceful producers of the latter, which requires them to possess a large number of resistance genes in their antibiotic biosynthesis gene clusters (Peterson and Kaur, 2018). We succeeded to isolate 77 actinobacterial strains covering 10 genera, of which nine are considered rare. The genus Micromonospora is a proliﬁc producer of diverse classes of antibiotic compounds, which have not yet reached a saturation point in discoveries yet (Boumehira et al., 2016). Asanoa is a rare genus within Micromonosporaceae and besides its frequent association to plants, speciﬁcally medicinal plants, few data are available on its antimicrobial potential (Qin et al., 2009; Niemhom et al., 2016). Several species from genus Actinokineospora are in recent focus of bioprospecting for their antimicrobial and antitrypanosomal activities (Abdelmohsen et al., 2014; Intra et al., 2016). More than a third of the described Nocardia species are opportunistic pathogens, however, they FIGURE 2 \| Maximum composite likelihood analysis clustering of Streptomyces isolates from Edelweiss rhizosphere. Phylogentic tree reconstruction was performed with Tamura (1992) as best ﬁtting evolutionary model with invariant sites and Gamma distribution using Mega software version 7.0.18 (Kumar et al., 2016). Metabarcoding of Plant Tissues Residual plots revealed that this was primarily driven by a dominance of Microbacteriaceae and Micrococcaceae among the leaf and inﬂorescence tissues, while taxonomic distribution was more balanced and diverse in belowground tissues (Supplementary Figure 2). Diﬀerences in taxonomic distribution among belowground tissues were non-signiﬁcant. FIGURE 2 \| Maximum composite likelihood analysis clustering of Streptomyces isolates from Edelweiss rhizosphere. Phylogentic tree reconstruction was performed with Tamura (1992) as best ﬁtting evolutionary model with invariant sites and Gamma distribution using Mega software version 7.0.18 (Kumar et al., 2016). Further speciﬁcations of the phylogeny are congruent to Figure 1. DISCUSSION Further speciﬁcations of the phylogeny are congruent to Figure 1. in these genera into the same OTU based on strong sequence conservation among isolates in the V3–V4 region sequenced using NGS technology. November 2019 \| Volume 10 \| Article 2531 Frontiers in Microbiology \| www.frontiersin.org 7 Actinobacteria Associated to Edelweiss Oberhofer et al. FIGURE 3 \| Continued rontiers in Microbiology \| www.frontiersin.org 8 November 2019 \| Volume 10 \| Article 2531 FIGURE 3 \| Continued FIGURE 3 \| Continued November 2019 \| Volume 10 \| Article 2531 Frontiers in Microbiology \| www.frontiersin.org 8 Actinobacteria Associated to Edelweiss Actinobacteria Associated to Edelweiss Oberhofer et al. FIGURE 3 \| (A) ML analysis of combined sequences from Actinobacterial rhizosphere isolates and metagenomic data. The phylogenetic tree includes 16S rDNA sequences derived from rhizosphere isolates (culture, light gray), their closest type strains as references (black) and metabarcoding OTUs (dark gray). Cultured genera are named in the outer ring and highlighted by color coding, based on the last common ancestor spanning all reference sequences assigned to each genus. All indicated genera are supported by bootstrap values >70, except Streptomyces. The outermost four concentric rings indicate presence of each OTU according to tissue: root (yellow), rhizome (red), leaf (burgundy), inﬂorescence (green). This graphic was produced using GraPhlAn (Asnicar et al., 2015). (B) Total numbers of OTUs according to tissue with color-coding as in panel (A). FIGURE 4 \| Actinobacteria diversity in different plant tissues of Edelweiss based on metabarcoding data. Tissues of the host plant Leontopodium nivale subsp. alpinum are illustrated on the x-axis (inﬂorescence, leaf, rhizome, and root) and sequence data are based on presence/absence coding for each OTU. Taxonomic assignments were performed with RDB classiﬁer and Silva database (Wang et al., 2007) and reported at the taxonomic level of families. also produce industrially important antibiotic compounds and enzymes (Tanaka et al., 1997; Barka et al., 2016). Leifsonia h b i l t d f Chi d I di di i l l t endophyte, e.g., in ginseng (Vendan et al., 2010) and is known to produce pigments with antimicrobial activity and other antibiotic d (P l t l 2013 U d i d K i h i FIGURE 4 \| Actinobacteria diversity in different plant tissues of Edelweiss based on metabarcoding data. Tissues of the host plant Leontopodium nivale subsp. FIGURE 3 \| (A) ML analysis of combined sequences from Actinobacterial rhizosphere isolates and metagenomic data. The phylogenetic tree includes 16S rDNA sequences derived from rhizosphere isolates (culture, light gray), their closest type strains as references (black) and metabarcoding OTUs (dark gray). Cultured genera are named in the outer ring and highlighted by color coding, based on the last common ancestor spanning all reference sequences assigned to each genus. All indicated genera are supported by bootstrap values >70, except Streptomyces. The outermost four concentric rings indicate presence of each OTU according to tissue: root (yellow), rhizome (red), leaf (burgundy), inﬂorescence (green). This graphic was produced using GraPhlAn (Asnicar et al., 2015). (B) Total numbers of OTUs according to tissue with color-coding as in panel (A). DISCUSSION alpinum are illustrated on the x-axis (inﬂorescence, leaf, rhizome, and root) and sequence data are based on presence/absence coding for each OTU. Taxonomic assignments were performed with RDB classiﬁer and Silva database (Wang et al., 2007) and reported at the taxonomic level of families. also produce industrially important antibiotic compounds and endophyte, e.g., in ginseng (Vendan et al., 2010) and is known FIGURE 4 \| Actinobacteria diversity in different plant tissues of Edelweiss based on metabarcoding data. Tissues of the host plant Leontopodium nivale subsp. alpinum are illustrated on the x-axis (inﬂorescence, leaf, rhizome, and root) and sequence data are based on presence/absence coding for each OTU. Taxonomic assignments were performed with RDB classiﬁer and Silva database (Wang et al., 2007) and reported at the taxonomic level of families. also produce industrially important antibiotic compounds and enzymes (Tanaka et al., 1997; Barka et al., 2016). Leifsonia has been isolated from Chinese and Indian medicinal plants, but information depth on natural products from this genus refer to the presence or absence of synthetase classes and antibiotic sensitivity patterns (Qiu et al., 2007; Passari et al., 2015). Micrococcus can have plant growth promoting abilities as endophyte, e.g., in ginseng (Vendan et al., 2010) and is known to produce pigments with antimicrobial activity and other antibiotic compounds (Palomo et al., 2013; Umadevi and Krishnaveni, 2013). Kitasatospora is recognized as versatile producer of secondary metabolites of highly diverse nature, such as anti- tumor agents, immune modulators, anti-virals, herbicidals, and anti-protozoan compounds (Takahashi, 2017). November 2019 \| Volume 10 \| Article 2531 Frontiers in Microbiology \| www.frontiersin.org Frontiers in Microbiology \| www.frontiersin.org 9 Actinobacteria Associated to Edelweiss Oberhofer et al. available promote a selective eﬀect of host plant species, especially within the same site (Sheng et al., 2010; Ciccazzo et al., 2014; Chang et al., 2018). Our data is preliminary in this respect and further studies including multiple sampling sites are required to address possible site eﬀects of encountered microbiota. Generally, Streptomyces, Micromonospora, and Nocardia commonly occur as endophytes in plants (Qin et al., 2010) and are also part of rhizo- and endosphere assemblies in L. nivale subsp. alpinum in this study. Artemisia annua (Asteraceae), a related medicinal plant, shares the endophytic genera Micrococcus, Nocardia, and Streptomyces (Li et al., 2011; Figure 3). Taechowisan et al. DISCUSSION (2003) isolated Actinobacterial endophytes from 36 medicinal plants and retrieved only four diﬀerent genera including all of the above mentioned common genera plus Microbispora. In contrast, the genera Asanoa, Aktinokineospora, Mycobacterium, Micrococcus, Leifsonia, Microbacterium, and Kitasatospora are less commonly encountered. From a bioprospecting perspective, all Actinobacteria isolated in this study but Streptomyces can be considered as rare and promising candidates for further research (Kurtböke, 2003). Metabarcoding data highlight the presence of numerous yet undescribed endophytic Actinobacteria belonging to deep rooting clades A, B, and D, which were detected in roots and rhizomes of Edelweiss plants (Figure 3 and Supplementary Figure 1), but have rarely been isolated from plants before with the exception of Solirubrobacter phytolaccaceae, S. ginsenosidimutans, Patulibacter brassicae, and P. ginsengiterrae (An et al., 2011; Kim et al., 2012; Wei et al., 2014; Jin et al., 2016). Based on these insights, future investigations may speciﬁcally consider isolation of Actinobacteria from its belowground tissues, which bear large amounts of this uninterrogated diversity. Selective isolation eﬀorts may be tailored to targeting speciﬁcally clades A, B, and D, which promise larger taxonomical depth than currently known and would greatly expand our knowledge on Actinobacterial diversity, potentially including many new producers of novel metabolites. Host plant species exert a strong selective eﬀect on their rhizosphere and endophyte microbiome (Ofek-Lalzar et al., 2014; Kumar et al., 2017), whereby endophytes often originate in the rhizosphere soil (Compant et al., 2010; Hardoim et al., 2015). Therefore, sequence data from rhizosphere and endosphere of the host plant L. nivale subsp. alpinum were jointly analyzed using a phylogenetic approach (Figure 3) to explore the endophytic Actinobacteria community. Pooling plant individuals among the investigated tissues prevents conclusions on diﬀerences of Actinobacteria communities associated to each plant specimen, however, this exceeded the focus of this study. Actinobacteria genera were found in all investigated plant organs with decreasing diversity from roots (292 OTUs), rhizomes (213), leaves (29), and inﬂorescences (11) based on a cutoﬀat 97% identity (Figure 3B). Rhizosphere isolates were well-represented among endophyte OTUs (Figure 3A), however, limited resolution of endophyte OTUs due to short sequence length and diﬀering error rates do not allow a numerical estimation of the overlap between endo- and rhizosphere Actinobacteria. Rhizosphere isolated exclusively occurred in clade C (Actinomycetales), but were absent from clades A (Rubrobacteridae and Thermoleophilales), B (Acidomicrobiales), and D (Unclassiﬁed) (Figure 3A). DISCUSSION Rubrobacteridae and Acidomicrobiales are known for the formation of micro-colonies of less than 25 µm with extremely slow growth rates supporting an isolation bias against this clade (Davis et al., 2011). Thermoleophilales are typically extremophiles. Clades A and B contain members of six assigned genera: Conexibacter, Solirubrobacter, and Thermoleophilum (clade A), Aciditerrimonas, Iamia, and Illumatobacter (clade B) as well as a large proportion of unclassiﬁed OTUs (Figure 3 and Supplementary Figure 1). Members of clade A and B have been rarely isolated from plants, but do occur in metagenomic endophyte studies (Pereira et al., 2017; Wemheuer et al., 2018). This suggests a pervasive culture bias against members of both clades as a more likely scenario rather than a selective enrichment of members of clade A and B in the endosphere. A culture and/or study bias is also supported by the uneven distribution of OTUs based on genus-level taxonomic assignment with almost 90% of assigned genera housed in clade C and is supportive of the notion that large sections of the phylogenetic diversity of Actinobacteria remain yet to be described (Gao and Gupta, 2012). DATA AVAILABILITY STATEMENT The datasets generated for this study can be found in the GenBank MH513505 – MH513581 (publication date July 15, 2019), https://github.com/JackyHess/Leontopodium_metagen, European Nucleotide Archive PRJEB32100 (ERP114738), alignments and phylogenies are made available at Dryad Digital Depository6. On a higher taxonomic level, Actinobacteria family diversity based on metabarcoding data suggests two sub-groups within diﬀerent plant tissues: highly diverse belowground communities in rhizomes and roots and less diverse communities in aboveground tissues dominated by diﬀerent families of Actinobacteria (Figure 4). In order to conﬁrm diﬀerences in taxonomic distribution among tissues and assess inter- individual variation in endophytic diversity, fully replicated metabarcoding designs will be required. 6https://doi.org/10.5061/dryad.mkkwh70vg Frontiers in Microbiology \| www.frontiersin.org REFERENCES A., Vatsa, P., Sanchez, L., Gaveau-Vaillant, N., Jacquard, C., Klenk, H.- P., et al. (2016). Taxonomy, physiology, and natural products of actinobacteria. Microbiol. Mol. Biol. Rev. 80, 1–43. doi: 10.1128/MMBR.00019-15 Haas, B. J., Gevers, D., Earl, A. M., Feldgarden, M., Ward, D. V., Giannoukos, G., et al. (2011). 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Safe-site eﬀects on rhizosphere bacterial communities in a high-altitude alpine environment. Biomed. Res. Int. 2014, 480170–480179. doi: 10.1155/2014/ 480170 Hörandl, E., Dobeš, C., Suda, J., Vít, P., Urfus, T., Temsch, E. M., et al. (2011). Apomixis is not prevalent in subnival to nival plants of the European Alps. Ann. Bot. 108, 381–390. doi: 10.1093/aob/mcr142 Hütsch, B. W., Augustin, J., and Merbach, W. (2002). Plant rhizodeposition — an important source for carbon turnover in soils. J. Plant Nutr. Soil Sci. 165, 397–407. Compant, S., Clement, C., and Sessitsch, A. (2010). REFERENCES Essarioui, A., LeBlanc, N., Kistler, H. C., and Kinkel, L. L. (2017). Plant community richness mediates inhibitory interactions and resource competition between Streptomyces and Fusarium populations in the rhizosphere. Microb. Ecol. 74, 157–167. doi: 10.1007/s00248-016-0907-5 Abdelmohsen, U., Cheng, C., Viegelmann, C., Zhang, T., Grkovic, T., Ahmed, S., et al. (2014). 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Rev. 76, 66–112. doi: 10.1128/MMBR.05011-5011 An, D. S., Wang, L., Kim, M. S., Bae, H. M., Lee, S. T., and Im, W. T. (2011). Solirubrobacter ginsenosidimutans sp. nov., isolated from soil of a ginseng ﬁeld. Int. J. Syst. Evol. Mircrobiol. 61, 2606–2609. doi: 10.1099/ijs.0.028431-28430 Genbank Submission. SUB4176280. Accession Numbers of Actinobacterial Rhizosphere Isolates from Leontopodium Nivale subsp. alpinum (MH513505– MH513581). Github: https://github.com/JackyHess/Leontopodium_metagen Asnicar, F., Weingart, G., Tickle, T. L., Huttenhower, C., and Segata, N. (2015). Compact graphical representation of phylogenetic data and metadata with GraPhlAn. PeerJ 3:e1029. doi: 10.7717/peerj.1029 Bais, H. P., Weir, T. L., Perry, L. G., Gilroy, S., and Vivanco, J. M. (2006). The role of root exudates in rhizosphere interactions with plants and other organisms. Ann. Rev. Plant Biol. 57, 233–266. doi: 10.1146/annurev.arplant.57.032905.105159 Golinska, P., Wypij, M., Agarkar, G., Rathod, D., Dahm, H., and Rai, M. (2015). Endophytic actinobacteria of medicinal plants: diversity and bioactivity. Antonie Van Leeuwenhoek 108, 267–289. doi: 10.1007/s10482-015- 0502-7 Baltz, R. (2008). Renaissance in antibacterial discovery from actinomycetes. Curr. Opin. Pharmacol. 8, 557–563. doi: 10.1016/j.coph.2008. 04.008 Gottel, N. R., Castro, H. F., Kerley, M., Yang, Z., Pelletier, D. A., Podar, M., et al. (2011). Distinct microbial communities within the endosphere and rhizosphere of Populus deltoides roots across contrasting soil types. Appl. Environ. Microb. 77, 5934–5944. doi: 10.1128/AEM.05255-11 Barka, E. FUNDING appreciate Andrea Kodym for the plant surface sterilization and thank Lindsey A. Segalla for helpful comments on the manuscript. This research was supported by the University of Vienna. AUTHOR CONTRIBUTIONS MO, SZ, CW, TR, and JH contributed to the concept and design of this research. TR and JH conducted the bioinformatics analyses. MO, ML, and FG isolated Actinobacteria from the rhizosphere soil and genotyped them. MO, JH, and ML analyzed the phylogenetic relationship of isolates. MO, JH, and SZ drafted the manuscript. All authors critically revised it and agreed to all aspects of the work presented. This study describes the isolation of Actinobacteria from the rhizosphere of an alpine medicinal plant, which will be explored for drug discovery by genome- and culture-based bioprospecting approaches in the future. Moreover, our results provide an initial view of taxonomic representation of Actinobacteria occurring endogenously in L. nivale subsp. alpinum. Few studies are available on Actinobacteria associated to alpine plants, but those November 2019 \| Volume 10 \| Article 2531 Frontiers in Microbiology \| www.frontiersin.org 10 Actinobacteria Associated to Edelweiss Oberhofer et al. The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2019.02531/full#supplementary-material The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2019.02531/full#supplementary-material SUPPLEMENTARY MATERIAL We gratefully acknowledge Franz Tod from the Department of Botany and Biodiversity, Vienna, for the collection of the experimental rhizosphere soil and the plants. We The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2019.02531/full#supplementary-material REFERENCES 33, 1870–1874. doi: 10.1093/molbev/msw054 Quintana, E. T., Badillo, R. 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https://openalex.org/W2101319683	https://www.scielo.br/j/ibju/a/XYrXZWgh5RK6xctZKxtXpyJ/?lang=en&format=pdf	English	null	Identification of mechanisms involved in the relaxation of rabbit cavernous smooth muscle by a new nitric oxide donor ruthenium compound	International Braz J Urol	2,012	cc-by	5,486	Vol. 38 (5): 687-694, September - October, 2012 Vol. 38 (5): 687-694, September - October, 2012 ORIGINAL Article ABSTRACT L- -cysteine failed to modify the relaxations caused by the agents. Hydroxicobalamine significantly diminished the relaxation effect of Rut-Caf. Glibenclamide significantly increased the efficacy of Rut-Caf (pEC50 4.09 x 7.09). There were no alterations of po- tency or maximum effect of the substances with the addition of the other ion channel blockers. Rut-Caf induced production of significant amounts of cGMP and cAMP du- ring the relaxation process. Accepted after revision: July 27, 2012 Conclusions: In conclusion, Rut-Caf causes relaxation of smooth muscle of corpus cavernosum by means of activation of sGC with intracellular production of cGMP and cAMP; and also by release of NO in the intracellular environment. Rut-Caf releases the NO free radical and it does not act directly on the potassium ion channels. Identification of mechanisms involved in the relaxation of rabbit cavernous smooth muscle by a new nitric oxide donor ruthenium compound João Batista Gadelha de Cerqueira, Lúcio Flávio Gonzaga-Silva, Francisco Ordelei Nascimento da Silva, João Victor Medeiros de Cerqueira, Ricardo Reges Maia Oliveira, Maria Elisabete Amaral de Moraes, Nilberto Robson Falcão do Nascimento Departamento de Cirurgia (JBGC, LFGS, RRMO), Departamento de Química Orgânica e Inorgânica (FONS), Departamento de Fisiologia e Farmacologia (MEAM) Universidade Federal do Ceará, Ceará, Brazil and Instituto Superior de Ciências Biomédicas- Universidade Estadual do Ceará (JVMC, NRFN), Ceará, Brazil ABSTRACT Key words: Nitric oxide; Nitrosyl-ruthenium complex; Endothelium, Vascular; Potassium Channel Int Braz J Urol. 2012; 38: 687-94 __________________ Submitted for publication: March 08, 2012 __________________ Accepted after revision: July 27, 2012 Key words: Nitric oxide; Nitrosyl-ruthenium complex; Endothelium, Vascular; Potassium Channel Purpose: The aim of this study was to evaluate the relaxation in vitro of cavernous smooth muscle induced by a new NO donor of the complex nitrosil-ruthenium, named trans-[Ru(NH3)4(caffeine)(NO)]C13 (Rut-Caf) and sodium nitroprusside (SNP). 4 13 Materials and Methods: The tissues, immersed in isolated bath systems, were pre-con- tracted with phenilephrine (PE) (1 µM) and then concentration-response curves (10-12 p p ( ) ( µ ) p ( - 10-4 M) were obtained. To clarify the mechanism of action involved, it was added to the baths ODQ (10 µM, 30 µM), oxyhemoglobin (10 µM), L-cysteine (100 µM), hydro- xicobalamine (100 µM), glibenclamide, iberotoxin and apamine. Tissue samples were frozen in liquid nitrogen to measure the amount of cGMP and cAMP produced. - 10-4 M) were obtained. To clarify the mechanism of action involved, it was added to the baths ODQ (10 µM, 30 µM), oxyhemoglobin (10 µM), L-cysteine (100 µM), hydro- xicobalamine (100 µM), glibenclamide, iberotoxin and apamine. Tissue samples were frozen in liquid nitrogen to measure the amount of cGMP and cAMP produced. Submitted for publication: March 08, 2012 Results: The substances provoked significant relaxation of the cavernous smooth mus- cle. Both Rut-Caf and SNP determined dose-dependent relaxation with similar potency (pEC50) and maximum effect (Emax). The substances showed activity through activation of the soluble guanylyl cyclase (sGC), because the relaxations were inhibited by ODQ. Oxyhemoglobin significantly diminished the relaxation effect of the substances. L- -cysteine failed to modify the relaxations caused by the agents. Hydroxicobalamine significantly diminished the relaxation effect of Rut-Caf. Glibenclamide significantly increased the efficacy of Rut-Caf (pEC50 4.09 x 7.09). There were no alterations of po- tency or maximum effect of the substances with the addition of the other ion channel blockers. Rut-Caf induced production of significant amounts of cGMP and cAMP du- ring the relaxation process. Results: The substances provoked significant relaxation of the cavernous smooth mus- cle. Both Rut-Caf and SNP determined dose-dependent relaxation with similar potency (pEC50) and maximum effect (Emax). The substances showed activity through activation of the soluble guanylyl cyclase (sGC), because the relaxations were inhibited by ODQ. Oxyhemoglobin significantly diminished the relaxation effect of the substances. Accepted after revision: July 27, 2012 ibju \| Identification of mechanisms involved in the relaxation of rabbit cavernous smooth muscle ibju \| Identification of mechanisms involved in the relaxation of rabbit cavernous smooth muscle acetylcysteine-etylester (SNACET), was utilized in studies with strips of human corpus cavernosum mounted in isolated bath systems, proving to be potentially useful for tissue relaxation (10). rotransmitter that mediates penile erection in all animals (2). NO activates soluble guanylate cyclase (sGC), which induces production of cyclic guano- sine monophosphate (cGMP) from guanosine tri- phosphate (GTP). The cGMP acts in intracellular effectors, like protein kinase G (PKG), which cause diminishment of intracellular calcium and disas- sociation of actin and myosin fibers, ultimately leading to relaxation of the smooth muscle (2). SNP has already been utilized intracavern- ously in vivo, in both human and animals, to in- duce penile erection. In dogs and monkeys, SNP induced dose-related erection, without causing hypotension (11). In patients with ED, SNP pro- voked erection of shorter duration than did pa- paverine, without side effects - like hypotension, injection site pain, or priapism (11,12). NO donors are substances that release NO, either in vivo or in vitro. One of these donors, sodium nitroprusside (SNP), is a powerful vasodi- lator utilized in patients with hypertensive crisis. Nevertheless, it is extremely labile, induces toler- ance and releases cyanide, which is toxic to the endothelium (3). Ruthenium compounds, which are NO do- nors, have been tested in vitro and showed a simi- lar relaxation to SNP on rat aorta smooth muscle (13). These compounds theoretically have the po- tential to be better than SNP because they do not release cyanide nor they react with the superox- ide anion, forming peroxynitrite. However, further experimental studies are necessary to confirm this theory. Furthermore, they are stable at physiologic pH and soluble in water (13). NO is a potent vasodilator synthesized by neurons, endothelial cells, leucocytes and platelets among others. It is released by the autonomous nerve terminals, and by the vascular and sinusoidal endothelium when stimulated by acetylcholine (4). Synthesis of NO is catalyzed by NOS, which converts l-arginine and oxygen to l-citrul- ine and NO. NOS exists as three isoforms in mam- mals: nNOS and eNOS are preferentially expressed in neurons and endothelial cells, respectively, and iNOS in virtually all cell types. All three NOS iso- forms have been identified in the corpus caver- nosum, with nNOS and eNOS being considered responsible for initiating and sustaining erection, respectively (5). ibju \| Identification of mechanisms involved in the relaxation of rabbit cavernous smooth muscle Down-regulation of nNOS expres- sion has been found in the corpus cavernosum of aging rats (6), a model in which corpus cavernous smooth muscle relaxation is impaired (7). The aim of this study was to evaluated the relaxation in vitro of cavernous smooth muscle induced by a new NO donor of the complex nitro- syl-ruthenium, named trans-[Ru(NH3)4(caffeine) (NO)]C13 (Rut-Caf) and sodium nitroprusside (SNP). INTRODUCTION tion induced by adrenergic excitation to maintain penile flaccidity (1). Nitric oxide (NO), the endo- thelium-derived relaxation factor discovered by Palmer and Moncada, is the main inhibitory neu- Human corpus cavernosum contains vas- cular smooth muscle, kept under tonic contrac- 687 ibju \| Identification of mechanisms involved in the relaxation of rabbit cavernous smooth muscle ibju \| Identification of mechanisms involved in the relaxation of rabbit cavernous smooth muscle transducer, while the other end was attached to a mobile unit that allowed tension adjustment. Experiment 8: The RbCC strips were frozen in liquid nitrogen for dosage of cGMP and cAMP. The method of non-acetylation was utilized and samples were separated for dosage of proteins ac- cording to the Bradford method (1976) (15). Tri- chloroacetic acid (TCA) was added to the tissue macerate, resulting in a final TCA concentration of 10%. After centrifugation, the supernatant was washed with water saturated diethylic ether. This process was repeated six times. After the wash- ings, the samples were dried in a nitrogen atmo- sphere at 60ºC, and then suspended again in the assay buffer of the immunoenzymatic kit for dos- age of cGMP and cAMP. The tissues rested for one hour. Tension was calibrated and the solution renewed every 15 minutes. Alterations on tension were registered on a polygraph (Gemini 7070, Ugo-Basile, Italy). Eight different experimental protocols were per- formed, as outlined next. Experiment 1: After pre-contraction with phenilephrine (1 µM), rising concentrations (10-12 - 10-3 M) of SNP (as a control) and Rut-Caf were administered to the baths and relaxation concen- tration-response curves were obtained. Experiment 2: To evaluate the NO libera- tion profile for Rut-Caf, oxyhemoglobin (10 µM) was added to the baths, 30 minutes before pre- contraction with phenilephrine (1 µM). The relaxant effect of the substances was measured from the maximal contraction plateau induced by phenilephrine, and expressed as per- centages of contraction diminishment. The maxi- mal effect (Emax) was considered as the maximal amplitude response induced by the relaxant agents on the concentration-response curves. The drug concentrations that induced 50% maximal relax- ation (pEC50) were determined after logarithmic transformation of concentration-response normal curves, and expressed as the negative logarithmic of values for each tissue (pEC0). Percentages of contraction diminishment superior to 50% were considered significant. Experiment 3: To evaluate possible anion nitroxyl (NO-) liberation by Rut-Caf during the re- laxation process, L-cysteine (100 µM), a specific NO- remover was added to the baths, 30 minutes before pre-contraction with phenilephrine (1 µM). Experiment 4: To evaluate the contribu- tion of metabolically activated potassium chan- nels (KATP) on the relaxation process, one KATP blocker - glibenclamide (1 µM) - was added to the baths, 30 minutes before pre-contraction with phenilephrine (1 µM). ibju \| Identification of mechanisms involved in the relaxation of rabbit cavernous smooth muscle i The data were expressed as averages ± stan- dard deviation. Statistical analysis was performed with ANOVA, followed by the Tukey-Kramer test. Values of p < 0.05 were considered significant. Experiment 5: To evaluate the contribu- tion of high, medium and low conductivity calci- um-activated potassium channels (KCA) on the re- laxation, two KCA blockers - iberotoxin (1 µM) and apamine (0.1 µM) - were added to the baths, 30 minutes before pre-contraction with phenileph- rine (1 µM). MATERIALS AND METHODS The study was approved by the local Com- mittee of Ethics on Animal Use in Research. Adult male New Zealand rabbits, weight 2 - 3 kg, were used. After anesthesia, the penis was removed entirely, and placed in Krebs-Henseleit solution. Cavernous tissue was dissected free of connective tissue and albuginea, providing two corpus caver- nosum strips (1 cm) from each penis. Endothelial dysfunction is present in a large group of patients with erectile dysfunction (ED) and co-morbidities like hypertension and diabetes. This syndrome is characterized by an endogenous NO production deficiency (8). About 56% of patients in this group show resistance to current ED treatment with phosphodiesterase 5 (PDE-5) inhibitors (9). Research for new drugs that increase bioavailability of endogenous NO is a permanent challenge. These strips were mounted in isolated baths (10 mL) containing Krebs-Henseleit solution. This solution contains Na, K, Cl, Ca, MgSO4, HCO3, PO4, glucose, albumin, and tromethamine (THAM) and was used to maintain tissues during experi- ments (14). The strips were aired with a mixture of O2 (95%) and CO2 (5%), pH 7.4, 37°C. Tissues were placed vertically, with tension of 1g. One extremity was connected to an isometric power Recently, new NO donor compounds with higher stability and less toxicity have been sub- ject of research. One group of such substances, S-nitroso-glutathione (GSNO) and S-nitroso-N- 688 ibju \| Identification of mechanisms involved in the relaxation of rabbit cavernous smooth muscle ibju \| Identification of mechanisms involved in the relaxation of rabbit cavernous smooth muscle 0.915). The Emax value for Rut-Caf was 77% and for Rut-Caf + L-cysteine was 80% (Figure-1). 0.915). The Emax value for Rut-Caf was 77% and for Rut-Caf + L-cysteine was 80% (Figure-1). 0.915). The Emax value for Rut-Caf was 77% and for Rut-Caf + L-cysteine was 80% (Figure-1). mg. Rut-Caf generated 143.52 pmoL/mg (p < 0.01) and SNP 43.50 pmoL/mg (p < 0.05) (Figure-4). Finally, Rut-Caf (100 µM) produced cAMP in the corpus cavernosum in quantity similar to that of forskolin (10 µM) and significantly supe- rior to the control amount (p < 0.001) (Figure-5). mg. Rut-Caf generated 143.52 pmoL/mg (p < 0.01) and SNP 43.50 pmoL/mg (p < 0.05) (Figure-4). Glibenclamide modified potency and effi- cacy of Rut-Caf, as reflected by pEC50 values for Rut-Caf of 5.2 ± 0.2 and Rut-Caf + glibenclamide of 7.3 ± 0.2 (p = 0.016). Emax value increased from 68 ± 8.9% to 92 ± 7% (p = 0.060). Glibenclamide modified potency and effi- cacy of Rut-Caf, as reflected by pEC50 values for Rut-Caf of 5.2 ± 0.2 and Rut-Caf + glibenclamide of 7.3 ± 0.2 (p = 0.016). Emax value increased from 68 ± 8.9% to 92 ± 7% (p = 0.060). Finally, Rut-Caf (100 µM) produced cAMP in the corpus cavernosum in quantity similar to that of forskolin (10 µM) and significantly supe- rior to the control amount (p < 0.001) (Figure-5). DISCUSSION Tissue incubation with ibero-apamin did not interfere with Rut-Caf relaxations. The pEC50 for Rut-Caf was 4.7 ± 0.2, compared to 4.8 ± 0.3 for Rut-Caf + ibero-apamin (p = 0.779) (Figure-2). Tissue incubation with ibero-apamin did not interfere with Rut-Caf relaxations. The pEC50 for Rut-Caf was 4.7 ± 0.2, compared to 4.8 ± 0.3 for Rut-Caf + ibero-apamin (p = 0.779) (Figure-2). Hydroxocobalamin almost abolished the relaxant effects of Rut-Caf. The pEC50 for Rut-Caf was 4.9 ± 0.1, while pEC50 for Rut-Caf + hydroxo- This study evaluated a new ruthenium compound and its capability to promote relax- ation of cavernous smooth muscle. Hydroxocobalamin almost abolished the relaxant effects of Rut-Caf. The pEC50 for Rut-Caf was 4.9 ± 0.1, while pEC50 for Rut-Caf + hydroxo- The substance Rut-Caf provoked sig- nificant relaxation of cavernous smooth muscle Figure 1 - Effect of Rut-Caf upon rabbit corpus cavernosum strips with and without pretreatment with 10 μM/L oxyhemo- globin, 1 mmoL/L L-cysteine or 0.1 mmoL/L hydroxocobalamin. Concentration/response curves were plotted for graded concentrations (10-12 to 10-3 M) of Rut-Caf. Results were expressed as average ± standard error based on seven experi- ments. The statistical significance was verified with ANOVA followed by Buonferroni’s test. cobalamin was 5.5 ± 0.2 (p = 0.713). As to Emax, values were respectively 85.3 ± 9.4% and 21.3 ± 2.2% (p < 0.001). The relaxation induced by Rut-Caf was sig- nificantly inhibited by ODQ. This is evidenced by the Emax values for Rut-Caf - 58 ± 9.2% – and Rut-Caf + ODQ - 34.6 ± 3.7% (p = 0.038) (Figure-3). Both SNP and Rut-Caf produced cGMP in the cavernous tissue in amounts significantly higher than the basal control value of 8.26 pmoL/ (Emax = 80%) with potency similar to SNP; how- ever, with lower maximum effect. The most prob- able hypothesis is that SNP acts on relaxation of vascular smooth muscle activating the sGC and as a hyperpolarizing agent through direct activa- tion of potassium ion channels and Rut-Caf acts mainly on activation of sGC (16). The soluble isoform sGC plays a pivotal role in erectile function because it provides the link between NO and cGMP, which represent the p<0.05 vs. Rut-Caf without oxyhemoblin pretreatment. p<0.05 vs. Rut-Caf without hydroxocobalamin pretreatment. p<0.05 vs. Rut-Caf without oxyhemoblin pretreatment. p<0.05 vs. Rut-Caf without hydroxocobalamin pretreatment. p<0.05 vs. Rut-Caf without oxyhemoblin pretreatment. p<0.05 vs. Rut-Caf without hydroxocobalamin pretreatment. p<0.05 vs. RESULTS Experiment 6: To evaluate the way NO is involved in the relaxation, hydroxocobalamin (0.1 mM), a NO remover, was added to the baths, 30 minutes before pre-contraction with phenileph- rine (1 µM). The SNP Emax value was 100%, and its pEC50 was 6.9 ± 0.2. For Rut-Caf, Emax value was 72.6 ± 6.6%, and pEC50 was 6.8 ± 0.2. There was no significant difference in potency between the two substances (p = 0.851). Experiment 7: To determine the activa- tion of sGC by Rut-Caf, the agent 1H-[1,2,4] oxadiazole[4,3-α]quinoxalin-1-one (ODQ) - a sGC blocker - was added to the baths, 30 minutes be- fore pre-contraction with phenilephrine (1 µM). The relaxation curve induced by Rut-Caf was dislocated below with oxyhemoglobin, while the maximal response for Rut-Caf, in this setting, was 49.8 ± 6.5%. Incubation of tissues with L-cysteine did not provoke any relaxation alterations. The values of pEC50 for Rut-Caf and Rut-Caf + L-cysteine were respectively 6.8 ± 0.2 and 6.9 ± 0.2 (p = In all the experiments, relaxation concen- tration-response curves were obtained according to rising concentrations (10-12 - 10-3 M) of Rut-Caf. 689 DISCUSSION Rut-Caf without oxyhemoblin pretreatment. p<0.05 vs. Rut-Caf without hydroxocobalamin pretreatment. cobalamin was 5.5 ± 0.2 (p = 0.713). As to Emax, values were respectively 85.3 ± 9.4% and 21.3 ± 2.2% (p < 0.001). (Emax = 80%) with potency similar to SNP; how- ever, with lower maximum effect. The most prob- able hypothesis is that SNP acts on relaxation of vascular smooth muscle activating the sGC and as a hyperpolarizing agent through direct activa- tion of potassium ion channels and Rut-Caf acts mainly on activation of sGC (16). The relaxation induced by Rut-Caf was sig- nificantly inhibited by ODQ. This is evidenced by the Emax values for Rut-Caf - 58 ± 9.2% – and Rut-Caf + ODQ - 34.6 ± 3.7% (p = 0.038) (Figure-3). p g Both SNP and Rut-Caf produced cGMP in the cavernous tissue in amounts significantly higher than the basal control value of 8.26 pmoL/ The soluble isoform sGC plays a pivotal role in erectile function because it provides the link between NO and cGMP, which represent the 690 ibju \| Identification of mechanisms involved in the relaxation of rabbit cavernous smooth muscle Figure 2 - A. Relaxation induced by Rut-Caf in rabbit corpus cavernosum strips pre-contracted with 1 μM phenylephrine with and without glibenclamide pretreatment. Results were expressed as average ± standard error based on seven experiments. B. Relaxation induced by Rut-Caf in rabbit corpus cavernosum strips pre-contracted with 1 μM phenylephrine with and without iberiotoxin/apamin pretreatment. Results were expressed as average ± standard error based on seven experiments. a b a a b b b extracellular and intracellular signaling mole- cules, respectively, in physiologic erection (17). ing its maximum effect, due to intracellular re- lease of NO. Bonaventura et al., studying substances that also belong to the nitrosyl-ruthenium com- plex, in rabbit aorta rings, demonstrated a maxi- mal relaxation effect of 102% with pEC50 of 6.61 ± 0.09. These data confirm, as showed in this study, that ruthenium compounds are powerful vasodilators (13). In this in vitro study, the addition of oxy- hemoglobin to the organ baths reduced the maxi- mum effect of Rut-Caf, but could not abolish it. Therefore, the relaxant effect of this substance is also probably due to the intracellular release of NO (13). DISCUSSION Figure 3 - Concentration/response curves showing relaxation induced by Rut-Caf in rabbit corpus cavernosum strips with and without pre-treatment with 10 μM ODQ or 30 μM ODQ. Results were expressed as average ± standard error based on seven experiments. Figure 4 - cGMP dosage of rabbit corpus cavernosum strips exposed to saline solution (control), 100 μM Rut-Caf or 3 µM SNP (positive control). Figure 5 - cAMP dosage of rabbit corpus cavernosum strips exposed to saline solution (control), 100 μM Rut-Caf or 10 µM forscolin (positive control). p < 0.05 vs. Rut-Caf with 10 μM ODQ pretreatment p < 0.01 vs. Rut-Caf with 30 μM ODQ pretreatment seven experiments. p < 0.05 vs. Rut-Caf with 10 μM ODQ pretreatment p < 0.01 vs. Rut-Caf with 30 μM ODQ pretreatment p < 0.05 vs. Rut-Caf with 10 μM ODQ pretreatment p < 0.01 vs. Rut-Caf with 30 μM ODQ pretreatment p < 0.05 vs. Rut-Caf with 10 μM ODQ pretreatment p < 0.01 vs. Rut-Caf with 30 μM ODQ pretreatment Figure 4 - cGMP dosage of rabbit corpus cavernosum strips exposed to saline solution (control), 100 μM Rut-Caf or 3 µM SNP (positive control). Figure 4 - cGMP dosage of rabbit corpus cavernosum strips exposed to saline solution (control), 100 μM Rut-Caf or 3 µM SNP (positive control). Figure 5 - cAMP dosage of rabbit corpus cavernosum strips exposed to saline solution (control), 100 μM Rut-Caf or 10 µM forscolin (positive control). Figure 5 - cAMP dosage of rabbit corpus cavernosum strips exposed to saline solution (control), 100 μM Rut-Caf or 10 µM forscolin (positive control). mover, hydroxocobalamin, to the baths signifi- cantly decreased the relaxation induced by Rut- Caf. In contrast with Bonaventura et al. findings, Rut-Caf does not release the nitroxyl anion dur- ing relaxation. In consequence, probably its re- laxation effect is related to release of the NO free radical. laxation response provoked by Rut-Caf. This dem- onstrates the action of the substance studied on the NO-cGMP cell signaling system. According to the studies with compounds of the group nitrosyl-ruthenium, it was observed that these substances act by means of donation of free NO and nitroxyl anion (NO-) (13). Utiliz- ing a specific nitroxyl anion remover, L-cysteine, the potential of relaxation remained unchanged. However, the addition of the NO free radical re- Smooth muscle has neither a T-tubule system nor a well-developed sarcoplasmatic re- ticulum. DISCUSSION McDonald and Murad demonstrated that NO acts on the relaxation of vascular smooth muscle through activation of soluble guanylate cyclase, increasing the synthesis and bioavailabil- ity of intracellular cGMP (18). In this study, tis- sue incubation with a specific inhibitor of soluble guanylate cyclase (ODQ; 10 µM) abolished the re- In vivo, NO arranges with hemoglobin creating a stable iron-nitrosyl complex that does not release NO. Bonaventura et al., evaluating the effects of ruthenium compounds in rabbit aorta rings in the presence of oxyhemoglobin, observed a decreased potency without significantly modif- 691 ibju \| Identification of mechanisms involved in the relaxation of rabbit cavernous smooth muscle laxation response provoked by Rut-Caf. This dem- onstrates the action of the substance studied on the NO-cGMP cell signaling system. According to the studies with compounds of the group nitrosyl-ruthenium, it was observed that these substances act by means of donation of free NO and nitroxyl anion (NO-) (13). Utiliz- ing a specific nitroxyl anion remover, L-cysteine, the potential of relaxation remained unchanged. mover, hydroxocobalamin, to the baths signifi- cantly decreased the relaxation induced by Rut- Caf. In contrast with Bonaventura et al. findings, Rut-Caf does not release the nitroxyl anion dur- ing relaxation. In consequence, probably its re- laxation effect is related to release of the NO free radical. Smooth muscle has neither a T-tubule system nor a well-developed sarcoplasmatic re- Figure 3 - Concentration/response curves showing relaxation induced by Rut-Caf in rabbit corpus cavernosum strips with and without pre-treatment with 10 μM ODQ or 30 μM ODQ. Results were expressed as average ± standard error based on seven experiments. Figure 4 - cGMP dosage of rabbit corpus cavernosum strips exposed to saline solution (control), 100 μM Rut-Caf or 3 µM SNP (positive control). Figure 5 - cAMP dosage of rabbit corpus cavernosum strips exposed to saline solution (control), 100 μM Rut-Caf or 10 µM forscolin (positive control). p < 0.05 vs. Rut-Caf with 10 μM ODQ pretreatment p < 0.01 vs. Rut-Caf with 30 μM ODQ pretreatment ibju \| Identification of mechanisms involved in the relaxation of rabbit cavernous smooth muscle Figure 3 - Concentration/response curves showing relaxation induced by Rut-Caf in rabbit corpus cavernosum strips with and without pre-treatment with 10 μM ODQ or 30 μM ODQ. Results were expressed as average ± standard error based on seven experiments. ibju \| Identification of mechanisms involved in the relaxation of rabbit cavernous smooth muscle ibju \| Identification of mechanisms involved in the relaxation of rabbit cavernous smooth muscle relaxation process, was demonstrated in studies (10,21). important role, and calcium must enter the cyto- plasm through the plasma membrane during an action potential. Similar to these studies, it was demon- strated that the production of cGMP induced by Rut-Caf in the relaxation of cavernous smooth muscle was significantly higher than that induced by saline solution and like that produced by SNP. A direct hyperpolarizing action of the Rut- Caf was not observed. The addition of calcium ac- tivated potassium ion channels of high, medium and low conductivity inhibitors did not modify the relaxation effect of Rut-Caf. When glibenclamide, which is an ATP dependent potassium channel blocker, was added to the baths, an increase on the efficacy of Rut-Caf was observed (pEC50 4.04 x 7.69, p < 0.05). It was also observed production of cAMP induced by Rut-Caf during the relaxation pro- cess. Uckert et al. demonstrated the interaction of cGMP-cAMP cell signaling systems in the re- laxation of cavernous smooth muscle (22). These authors demonstrated the presence of cAMP-spe- cific phosphodiesterases, PDE-3 and PDE-5 in the cavernous endothelium. They also noted that the cAMP specific kinase PKA inhibited the relaxation induced by PDE-5 inhibitors, specific to cGMP, characterizing the integration of the cGMP-cAMP cell signaling systems on the cavernous smooth muscle relaxation. Although the mechanisms for that interaction are not clear, the authors have proposed that the increase in cAMP would be sec- ondary to the drop in the level of cGMP, which would diminish the activity of PDE-3, increasing the level of cAMP. So, the regulation of cAMP- specific phosphodiesterases by cGMP could ex- plain the increase of cAMP observed in this study, as a direct effect of Rut-Caf in the relaxation of cavernous smooth muscle. The results obtained in this study were similar to the findings of Lee and Kang in human corpus cavernosum (19). These researchers, study- ing the effect of relaxation of a NO donor - SIN- 1, could observe an increase in the probability of opening of calcium activated potassium channels in an indirect way, through activation of soluble guanylate cyclase and activation of the cGMP protein kinase. However, it was not demonstrated direct action of SIN-1 on the ion channels studied. The best explanation for the efficacy of Rut-Caf would be the action of glibenclamide as a reducing agent. ibju \| Identification of mechanisms involved in the relaxation of rabbit cavernous smooth muscle Bates et al. showed that the bioactivation of NO in biological medium needs the presence of a reducing agent. In consequence, glibenclamide acting as a reducing agent increase the efficacy of Rut-Caf (3). This hypothesis was supported by the spectroscopic profile. The product formed (UV- Vis) is compatible with the formation of the spe- cies Rut-Caf-H2O. Initially, there is a reduction of NO0 by glibenclamide with posterior liberation of the metal coordination sphere. In conclusion, this suggests that amplification in the presence of glib- enclamide occurs due to reduction of NO+ present in the metal coordination sphere, increasing the concentration of NO0 in the reactive environment. Lindaman et al. studied the gallbladder smooth muscle in vitro. They noted that caffeine acting as an unspecific phosphodiesterases inhibi- tor 1 to 5, increased the production of cGMP and cAMP in the relaxation process (23). Similarly, the presence of caffeine in the structure of Rut-Caf could contribute to the increase of cAMP pro- duced by that substance. It should be emphasized that the results of this study were obtained in in vitro experimental model and future studies in vivo and clinical stud- ies are necessary in order to confirm them. Although it is known that ion channels ef- fectively act on the control of the basal tone of the cavernous smooth muscle (1), they do not partici- pate in the relaxation process induced by Rut-Caf. DISCUSSION Therefore extracellular calcium plays an 692 REFERENCES 15. Bradford MM: A rapid and sensitive method for the quantita- tion of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976; 72: 248-54. 1. Andersson KE: Pharmacology of penile erection. Pharmacol Rev. 2001; 53: 417-50. 1. Andersson KE: Pharmacology of penile erection. Pharmacol Rev. 2001; 53: 417-50. 2. Lohse MJ, Förstermann U, Schmidt HH: Pharmacology of NO:cGMP signal transduction. Naunyn Schmiedebergs Arch Pharmacol. 1998; 358: 111-2. 2. Lohse MJ, Förstermann U, Schmidt HH: Pharmacology of NO:cGMP signal transduction. Naunyn Schmiedebergs Arch Pharmacol. 1998; 358: 111-2. 16. Bolotina VM, Najibi S, Palacino JJ, Pagano PJ, Cohen RA: Nitric oxide directly activates calcium-dependent potassium channels in vascular smooth muscle. Nature. 1994; 368: 850-3. 3. Bates JN, Baker MT, Guerra R Jr, Harrison DG: Nitric oxide generation from nitroprusside by vascular tissue. Evidence that reduction of the nitroprusside anion and cyanide loss are required. Biochem Pharmacol. 1991; 42 (Suppl:) S157- 65. 3. Bates JN, Baker MT, Guerra R Jr, Harrison DG: Nitric oxide generation from nitroprusside by vascular tissue. Evidence that reduction of the nitroprusside anion and cyanide loss are required. Biochem Pharmacol. 1991; 42 (Suppl:) S157- 65. 17. Andersson KE: Neurophysiology/pharmacology of erection. Int J Impot Res. 2001; 13 (Suppl 3): S8-S17. 18. McDonald LJ, Murad F: Nitric oxide and cyclic GMP signal- ing. Proc Soc Exp Biol Med. 1996; 211: 1-6. 4. Ignarro LJ: The unique role of nitric oxide as a signaling molecule in the cardiovascular system. Ital Heart J. 2000; 1 (Suppl 3): S28-9. 4. Ignarro LJ: The unique role of nitric oxide as a signaling molecule in the cardiovascular system. Ital Heart J. 2000; 1 (Suppl 3): S28-9. 19. Lee SW, Kang TM: Effects of nitric oxide on the Ca2+-activat- ed potassium channels in smooth muscle cells of the human corpus cavernosum. Urol Res. 2001; 29: 359-65. 5. Hurt KJ, Musicki B, Palese MA, Crone JK, Becker RE, Moriar- ity JL, et al.: Akt-dependent phosphorylation of endothelial nitric-oxide synthase mediates penile erection. Proc Natl Acad Sci U S A. 2002; 99: 4061-6. 20. Arnold WP, Mittal CK, Katsuki S, Murad F: Nitric oxide acti- vates guanylate cyclase and increases guanosine 3’:5’-cyclic monophosphate levels in various tissue preparations. Proc Natl Acad Sci U S A. 1977; 74: 3203-7. 6. Carrier S, Nagaraju P, Morgan DM, Baba K, Nunes L, Lue TF: Age decreases nitric oxide synthase-containing nerve fibers in the rat penis. J Urol. ibju \| Identification of mechanisms involved in the relaxation of rabbit cavernous smooth muscle ibju \| Identification of mechanisms involved in the relaxation of rabbit cavernous smooth muscle in the intra-cellular environment. Rut-Caf releases the NO free radical and it does not act directly on the potassium ion channels. 12. Shamloul R, Atteya A, Elnashaar A, Gadallah A, Zohdy W, Abdelsalam W: Intracavernous sodium nitroprusside (SNP) versus papaverine/phentolamine in erectile dysfunction: a comparative study of short-term efficacy and side-effects. J Sex Med. 2005; 2: 117-20. 13. Bonaventura D, de Lima RG, Vercesi JA, da Silva RS, Bend- hack LM: Comparison of the mechanisms underlying the relaxation induced by two nitric oxide donors: sodium nitro- prusside and a new ruthenium complex. Vascul Pharmacol. 2007; 46: 215-22. CONFLICT OF INTEREST None declared. 14. Palea S, Barras M: Comparison of the relaxant effects of alfu- zosin, phentolamine and sildenafil on rabbit isolated corpus cavernosum. BJU Int. 2003; 91: 873-7. CONCLUSIONS The production of intracellular cGMP from activation of sGC by NO in different tissues was initially demonstrated by Arnold et al. (20). The production of cGMP in corpus cavernosum of animals and humans by NO donors, during the In conclusion, Rut-Caf causes relaxation of smooth muscle of corpus cavernosum by means of activation of sGC with intracellular production of cGMP and cAMP; and also by release of NO 693 Dr. João Batista Gadelha de Cerqueira Rua Paula Ney, 599 /302, Aldeota Fortaleza, Ceará, 60140-200, Brazil E-mail: joaogadelhac@gmail.com REFERENCES 1997; 157: 1088-92. 21. Kim NN, Huang YH, Goldstein I, Bischoff E, Traish AM: In- hibition of cyclic GMP hydrolysis in human corpus caver- nosum smooth muscle cells by vardenafil, a novel, selec- tive phosphodiesterase type 5 inhibitor. Life Sci. 2001; 69: 2249-56. 7. Cartledge J, Minhas S, Eardley I: The role of nitric oxide in penile erection. Expert Opin Pharmacother. 2001; 2: 95-107. 8. Feldman HA, Goldstein I, Hatzichristou DG, Krane RJ, McKinlay JB: Impotence and its medical and psychosocial correlates: results of the Massachusetts Male Aging Study. J Urol. 1994; 151: 54-61.i 22. Uckert S, Hedlund P, Waldkirch E, Sohn M, Jonas U, Anders- son KE, et al.: Interactions between cGMP- and cAMP-path- ways are involved in the regulation of penile smooth muscle tone. World J Urol. 2004; 22: 261-6. 9. Rendell MS, Rajfer J, Wicker PA, Smith MD: Sildenafil for treatment of erectile dysfunction in men with diabetes: a randomized controlled trial. Sildenafil Diabetes Study Group. JAMA. 1999; 281: 421-6. 23. Lindaman BA, Hinkhouse MM, Conklin JL, Cullen JJ: The ef- fect of phosphodiesterase inhibition on gallbladder motility in vitro. J Surg Res. 2002; 105: 102-8. 10. Seidler M, Uckert S, Waldkirch E, Stief CG, Oelke M, Tsikas D, et al.: In vitro effects of a novel class of nitric oxide (NO) donating compounds on isolated human erectile tissue. Eur Urol. 2002; 42: 523-8. 11. Trigo-Rocha F, Martinez-Piñeiro L, Donatucci CF, Hsu GL, Lue TF, Tanagho EA: Sodium nitroprusside: physiologic ef- fects as a nitric oxide donor in three species. Int J Impot Res. 1995; 7: 49-56. Dr. João Batista Gadelha de Cerqueira Rua Paula Ney, 599 /302, Aldeota Fortaleza, Ceará, 60140-200, Brazil E-mail: joaogadelhac@gmail.com 694
https://openalex.org/W4362424458	https://aacr.figshare.com/articles/journal_contribution/Supplementary_Data_from_Neuroblastoma_Formation_Requires_Unconventional_CD4_T_Cells_and_Arginase-1_Dependent_Myeloid_Cells/22429705/1/files/39876037.pdf	Latin	null	Supplementary Data from Neuroblastoma Formation Requires Unconventional CD4 T Cells and Arginase-1–Dependent Myeloid Cells	null	2,023	cc-by	4,472	Met Ala Arg Asp Ile Ser Arg intron Met Ala Arg Asp Ile Tyr Arg intron * * * HDR changes to block Cas9 WT ALK ALKY1282S ALK1178L TTGATTACGCCCTCTGCCCTGTAGCAAACTTAACCACCGGAACATAGTCCGATGCATTGGTGTGAGTCTACAAGCCCTGCCCCGCTTCATCCTGCTGGAACTCATGGCTGGCGGAGACC intron 22-23 K L N H Q I N I V L R C V G S Q L A P I L L L M E R F Nsi I a b GCTAATTCAACTCCTGCCAAGCCTGCCCAGTCTATGCATCGCCCCAGGAAGGTTGACCTCAGGTTCAGCCAGAGACCCCTCCACCCATATCCACAGAATATAGCTTCACAGCGTGA 380 390 400 410 420 430 440 450 460 470 480 490 GGCTCAGTTAAATTTGATTACGCCCTCTGCCCTGTAGCAAACTTAACCACCAGAACATAGTCCGATGCATTGGTGTGAGTCTACAAGCCCTGCCCCGCTTCATCCTGCTGGAAC 40 550 560 570 580 590 600 610 620 630 640 650 GCCCGGTGAGTGAGAGGCAGTCTTCTCTCCAGGGTTCATGTCAGCGGGAGCGGGTTGCAAGGGAGGTAACTGTGCTTGGTGGATTCCTGGGAGTCGGCCCTGTGGGCTCCT 0.0 0.5 1.0 1.5 2.0 Cd3e 0.0 0.5 1.0 1.5 2.0 2.5 Cd8a 0.0 0.5 1.0 1.5 Cd4 0.0 0.5 1.0 1.5 2.0 Foxp3 0 1 2 3 4 Gzma 0 1 2 3 Gzmb 0.0 0.5 1.0 1.5 2.0 Prf1 0 1 2 3 Vcam1 0 1 2 3 4 Hmox1 0 1 2 3 4 Lgals3 0 1 2 3 4 Apoc1 0 1 2 Ifngr1 0 1 2 Mycn 0 1 2 Ncam1 0.0 0.5 1.0 1.5 2.0 Rtn4 0 1 2 3 Th 0 1 2 3 4 Cd14 0.0 0.5 1.0 1.5 2.0 2.5 Ccr2 0 1 2 3 Cd209a 0.0 0.5 1.0 1.5 2.0 Itgax 0.0 0.5 1.0 1.5 2.0 Batf3 0 1 2 3 4 5 Lyz2 0 1 2 3 Cd68 0 1 2 3 Il12b 0 1 2 3 4 Ccl22 0.0 0.5 1.0 1.5 Flt3 0.0 0.5 1.0 1.5 2.0 Relb 0.0 0.5 1.0 1.5 Etv3 tSNE_1 tSNE_2 3 6 0 5 8 15 11 12 17 13 1 2 4 14 16 22 10 9 18 21 7 20 23 19 −25 0 25 −40 −20 0 20 40 tSNE_1 tSNE_2 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 0 1 2 Ncam1 0 1 2 3 Th 0 1 2 Mycn 0.0 0.5 1.0 1.5 2.0 2.5 Tubb2a 0. 0. 1. 1. 2. Ddc 0 1 2 3 Dbh 0 1 2 Cadm1 0 1 2 3 Fscn1 0.0 0.5 1.0 1.5 2.0 Pfn2 0 1 2 Fdps 0.0 0.5 1.0 1.5 2.0 Rab3b 0 1 2 3 4 Cd9 0.0 0.5 1.0 1.5 2.0 Tagln3 0 1 2 Dpysl3 0 1 2 Ptms 0 1 2 3 4 Stmn2 0.0 0.5 1.0 1.5 Fxyd6 0 1 2 3 4 Tuba1a 0 1 2 3 Uchl1 0 1 2 3 Tubb3 0.0 0.4 0.8 1.2 1.6 Pirt 0.0 0.4 0.8 1.2 1.6 Tmeff2 0.0 0.5 1.0 1.5 Dst 0 1 2 3 Stmn4 0 1 2 Hmgb3 tSNE_1 tSNE_2 d e c 0 20 40 B cells CD4 CD8 NK cells Treg Frequency of non-tumor cells 0 4 8 12 Macrophages Monocytes Inflammatory Monocytes Conventional Monocytes Neutrophils CellType Tumor Alk−1 Alk−2 Alk−3 f Met Ala Arg Asp Ile Ser Arg intron Met Ala Arg Asp Ile Tyr Arg intron * * HDR changes to block Cas9 WT ALK ALKY1282S ALK1178L TTGATTACGCCCTCTGCCCTGTAGCAAACTTAACCACCGGAACATAGTCCGATGCATTGGTGTGAGTCTACAAGCCCTGCCCCGCTTCATCCTGCTGGAACTCATGGCTGGCGGAGACC intron 22-23 K L N H Q I N I V L R C V G S Q L A P I L L L M E R F Nsi I a b GCTAATTCAACTCCTGCCAAGCCTGCCCAGTCTATGCATCGCCCCAGGAAGGTTGACCTCAGGTTCAGCCAGAGACCCCTCCACCCATATCCACAGAATATAGCTTCACAGCGTGA 380 390 400 410 420 430 440 450 460 470 480 490 GGCTCAGTTAAATTTGATTACGCCCTCTGCCCTGTAGCAAACTTAACCACCAGAACATAGTCCGATGCATTGGTGTGAGTCTACAAGCCCTGCCCCGCTTCATCCTGCTGGAAC 40 550 560 570 580 590 600 610 620 630 640 650 GCCCGGTGAGTGAGAGGCAGTCTTCTCTCCAGGGTTCATGTCAGCGGGAGCGGGTTGCAAGGGAGGTAACTGTGCTTGGTGGATTCCTGGGAGTCGGCCCTGTGGGCTCCT 0.0 0.5 1.0 1.5 2.0 Cd3e 0.0 0.5 1.0 1.5 2.0 2.5 Cd8a 0.0 0.5 1.0 1.5 Cd4 0.0 0.5 1.0 1.5 2.0 Foxp3 0 1 2 3 4 Gzma 0 1 2 3 Gzmb 0.0 0.5 1.0 1.5 2.0 Prf1 0 1 2 3 Vcam1 0 1 2 3 4 Hmox1 0 1 2 3 4 Lgals3 0 1 2 3 4 Apoc1 0 1 2 Ifngr1 0 1 2 Mycn 0 1 2 Ncam1 0.0 0.5 1.0 1.5 2.0 Rtn4 0 1 2 3 Th 0 1 2 3 4 Cd14 0.0 0.5 1.0 1.5 2.0 2.5 Ccr2 0 1 2 3 Cd209a 0.0 0.5 1.0 1.5 2.0 Itgax 0.0 0.5 1.0 1.5 2.0 Batf3 0 1 2 3 4 5 Lyz2 0 1 2 3 Cd68 0 1 2 3 Il12b 0 1 2 3 4 Ccl22 0.0 0.5 1.0 1.5 Flt3 0.0 0.5 1.0 1.5 2.0 Relb 0.0 0.5 1.0 1.5 Etv3 tSNE_1 tSNE_2 3 6 0 5 8 15 11 12 17 13 1 2 4 14 16 22 10 9 18 21 7 20 23 19 −25 0 25 −40 −20 0 20 40 tSNE_1 tSNE_2 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 0 1 2 Ncam1 0 1 2 3 Th 0 1 2 Mycn 0.0 0.5 1.0 1.5 2.0 2.5 Tubb2a 0. 0. 1. 1. 2. Supplementary Figure S3 Met Ala Arg Asp Ile Ser Arg intron Met Ala Arg Asp Ile Tyr Arg intron * * HDR changes to block Cas9 WT ALK ALKY1282S ALK1178L TTGATTACGCCCTCTGCCCTGTAGCAAACTTAACCACCGGAACATAGTCCGATGCATTGGTGTGAGTCTACAAGCCCTGCCCCGCTTCATCCTGCTGGAACTCATGGCTGGCGGAGACC intron 22-23 K L N H Q I N I V L R C V G S Q L A P I L L L M E R F Nsi I a b GCTAATTCAACTCCTGCCAAGCCTGCCCAGTCTATGCATCGCCCCAGGAAGGTTGACCTCAGGTTCAGCCAGAGACCCCTCCACCCATATCCACAGAATATAGCTTCACAGCGTGA 380 390 400 410 420 430 440 450 460 470 480 490 GGCTCAGTTAAATTTGATTACGCCCTCTGCCCTGTAGCAAACTTAACCACCAGAACATAGTCCGATGCATTGGTGTGAGTCTACAAGCCCTGCCCCGCTTCATCCTGCTGGAAC 40 550 560 570 580 590 600 610 620 630 640 650 GCCCGGTGAGTGAGAGGCAGTCTTCTCTCCAGGGTTCATGTCAGCGGGAGCGGGTTGCAAGGGAGGTAACTGTGCTTGGTGGATTCCTGGGAGTCGGCCCTGTGGGCTCCT 0.0 0.5 1.0 1.5 2.0 Cd3e 0.0 0.5 1.0 1.5 2.0 2.5 Cd8a 0.0 0.5 1.0 1.5 Cd4 0.0 0.5 1.0 1.5 2.0 Foxp3 0 1 2 3 4 Gzma 0 1 2 3 Gzmb 0.0 0.5 1.0 1.5 2.0 Prf1 0 1 2 3 Vcam1 0 1 2 3 4 Hmox1 0 1 2 3 4 Lgals3 0 1 2 3 4 Apoc1 0 1 2 Ifngr1 0 1 2 Mycn 0 1 2 Ncam1 0.0 0.5 1.0 1.5 2.0 Rtn4 0 1 2 3 Th 0 1 2 3 4 Cd14 0.0 0.5 1.0 1.5 2.0 2.5 Ccr2 0 1 2 3 Cd209a 0.0 0.5 1.0 1.5 2.0 Itgax 0.0 0.5 1.0 1.5 2.0 Batf3 0 1 2 3 4 5 Lyz2 0 1 2 3 Cd68 0 1 2 3 Il12b 0 1 2 3 4 Ccl22 0.0 0.5 1.0 1.5 Flt3 0.0 0.5 1.0 1.5 2.0 Relb 0.0 0.5 1.0 1.5 Etv3 tSNE_1 tSNE_2 3 6 0 5 8 15 11 12 17 13 1 2 4 14 16 22 10 9 18 21 7 20 23 19 −25 0 25 −40 −20 0 20 40 tSNE_1 tSNE_2 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 0 1 2 Ncam1 0 1 2 3 Th 0 1 2 Mycn 0.0 0.5 1.0 1.5 2.0 2.5 Tubb2a 0. 0. 1. 1. 2. Ddc 0 1 2 3 Dbh 0 1 2 Cadm1 0 1 2 3 Fscn1 0.0 0.5 1.0 1.5 2.0 Pfn2 0 1 2 Fdps 0.0 0.5 1.0 1.5 2.0 Rab3b 0 1 2 3 4 Cd9 0.0 0.5 1.0 1.5 2.0 Tagln3 0 1 2 Dpysl3 0 1 2 Ptms 0 1 2 3 4 Stmn2 0.0 0.5 1.0 1.5 Fxyd6 0 1 2 3 4 Tuba1a 0 1 2 3 Uchl1 0 1 2 3 Tubb3 0.0 0.4 0.8 1.2 1.6 Pirt 0.0 0.4 0.8 1.2 1.6 Tmeff2 0.0 0.5 1.0 1.5 Dst 0 1 2 3 Stmn4 0 1 2 Hmgb3 tSNE_1 tSNE_2 d e c 0 20 40 B cells CD4 CD8 NK cells Treg Frequency of non-tumor cells 0 4 8 12 Macrophages Monocytes Inflammatory Monocytes Conventional Monocytes Neutrophils CellType Tumor Alk−1 Alk−2 Alk−3 f Ddc 0 1 2 3 Dbh 0 1 2 Cadm1 0 1 2 3 Fscn1 0.0 0.5 1.0 1.5 2.0 Pfn2 0 1 2 Fdps 0.0 0.5 1.0 1.5 2.0 Rab3b 0 1 2 3 4 Cd9 0.0 0.5 1.0 1.5 2.0 Tagln3 0 1 2 Dpysl3 0 1 2 Ptms 0 1 2 3 4 Stmn2 0.0 0.5 1.0 1.5 Fxyd6 0 1 2 3 4 Tuba1a 0 1 2 3 Uchl1 0 1 2 3 Tubb3 0.0 0.4 0.8 1.2 1.6 Pirt 0.0 0.4 0.8 1.2 1.6 Tmeff2 0.0 0.5 1.0 1.5 Dst 0 1 2 3 Stmn4 0 1 2 Hmgb3 tSNE_1 tSNE_2 d e c 0 20 40 B cells CD4 CD8 NK cells Treg Frequency of non-tumor cells 0 4 8 12 Macrophages Monocytes Inflammatory Monocytes Conventional Monocytes Neutrophils CellType Tumor Alk−1 Alk−2 Alk−3 f Met Ala Arg Asp Ile Ser Arg intron Met Ala Arg Asp Ile Tyr Arg intron * * HDR changes to block Cas9 WT ALK ALKY1282S ALK1178L TTGATTACGCCCTCTGCCCTGTAGCAAACTTAACCACCGGAACATAGTCCGATGCATTGGTGTGAGTCTACAAGCCCTGCCCCGCTTCATCCTGCTGGAACTCATGGCTGGCGGAGACC intron 22-23 K L N H Q I N I V L R C V G S Q L A P I L L L M E R F Nsi I a b GCTAATTCAACTCCTGCCAAGCCTGCCCAGTCTATGCATCGCCCCAGGAAGGTTGACCTCAGGTTCAGCCAGAGACCCCTCCACCCATATCCACAGAATATAGCTTCACAGCGTGA 380 390 400 410 420 430 440 450 460 470 480 490 GGCTCAGTTAAATTTGATTACGCCCTCTGCCCTGTAGCAAACTTAACCACCAGAACATAGTCCGATGCATTGGTGTGAGTCTACAAGCCCTGCCCCGCTTCATCCTGCTGGAAC 0 550 560 570 580 590 600 610 620 630 640 650 GCCCGGTGAGTGAGAGGCAGTCTTCTCTCCAGGGTTCATGTCAGCGGGAGCGGGTTGCAAGGGAGGTAACTGTGCTTGGTGGATTCCTGGGAGTCGGCCCTGTGGGCTCCT 3 6 0 5 8 15 11 12 17 13 1 2 4 14 16 22 10 9 18 21 7 20 23 19 −25 0 25 −40 −20 0 20 40 tSNE_2 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 0 1 2 Ncam1 0 1 2 3 Th 0 1 2 Mycn 0.0 0.5 1.0 1.5 2.0 2.5 Tubb2a 0 0 1 1 2 Ddc 0 1 2 3 Dbh 0 1 2 Cadm1 0 1 2 3 Fscn1 0.0 0.5 1.0 1.5 2.0 Pfn2 0 2 Fdps 0.0 0.5 1.0 1.5 2.0 Rab3b 0 1 2 3 4 Cd9 0.0 0.5 1.0 1.5 2.0 Tagln3 0 1 2 Dpysl3 0 2 Ptms 0 1 2 3 4 Stmn2 0.0 0.5 1.0 1.5 Fxyd6 0 1 2 3 4 Tuba1a 0 1 2 3 Uchl1 0 2 3 Tubb3 0.0 0.4 0.8 1.2 1.6 Pirt 0.0 0.4 0.8 1.2 1.6 Tmeff2 0.0 0.5 1.0 1.5 Dst 0 1 2 3 Stmn4 0 2 Hmgb3 tSNE_1 tSNE 2 d c ATGGCTGGCGGAGACC M 0 1 2 Ncam1 0 1 2 3 Th 0 1 2 Mycn 0.0 0.5 1.0 1.5 2.0 2.5 Tubb2a 0.0 0.5 1.0 1.5 2.0 Ddc 0 1 2 3 Dbh 0 1 2 Cadm1 0 1 2 3 Fscn1 0.0 0.5 1.0 1.5 2.0 Pfn2 0 1 2 Fdps 0.0 0.5 1.0 1.5 2.0 Rab3b 0 1 2 3 4 Cd9 0.0 0.5 1.0 1.5 2.0 Tagln3 0 1 2 Dpysl3 0 1 2 Ptms 0 1 2 3 4 Stmn2 0.0 0.5 1.0 1.5 Fxyd6 0 1 2 3 4 Tuba1a 0 1 2 3 Uchl1 0 1 2 3 Tubb3 0.0 0.4 0.8 1.2 1.6 Pirt 0.0 0.4 0.8 1.2 1.6 Tmeff2 0.0 0.5 1.0 1.5 Dst 0 1 2 3 Stmn4 0 1 2 Hmgb3 d d Met Ala Arg Asp Ile Ser Arg intron Met Ala Arg Asp Ile Tyr Arg intron * * HDR changes to block Cas9 WT ALK ALKY1282S a GCTAATTCAACTCCTGCCAAGCCTGCCCAGTCTATGCATCGCCCCAGGAAGGTTGACCTCAGGTTCAGCCAGAGACCCCTCCACCCATATCCACAGAATATAGCTTCACAGCGTGA g Asp Ile Ser Arg intron * * HDR changes to block Cas9 ALKY1282S ALK1178L GTGTGAGTCTACAAGCCCTGCCCCGCTTCATCCTGCTGGAACTCATGGCTGGCGGAGACC L V G S Q L A P I L L L M E R F GACCCCTCCACCCATATCCACAGAATATAGCTTCACAGCGTGA 460 470 480 490 GTGTGAGTCTACAAGCCCTGCCCCGCTTCATCCTGCTGGAAC 620 630 640 650 GTGCTTGGTGGATTCCTGGGAGTCGGCCCTGTGGGCTCCT 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 0 1 2 Ncam1 0 1 2 3 Th 0 1 2 Mycn 0.0 0.5 1.0 1.5 2.0 2.5 Tubb2a 0.0 0.5 1.0 1.5 2.0 Ddc 0 1 2 3 Dbh 0 1 2 Cadm1 0 1 2 3 Fscn1 0.0 0.5 1.0 1.5 2.0 Pfn2 0 1 2 Fdps 0.0 0.5 1.0 1.5 2.0 Rab3b 0 1 2 3 4 Cd9 0.0 0.5 1.0 1.5 2.0 Tagln3 0 1 2 Dpysl3 0 1 2 Ptms 0 1 2 3 4 Stmn2 0.0 0.5 1.0 1.5 Fxyd6 0 1 2 3 4 Tuba1a 0 1 2 3 Uchl1 0 1 2 3 Tubb3 0.0 0.4 0.8 1.2 1.6 Pirt 0.0 0.4 0.8 1.2 1.6 Tmeff2 0.0 0.5 1.0 1.5 Dst 0 1 2 3 Stmn4 0 1 2 Hmgb3 tSNE_1 tSNE_2 d a ALK1178L TTGATTACGCCCTCTGCCCTGTAGCAAACTTAACCACCGGAACATAGTCCGATGCATTGGTGTGAGTCTACAAGCCCTGCCCCGCTTCATCCTGCTGGAACTCA intron 22-23 K L N H Q I N I V L R C V G S Q L A P I L L L E R F Nsi I b 390 400 410 420 430 440 450 460 470 480 490 GGCTCAGTTAAATTTGATTACGCCCTCTGCCCTGTAGCAAACTTAACCACCAGAACATAGTCCGATGCATTGGTGTGAGTCTACAAGCCCTGCCCCGCTTCATCCTGCTGGAAC 550 560 570 580 590 600 610 620 630 640 650 b 39 GCCCGGTGAGTGAGAGGCAGTCTTCTCTCCAGGGTTCATGTCAGCGGGAGCGGGTTGCAAGGGAGGTAACTGTGCTTGGTGGATTCCTGGGAGTCGGC 3 6 0 5 8 15 11 12 17 13 1 2 4 14 16 22 10 9 18 21 7 20 23 19 −25 0 25 −40 −20 0 20 40 tSNE_1 tSNE_2 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 c c 0.0 0.5 1.0 1.5 2.0 Cd3e 0.0 0.5 1.0 1.5 2.0 2.5 Cd8a 0.0 0.5 1.0 1.5 Cd4 0.0 0.5 1.0 1.5 2.0 Foxp3 0 1 2 3 4 Gzma 0 1 2 3 Gzmb 0.0 0.5 1.0 1.5 2.0 Prf1 0 1 2 3 Vcam1 0 1 2 3 4 Hmox1 0 1 2 3 4 Lgals3 0 1 2 3 4 Apoc1 0 1 2 Ifngr1 0 1 2 Mycn 0 1 2 Ncam1 0.0 0.5 1.0 1.5 2.0 Rtn4 0 1 2 3 Th 0 1 2 3 4 Cd14 0.0 0.5 1.0 1.5 2.0 2.5 Ccr2 0 1 2 3 Cd209a 0.0 0.5 1.0 1.5 2.0 Itgax 0.0 0.5 1.0 1.5 2.0 Batf3 0 1 2 3 4 5 Lyz2 0 1 2 3 Cd68 0 1 2 3 Il12b 0 1 2 3 4 Ccl22 0.0 0.5 1.0 1.5 Flt3 0.0 0.5 1.0 1.5 2.0 Relb 0.0 0.5 1.0 1.5 Etv3 tSNE_1 tSNE_2 tSNE_1 _ e 0 20 40 B cells CD4 CD8 NK cells Treg Frequency of non-tumor cells 0 4 8 12 Macrophages Monocytes Inflammatory Monocytes Conventional Monocytes Neutrophils CellType Tumor Alk−1 Alk−2 Alk−3 f 0 20 40 B cells CD4 CD8 NK cells Treg Frequency of non-tumor cells 0 4 8 12 Macrophages Monocytes Inflammatory Monocytes Conventional Monocytes Neutrophils CellType Tumor Alk−1 Alk−2 Alk−3 f 0.0 0.5 1.0 1.5 2.0 Cd3e 0.0 0.5 1.0 1.5 2.0 2.5 Cd8a 0.0 0.5 1.0 1.5 Cd4 0.0 0.5 1.0 1.5 2.0 Foxp3 0 1 2 3 4 Gzma 0 1 2 3 Gzmb 0.0 0.5 1.0 1.5 2.0 Prf1 0 1 2 3 Vcam1 0 1 2 3 4 Hmox1 0 1 2 3 4 Lgals3 0 1 2 3 4 Apoc1 0 1 2 Ifngr1 0 1 2 Mycn 0 1 2 Ncam1 0.0 0.5 1.0 1.5 2.0 Rtn4 0 1 2 3 Th 0 1 2 3 4 Cd14 0.0 0.5 1.0 1.5 2.0 2.5 Ccr2 0 1 2 3 Cd209a 0.0 0.5 1.0 1.5 2.0 Itgax 0.0 0.5 1.0 1.5 2.0 Batf3 0 1 2 3 4 5 Lyz2 0 1 2 3 Cd68 0 1 2 3 Il12b 0 1 2 3 4 Ccl22 0.0 0.5 1.0 1.5 Flt3 0.0 0.5 1.0 1.5 2.0 Relb 0.0 0.5 1.0 1.5 Etv3 e f f e Frequency of non-tumor cells Supplementary Figure S1 Supplementary Figure S1 c −25 0 25 −25 0 25 tSNE_1 tSNE_2 0 1 2 3 4 B2m a b 40 60 80 100 cent incidence 40 60 80 100 cent survival P=0.9113 P=0.7456 NK Depletion a −25 0 25 −25 0 25 tSNE_1 tSNE_2 0 1 2 3 4 B2m a B2m tSNE_2 Supplementary Figure S2 c −25 0 25 tSNE_1 b 0 20 40 60 80 0 20 40 60 80 100 Time (days) Percent incidence 0 20 40 60 80 0 20 40 60 80 100 Time (days) Percent survival Control (n=21) Treatment (n=15) P=0.9113 P=0.7456 NK Depletion b b c 0 20 40 60 80 0 20 40 60 80 100 Time (days) Percent incidence 0 20 40 60 80 0 20 40 60 80 100 Time (days) Percent survival Control (n=21) Treatment (n=15) P=0.9113 P=0.7456 NK Depletion c Percent survival Supplementary Figure S2 95.0 0.53 81.8 0.98 a a 95.0 0.53 Supplementary Figure S3 FCS SSC FSC-A FSC-H Ly6-G (FITC) SSC CD11b (PacBlue) Ly6G (FITC) MHCII Ly6C A Monocytes B Activated monocytes E Eos (Siglec F+) D Mφ Eos C Mφ C Mφ E Eos (Siglec F+) A Monocytes D Mφ Eos b c CD11b+, Ly6C-, MHCII+ Ly6G D+E fractions 0 2 4 6 8 10 Blood Monocytes (%) * AlkY1282S/+; TH-MYCN+; Ccr2-/- AlkY1282S/+; TH-MYCN+ d 0.00 0.05 0.10 0.15 0.20 0.00 0.25 0.50 0.75 1.00 proportion of classification score contributed by macrophages proportion of classification score Signal B cells CD4+ T cells CD8+ T cells a 0.00 0.05 0.10 0.15 0.20 0.00 0.25 0.50 0.75 1.00 proportion of classification score contributed by macrophages proportion of classification score Signal B cells CD4+ T cells CD8+ T cells a 0.00 0.05 0.10 0.15 0.20 0.00 0.25 0.50 0.75 1.00 proportion of classification score contributed by macrophages proportion of classification score Signal B cells CD4+ T cells CD8+ T cells a a FCS SSC FSC-A FSC-H Ly6-G (FITC) SSC CD11b (PacBlue) Ly6G (FITC) MHCII Ly6C A Monocytes B Activated monocytes E Eos (Siglec F+) D Mφ Eos C Mφ MHCII (PE) Ly6C (APC) AlkF1174L/+; TH-MYCN+ 8.85 16.8 11.8 14.4 37.4 C Mφ E Eos (Siglec F+) A Monocytes D Mφ Eos b c 0 10 20 30 40 % of the CD11b+ fraction CD11b+, Ly6C-, MHCII+, Ly6G- 0 20 40 60 80 100 % of the CD11b+ fraction D+E fractions AlkMUT; TH-MYCN+; Ccr2-/- AlkMUT; TH-MYCN+ AlkMUT; TH-MYCN+; Stat6-/- AlkMUT; TH-MYCN+; Ccr2-/- AlkMUT; TH-MYCN+ * * AlkY1282S/+; TH-MY MHCII (PE) Ly6C (APC) 1.29 46.2 25 0 2 4 6 8 10 Blood Monocytes (%) AlkY1282S/+; TH-MYCN+; Ccr2-/- AlkY1282S/+; TH-MYCN+ d 0.00 0.05 0.10 0.15 0.00 0.25 0.50 0.75 1.00 proportion of classification score contributed by macrophages proportion of classification scor Signal B cells CD4+ T cells CD8+ T cells FCS SSC FSC-A FSC-H Ly6-G (FITC) SSC CD11b (PacBlue) Ly6G (FITC) A Monocytes B Activated monocytes C Mφ A Monocytes b c 0 2 4 6 8 10 Blood Monocytes (%) AlkY1282S/+; TH-MYCN+; Ccr2-/- AlkY1282S/+; TH-MYCN+ b 0 2 4 6 8 10 Blood Monocytes (%) AlkY1282S/+; TH-MYCN+; Ccr2-/- AlkY1282S/+; TH-MYCN+ b FCS SSC FSC-A FSC-H Ly6-G (FITC) SSC CD11b (PacBlue) Ly6G (FITC) b c 0 2 4 6 8 10 Blood Monocytes (%) YCN+; YCN+ FCS SSC FSC-A FSC-H Ly6-G (FITC) SSC CD11b (PacBlue) Ly6G (FITC) MHCII Ly6C A Monocytes B Activated monocytes E Eos (Siglec F+) D Mφ Eos C Mφ MHCII (PE) Ly6C (APC) AlkF1174L/+; TH-MYCN+ 8.85 16.8 11.8 14.4 37.4 C Mφ E Eos (Siglec F+) A Monocytes D Mφ Eos b c 0 10 20 30 40 % of the CD11b+ fraction CD11b+, Ly6C-, MHCII+, Ly6G- 0 20 40 60 80 100 % of the CD11b+ fraction D+E fractions AlkMUT; TH-MYCN+; Ccr2-/- AlkMUT; TH-MYCN+ AlkMUT; TH-MYCN+; Stat6-/- AlkMUT; TH-MYCN+; Ccr2-/- AlkMUT; TH-MYCN+ * * AlkY1282S/+; TH-MYCN+; Cc MHCII (PE) Ly6C (APC) 1.29 1.8 46.2 25.3 1 0 2 4 6 8 10 Blood Monocytes (%) ** AlkY1282S/+; TH-MYCN+; Ccr2-/- AlkY1282S/+; TH-MYCN+ d FCS SSC FSC-A FSC-H c c CD11b (PacBlue) Ly6G (FITC) FSC-H Ly6-G (FITC) C Mφ E Eos (Siglec F+) A Monocytes D Mφ Eos MHCII Ly6C A Monocytes B Activated monocytes E Eos (Siglec F+) D Mφ Eos C Mφ Supplementary Figure S4 MHCII L MHCII (PE) Ly6C (APC) AlkF1174L/+; TH-MYCN+ 8.85 16.8 11.8 14.4 37.4 0 10 20 30 40 % of the CD11b+ fraction CD11b+, Ly6C-, MHCII+, Ly6G- 0 20 40 60 80 100 % of the CD11b+ fraction D+E fractions AlkMUT; TH-MYCN+; Ccr2-/- AlkMUT; TH-MYCN+ AlkMUT; TH-MYCN+; Stat6-/- AlkMUT; TH-MYCN+; Ccr2-/- AlkMUT; TH-MYCN+ * * AlkY1282S/+; TH-MYCN MHCII (PE) Ly6C (APC) 1.29 46.2 25.3 d Supplementary Figure S4 0 10 20 30 40 % of the CD11b+ fraction CD11b+, Ly6C-, MHCII+, Ly6G- 0 20 40 60 80 100 % of the CD11b+ fraction D+E fractions AlkMUT; TH-MYCN+; Ccr2-/- AlkMUT; TH-MYCN+ AlkMUT; TH-MYCN+; Stat6-/- AlkMUT; TH-MYCN+; Ccr2-/- AlkMUT; TH-MYCN+ * * d 0 10 20 30 40 % of the CD11b+ fraction CD11b+, Ly6C-, MHCII+, Ly6G- 0 20 40 60 80 100 % of the CD11b+ fraction D+E fractions AlkMUT; TH-MYCN+; Ccr2-/- AlkMUT; TH-MYCN+ AlkMUT; TH-MYCN+; Stat6-/- AlkMUT; TH-MYCN+; Ccr2-/- AlkMUT; TH-MYCN+ * * d d MHCII (PE) Ly6C (APC) AlkF1174L/+; TH-MYCN+ 8.85 16.8 11.8 14.4 37.4 AlkY1282S/+; TH-MYCN+; Ccr2-/- MHCII (PE) Ly6C (APC) 1.29 1.81 46.2 25.3 16.2 Supplementary Figure S4 d f 10 5 5.5 10 5 10 5 c −20 −10 0 10 20 −20 0 20 40 tSNE_1 tSNE_2 Arg1 −20 −10 0 10 20 −20 0 20 40 tSNE_1 tSNE_2 Il4ra −20 −10 0 10 20 −20 0 20 40 tSNE_1 tSNE_2 Arg1_Il4ra 2.5 5.0 7.5 10.0 2 4 6 8 10 Arg1 Il4ra Color threshold: 0.5 −20 −10 0 10 20 −20 0 20 40 tSNE_1 tSNE_2 Arg1 −20 −10 0 10 20 −20 0 20 40 tSNE_1 tSNE_2 S100a9 −20 −10 0 10 20 −20 0 20 40 tSNE_1 tSNE_2 Arg1_S100a9 2.5 5.0 7.5 10.0 2 4 6 8 10 Arg1 S100a9 Color threshold: 0.5 CD68 CD163 S100 ARG1 e 0 105 Arg1 PE 0 1.0M 2.0M 3.0M 4.0M SSC-A 0 106 CD68 FITC 0 -10 4 104 105 106 CCR2 APC 0 105 Arg1 PE 0 1.0M 2.0M 3.0M 4.0M SSC-A 0 105 Arg1 PE 0 1.0M 2.0M 3.0M 4.0M SSC-A 0 106 CD68 FITC 0 -10 4 104 105 106 CCR2 APC 0 105 Arg1 PE 0 1.0M 2.0M 3.0M 4.0M SSC-A 0 105 Arg1 PE 0 1.0M 2.0M 3.0M 4.0M SSC-A 0 106 CD68 FITC 0 -10 4 104 105 106 CCR2 APC 0 105 Arg1 PE 0 1.0M 2.0M 3.0M 4.0M SSC-A CD45- CD45- CD45- CD45+ CD45+ CD45+ 0 106 CD68 FITC 0 105 106 Arg1 PE 0 106 CD68 FITC 0 105 106 Arg1 PE 0 106 CD68 FITC 0 105 106 Arg1 PE b a c 0 106 CD68 FITC 0 105 106 Arg1 PE 0 106 CD68 FITC 0 105 106 Arg1 PE 0 106 CD68 FITC 0 105 106 Arg1 PE b 0 106 CD68 FITC 0 105 106 Arg1 PE b b 0 105 Arg1 PE 0 1.0M 2.0M 3.0M 4.0M SSC-A CD45- 0 105 Arg1 PE 0 1.0M 2.0M 3.0M 4.0M SSC-A CD45- a 0 106 CD68 FITC 0 105 106 Arg1 PE 0 106 CD68 FITC 0 105 106 Arg1 PE a 0 105 Arg1 PE 0 1.0M 2.0M 3.0M 4.0M SSC-A CD45- 0 105 Arg1 PE 0 1.0M 2.0M 3.0M 4.0M SSC-A CD45+ 0 105 Arg1 PE 0 1.0M 2.0M 3.0M 4.0M SSC-A CD45+ 0 105 Arg1 PE 0 1.0M 2.0M 3.0M 4.0M SSC-A CD45+ c c CD68 CD163 S100 ARG1 Supplementary Figure S5 d f CD11b+ Ly6G- 3.71 0 10 4 CD11b Pacific Blue 0 10 3 10 4 10 5 Ly6G PerCP/Cy5.5 Ly6C+ 43.5 0 10 3 10 5 Ly6C FITC 0 10 3 10 4 10 5 SSC Ly6C+ CD124+ 1.96 0 10 4 CD124 (IL4R) PE 0 10 3 10 4 10 5 Ly6C FITC CD11b+ Ly6G- 10.4 0 10 4 CD11b Pacific Blue 0 10 3 10 4 10 5 Ly6G PerCP/Cy5.5 Ly6C+ 36.7 0 10 3 10 5 Ly6C FITC 0 10 3 10 4 10 5 SSC Ly6C+ CD124+ 0.62 0 10 4 CD124 (IL4R) PE 0 10 3 10 4 10 5 Ly6C FITC CD11b+ Ly6G- 5.94 0 10 4 CD11b Pacific Blue 0 10 3 10 4 10 5 Ly6G PerCP/Cy5.5 Ly6C+ 30.8 0 10 3 10 5 Ly6C FITC 0 10 3 10 4 10 5 SSC Ly6C+ CD124+ 3.78 0 10 4 CD124 (IL4R) PE 0 10 3 10 4 10 5 Ly6C FITC −20 −10 0 10 20 −20 0 20 40 tSNE_1 tSNE_2 Arg1 −20 −10 0 10 20 −20 0 20 40 tSNE_1 tSNE_2 Il4ra −20 −10 0 10 20 −20 0 20 40 tSNE_1 tSNE_2 Arg1_Il4ra 2.5 5.0 7.5 10.0 2 4 6 8 10 Arg1 Il4ra Color threshold: 0.5 −20 −10 0 10 20 −20 0 20 40 tSNE_1 tSNE_2 Arg1 −20 −10 0 10 20 −20 0 20 40 tSNE_1 tSNE_2 S100a9 −20 −10 0 10 20 −20 0 20 40 tSNE_1 tSNE_2 Arg1_S100a9 S100a9 e 0 105 Arg1 PE 0 106 CD68 FITC 0 -10 4 104 105 106 CCR2 APC 0 105 Arg1 PE 0 106 CD68 FITC 0 -10 4 104 105 106 CCR2 APC 0 105 Arg1 PE 0 106 CD68 FITC 0 -10 4 104 105 106 CCR2 APC 0 106 CD68 FITC 0 -10 4 104 105 106 CCR2 APC 0 106 CD68 FITC 0 -10 4 104 105 106 CCR2 APC 0 106 CD68 FITC 0 -10 4 104 105 106 CCR2 APC CD68 FITC d −20 −10 0 10 20 −20 0 20 40 tSNE_1 tSNE_2 Arg1 −20 −10 0 10 20 −20 0 20 40 tSNE_1 tSNE_2 Il4ra −20 −10 0 10 20 −20 0 20 40 tSNE_1 tSNE_2 Arg1_Il4ra 2.5 5.0 7.5 10.0 2 4 6 8 10 Arg1 Il4ra Color threshold: 0.5 −20 −10 0 10 20 −20 0 20 40 tSNE_1 tSNE_2 Arg1 −20 −10 0 10 20 −20 0 20 40 tSNE_1 tSNE_2 S100a9 −20 −10 0 10 20 −20 0 20 40 tSNE_1 tSNE_2 Arg1_S100a9 2.5 5.0 7.5 10.0 2 4 6 8 10 Arg1 S100a9 Color threshold: 0.5 e CD68 FITC CD68 FITC CD68 FITC d −20 −10 0 10 20 −20 0 20 40 tSNE_1 tSNE_2 Arg1 −20 −10 0 10 20 −20 0 20 40 tSNE_1 tSNE_2 Il4ra −20 −10 0 10 20 −20 0 20 40 tSNE_1 tSNE_2 Arg1_Il4ra 2.5 5.0 7.5 10.0 2 4 6 8 10 Arg1 Il4ra Color threshold: 0.5 −20 −10 0 10 20 −20 0 20 40 tSNE_1 tSNE_2 Arg1 −20 −10 0 10 20 −20 0 20 40 tSNE_1 tSNE_2 S100a9 −20 −10 0 10 20 −20 0 20 40 tSNE_1 tSNE_2 Arg1_S100a9 2.5 5.0 7.5 10.0 2 4 6 8 10 Arg1 S100a9 Color threshold: 0.5 d Arg1 e e Ly6C+ 43.5 0 10 3 10 5 Ly6C FITC 0 10 3 10 4 10 5 SSC f CD11b+ Ly6G- 3.71 0 10 4 CD11b Pacific Blue 0 10 3 10 4 10 5 Ly6G PerCP/Cy5.5 CD11b+ Ly6G- 3.71 0 10 4 CD11b Pacific Blue 0 10 3 10 4 10 5 Ly6G PerCP/Cy5.5 f Ly6C FITC Ly6C+ 36.7 0 10 3 10 5 0 10 3 10 4 10 5 SSC CD11b Pacific Blue CD11b+ Ly6G- 10.4 4 0 10 3 10 4 10 5 Ly6G PerCP/Cy5.5 Supplementary Figure S5
https://openalex.org/W4311716388	https://digitalcommons.wustl.edu/cgi/viewcontent.cgi?article=2045&context=oa_4	English	null	Future Newborns with Opioid-Induced Neonatal Abstinence Syndrome (NAS) Could Be Assessed with the Genetic Addiction Risk Severity (GARS) Test and Potentially Treated Using Precision Amino-Acid Enkephalinase Inhibition Therapy (KB220) as a Frontline Modality Instead of Potent Opioids	Journal of personalized medicine	2,022	cc-by	18,050	Future newborns with opioid-induced neonatal abstinence Future newborns with opioid-induced neonatal abstinence syndrome (NAS) could be assessed with the genetic addiction syndrome (NAS) could be assessed with the genetic addiction risk severity (GARS) test and potentially treated using precision risk severity (GARS) test and potentially treated using precision amino-acid enkephalinase inhibition therapy (KB220) as a amino-acid enkephalinase inhibition therapy (KB220) as a frontline modality instead of potent opioids frontline modality instead of potent opioids Mauro Ceccanti Sapienza University of Rome Mark S Gold Washington University School of Medicine in St. Louis et al. Follow this and additional works at: https://digitalcommons.wustl.edu/oa_4 Part of the Medicine and Health Sciences Commons Washington University School of Medicine Washington University School of Medicine Digital Commons@Becker Digital Commons@Becker 2020-Current year OA Pubs Open Access Publications 12-6-2022 Future newborns with opioid-induced neonatal abstinence Future newborns with opioid-induced neonatal abstinence syndrome (NAS) could be assessed with the genetic addiction syndrome (NAS) could be assessed with the genetic addiction risk severity (GARS) test and potentially treated using precision risk severity (GARS) test and potentially treated using precision amino-acid enkephalinase inhibition therapy (KB220) as a amino-acid enkephalinase inhibition therapy (KB220) as a frontline modality instead of potent opioids frontline modality instead of potent opioids Mauro Ceccanti Sapienza University of Rome Mark S Gold Washington University School of Medicine in St. Louis et al. Washington University School of Medicine Washington University School of Medicine Digital Commons@Becker Digital Commons@Becker 2020-Current year OA Pubs Open Access Publications 12-6-2022 Future newborns with opioid-induced neonatal abstinence Future newborns with opioid-induced neonatal abstinence syndrome (NAS) could be assessed with the genetic addiction syndrome (NAS) could be assessed with the genetic addiction risk severity (GARS) test and potentially treated using precision risk severity (GARS) test and potentially treated using precision amino-acid enkephalinase inhibition therapy (KB220) as a amino-acid enkephalinase inhibition therapy (KB220) as a frontline modality instead of potent opioids frontline modality instead of potent opioids Mauro Ceccanti Sapienza University of Rome Mark S Gold Washington University School of Medicine in St. Louis et al. Open Access Publications Open Access Publications Recommended Citation Recommended Citation Ceccanti, Mauro; Gold, Mark S; and et al., "Future newborns with opioid-induced neonatal abstinence syndrome (NAS) could be assessed with the genetic addiction risk severity (GARS) test and potentially treated using precision amino-acid enkephalinase inhibition therapy (KB220) as a frontline modality instead of potent opioids." Journal of Personalized Medicine. 12, 12. 2015 (2022). https://digitalcommons.wustl.edu/oa_4/1063 This Open Access Publication is brought to you for free and open access by the Open Access Publications at Digital Commons@Becker. It has been accepted for inclusion in 2020-Current year OA Pubs by an authorized administrator of Digital Commons@Becker. For more information, please contact vanam@wustl.edu. Journal of Personalized Medicine Journal of Personalized Medicine Future Newborns with Opioid-Induced Neonatal Abstinence Syndrome (NAS) Could Be Assessed with the Genetic Addiction Risk Severity (GARS) Test and Potentially Treated Using Precision Amino-Acid Enkephalinase Inhibition Therapy (KB220) as a Frontline Modality Instead of Potent Opioids Louis, MO 63110, USA * Correspondence: drd2gene@gmail.com (K.B.); ashim6786@gmail.com (A.G.) Citation: Ceccanti, M.; Blum, K.; Bowirrat, A.; Dennen, C.A.; Braverman, E.R.; Baron, D.; Mclaughlin, T.; Giordano, J.; Gupta, A.; Downs, B.W.; et al. Future Newborns with Opioid-Induced Neonatal Abstinence Syndrome (NAS) Could Be Assessed with the Genetic Addiction Risk Severity (GARS) Test and Potentially Treated Using Precision Amino-Acid Enkephalinase Inhibition Therapy (KB220) as a Frontline Modality Instead of Potent Opioids. J. Pers. Med. 2022, 12, 2015. https:// doi.org/10.3390/jpm12122015 Academic Editors: Marco U. Future Newborns with Opioid-Induced Neonatal Abstinence Syndrome (NAS) Could Be Assessed with the Genetic Addiction Risk Severity (GARS) Test and Potentially Treated Using Precision Amino-Acid Enkephalinase Inhibition Therapy (KB220) as a Frontline Modality Instead of Potent Opioids Scalese and Valentina Vengeliene Received: 29 August 2022 Accepted: 25 November 2022 Published: 6 December 2022 2 The Kenneth Blum Behavioral & Neurogenetic Institute, Austin, TX 78701, USA 2 The Kenneth Blum Behavioral & Neurogenetic Institute, Austin, TX 78701, USA g 3 Division of Addiction Research & Education, Center for Mental Health & Sports, Exercise and Global Mental 3 Division of Addiction Research & Education, Center for Mental Health & Sports, Exercise and Global Men Health, Western University Health Sciences, Pomona, CA 91766, USA 4 Institute of Psychology, ELTE Eötvös Loránd University, Egyetem tér 1-3, H-1053 Budapest, Hungary 5 Department of Psychiatry, School of Medicine, University of Vermont, Burlington, VT 05405, USA 6 Department of Psychiatry, Wright State University Boonshoft School of Medicine and Dayton VA Medical Centre, Dayton, OH 45324, USA p y y y g 6 Department of Psychiatry, Wright State University Boonshoft School of Medicine and Dayton VA Medical Centre, Dayton, OH 45324, USA 7 Reward Deﬁciency Clinics of America, Austin, TX 78701, USA 8 Center for Genomics and Applied Gene Technology, Institute of Integrative Omics and applied 9 Department of Precision Behavioral Management, Transplicegen Therapeutics, Inc., LLC., Austin, TX 78701, USA 10 Department of Molecular Biology and Adelson School of Medicine, Ariel University, Ariel 40700, Israel Department of Molecular Biology and Adelson School of Medicine, Ariel University, Ariel 40700, Israel 11 Department of Family Medicine, Jefferson Health Northeast, Philadelphia, PA 19107, USA 11 Department of Family Medicine, Jefferson Health Northeast, Philadelphia, PA 19107, USA 12 Ketamine Infusion Clinic of South Florida, Pompano Beach, FL 33062, USA 3 Future Biologics, Lawrenceville, GA 30043, USA 14 Department of Pharmaceutical Sciences, Southern University College of Pharmacy, Houston, TX 77004, USA 15 Department of Genetics, Ecology and Evolution, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte 31270-901, Brazil 16 Center for Pain and the Brain (PAIN Group), Department of Anesthesiology, Critical Care & Pain Medicine, Boston Children’s Hospital, Harvard School of Medicine, Boston, MA 02115, USA 17 Behavioral Neuropharmacology and Neuroimaging Laboratory, Clinical Research Institute on Addictions, Department of Pharmacology and Toxicology, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, NY 14203, USA 17 Behavioral Neuropharmacology and Neuroimaging Laboratory, Clinical Research Institute on Addictions, Department of Pharmacology and Toxicology, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, NY 14203, USA 18 Department of Psychiatry, South Texas Veteran Health Care System, Audie L. Future Newborns with Opioid-Induced Neonatal Abstinence Syndrome (NAS) Could Be Assessed with the Genetic Addiction Risk Severity (GARS) Test and Potentially Treated Using Precision Amino-Acid Enkephalinase Inhibition Therapy (KB220) as a Frontline Modality Instead of Potent Opioids Scalese Valentina Vengeliene eived: 29 August 2022 epted: 25 November 2022 lished: 6 December 2022 lisher’s Note: MDPI stays neutral h regard to jurisdictional claims in lished maps and institutional afﬁl- 1 Società Italiana per il Trattamento dell’Alcolismo e le sue Complicanze (SITAC), ASL Roma1, Sapienza University of Rome, 00185 Rome, Italy 2 The Kenneth Blum Behavioral & Neurogenetic Institute, Austin, TX 78701, USA 3 Division of Addiction Research & Education, Center for Mental Health & Sports, Exercise and Global Mental Health, Western University Health Sciences, Pomona, CA 91766, USA 4 Institute of Psychology, ELTE Eötvös Loránd University, Egyetem tér 1-3, H-1053 Budapest, Hungary 5 Department of Psychiatry, School of Medicine, University of Vermont, Burlington, VT 05405, USA 6 Department of Psychiatry, Wright State University Boonshoft School of Medicine and Dayton VA Medical Centre, Dayton, OH 45324, USA 7 Reward Deﬁciency Clinics of America, Austin, TX 78701, USA 8 Center for Genomics and Applied Gene Technology, Institute of Integrative Omics and applied Biotechnology (IIOAB), Nonakuri, Purba Medinipur, West Bengal 721172, India 9 Department of Precision Behavioral Management, Transplicegen Therapeutics, Inc., LLC., Austin, TX 78701, USA 10 Department of Molecular Biology and Adelson School of Medicine, Ariel University, Ariel 40700, Israel 11 Department of Family Medicine, Jefferson Health Northeast, Philadelphia, PA 19107, USA 12 Ketamine Infusion Clinic of South Florida, Pompano Beach, FL 33062, USA 13 Future Biologics, Lawrenceville, GA 30043, USA 14 Department of Pharmaceutical Sciences, Southern University College of Pharmacy, Houston, TX 77004, USA 15 Department of Genetics, Ecology and Evolution, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte 31270-901, Brazil 16 Center for Pain and the Brain (PAIN Group), Department of Anesthesiology, Critical Care & Pain Medicine, Boston Children’s Hospital, Harvard School of Medicine, Boston, MA 02115, USA 17 Behavioral Neuropharmacology and Neuroimaging Laboratory, Clinical Research Institute on Addictions, Department of Pharmacology and Toxicology, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, NY 14203, USA 18 Department of Psychiatry, South Texas Veteran Health Care System, Audie L. Murphy Memorial VA Hospital, Long School of Medicine, University of Texas Medical Center, San Antonio, TX 78229, USA 19 Neurogenesis Project, Jacksonville, FL 32223, USA 20 Department of Psychiatry, Washington University School of Medicine, St. Future Newborns with Opioid-Induced Neonatal Abstinence Syndrome (NAS) Could Be Assessed with the Genetic Addiction Risk Severity (GARS) Test and Potentially Treated Using Precision Amino-Acid Enkephalinase Inhibition Therapy (KB220) as a Frontline Modality Instead of Potent Opioids Mauro Ceccanti 1 , Kenneth Blum 2,3,4,5,6,7,8,9,10,* , Abdalla Bowirrat 10 , Catherine A. Dennen 11, Eric R. Braverman 2, David Baron 3, Thomas Mclaughlin 7, John Giordano 2,12, Ashim Gupta 13,* , Bernard W. Downs 2 , Debasis Bagchi 2,14 , Debmalya Barh 8,15 , Igor Elman 16, Panayotis K. Thanos 17, Rajendra D. Badgaiyan 18 , Drew Edwards 19 and Mark S. Gold 20 Mauro Ceccanti 1 , Kenneth Blum 2,3,4,5,6,7,8,9,10,* , Abdalla Bowirrat 10 , Catherine A. Dennen 11, Eric R. Braverman 2, David Baron 3, Thomas Mclaughlin 7, John Giordano 2,12, Ashim Gupta 13,* , Bernard W. Downs 2 , Debasis Bagchi 2,14 , Debmalya Barh 8,15 , Igor Elman 16, Panayotis K. Thanos 17, Rajendra D. Badgaiyan 18 , Drew Edwards 19 and Mark S. Gold 20 1 Società Italiana per il Trattamento dell’Alcolismo e le sue Complicanze (SITAC), ASL Roma1, Sapienza University of Rome, 00185 Rome, Italy ation: Ceccanti, M.; Blum, K.; wirrat, A.; Dennen, C.A.; verman, E.R.; Baron, D.; aughlin, T.; Giordano, J.; Gupta, Downs, B.W.; et al. Future wborns with Opioid-Induced natal Abstinence Syndrome AS) Could Be Assessed with the etic Addiction Risk Severity RS) Test and Potentially Treated ng Precision Amino-Acid ephalinase Inhibition Therapy 220) as a Frontline Modality ead of Potent Opioids. J. Pers. . 2022, 12, 2015. https:// org/10.3390/jpm12122015 demic Editors: Marco U. Future Newborns with Opioid-Induced Neonatal Abstinence Syndrome (NAS) Could Be Assessed with the Genetic Addiction Risk Severity (GARS) Test and Potentially Treated Using Precision Amino-Acid Enkephalinase Inhibition Therapy (KB220) as a Frontline Modality Instead of Potent Opioids Murphy Memorial VA Hospital, Long School of Medicine, University of Texas Medical Center, San Antonio, TX 78229, USA 19 Neurogenesis Project, Jacksonville, FL 32223, USA 18 Department of Psychiatry, South Texas Veteran Health Care System, Audie L. Murphy Memorial VA Hospital, Long School of Medicine, University of Texas Medical Center, San Antonio, TX 78229, USA 19 Neurogenesis Project, Jacksonville, FL 32223, USA Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. g j 20 Department of Psychiatry, Washington University School of Medicine, St. Louis, MO 63110, USA * Correspondence: drd2gene@gmail.com (K.B.); ashim6786@gmail.com (A.G.) g j 20 Department of Psychiatry, Washington University School of Medicine, St. Louis, MO 63110, USA * Correspondence: drd2gene@gmail.com (K.B.); ashim6786@gmail.com (A.G.) Abstract: In this nonsystematic review and opinion, including articles primarily selected from PubMed, we examine the pharmacological and nonpharmacological treatments of neonatal abstinence syndrome (NAS) in order to craft a reasonable opinion to help forge a paradigm shift in the treatment and prevention of primarily opioid-induced NAS. Newborns of individuals who use illicit and licit substances during pregnancy are at risk for withdrawal, also known as NAS. In the US, the reported prevalence of NAS has increased from 4.0 per 1000 hospital births in 2010 to 7.3 per 1000 hospital births in 2017, which is an 82% increase. The management of NAS is varied and involves a combination of nonpharmacologic and pharmacologic therapy. The preferred ﬁrst-line pharmacological treatment for NAS is opioid therapy, speciﬁcally morphine, and the goal is the short-term improvement in NAS symptomatology. Nonpharmacological therapies are individualized and typically focus on general Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/jpm J. Pers. Med. 2022, 12, 2015. https://doi.org/10.3390/jpm12122015 J. Pers. Med. 2022, 12, 2015 2 of 23 care measures, the newborn–parent/caregiver relationship, the environment, and feeding. When used appropriately, nonpharmacologic therapies can help newborns with NAS avoid or reduce the amount of pharmacologic therapy required and the length of hospitalization. In addition, genetic polymorphisms of the catechol-o-methyltransferase (COMT) and mu-opioid receptor (OPRM1) genes appear to affect the length of stay and the need for pharmacotherapy in newborns with prenatal opioid exposure. 1. Introduction In this nonsystematic review and opinion, we primarily selected articles from PubMed to craft a reasonable opinion to help forge a paradigm shift in the treatment and prevention of primarily opioid-induced neonatal abstinence syndrome (NAS). Unfortunately, NAS from exposure to opioids in utero has reached epidemic levels worldwide. In infants, it is well known that nonpharmacologic modalities are the standard of care. However, pharmacotherapy is often required for the treatment of NAS. This article examines the current standard-of-care nonpharmacological and pharmacological treatments and explores additional potential alternative nonpharmacological treatments that could improve NAS treatment outcomes. Future Newborns with Opioid-Induced Neonatal Abstinence Syndrome (NAS) Could Be Assessed with the Genetic Addiction Risk Severity (GARS) Test and Potentially Treated Using Precision Amino-Acid Enkephalinase Inhibition Therapy (KB220) as a Frontline Modality Instead of Potent Opioids Therefore, based on this extensive literature and additional research, this team of coauthors suggests that, in the future, in addition to the current nonpharmacological therapies, patients with opioid-induced NAS should undergo genetic assessment (i.e., the genetic addiction risk severity (GARS) test), which can subsequently be used to guide DNA-directed precision amino-acid enkephalinase inhibition (KB220) therapy as a frontline modality instead of potent opioids. Keywords: neonatal abstinence syndrome (NAS); reward deﬁciency syndrome (RDS); genetic addic- tion risk severity (GARS); hypodopaminergia; dopamine homeostasis; opioids and alcohol common mechanism 1.1. Neonatal Abstinence Syndrome (NAS) Newborns of individuals who use illicit and licit substances during pregnancy are at risk for withdrawal, also known as NAS. NAS is a spectrum of newborn neurobehavioral dysregulation symptoms that are complex, variable, and poorly understood. Although NAS is most frequently linked to opioid exposure, it can be related to other substances such as nicotine, methamphetamine, benzodiazepines, selective serotonin reuptake inhibitors (SSRIs), etc. [1,2]. The most common characteristic signs and symptoms of NAS indicate dysfunction in any of the following domains: motor and tone control, state control and attention, sensory integration, and autonomic functioning [3]. The onset of the signs and symptoms of NAS varies, depending on the history of substance exposure and the half-life of substance elimination. For opioids in particular, withdrawal can be delayed up to ﬁve days after birth or longer [4]. The presence and severity of these signs and symptoms pro- vide the foundation for scoring systems (i.e., the Modiﬁed Finnegan Neonatal Abstinence Score, the Johns Hopkins Bayview Medical Center neonatal abstinence scoring sheet and initiation of treatment, etc.) that are used to make treatment decisions in newborns with NAS. The long-term consequences of NAS can include, but are not limited to, behavioral problems, neurodevelopmental delays, and when untreated, death [5]. 2. Treatment of NAS The management of NAS is varied and involves a combination of nonpharmaco- logic and pharmacologic therapy. The treatment goals for NAS include preventing NAS- associated complications and restoring normal newborn activities, such as nutrition intake, weight gain, sleep, and adjustment to the social environment [11]. As with all pharma- cological treatments, the potential risks and beneﬁts of treatment must be considered for each patient. For example, some disadvantages include prolonged hospitalizations and drug exposure, while some advantages include the relief of withdrawal symptoms and the prevention of potential complications. 1.2. Epidemiology and Economic Issues Illicit drug use in the United States (US) has been steadily increasing and has reached unprecedented levels [6,7]. According to data from the National Survey on Drug Abuse and Health (NSDUH), in 2020 approximately 59.3 million individuals living in the US had used illicit drugs in the past year [6]. In addition, provisional data from the Centers for Disease Control and Prevention (CDC) revealed that there were approximately 107,622 drug overdose deaths in the US in 2021 [8]. This increase in illicit drug use in the US has resulted in an increase in NAS. In the US, the reported prevalence of NAS has increased from 4.0 J. Pers. Med. 2022, 12, 2015 3 of 23 3 of 23 per 1000 hospital births in 2010 to 7.3 per 1000 hospital births in 2017, which is an 82% increase [9]. In a study by Patrick et al. [10], birth and economic data from 580 counties over 7 years were reviewed. These included 1803 metropolitan country-years, 1268 rural country-years, and 927 remote county-years. An assessment inclusive of these years found 6,302,497 births with 47,224 births diagnosed with NAS. Speciﬁcally, the median NAS range was 7.1 per 1000 hospital births. Notably, individuals diagnosed with NAS are associated with the 10-year unemployment rate. Victims of NAS in this global investigation presented in the lowest unemployment quartile. 2.1. Nonpharmacological Interventions Nonpharmacologic care should be initiated at birth for all substance-exposed new- borns, and the parent/caregiver should also be actively involved. It should continue throughout the newborn’s hospitalization and after discharge, regardless of the need for pharmacologic treatment. When used appropriately, nonpharmacologic therapies can help newborns with NAS avoid or reduce the amount of pharmacologic therapy required. However, they do not serve as a substitute for pharmacotherapy when it is necessary. Nonpharmacologic therapies entail individualized assessments of the newborn and parent/caregiver’s functioning; the environment, to determine speciﬁc newborn– parent/caregiver dyad triggers for dysregulation; and adaptative responses to the envi- ronment to reduce physiological and neurobehavioral symptoms and promote newborn– parent/caregiver dyadic regulation [3]. General care measures [3,12]: General care measures in NAS include identifying the signs, symptoms, and triggers of physiological behavioral dysregulation and individualizing the care of the newborn based on these observations as well as promoting organization, competence, and physiological stability in newborns by identifying techniques that improve symptomatology that are speciﬁc to each newborn. For example, gentle vertical rocking can help reduce excessive irritability. Moreover, tremors and hypertonicity can be reduced by utilizing swaddling and positioning (i.e., the side-lying C position), which decrease motoric hyperactivity and allow newborns to organize their behaviors and become calm. Nonpharmacologic interventions in this domain include assessing parental function- ing and interaction with the newborn to help reduce dysregulation and promote dyadic synchronization [13]; educating the parent/caregiver on how to identify the signs of with- drawal; teaching the parent/caregiver about their newborn’s sensitivities; helping the parent/caregiver develop strategies and respond to the newborn in a manner that re- duces the newborn’s dysregulation and expression of NAS; aiding the parent/caregiver in understanding their feelings surrounding their newborn’s functioning so they can re- spond more appropriately; and managing maternal issues such as mental illness, limited health care access, intimate partner violence, etc., in order to maintain a healthy newborn– parent/caregiver relationship, which is vital for the newborn’s development [5]. Environment [12]: J. Pers. Med. 2022, 12, 2015 4 of 23 Nonpharmacologic interventions in this domain include identifying potential sen- sory and environmental input sources of dysregulation for newborns and altering the environment to minimize their effects and dysregulation. For example, a newborn who becomes hypertonic or irritable with eye contact might need the parent/caregiver to avoid eye contact while feeding, handling, or performing other activities together. 2.1. Nonpharmacological Interventions In addition, a newborn who becomes easily overstimulated by noise can be cared for in a quiet area. Finally, rooming-in (i.e., the colocation of the newborn and the parent/caregiver after deliv- ery and beyond) has been found to reduce NAS severity [12,14,15] and is recommended in the inpatient setting. Feeding: g For newborns with NAS, formula feeding should not necessarily be the default. In fact, breastfeeding has been shown to be successful in some individuals with opioid use disorder (OUD) [12,16–18]. Recommendations involving an individual’s suitability for breastfeeding should be tailored for individuals with one or more of the following traits: the concurrent usage of other prescription medications; participation in prenatal care and/or substance use disorder (SUD) treatment during or after the second trimester; and relapse during the third trimester with abstinence maintained for 30 days prior to delivery. Breastfeeding by methadone-maintained individuals seems to be safe and can lessen the severity of NAS and the necessity for pharmacological intervention [19–22]. The concentrations of methadone have been found to be low in human breast milk (range: 21–462 ng/mL) and do not appear to be associated with the parent’s methadone dose [19]. The low concentrations of methadone found in human breast milk are unlikely to have a signiﬁcant impact on the newborn’s display of NAS, and other breastfeeding-related variables could be responsible for the decreased severity of NAS in breastfed infants of methadone-maintained individuals. In addition, buprenorphine is excreted in low concentrations into human breast milk and seems to be safe for newborns of buprenorphine- maintained individuals [23,24]. 2.2. Pharmacological Interventions Pharmacological management is initiated for newborns who display signiﬁcant signs and symptoms of NAS despite adequate and personalized nonpharmacological care. The goal of pharmacological management is a short-term improvement in NAS symptomatol- ogy. Currently, opioid therapy is the preferred ﬁrst-line treatment for NAS. This is based on limited data that show opioid therapy reduces the need for additional medications and shortens hospital stays [5,12,25–28]. Morphine and methadone are the preferred opioids, and the selection is based on the clinician/hospital. Morphine is typically the preferred agent of the two, while methadone is considered a reasonable alternative. However, there have been studies that indicate that methadone minimally reduces hospital stay and treat- ment duration when compared to morphine [29,30]. Buprenorphine is another agent that has been used and appears to be effective in the treatment of NAS [31,32]. However, its use in newborns is limited due to the high ethanol content (30%) and its challenging sublingual administration [12]. A 2020 systematic review by Zankl et al. identiﬁed 16 trials including 1110 infants [5]. In one of the trials (N = 80 infants), morphine was compared to supportive care alone, and the results showed that morphine increased the length of treatment and hospitalization but shortened the time needed to regain birthweight. In trials that compared morphine to methadone (two trials, N = 147 infants), it was found that they both had comparable rates of breastfeeding success, length of hospitalization, and treatment failure. In trials that compared morphine to buprenorphine (three trials, N = 113 infants), it was found that they both had comparable rates of treatment failure, but the length of hospitalization was shorter in the buprenorphine group. In a separate network meta-analysis utilizing both indirect and direct comparisons (18 trials, N = 1072 infants), six medications were evaluated, including morphine, methadone, buprenorphine, clonidine, phenobarbital, and DTO. Morphine and methadone were associated with the lowest rates of treatment failure, J. Pers. Med. 2022, 12, 2015 5 of 23 but the differences were not statistically signiﬁcant [33]. Additionally, buprenorphine was found to have the shortest length of hospitalization. but the differences were not statistically signiﬁcant [33]. Additionally, buprenorphine was found to have the shortest length of hospitalization. 2.2. Pharmacological Interventions g p In addition, according to a meta-analysis by Cleary et al., there were no statistically signiﬁcant differences in the incidence of NAS in newborns of women on higher doses of opioids when compared to lower doses in studies that used an objective NAS scoring system and prospective studies [34]. Similarly, Bakstad et al. reported that the maternal methadone or buprenorphine dose was not predictive of the occurrence or need for NAS treatment in newborns [35]. A second medication is sometimes required in newborns who have severe NAS that is not sufﬁciently controlled with a single agent [5,33,36,37]. The two most commonly used second-line medications are clonidine and phenobarbital. Typically, clonidine is the preferred second-line medication due to concerns regarding phenobarbital’s adverse effects, including oversedation, a high alcohol content, challenges with weaning substance- exposed newborns from phenobarbital, and phenobarbital’s potential long-term impacts on neurodevelopment based on animal studies [12,38–41]. In addition, the concurrent use of phenobarbital and clonidine appears to reduce the consequences of opioid-induced negative neuronal development in newborns with NAS [36,42]. g p Agthe et al. found that, in a clinical trial (N = 80) with newborns who had intrauterine exposure to heroin or methadone, the addition of clonidine to standard opioid therapy was found to decrease pharmacological treatment (11 vs. 15 days) when compared to placebo [43]. The placebo group also required higher dosages of opioid therapy. No signiﬁcant short-term complications, such as bradycardia, hypotension, hypertension, or oxygen desaturations, were observed in either group. However, the clonidine group had three deaths (sudden infant death syndrome (SIDS), myocarditis, and homicide). Johnson et al. compared the use of phenobarbital and opioid therapy together to opioid therapy alone and found that the combined therapy decreased the length of hospitalization and the duration of symptoms when compared to opioid therapy alone [44]. However, despite the use of phenobarbital in the treatment of NAS, no safety proﬁle has been established, and the alcohol content remains a concern [45]. Finally, in a retrospective multicenter study (N = 563) by Merhar et al., it was found that the length of hospitalization and morphine treatment was shorter for newborns who were treated with a combined therapy of morphine and phenobarbital compared to those who were treated with a combined therapy of clonidine and morphine [37]. However, more newborns were discharged on phenobarbital than clonidine (78% vs. 29%). 2.4. A Case in Favor of Non-Opioid Treatment in NAS Opioid pharmacokinetics are inﬂuenced in neonates by a higher body water content that can alter drug distribution and metabolic processes that are not mature and can lead to low plasma protein and liver enzyme activity. For example, these factors could affect cleared metabolites, resulting in a prolonged half-life of opioids. This is further complicated by reduced renal excretion, which might be due to immature tubular secretion, glomerular ﬁltration, and reabsorption [51]. In addition, animal experiments have shown that an immature blood–brain barrier in neonates may result in an augmented sensitivity to opioids [52]. An argument against the utilization of opioids to treat NAS has been espoused by some investigators globally [53–56], and a word search for “acupuncture and NAS” revealed 102 PubMed listings (26 January 2022). Following an extensive review of the literature, including the Cochrane Databases, Jackson et al. [53] reported that acupuncture is a safe and effective nonpharmacological alternative to potent opioids for the treatment of NAS. p g p p Similarly, while not in neonates but in adults, our laboratory observed a signiﬁcant attenuation of opioid withdrawal symptoms with a well-researched nutraceutical com- plex prodopaminergic regulator (KB220Z) as an aqua-power liquid variant [57]. Out of 17 heavily opioid-dependent patients during detoxiﬁcation, only three received buprenor- phine/naloxone (Bup/Nx) along with KB220Z. We ﬁrst used a dose of KB220Z of two ounces (oz) twice daily before meals, along with other detoxifying agents, including cloni- dine, benzodiazepines, and gabapentin. The dose of KB220Z was maintained for six days in ﬁve patients. Then, in a second scenario, we employed a higher dose of four oz every six hours over six days. The higher dose was utilized in another 12 patients. Only three people relapsed with these two protocols during the ﬁrst two weeks of the experiment. Importantly, the remaining 82% were maintained on KB220Z. Speciﬁcally, these subjects were maintained without any additional Bup/Nx for a minimum of 120 days and, in one subject, 214 days. One limitation is the inability to interpret these results or make any conclusions regarding speciﬁc KB220Z effects. If further conﬁrmed in more extensive studies, using KB220Z for opiate/opioid detox- iﬁcation may provide a novel way to eliminate the need for addictive opioids during withdrawal and detoxiﬁcation. This paradigm shift, which requires extensive research, may translate to a reduction in universally employing powerful and addictive opioids to treat OUD and NAS [58]. 2.3. Neurodevelopmental Issues with Opioid Treatment Czynski et al. [46] reported that the prevalence of NAS has increased by 333% over the last two decades, which translates to approximately one infant born every 15 min in the United States [47]. This unfortunate statistic reveals that 50–80% of newborn infants exposed to opioids in utero develop NAS. Along these lines, Boardman et al. [48] suggested that a literature summary of 40 years necessitated a reassessment of ways to treat NAS without opioids, even during withdrawal periods. These investigators identiﬁed knowl- edge gaps and urged the scientiﬁc community to re-evaluate childhood clinical outcomes such as infant brain development and visual and long-term neurocognitive function. Van den Hoogen et al. [49], assessing the behavioral and cFos responses, known to be a marker for neuronal activation in neonatal animals withdrawing from opioids, found increased cFos expression in spinally projecting neurons within the periaqueductal grey (PAG), locus coeruleus, and rostral ventromedial medulla (RVM). They also observed that the narcotic antagonist naloxone precipitated profound withdrawal symptoms across all developmental levels and stages within several key brainstem regions. Another example of neurode- velopmental issues linked to opioids was investigated by others [46], involving mothers maintained on methadone or buprenorphine but randomized to morphine vs. methadone. Czynski et al. [46] reported that adding phenobarbital to the treatment routine resulted in several medical problems, suggesting that sedative hypnotics may not be an appropriate J. Pers. Med. 2022, 12, 2015 6 of 23 modality in these NAS cases. Finally, Witt et al. [50] evaluated long-term childhood and infant mortality involving 1900 individuals diagnosed with NAS and 12,283 controls. The results indicated that NAS-diagnosed children were readmitted to the hospital within ﬁve years of life more frequently when compared to non-NAS controls. Most perplexing was the ﬁnding that in NAS patients there was an unadjusted signiﬁcant increased mortality risk ratio of 1.94 (95% CI 0.99–3.80). Witt and associates [50] concluded that childhood readmission due to NAS “argues” for innovative (possibly nonpharmacological) early interventions to prevent morbidity and possibly mortality. 2.5. Snapshot of Dopaminergic Mechanisms in Addiction Undoubtedly, substance and nonsubstance behavioral addiction is a complex genetic and epigenetic disease that afﬂicts millions worldwide. Clinically, a major issue biologi- cally is a breakdown in the function of the brain’s reward circuitry in many cases, even in newborns, especially as a function of speciﬁc known genetic risk variants as antecedents compounded by the epigenetic effects of in utero opioids that impact behavior. It is of interest that much of our knowledge of the neurobiological underpinnings of human be- havior regarding “reward dysfunction” has been derived from animal research. Poisson et al. [59] recently reviewed the evidence related to the critical role of striatal dopamine (DA) in all addictive behaviors. While our laboratory has been at the forefront of DA genetics and its association with not only severe alcoholism but also general reward deﬁ- J. Pers. Med. 2022, 12, 2015 7 of 23 7 of 23 ciency [60,61], Poisson and associates [59] contributed to the identiﬁcation of speciﬁc and distinct mesostriatal and nigrostriatal DA circuit functions in substance use disorder (SUD). Although at least seven major neurotransmitter systems (serotoninergic, endorphinergic, GABAergic, glutamatergic, opioidergic, acetylcholinergic, and dopaminergic) are involved, striatal dopamine is essential for controlling one’s craving behaviors, impairments in de- cision making that underlie several risk-taking behaviors, anti-socialization, and overall compulsive and impulsive behaviors (Figure 1). ciency [60,61], Poisson and associates [59] contributed to the identiﬁcation of speciﬁc and distinct mesostriatal and nigrostriatal DA circuit functions in substance use disorder (SUD). Although at least seven major neurotransmitter systems (serotoninergic, endorphinergic, GABAergic, glutamatergic, opioidergic, acetylcholinergic, and dopaminergic) are involved, striatal dopamine is essential for controlling one’s craving behaviors, impairments in de- cision making that underlie several risk-taking behaviors, anti-socialization, and overall compulsive and impulsive behaviors (Figure 1). Figure 1. Behavioral models are used to classify the phenotypes of substance use disorder (SUD (Top) The behavioral criteria of SUDs (circled letters) can be sorted into three main categorie impaired control of substance use (Group I), impaired social behavior (Group II), and risky substan use (Group III). (Left) Common rodent experimental models and the SUD criteria that are thought best approximate them. Note that most models capture multiple SUD features. 2.5. Snapshot of Dopaminergic Mechanisms in Addiction (Right) Mesostriat circuits (light purple), including dopamine projections from the ventral tegmental area (VTA) to th nucleus accumbens (NAc), and nigrostriatal circuits (dark purple), including dopamine projections the dorsomedial (DMS) and dorsolateral striatum (DLS), generally have dissociable roles in differe components of major SUD models. The middle panels list the most clearly deﬁned roles for the two systems in each SUD category. Figure 1. Behavioral models are used to classify the phenotypes of substance use disorder (SUD). Figure 1. Behavioral models are used to classify the phenotypes of substance use disorder (SUD). (Top) The behavioral criteria of SUDs (circled letters) can be sorted into three main categories: impaired control of substance use (Group I), impaired social behavior (Group II), and risky substance use (Group III). (Left) Common rodent experimental models and the SUD criteria that are thought to best approximate them. Note that most models capture multiple SUD features. (Right) Mesostriatal circuits (light purple), including dopamine projections from the ventral tegmental area (VTA) to the nucleus accumbens (NAc), and nigrostriatal circuits (dark purple), including dopamine projections to the dorsomedial (DMS) and dorsolateral striatum (DLS), generally have dissociable roles in different components of major SUD models. The middle panels list the most clearly deﬁned roles for these two systems in each SUD category. As Poisson et al. [59] pointed out, most of the brain’s dopamine neurons are in two midbrain regions (Figure 1): the ventral tegmental area (VTA) and the substantia nigra pars compacta (SNc). Others revealed that DA neurons in the VTA mainly project to the ventral striatum, speciﬁcally the nucleus accumbens (NAc) core and shell. The J. Pers. Med. 2022, 12, 2015 8 of 23 8 of 23 NAc shell comprises the mesostriatal pathway and links to certain frontal regions in the prefrontal cortex, pallidum, and amygdala [62,63]. The work of Nestler’s group [64] and others [65,66] has shown that DA neurons in the VTA intermingle with GABAergic and glutamatergic neurons. In contrast, the SNc DA neurons project to the dorsomedial (DMS) and dorsolateral (DLS) striatum almost exclusively and comprise a well-known nigrostriatal system [67–69]. Importantly, in the striatum, Gerfen [70] showed that DA neurons contact GABAergic medium spiny neurons (MSNs) that contain excitatory type 1 (D1-MSNs) or inhibitory type 2 (D2-MSNs) DA receptors. Kupchik et al. [71] have further conﬁrmed this work. 2.5. Snapshot of Dopaminergic Mechanisms in Addiction The primary role of DA’s modulatory effect on striatal activity due to these outputs involves the control of speciﬁc behaviors (such as motivation and reward learning). It is indeed well known that most highly addictive psychoactive drugs (such as cocaine, alcohol, and morphine) cause the release of DA in the NAc and other striatal regions. According to Collins and Saunders [72], based on terminal mechanisms, DA release may play an essential role in many infractions related to aberrant drug use and cravings and even drug reinstatement or relapse, the cornerstone of unwanted SUD [73–77]. A review of the literature revealed that DA neurons across the VTA and SNc circuitry impact a wide array of behavioral functions, showing signiﬁcant overlap or co-occurrence across many reward-related behaviors [78–81]. Mesostriatal DA neurons contribute to the execution of goal-directed behaviors and learning. However, nigrostriatal DA, speciﬁcally in the DLS, impacts movement control and even the execution of rigid habitual actions that translate to addiction heterogeneity [82–87]. It is important to recognize that DA has a powerful effect on many behaviors that, when impaired, induce in the reward circuitry maladapted dysfunctional behaviors and addiction, including poor decision making, a prominent underpinning of compulsive behaviors [88–119]. 2.6. Evidence-Based Prodopaminergic Regulation (KB220) Genetic polymorphisms of the catechol-o-methyltransferase (COMT) and mu-opioid receptor (OPRM1) genes appear to affect the length of stay and the need for pharmacother- apy in newborns with prenatal opioid exposure [120]. These ﬁndings are consistent with data from adult studies that demonstrated that variations in these genes are associated with adult opioid dependence variability [121]. Epigenetic modiﬁcations to the OPRM1 gene have also been linked to the severity of NAS [122]. Thus, it appears prudent to incorporate genetic testing in order to reveal reward circuitry gene polymorphisms, especially those associated with dopaminergic pathways and opioid receptors, as a means of improving NAS treatment outcomes [123]. Therefore, after a decade of attempting to reduce severe NAS symptomatology with nonpharmacological approaches, including acupuncture and transcranial stimula- tion [124,125], we propose adding the complex prodopaminergic regulator (KB220Z) based on genetic assessment (i.e., the genetic addiction risk severity (GARS) test), along with other non-opioid modalities. KB220Z is a formulation of enkephalin, enkephalinase inhibitors, and dopamine-releasing neuronutrients that is utilized to induce dopamine homeostasis for the detoxiﬁcation and treatment of individuals genetically predisposed to developing addic- tive and compulsive behaviors known as reward deﬁciency syndrome (RDS) (Table 1) [61]. The formulations are based on the results of the GARS test, which evaluates the presence of reward genes and risk alleles and can successfully stratify the potential for developing OUD-related risks (Table 1) [61]. In addition, second-line non-opioid pharmacological agents such as clonidine [126] could also be used in the short term, followed by longer-term dopamine regulation with KB220 variants. J. Pers. Med. 2022, 12, 2015 9 of 23 Table 1. Neuroadaptagen amino-acid therapy (NAAT) . GARS Listed Nutrient Neuroadaptagen Target D-Phenylalanine Opioid peptides L-Phenylalanine Dopamine L-tryptophan Serotonin L-Tyrosine Dopamine L-Glutamine (low dose) GABA Chromium Serotonin Rhodiola Rosea COMT/MOA Passionﬂower (low dose) Benzodiazepine = GABA complex Pyridoxine Enzyme Catalyst Table 1 describes the ingredients and proposed neurotransmitter targets of KB220. Table 1. Neuroadaptagen amino-acid therapy (NAAT) *. In the most recent reiteration, additional nutrients have been added to the formula, such as β-nicotinamide adenine dinucleotide (β-NAD) to function as a catalyst for dopamine synthesis [127] and N-acetyl-cysteine [128] to help promote glutaminergic drive in the VTA to release DA in the NAc. It is noteworthy that globally we are facing a signiﬁcant challenge in the increased utilization of opioids to reduce ongoing stress. In 2021, the USA had over 100,000 narcotic- overdose-induced fatalities [129]. 2.6. Evidence-Based Prodopaminergic Regulation (KB220) Certainly, NIAAA and NIDA continue to struggle with innovation to help reduce or eliminate this catastrophic epidemic. However, we are concerned with the current FDA-approved medication assistance treatments (MAT). The concern is that MAT works by blocking dopamine function and release at the preneuron in the NAc. While we understand short-term use to reduce harm [130], we oppose long-term use for tertiary treatment [131]. The research-based neuronutrient KB220, which has been intensely investigated in at least 38 studies, has demonstrated clinical beneﬁt [45]. The effects include, but are not limited to, reduced against medical advice (AMA) rates; reward system activation, including blood oxygen level dependent (BOLD) dopamine signaling; a reduction in craving behavior; relapse prevention; and the attenuation of stress, anger, and aggressive behaviors. It is noteworthy that our group has recently published an additional twelve studies utilizing KB220 variants [132]. Based on animal research and clinical trials, the prodopamine regulator KB220 shows promise in treating addiction and pain as well as opioid-induced withdrawal symptomatology [133]. While additional neurobiological and genetic studies are required to help understand the mechanism of action of this neuronutrient, possible studies related to NAS seem prudent. However, the evidence advocates for the induction of “dopamine homeostasis” [105]. We believe that the utilization of this nonpharmacological approach could enable an approach free from side effects that induces the “normalization” of brain neurotransmitter signaling epigenetically. Moreover, it is reasonable to predict that the utilization of this neuronutrient (KB220) could lead to improved function and the attenuation of NAS. It is vital that addiction researchers realize that providing opioids to treat opioid abuse is counterproductive and lacks ways to induce real potential prophylaxis. Instead, we propose long-term prophylaxis utilizing our concept of coupling GARS to help determine precision prodopaminergic regulation via KB220. With that said, we are encouraged by these results, as published over the last 50 years. We look forward to continued advancements in appropriate nutrigenomic solutions for the millions of victims of all addictions, including reward surfeit syndrome (RSS) in adolescents and RDS in adulthood [105] as well as addictions to substances such as drugs and food and to behaviors such as smoking, gambling, and gaming, especially in our next J. Pers. Med. 2022, 12, 2015 10 of 23 10 of 23 generation. Understanding these simple precepts may engender novel ways to treat NAS with nonpowerful opioids. 2.6. Evidence-Based Prodopaminergic Regulation (KB220) p p The neurological effects of KB220 in naïve rodents, uncovered in studies conducted by Marcelo Febo [134], showed BOLD activation using KB220 in regions of interest related to the brain reward circuitry. Speciﬁcally, there was a signiﬁcant increase in the functional connectivity of the NAc with the medial and lateral anterior thalamic nucleus and the surrounding somatosensory cortex (Figure 2). Another important ﬁnding revealed that KB220Z augmented the connectivity between corticothalamic areas and this region of the reward system. y Additionally, with KB220Z vs. placebo, when the anterior thalamic nucleus was the selected seed RIO, there was minimal evidence of connectivity observed outside this area. Febo et al. [134] found a signiﬁcant enhancement in connectivity with surrounding sensory cortical areas and the regions mentioned above, including the NAc (both left and right). A more substantial effect on the resting state functional connectivity (rsFC) in the dorsal hippocampus was of real interest. Furthermore, connectivity was increased between the left and right dorsal hippocampi, the upper limb somatosensory regions, the NAc and limbic cortical areas, and the anterior cingulate (Figure 2). Figure 2. Representative cross-correlation maps show ﬁve subjects: placebo-controlled and KB220Z- treated rats. The maps correspond to the resting-state connectivity for the NAc (highlighted in green in the atlas map above the ﬁgure; only the left seed is shown). Note the distributed but signiﬁcant connectivity between various brain regions and the NAc in the placebo subjects. KB220Z increased connectivity, especially between the left and right accumbens, dorsal striatum, and limbic cortical areas, such as the anterior cingulate, prelimbic, and infralimbic regions. Correlation maps for the representative subjects are presented at a threshold of 0.3 ≤z ≥1.2 [135] (with permission). Figure 2. Representative cross-correlation maps show ﬁve subjects: placebo-controlled and KB220Z- treated rats. The maps correspond to the resting-state connectivity for the NAc (highlighted in green in the atlas map above the ﬁgure; only the left seed is shown). Note the distributed but signiﬁcant connectivity between various brain regions and the NAc in the placebo subjects. KB220Z increased connectivity, especially between the left and right accumbens, dorsal striatum, and limbic cortical areas, such as the anterior cingulate, prelimbic, and infralimbic regions. Correlation maps for the representative subjects are presented at a threshold of 0.3 ≤z ≥1.2 [135] (with permission). 2.6. Evidence-Based Prodopaminergic Regulation (KB220) A follow-up study utilizing KB220Z was also administered to abstinent Chinese heroin abusers to help map the brain reward circuitry interaction potential in humans. Along these lines, it is noteworthy that Willuhn et al. [136] reported that cocaine use and even non-substance-related addictive behavior surge as dopaminergic function is decreased. Understanding that reduced or deﬁcient levels of brain DA enhance heroin- seeking behavior. Treatment strategies, including a DA agonist therapy that conserves dopamine function, could prevent relapse to opioids. The effect of KB220Z on the reward circuitry of ten heroin addicts undergoing pro- tracted abstinence for an average of 16.9 months was investigated [135]. Speciﬁcally, in a randomized, placebo-controlled crossover study of KB220Z, ﬁve subjects completed J. Pers. Med. 2022, 12, 2015 11 of 23 11 of 23 the triple-blinded experiment. Additionally, nine patients were genotyped utilizing the GARS test. the triple-blinded experiment. Additionally, nine patients were genotyped utilizing the GARS test. KB220Z induced an enhanced BOLD activation in caudate–accumbens–dopaminergic pathways compared to placebo following a one-hour acute administration. Moreover, KB220Z also attenuated the resting state activity in the putamen of abstinent heroin- dependent subjects. In the second phase of this preliminary investigation of all ten abstinent heroin-dependent patients, three brain regions of interest were signiﬁcantly activated from the resting state by KB220Z compared to placebo. Interestingly, augmented functional connectivity was observed in a putative network that included the cerebellum, medial frontal gyrus, dorsal anterior cingulate, NAc, occipital cortical area, and posterior cingulate. These results and other qEEG studies [137,138] sup- port the notion of a putative anticraving/anti-relapse role for KB220Z in opioid dependence by direct or indirect dopaminergic interaction. Preclinical experiments and human trials associated with KB220 variants have been published and reviewed [45]. Early formulations of KB220 increased brain enkephalin levels in rodents [136], reduced alcohol-seeking behavior in C57/BL mice [135], and converted ethanol-preferring C57/BL mice via pharmacogenetics to the same level of nonpreference as alcohol-averse DBA mice [136]. Thus, based on these and other animal and human studies [137–169], using KB220Z might be an ideal treatment for NAS, particularly to counteract underlying brain hypodopaminergia. 2.7. Common Neurochemical Mechanisms Related to Alcohol and Opiate/Opioid-Induced Withdrawal Symptomatology Wallace et al. [170] reported that as many as 47% of pregnant women misuse/abuse alcohol, and at least 6% misuse or abuse illegal drugs such as opioids. The European Monitoring Centre for Drugs and Drug Addiction has noted that approximately 500 thou- sand opioid-dependent Europeans are, unfortunately, on opioid maintenance substitution therapy (OMST) [171]. It is indeed a fact that about 30,000 opioid-dependent women have become pregnant [172]. The treatment of women involved with a combination of alcohol and opioid dependence is very complex and is a challenge that must be faced to attenuate the onslaught of unwanted NAS [173,174]. Since the early 1970s, Blum’s group has investigated the common neurochemical and genetic underpinnings of all addictive behaviors. One area of investigation by this group was a common mechanism among opiates, alcohol, and neurotransmitter involvement in withdrawal symptomology—the commonality concept related to condensation products derived from the identiﬁcation of in vivo isoquinoline formation. There is enough evidence to suggest that these condensation amines “link” to opiates. The message here is that when one imbibes alcohol, opiate-like isoquinolines are formed [175]. These isoquinolines induce a robust enhancement of ethanol-induced withdrawal symptoms (EIW) [176]. For example, a series of experiments revealed that the inhibition of catecholamine synthesis results in the potentiation of EIW [177]; haloperidol, a D2 dopamine receptor (DRD2), potentiates EIW [178]; serotonergic blockers potentiate EIW [179]; dopamine suppresses EIW [180]; morphine suppresses EIW [181,182]; naloxone inhibits alcohol dependence [183]; and clonidine enhances EIW [184–186]. Of interest is the ﬁnding that by employing quantitative electroencephalography (qEEG) as an imaging tool, Miller et al. [153] showed the impact of one formulation of KB220 as a putative activator of the mesolimbic system. These investigators [153] found that intravenous administration reduces or “normalizes” aberrant electrophysiological parameters of the brain reward circuitry region. Speciﬁcally, KB220 signiﬁcantly normalized widespread theta and alpha activity in alcoholics and heroin abusers, showing several neurotransmitter-linked polymorphic genes measured by the GARS test. The authors [153] suggested that the chronic activation of dopaminergic receptors, such as DRD2, will increase upregulation, induce an augmented “dopamine sensitivity,” and ultimately “enhance the sense of happiness,” speciﬁcally, for example, in carriers of the DRD2 A1 allele. J. Pers. Med. 2022, 12, 2015 12 of 23 12 of 23 2.8. 2.7. Common Neurochemical Mechanisms Related to Alcohol and Opiate/Opioid-Induced Withdrawal Symptomatology Genetic Addiction Risk Severity (GARS): Promoting the Early Identiﬁcation of Polymorphic Risk Alleles in RDS Since 1990, when our laboratory published the DRD2 Taq A1 allele and severe alco- holism association in JAMA, there has been an explosion of genetic candidate association studies, including genome-wide association studies (GWAS). To develop an accurate test to help identify those at risk for at least alcohol use disorder (AUD), Blum’s group developed the GARS test, consisting of ten genes and eleven associated risk alleles. To statistically validate the selection of the risk alleles measured by GARS for alcohol, we applied a strict analysis to studies that investigated the association of each polymorphism with AUD or AUD-related conditions published from 1990 until 2021. This analysis calculated the Hardy– Weinberg equilibrium of each polymorphism in cases and controls. Pearson’s χ2 test or Fisher’s exact test were applied to compare the gender, genotype, and allele distribution, if available. The statistical analyses found the 95% CI for the odds ratio (OR) and the post hoc risk for alcoholism prevalence to be an estimated 8% of the population, revealing a signiﬁcant detection. The likelihood ratio (LR) results also showed signiﬁcance for DRD2, DRD3, DRD4, dopamine transporter gene (DAT1), catechol-o-methyltransferase (COMT), mu opioid receptor gene (OPRM1), and serotonin transporter gene (5-HTT) at 5%. The United States and European patents on a ten-gene panel and eleven risk alleles had been issued prior to this statistical analysis. y One possible etiological root cause of addiction involves the identiﬁed neurotrans- mitter network function within the mesolimbic and prefrontal cortex (PFC) brain regions. It is essential that the scientiﬁc community recognizes that the subsequent regulation of the ﬁnal reward and motivational pathway of “wanting” translates to the physiological induction of “normal” neuronal dopamine release. The typical neuromodulating aspects of neurotransmission and its disruption from chronic exposure to drugs and behavioral addictions necessitate an approach that involves achieving “dopamine homeostasis,” es- pecially for AUD and other unwanted RDS behaviors [187–190]. Interestingly, Bidwell et al. [191], utilizing functional MRI imaging, found that during alcohol cueing DRD2 promoter methylation was strongly associated with responses to alcohol cues in many brain circuitry regions related to reward. In addition, the clinical metrics of AUD severity were positively associated with methylation of the promotor region of the DRD2 gene. 2.7. Common Neurochemical Mechanisms Related to Alcohol and Opiate/Opioid-Induced Withdrawal Symptomatology This ﬁnding details early work from our laboratory [60], whereby we suggested that the DRD2 A1 allele residing outside of the promotor region in the 3′ region of the genome, now known to be involved in the transcription process related to mRNA expression, is the root cause of severe alcoholism. Figure 3 displays this article’s primary tenant. It provides a schematic showing the interactive events related to coupling gentle dopaminergic agonists (not potent D2 agonists such as bromocriptine) with genetic risk testing as one way to induce homeostasis of the brain reward circuitry. Blum’s laboratory has worked to successfully develop the GARS test, an accurate genetic test to predict risk liability for RDS behaviors, including AUD. The association to determine risk using a clinical outcome, the addiction severity index media version (ASI-MV), was accomplished with the Institute of Behavioral Genetics, University of Colorado, Boulder. Ten reward candidate genes were selected to develop this patented GARS test. They included the dopamine receptors (DRD1, 2, 3, and 4); DAT1; 5-HTT, COMT, monoamine oxidase (MAO), gamma-aminobutyric acid (GABA), OPRM1, and some single nucleotide polymorphisms (SNPs) and point mutations, all chosen to reﬂect a hypodopaminergic trait. The functions of the chosen alleles of the ten genes were determined to negatively inﬂuence the net release of dopamine at the brain reward site. Thousands of association studies have provided conclusive evidence of RDS-speciﬁc risks. Unfortunately, our laboratory is the only group investigating this potentially important DNA-directed test that could be used early in life to identify speciﬁc polymorphic risk alleles. With that in mind, several studies and reviews have been published. A sampling J. Pers. Med. 2022, 12, 2015 13 of 23 13 of 23 of these peer-reviewed articles provides the fundamental rationale to enable the futuristic applications of the GARS in all RDS behaviors to help identify risk [60,61,192–212]. of these peer-reviewed articles provides the fundamental rationale to enable the futuristic applications of the GARS in all RDS behaviors to help identify risk [60,61,192–212]. Figure 3. Schematic of our proposed model to treat and identify genetic antecedents and to provide a way of inducing “dopamine homeostasis.” Figure 3. Schematic of our proposed model to treat and identify genetic antecedents and to provide a way of inducing “dopamine homeostasis.” In our opinion, subsequent large-scale genomics studies have had limited success in identifying alleles implicated in addiction and RDS. 2.7. Common Neurochemical Mechanisms Related to Alcohol and Opiate/Opioid-Induced Withdrawal Symptomatology Although GWAS and next-generation sequencing are valuable genetic tools, the primary reason for this known limited success is resolvable. For example, GWAS identiﬁes novel clusters of genes that may relate to an etiological root as a genetic antecedent to RDS behaviors such as AUD. However, we believe the next critical step following GWAS clusters is the subsequent convergence to individual candidate genes, despite their small contributions to the overall variance [213]. This precision addiction management technology (Figure 4) was developed to ac- curately identify genetic addiction risk severity using the GARS test and was awarded the ﬁrst USA and foreign patents. As previously mentioned, Blum, Noble, and asso- ciates [60,187,189] published the ﬁrst conﬁrmed association of the DRD2 gene A1 allele with severe alcoholism and other RDS behaviors. Following this work, Blum et al. devel- oped the GARS test and the prodopamine regulator, a precision DNA-guided nutraceutical neuronutrient (research ID code: KB220) (Figure 4). 14 of 23 14 of 23 J. Pers. Med. 2022, 12, 2015 Figure 4. Precision addiction management platform [210] (with permission). Figure 4. Precision addiction management platform [210] (with permission). 3. Conclusions Based on this extensive literature and additional research, we suggest that in the future, in addition to the current standard-of-care nonpharmacological therapies, patients with opioid-induced NAS should be assessed with the GARS test to guide DNA-directed precision amino-acid enkephalinase inhibition (KB220) therapy as a frontline modality instead of potent opioids (Figure 5). Figure 5. This schematic represents the paradigm shift required to circumvent the use of powerful opioids to treat opioid-induced NAS. We propose to couple the GARS test with DNA-directed precision KB220 therapy to detoxify and maintain patients with NAS using a non-opioid nutraceutical neuronutrient alternative. Figure 5. This schematic represents the paradigm shift required to circumvent the use of powerful opioids to treat opioid-induced NAS. We propose to couple the GARS test with DNA-directed precision KB220 therapy to detoxify and maintain patients with NAS using a non-opioid nutraceutical neuronutrient alternative. J. Pers. Med. 2022, 12, 2015 15 of 23 15 of 23 Author Contributions: Conceptualization, M.C. and K.B.; writing—original draft preparation, K.B. and M.C.; writing—review and editing, M.C., K.B., A.B., C.A.D., E.R.B., D.B. (David Baron), T.M., J.G., A.G., B.W.D., D.B. (Debasis Bagchi), D.B. (Debmalya Barh), I.E., P.K.T., R.D.B., D.E. and M.S.G.; supervision, K.B.; project administration, A.G. All authors have read and agreed to the published version of the manuscript. Funding: R.D.B. was the recipient of NIH R01NS073884, and K.B. with Marjorie Gondre-Lewis, Ph.D. (Howard University) was a recipient of R41 MD012318/MD/NIMHD NIH HHS/United States. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: The authors appreciate the expert edits of Margaret A. Madigan. Conﬂicts of Interest: K.B. is the inventor of GARS and the KB220 variants and is credited with domestic, foreign-issued, and pending patents. K.B. has entered into an exclusive licensing agreement with Ivitalize, Inc. The other authors declare no conﬂict of interest. References 1. Kocherlakota, P. Neonatal Abstinence Syndrome. Pediatrics 2014, 134, e547–e561. [CrossRef] [PubMed] y 2. 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https://openalex.org/W2607268158	https://europepmc.org/articles/pmc5402242?pdf=render	English	null	miR-195 Regulates Proliferation and Apoptosis through Inhibiting the mTOR/p70s6k Signaling Pathway by Targeting HMGA2 in Esophageal Carcinoma Cells	Disease markers	2,017	cc-by	7,237	Hindawi Disease Markers Volume 2017, Article ID 8317913, 9 pages https://doi.org/10.1155/2017/8317913 Hindawi Disease Markers Volume 2017, Article ID 8317913, 9 pages https://doi.org/10.1155/2017/8317913 Hindawi Disease Markers Volume 2017, Article ID 8317913, 9 pages https://doi.org/10.1155/2017/8317913 Yong Li, Dapeng Wu, Pei Wang, Xiaohui Li, and Gongning Shi Department of Cardiothoracic Surgery, Huaihe Hospital of Henan University, Kaifeng 475000, China Correspondence should be addressed to Gongning Shi; shiningccn@163.com Received 24 October 2016; Revised 13 February 2017; Accepted 13 March 2017; Published 9 April 2017 Copyright © 2017 Yong Li et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. miR-195 is related to tumorigenesis and frequently inhibits cell proliferation and promotes apoptosis in various cancers, including esophageal carcinoma (EC). The mTOR/p70s6k signaling pathway, which is the major target pathway for HMGA2, regulates the survival and cell proliferation of many tumors and is commonly active in EC. The relationships of miR-195, HMGA2, and the mTOR/p70s6k signaling pathway in EC, however, remain unknown. In the present study, we found that the miR-195 level was signiﬁcantly downregulated in EC tissues, while the mRNA expressions of HMGA2 were signiﬁcantly upregulated. Dual-luciferase reporter assay demonstrated that HMGA2 is a target of miR-195. MTT assay and ﬂow cytometry revealed that miR-195 overexpression inhibited cell proliferation and induced apoptosis by targeting HMGA2. We also found that HMGA2 restored the inhibitory eﬀect of miR-195 on phosphorylation of mTOR and p70S6K. Furthermore, rapamycin, a speciﬁc inhibitor of the mTOR/p70S6K signaling pathway, decreased the levels of Ki-67 and Bcl-2/Bax ratio, inhibited cell proliferation, and promoted apoptosis in EC cells. In conclusion, upregulation of miR-195 signiﬁcantly suppressed cell growth and induced apoptosis of EC cells via suppressing the mTOR/p70s6k signaling pathway by targeting HMGA2. 1. Introduction All cells were maintained in Dulbecco’s modiﬁed Eagle’s medium (DMEM; Genom, Hangzhou, China) supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA), 100 U/ml of penicillin, and 100 μg/ml of streptomycin (Gibco, Gaithersburg, MD, USA) at 37°C with 5% CO2. g y mTOR, the mammalian target of rapamycin, belongs to phosphoinositide 3-kinase- (PI3K-) related kinase family and is evolutionarily conserved [17, 18]. The mTOR/p70s6k signaling pathway regulates the survival and cell proliferation of many tumors and is commonly active in various cancers including EC [19, 20]. Therefore, mTOR has been regarded as a crucial target for the treatment of human cancer. The p70s6 kinase (p70s6k) is a major eﬀector of mTOR in the downstream of the mTOR/p70s6k signaling pathway, which is a major eﬀector in the downstream of the PI3K/AKT sig- naling pathway [21]. Research has indicated that the mTOR/p70s6k signaling pathway was activated in esopha- geal squamous cell carcinoma and rapamycin, suppressed mTOR expression and phosphorylation of p70s6k, arrested cell cycle at the G0/G1 phase, and induced apoptosis of EC cells [22]. 2.2. Cell Transfection. EC109 and EC9706 cells were seeded in 6-well plates and transfected with miR-195 mimics, miR- control, anti-miR-195, anti-miR-control, pcDNA-HMGA2, pcDNA empty vector, si-HMGA2, or si-control (GenePharma, Shanghai, China) by Lipofectamine™2000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol when the cells were grown to 80% conﬂuency. High-mobility group protein A2 (HMGA2), a major nonhistone chromosomal protein and DNA-binding tran- scription factor, interacts with many diﬀerent transcription factors, regulates the expression patterns of genes, and func- tions as a key regulator of foundational cellular processes, such as cell cycle, transformation, proliferation, and apopto- sis [23–25]. HMGA2 induces oncogenesis and is upregulated in various tumors [26, 27]. The PI3K/AKT/mTOR/p70s6k signaling pathway is the major target pathway associated with HMGA2 expression, and HMGA2 can activate the PI3K/AKT/mTOR/p70s6k signaling pathway directly by inducing phosphorylation of AKT, mTOR, and p70s6k in humanumbilicalcordblood-derivedstromalcells(hUCBSCs) [28]. However, the relationship between the mTOR/p70s6k signaling pathway and HMGA2 in EC is still unknown. In addition, our bioinformatics analysis predicted that HMGA2 is a putative target of miR-195. But no studies have been reported about the relationship between miR-195 and HMGA2 in EC. 2.3. Quantitative Real-Time PCR. Total RNA was isolated from fresh frozen samples of EC patients and cell lines using the Trizol Reagent (Invitrogen). cDNAs were synthesized from 500 ng of total RNA by using PrimeScript™one- step RT-PCR kit (TaKaRa). 1. Introduction mRNA [5, 6]. It is reported that miRs regulate the posttran- scriptional levels of more than 30% human genes [7]. miRs are closely related to diverse biological functions and patho- logic processes, including cell proliferation, diﬀerentiation, apoptosis, and self-renewal [8]. Studies have shown that miRs can act as oncogenes or tumor suppressors by targeting cancer-related genes in the development and carcinogenesis of EC and might be used as a diagnostic biomarker for human cancers [9–11]. For example, miR-337 was oncogenic and upregulated in EC cells and could markedly promote proliferation and inhibit apoptosis by suppressing PTEN expression [12]. miR-625 was reported to exhibit downregu- lation in EC and increase cell proliferation and invasion by targeting Sox2 [13]. miR-195 was demonstrated to inhibit cell proliferation and promote apoptosis in human cervical cancer and to suppress tumor growth and angiogenesis in breast cancer by diﬀerent function routes [14, 15]. A previous study has indicated that miR-195 was downregulated in esophageal squamous cell carcinoma and inhibited cell Esophageal carcinoma (EC), one of the most common and deadly malignance worldwide, is frequently diagnosed in East Asia, especially in Northern China [1, 2]. Although treatment technology has been improved, the prognosis of patients with EC remains unfavorable [3]. Moreover, EC has been associated with a low 5-year survival rate of approximately 10% due to a frequent local invasion and distant metastasis [4]. Therefore, it is urgently required to explore the molecular mechanism of carcinogenesis and a biomarker for early diagnosis of EC, which may provide new therapeutic strategies to improve the long-term survival of patients with EC. MicroRNAs (miRs), a class of small noncoding, single- stranded RNA with approximately 18–25 nucleotides, gener- ally regulate the posttranscriptional level of target messenger RNA (mRNA) expression by binding to the complementary domains of the 3′-untranslated region (UTR) of their target 2 Disease Markers Disease Markers proliferation and invasion by targeting Cdc42 [16]. However, the underlying molecular mechanism of miR-195 in EC remains largely unknown. or other esophageal surgeries before esophagectomy. The study was approved by the Local Research Ethics Committee of Huaihe Hospital of Henan University. Written informed consent was obtained from all patients or their guardians. Human EC cell lines (EC109 and EC9706) were obtained from the Chinese Science Institute (Shanghai, China). 1. Introduction The expression of miR-195 and HMGA2 was determined by real-time PCR reaction which was executed with SYBR Premix Ex Taq II (TaKaRa) on a CFX 96 real-time PCR thermocycler (TaKaRa). U6 small nuclear RNA (snRNA) expression was used as the internal control. 2.4. Dual-Luciferase Assay. The 3′-UTR of HMGA2 contain- ing putative miR-195 binding sites (HMGA2-3′-UTR-wt) predicted by TargetScan (http://www.targetscan.org/) and a mutant miR-195 binding site (HMGA2-3′-UTR-mut) was synthesized and cloned into the pMIR-REPORT Luciferase Expression Reporter Vector (Invitrogen). For reporter assays, 50 pmol miR-195 or miR-control was cotransfected with 50 ng of pMIR-REPORT HMGA2-3′-UTR-wt or pMIR- REPORT HMGA2-3′-UTR-mut into EC109 and EC9706 cells placed in 24-well plates using Lipofectamine 2000 (Invitrogen). At 24 h after transfection, the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) was used to analyze luciferase activities and Renilla activity was used as the internal control. In our study, we veriﬁed the expression of miR-195 and HMGA2 in EC tissues and adjacent normal epithelium tis- sues. Then, we studied the relationship between miR-195 and HMGA2. Finally, we investigated the role of miR-195, HMGA2, and the mTOR/p70S6K signaling pathway in the regulation of cell proliferation and apoptosis in EC. 2. Materials and Methods (a) The expression of miR-195 in ﬁfteen surgically resected EC tissues and adjacent normal epithelium tissues was examined. (b) Correlation between miR-195 and HMGA2 mRNA expression in EC tissues. miR-195 and HMGA2 mRNA expression proﬁles were determined by qRT-PCR. The relative expression of miR-195 and HMGA2 mRNA was normalized to the U6 snRNA. Values are presented as the mean ± SD, n = 3. ∗∗∗P < 0 001. compared with that in adjacent normal epithelium tissues. Figure 1(b) showed a statistically signiﬁcant negative correla- tion between HMGA2 mRNA and miR-195 expressions in 15 EC tissues. corresponding horseradish peroxidase- (HRP-) conjugated secondary antibody (Santa Cruz Biotechnology) for 2 h. Pro- tein bands were ﬁnally visualized using chemiluminescence (Millipore, MA, USA) and normalized to β-actin. 2.6. Cell Proliferation Assay. EC109 and EC9706 cells were seeded into 96-well plates (BD Biosciences, San Jose, CA, USA) and were incubated in an incubator of 5% CO2 at 37°C. At 24 h or 48h after transfection with miR- 195 mimics, miR-control, pcDNA, or pcDNA-HMGA2, cells were incubated with 3-(4,5)-dimethylthiahiazo(-z-y1)- 3,5-di-phenytetrazoliumromide (MTT) (20μl of 5 mg/ml) (KeyGen BioTech, Nanjing, China) reagent for 4 h at 37°C. Following incubation, the supernatants were then discarded and 150 μl of dimethyl sulfoxide (Invitrogen) was used to solubilize the purple precipitates of formazan. The absor- bances of each well at 490 nm were detected using an auto- matic micro plate reader (Gene, HK). The experiments were repeated in triplicates. 2.6. Cell Proliferation Assay. EC109 and EC9706 cells were seeded into 96-well plates (BD Biosciences, San Jose, CA, USA) and were incubated in an incubator of 5% CO2 at 37°C. At 24 h or 48h after transfection with miR- 195 mimics, miR-control, pcDNA, or pcDNA-HMGA2, cells were incubated with 3-(4,5)-dimethylthiahiazo(-z-y1)- 3,5-di-phenytetrazoliumromide (MTT) (20μl of 5 mg/ml) (KeyGen BioTech, Nanjing, China) reagent for 4 h at 37°C. Following incubation, the supernatants were then discarded and 150 μl of dimethyl sulfoxide (Invitrogen) was used to solubilize the purple precipitates of formazan. The absor- bances of each well at 490 nm were detected using an auto- matic micro plate reader (Gene, HK). The experiments were repeated in triplicates. 3.2. miR-195 Directly Regulates HMGA2 Expression by Binding to the 3′-UTR of HMGA2 in EC Cell Lines. The pre- dicted HMGA2 3′-UTR binding site for miR-195 is shown in Figure 2(a) based on the TargetScan software and bioinfor- matics analysis. 2. Materials and Methods To identify whether miR-195 could directly target the HMGA2 gene, we constructed luciferase reporter vectors pMIR-REPORT HMGA2-3′-UTR-wt and pMIR- REPORT HMGA2-3′-UTR-mut containing the luciferase coding sequence in the downstream of the 3′-UTR of HMGA2. The luciferase reporter system showed that miR-195 decreased approximately half of the luciferase activity of pMIR-REPORT HMGA2-3′-UTR-wt compared with that of the miR-control groups, while no obvious inhibitory eﬀect on luciferase activity was observed in that of pMIR-REPORT HMGA2-3′-UTR-mut for EC109 and EC9706 cells (Figures 2(b) and 2(c)). The results indicated that HMGA2 was a target of miR-195. Furthermore, to conﬁrm whether miR-195 downregulated HMGA2 expres- sion, EC109 and EC9706 cells were transfected with miR- 195 mimics, anti-miR-195, or miR-control. As shown in Figures 2(d) and 2(e), the expression of HMGA2 mRNA was signiﬁcantly reduced by miR-195 overexpression but strikingly improved by miR-195 downregulation in EC109 and EC9706 cells. Besides, the HMGA2 protein expres- sion was signiﬁcantly decreased by miR-195 overexpression but signiﬁcantly increased by miR-195 downregulation in both EC109 (Figures 2(f) and 2(g)) and EC9706 cells (Figures 2(h) and 2(i)). These data suggested that miR-195 could speciﬁcally bind to the 3′-UTR of HMGA2 and nega- tively regulate HMGA2 expression. 2.7. Flow Cytometric Detection of Apoptosis. EC109 and EC9706 cells were seeded into 96-well plates (BD Biosci- ences, San Jose, CA, USA) and were incubated in an incuba- tor of 5% CO2 at 37°C. At 48 h after transfection with miR- 195 mimics, miR-control, pcDNA, or pcDNA-HMGA2, cells were collected and stained with Annexin V-FITC/PI (BestBio Biotechnology Co., Ltd., Shanghai, China) according to the manufacturer’s instructions. Finally, the apoptosis of cells were determined using ﬂow cytometer (Becton Dickinson, San Jose, CA, USA). 2.8. Statistical Analysis. All data were analyzed by SPSS 13.0 software. Values were expressed as means ±SEM or mean ± SD. Statistical comparisons were calculated with Student’s t-test or one-way analysis of variance (ANOVA). P values less than 0.05 were considered to be statistically signiﬁcant. 2.8. Statistical Analysis. All data were analyzed by SPSS 13.0 software. Values were expressed as means ±SEM or mean ± SD. Statistical comparisons were calculated with Student’s t-test or one-way analysis of variance (ANOVA). P values less than 0.05 were considered to be statistically signiﬁcant. 3.3. miR-195 Overexpression Inhibits Cell Proliferation and Induces Apoptosis in EC Cell Lines by Targeting HMGA2. 2. Materials and Methods 2.5. Western Blot Analysis. EC109 and EC9706 cells were collected and total cell lysate was obtained by using RIPA buﬀer (ZhongShan JinQiao, Beijing, China). BCA protein assay kits (CoWin Biotechnology, Beijing, China) were used to detect the protein concentrations. Equal amounts of protein extracts from diﬀerent groups were subjected to 12% SDS-PAGE and transferred onto nitrocellulose membranes (Millipore, Boston, MA, USA). After blocking in 5% nonfat milk for 1 h, the membranes were probed with indicated primary antibodies against HMGA2, t-p70s6k, p-p70s6k, t-mTOR, p-mTOR, Ki-67, Bcl-2, Bax, and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and incu- bated overnight at 4°C. After washing with TBS three times, the immunoblotted membranes were further incubated with 2.1. Clinical Specimens and Cell Lines. Fifteen surgically resected EC tissues and adjacent normal epithelium tissues were obtained from Huaihe Hospital of Henan University. The EC tissues and adjacent normal epithelium tissues were immediately collected after surgical resection, immersed with RNAlater reagent (TaKaRa, Dalian, China) for 30 min and subsequently frozen at −80°C until use. The primary tumor tissues were conﬁrmed by pathological analysis performed by a pathologist and classiﬁed according to the World Health Organization classiﬁcation. The tumor cellularity of the selected EC samples was more than 70%. None of the patients with EC had received radiotherapy, chemotherapy, 3 Disease Markers 3 0 Normal Tumor 1 2 3 4 5 ⁎⁎⁎ Relative miR‒195 levels (a) 0 Normal Tumor 1 2 3 4 5 ⁎⁎⁎ Relative miR‒195 levels 2.5 r = ‒0.795, P = 0.0004 2.0 1.5 1.0 0.5 0.0 0.0 0.3 0.6 0.9 1.2 1.5 1.8 Relative HMGA2 mRNA Relative miR‒195 expression (a) (b) Figure 1: miR-195 is downregulated and HMGA2 is upregulated in human EC tissues. (a) The expression of miR-195 in ﬁfteen surgically resected EC tissues and adjacent normal epithelium tissues was examined. (b) Correlation between miR-195 and HMGA2 mRNA expression in EC tissues. miR-195 and HMGA2 mRNA expression proﬁles were determined by qRT-PCR. The relative expression of miR-195 and HMGA2 mRNA was normalized to the U6 snRNA. Values are presented as the mean ± SD, n = 3. ∗∗∗P < 0 001. 2.5 r = ‒0.795, P = 0.0004 2.0 1.5 1.0 0.5 0.0 0.0 0.3 0.6 0.9 1.2 1.5 1.8 Relative HMGA2 mRNA Relative miR‒195 expression (b) (b) (a) Figure 1: miR-195 is downregulated and HMGA2 is upregulated in human EC tissues. 2. Materials and Methods In order to explore the role of miR-195 overexpression on the viability and apoptosis of EC cell lines, EC109 and EC9706 cells were transfected with miR-control or miR-195 mimics or cotransfected miR-195 mimics with pcDNA or pcDNA-HMGA2. As expected, pcDNA-HMGA2 3. Results 3.1. miR-195 and HMGA2 Expressions in Human EC Tissues. qRT-PCR was used to analyze the expression levels of miR- 195 and HMGA2 mRNA in 15 EC tissues. As shown in Figure 1(a), miR-195 expression was lower in EC tissues as Disease Markers HMGA2 3′UTR‒MUT 5′…CUAGUACUUAUUACACUUAGAUG… (a) 1.5 1.0 0.5 0.0 wt EC109 mut Normalized luciferase activity ⁎⁎⁎ miR‒control miR‒195 mimics wt EC9706 mut 1.5 1.0 0.5 0.0 Normalized luciferase activity ⁎⁎⁎ miR‒control miR‒195 mimics EC109 2.5 2.0 1.5 1.0 0.5 0.0 Relative HMGA2 mRNA expression ⁎⁎⁎ ⁎⁎ NC miR‒control miR‒195 mimics Anti‒miR‒195 (b) (c) (d) EC9706 3 2 1 0 Relative HMGA2 mRNA expression ⁎⁎⁎ ⁎⁎ NC miR‒control miR‒195 mimics Anti‒miR‒195 HMGA2 EC109 훽‒actin NC miR‒control miR‒195 mimics Anti‒miR‒195 EC109 2.5 2.0 1.5 1.0 0.5 0.0 Relative HMGA2 protein levels ⁎⁎⁎ ⁎⁎ NC miR‒control miR‒195 mimics Anti‒miR‒195 (e) (f) (g) HMGA2 EC9706 훽‒actin NC miR‒control miR‒195 mimics Anti‒miR‒195 EC9706 2.5 2.0 1.5 1.0 0.5 0.0 NC miR‒control miR‒195 mimics Anti‒miR‒195 Relative HMGA2 protein levels ⁎⁎⁎ ⁎⁎ (h) (i) Figure 2: miR-195 directly targets the 3′-UTR of HMGA2 to regulate HMGA2 expression in EC cell lines. (a) Prediction of the HMG mRNA 3′-UTR sites targeted by miR-195. The underline showed the mutated nucleotides on the 3′-UTR of HMGA2. Relative lucifer activity in (b) EC109 and (c) EC9706 cells. Cells were transfected with pMIR-REPORT vector containing HMGA2-3′-UTR-wt HMGA2-3′-UTR-mut together with miR-195 mimics or miR-control for 24 h. Renilla activity was used as the internal control. T expressions of HMGA2 mRNA in (d) EC109 and (e) EC9706 cells transfected with miR-195 mimics, anti-miR-195, or miR-control w detected by qRT-PCR. The expressions of HMGA2 protein in (f and g) EC109 and (h and i) EC9706 cells transfected with miR- mimics, anti-miR-195, or miR-control were detected by western blot. The expressions of HMGA2 mRNA and protein were normalized U6 snRNA and β-actin. Values are presented as the mean ± SD, n = 3. ∗∗P < 0 01, ∗∗∗P < 0 001. (i) (h) Figure 2: miR-195 directly targets the 3′-UTR of HMGA2 to regulate HMGA2 expression in EC cell lines. (a) Prediction of the HMGA2 mRNA 3′-UTR sites targeted by miR-195. The underline showed the mutated nucleotides on the 3′-UTR of HMGA2. Relative luciferase activity in (b) EC109 and (c) EC9706 cells. Cells were transfected with pMIR-REPORT vector containing HMGA2-3′-UTR-wt or HMGA2-3′-UTR-mut together with miR-195 mimics or miR-control for 24 h. 3. Results Renilla activity was used as the internal control. The expressions of HMGA2 mRNA in (d) EC109 and (e) EC9706 cells transfected with miR-195 mimics, anti-miR-195, or miR-control were detected by qRT-PCR. The expressions of HMGA2 protein in (f and g) EC109 and (h and i) EC9706 cells transfected with miR-195 mimics, anti-miR-195, or miR-control were detected by western blot. The expressions of HMGA2 mRNA and protein were normalized to U6 snRNA and β-actin. Values are presented as the mean ± SD, n = 3. ∗∗P < 0 01, ∗∗∗P < 0 001. dramatically reduced the inhibition eﬀect of miR-195 mimics on the expression of HMGA2 in EC109 (Figure 3(a)) and EC9706 cells (Figure 3(b)). After 24 h or 48 h of transfection, cell proliferation was detected by MTT assay. The results indicated that the miR-195 mimic signiﬁcantly inhibited cell proliferation in both EC109 and EC9706 5 Disease Markers 5 EC109 HMGA2 훽-actin miR-control miR-195 miR-195 + pcDNA miR-195 + pcDNA-HMGA2 EC9706 HMGA2 훽-actin miR-control miR-195 miR-195 + pcDNA miR-195 + pcDNA-HMGA2 (a) (b) 1.5 miR-control miR-195 miR-195+ pcDNA miR-195 + pcDNA-HMGA2 0.5 0.0 0 h 24 h 48 h EC109 1.0 OD value (휆= 490 nm) ⁎⁎ ⁎⁎ ⁎⁎ ⁎ ⁎ ⁎ miR-control miR-195 miR-195 + pcDNA miR-195 + pcDNA-HMGA2 0 h 24 h 48 h EC9706 1.5 0.5 0.0 1.0 OD value (휆= 490 nm) ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎⁎ (c) (d) Annexin V-FITC EC109 15 5 0 10 Apoptosis rate (%) PI miR-control 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 104 103 102 101 100 104 103 102 101 100 104 103 102 101 100 104 103 102 101 100 miR-195 miR-195 + pcDNA miR-195 + pcDNA-HMGA2 ⁎⁎⁎ ⁎⁎⁎ miR-control miR-195 miR-195 + pcDNA miR-195 + pcDNA-HMGA2 miR-control miR-195 EC9706 Annexin V-FITC 15 5 0 10 Apoptosis rate (%) PI miR-195 + pcDNA miR-195 miR-control miR-195 miR-195 + pcDNA miR-195 + pcDNA-HMGA2 + pcDNA-HMGA2 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 104 103 102 101 100 104 103 102 101 100 104 103 102 101 100 104 103 102 101 100 ⁎⁎⁎ ⁎⁎ (e) (f) Figure 3: Overexpression of HMGA2 relieved the eﬀect of miR-195 on proliferation and apoptosis in EC cell lines. 3. Results EC109 and EC9706 cells were transfected with miR-control or miR-195 mimics or cotransfected miR-195 mimics with pcDNA or pcDNA-HMGA2. The protein expressions of HMGA2 in EC109 (a) and EC9706 (b) cells were detected by western blot. Cell proliferation in EC109 (c) and EC9706 (d) cells was assessed by MTT assay at 24 and 48 h. Cell apoptosis in EC109 (e) and EC9706 (f) cells was detected by ﬂow cytometry via double staining of cells with Annexin V-FITC and propidium iodide (PI) at 48 h. Values are presented as the mean ± SD, n = 3. ∗∗P < 0 01, ∗∗∗P < 0 001. EC9706 HMGA2 훽-actin miR-control miR-195 miR-195 + pcDNA miR-195 + pcDNA-HMGA2 (b) (a) 1.5 0.5 0.0 0 h 24 h 48 h 1.0 OD value (휆= 490 nm) ⁎⁎ ⁎⁎ ⁎⁎ ⁎ ⁎ ⁎ 0 h 24 h 48 h 1.5 0.5 0.0 1.0 OD value (휆= 490 nm) ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎⁎ miR-control miR-195 miR-195+ pcDNA miR-195 + pcDNA-HMGA2 EC109 miR-control miR-195 miR-195 + pcDNA miR-195 + pcDNA-HMGA2 EC9706 (d) miR-control miR-195 miR-195 + pcDNA miR-195 + pcDNA-HMGA2 EC9706 (d) (c) EC109 15 5 0 10 Apoptosis rate (%) ⁎⁎⁎ ⁎⁎⁎ miR-control miR-195 miR-195 + pcDNA miR-195 + pcDNA-HMGA2 (c) (d) Annexin V-FITC PI 100 101 102 103 104 100 101 102 103 104 104 103 102 101 100 104 103 102 101 100 miR-195 + pcDNA miR-195 + pcDNA-HMGA2 (e) Annexin V-FITC PI miR-195 + pcDNA miR-195 + pcDNA-HMGA2 100 101 102 103 104 100 101 102 103 104 104 103 102 101 100 104 103 102 101 100 (f) miR-195 + pcDNA-HMGA2 (e) (f) Figure 3: Overexpression of HMGA2 relieved the eﬀect of miR-195 on proliferation and apoptosis in EC cell lines. EC109 and EC9706 cells were transfected with miR-control or miR-195 mimics or cotransfected miR-195 mimics with pcDNA or pcDNA-HMGA2. The protein expressions of HMGA2 in EC109 (a) and EC9706 (b) cells were detected by western blot. Cell proliferation in EC109 (c) and EC9706 (d) cells was assessed by MTT assay at 24 and 48 h. Cell apoptosis in EC109 (e) and EC9706 (f) cells was detected by ﬂow cytometry via double staining of cells with Annexin V-FITC and propidium iodide (PI) at 48 h. Values are presented as the mean ± SD, n = 3. ∗∗P < 0 01, ∗∗∗P < 0 001. 3.4. cells at 24 h and 48 h, whereas the cell proliferation inhi- bition by miR-195 mimic was alleviated due to the trans- fection of pcDNA-HMGA2 (Figures 3(c) and 3(d)). Then, we investigated the apoptosis of EC109 and EC9706 cells by ﬂow cytometry. The results showed that miR-195 mimics signiﬁcantly increased apoptosis in both EC109 and EC9706 cells, while the miR-195-induced apoptosis rate was reverted as a result of HMGA2 overexpression (Figures 3(e) and 3(f)). 3. Results HMGA2 Overexpression Recuperated the mTOR/p70S6K Signaling Pathway Suppressed by miR-195. Previous studies have indicated that the mTOR/p70S6K signaling pathway, a major target pathway associated with HMGA2, can be activated by HMGA2 via inducing the phosphorylation of mTOR and p70S6K in human umbilical cord blood- derived stromal cells (hUCBSCs) [28]. Since miR-195 directly targets HMGA2 and regulates the expression of HMGA2, we hypothesize that miR-195 regulates the mTOR/p70s6k cells at 24 h and 48 h, whereas the cell proliferation inhi- bition by miR-195 mimic was alleviated due to the trans- fection of pcDNA-HMGA2 (Figures 3(c) and 3(d)). Then, we investigated the apoptosis of EC109 and EC9706 cells by ﬂow cytometry. The results showed that miR-195 mimics signiﬁcantly increased apoptosis in both EC109 and EC9706 cells, while the miR-195-induced apoptosis rate was reverted as a result of HMGA2 overexpression (Figures 3(e) and 3(f)). 6 6 Disease Markers p-mTOR miR-control miR-195 miR-195 + pcDNA miR-195 + pcDNA-HMGA2 t-mTOR p-p70s6k t-p70s6k 훽-actin 2.0 miR-control miR-195 miR-195 + pcDNA miR-195+ pcDNA-HMGA2 1.5 1.0 0.5 0.0 Relative protein levels p-mTOR t-mTOR p-p70s6k t-p70s6k ⁎⁎⁎ ⁎⁎⁎ ⁎⁎⁎ ⁎⁎⁎ (a) (b) p-mTOR Anti-miR-control Anti-miR-195 Anti-miR-195 + si-control Anti-miR-195 + si-HMGA2 t-mTOR p-p70s6k t-p70s6k 훽-actin Anti-miR-control Anti-miR-195 Anti-miR-195 + si-control Anti-miR-195 + siHMGA2 3 2 1 0 Relative protein levels p-mTOR t-mTOR p-p70s6k t-p70s6k ⁎⁎⁎ ⁎⁎⁎ ⁎⁎⁎ ⁎⁎⁎ (c) (d) gure 4: miR-195 suppressed the mTOR/p70s6k signaling pathway by inhibiting HMGA2 expression in EC9706 cells. (a and b) Western ot analysis for relative proteins p-mTOR, t-mTOR, p-p70s6k, and t-p70s6k in EC9706 cells transfected with miR-control or miR-19 imics or cotransfected miR-195 mimics together with pcDNA or pcDNA-HMGA2. (c and d) Western blot analysis for relative protein mTOR, t-mTOR, p-p70s6k, and t-p70s6k in EC9706 cells transfected with anti-miR-control or anti-miR-195 or cotransfected anti-miR 95 together with si-control or si-HMGA2. β-actin was used as an internal control. Values are presented as the mean ± SD, n = 3 ∗P < 0 001. 3. Results p-mTOR miR-control miR-195 miR-195 + pcDNA miR-195 + pcDNA-HMGA2 t-mTOR p-p70s6k t-p70s6k 훽-actin 2.0 miR-control miR-195 miR-195 + pcDNA miR-195+ pcDNA-HMGA2 1.5 1.0 0.5 0.0 Relative protein levels p-mTOR t-mTOR p-p70s6k t-p70s6k ⁎⁎⁎ ⁎⁎⁎ ⁎⁎⁎ ⁎⁎⁎ (a) p-mTOR Anti-miR-control Anti-miR-195 Anti-miR-195 + si-control Anti-miR-195 + si-HMGA2 t-mTOR p-p70s6k t-p70s6k 훽-actin Anti-miR-control Anti-miR-195 Anti-miR-195 + si-control Anti-miR-195 + siHMGA2 3 2 1 0 Relative protein levels p-mTOR t-mTOR p-p70s6k t-p70s6k ⁎⁎⁎ ⁎⁎⁎ ⁎⁎⁎ ⁎⁎⁎ (c) (d) (c) (d) Figure 4: miR-195 suppressed the mTOR/p70s6k signaling pathway by inhibiting HMGA2 expression in EC9706 cells. (a and b) Western blot analysis for relative proteins p-mTOR, t-mTOR, p-p70s6k, and t-p70s6k in EC9706 cells transfected with miR-control or miR-195 mimics or cotransfected miR-195 mimics together with pcDNA or pcDNA-HMGA2. (c and d) Western blot analysis for relative proteins p-mTOR, t-mTOR, p-p70s6k, and t-p70s6k in EC9706 cells transfected with anti-miR-control or anti-miR-195 or cotransfected anti-miR- 195 together with si-control or si-HMGA2. β-actin was used as an internal control. Values are presented as the mean ± SD, n = 3. ∗∗∗P < 0 001. signaling pathway by inhibiting HMGA2 expression. EC9706 cells were transfected with miR-195 mimics or miR-195 mimics+pcDNA-HMGA2 or anti-miR-195 or anti-miR-195+si-HMGA2 to investigate the eﬀect of miR- 195 on mTOR and p70s6k. As presented in Figures 4(a) and 4(b), western blot showed that miR-195 overexpression obvi- ously suppressed the phosphorylation of mTOR and p70s6k, which was alleviated by HMGA2 overexpression. In addition, downregulation of miR-195 dramatically promoted the phos- phorylationof mTOR and p70s6k, which was inhibited by HMGA2 silencing (Figures 4(c) and 4(d)). These data suggested that HMGA2 overexpression strikingly relieved the inhibition eﬀect of miR-195 on the mTOR/p70S6K signaling pathway. EC9706 Cells. To investigate the role of the mTOR/p70S6K signaling pathway in cell proliferation and apoptosis, EC9706 cells were treated with 100 nmol/l rapamycin, a spe- ciﬁc inhibitor of the mTOR/p70S6K signaling pathway, for 24 h. MTT assay, ﬂow cytometry, and western blot were used to evaluate cell proliferation, apoptosis, and the related pro- tein of EC9706 cells, respectively. EC9706 cells treated with rapamycin showed a signiﬁcant decrease of cell viability (Figure 5(a)) and Ki-67 expression (Figure 5(b)) compared with those of the NC group. Moreover, rapamycin signiﬁ- cantly increased apoptosis rates (Figure 5(c)) and Bax/Bcl-2 ratio (Figure 5(d)) of EC9706 cells. Besides, rapamycin was found to have no obvious eﬀects on miR-195 and HMGA2 expression (Figures 5(e) and 5(f)). 3. Results (d) Western blot anal and Bax expression. (e) qRT-PCR was used to determine the expressions of miR-195 and HMGA2 mRNA. (f) Western blo HMGA2 t i i β ti d i t l t l V l t d th ± SD 3 ∗∗P < 0 01 ∗∗∗ NC Rapamycin 1.5 1.0 0.5 0.0 Apoptosis rate (%) ⁎⁎⁎ 훽-actin Bcl-2 Bcl-2 Bax Rapamycin NC Bax 2.5 ⁎⁎⁎ ⁎⁎⁎ 2.0 1.5 1.0 0.5 0.0 Relative protein levels (c) NC Rapamycin miR-195 HMGA2 mRNA 1.5 1.0 0.5 0.0 Relative mRNA expression levels (e) (d) HMGA2 훽-actin NC Rapamycin 1.5 1.0 0.5 0.0 Relative HMGA2 protein levels (f) (f) (e) Figure 5: Rapamycin inhibits the cell proliferation and induced apoptosis via suppression of the mTOR/p70S6K signaling pathway in EC9706 cells. EC9706 cells were treated with 100 nmol/l rapamycin for 24 h. (a) Cell viability was examined by MTT assay. (b) Western blot analysis of Ki-67 expression. (c) Apoptosis rates in EC9706 cells were assessed by ﬂow cytometry. (d) Western blot analysis of Bcl-2 and Bax expression. (e) qRT-PCR was used to determine the expressions of miR-195 and HMGA2 mRNA. (f) Western blot analysis of HMGA2 protein expression. β-actin was used as an internal control. Values are presented as the mean ± SD, n = 3. ∗∗P < 0 01, ∗∗∗P < 0 001. 3. Results All the data indicated that the mTOR/p70s6k signaling pathway may be involved in the proliferation and apoptosis modulated by miR-195 in EC9706 cells. 3.5. Inhibition of the mTOR/p70S6K Signaling Pathway Suppresses Cell Proliferation and Promotes Apoptosis in 7 NC Rapamycin ⁎⁎⁎ 1.5 1.0 0.5 0.0 OD value (휆 = 490 nm) 1.5 Ki67 훽-actin 1.0 0.5 0.0 NC Rapamycin Relative Ki67 protein levels ⁎⁎⁎ (a) (b) NC Rapamycin 1.5 1.0 0.5 0.0 Apoptosis rate (%) ⁎⁎⁎ 훽-actin Bcl-2 Bcl-2 Bax Rapamycin NC Bax NC Rapamycin 2.5 ⁎⁎⁎ ⁎⁎⁎ 2.0 1.5 1.0 0.5 0.0 Relative protein levels (c) (d) NC Rapamycin miR-195 HMGA2 mRNA 1.5 1.0 0.5 0.0 Relative mRNA expression levels HMGA2 훽-actin NC Rapamycin 1.5 1.0 0.5 0.0 Relative HMGA2 protein levels (e) (f) gure 5: Rapamycin inhibits the cell proliferation and induced apoptosis via suppression of the mTOR/p70S6K signaling pathway in C9706 cells. EC9706 cells were treated with 100 nmol/l rapamycin for 24 h. (a) Cell viability was examined by MTT assay. (b) Western ot analysis of Ki-67 expression. (c) Apoptosis rates in EC9706 cells were assessed by ﬂow cytometry. (d) Western blot analysis of Bcl-2 d Bax expression. (e) qRT-PCR was used to determine the expressions of miR-195 and HMGA2 mRNA. (f) Western blot analysis of MGA2 protein expression. β-actin was used as an internal control. Values are presented as the mean ± SD, n = 3. ∗∗P < 0 01, ∗∗∗P < 0 001. 7 isease Markers Disease Markers NC Rapamycin ⁎⁎⁎ 1.5 1.0 0.5 0.0 OD value (휆 = 490 nm) 1.5 Ki67 훽-actin 1.0 0.5 0.0 NC Rapamycin Relative Ki67 protein levels ⁎⁎⁎ (a) (b) 1.5 Ki67 훽-actin 1.0 0.5 0.0 NC Rapamycin Relative Ki67 protein levels ⁎⁎⁎ (b) (a) (b) NC Rapamycin 1.5 1.0 0.5 0.0 Apoptosis rate (%) ⁎⁎⁎ 훽-actin Bcl-2 Bcl-2 Bax Rapamycin NC Bax NC Rapamycin 2.5 ⁎⁎⁎ ⁎⁎⁎ 2.0 1.5 1.0 0.5 0.0 Relative protein levels (c) (d) NC Rapamycin miR-195 HMGA2 mRNA 1.5 1.0 0.5 0.0 Relative mRNA expression levels HMGA2 훽-actin NC Rapamycin 1.5 1.0 0.5 0.0 Relative HMGA2 protein levels (e) (f) Figure 5: Rapamycin inhibits the cell proliferation and induced apoptosis via suppression of the mTOR/p70S6K signaling EC9706 cells. EC9706 cells were treated with 100 nmol/l rapamycin for 24 h. (a) Cell viability was examined by MTT assay. blot analysis of Ki-67 expression. (c) Apoptosis rates in EC9706 cells were assessed by ﬂow cytometry. References [1] D. M. Parkin, F. Bray, J. Ferlay, and P. Pisani, “Estimating the world cancer burden: Globocan 2000,” International Journal of Cancer, vol. 94, no. 2, pp. 153–156, 2001. [2] A. Jemal, R. Siegel, J. Xu, and E. Ward, “Cancer statistics, 2010,” CA: A Cancer Journal for Clinicians, vol. 60, no. 5, pp. 277–300, 2010. pp g g The mTOR/p70s6k signaling pathway is implicated in cell proliferation, invasion, and metastasis in many cancers including squamous cell carcinomas of the head and neck [35], hepatocellular carcinomas [36], breast cancer [37], and EC [38]. Jia et al. showed that miR-223 restrained the cell proliferation via the mTOR/p70s6k signaling path- way by targeting IGF-1R in HeLa cells [39]. The present study revealed that overexpression of miR-195 suppressed the key components mTOR and p70S6K phosphorylation. HMGA2 overexpression rescued the inhibition of mTOR and p70S6K phosphorylation by miR-195 overexpression in EC9706 cells, whereas HMGA2 downregulation inhib- ited the promotion of mTOR and p70S6K phosphorylation by miR-195 downregulation. These results manifested that miR-195 inhibited the mTOR/p70s6k signaling pathway by targeting HMGA2. [3] R. Montesano, M. Holestein, and P. Hainaui, “Genetic alter- ations in esophageal cancer and their relevance to etiology and pathogenesis: a review,” International Journal of Cancer, vol. 69, no. 3, pp. 225–235, 1996. [4] T. Kim, S. R. Grobmyer, R. Smith et al., “Esophageal can- cer—the ﬁve year survivors,” Journal of Surgical Oncology, vol. 103, no. 2, pp. 179–183, 2011. [5] B. M. Ryan, A. I. Robles, and C. C. 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Authors’ Contributions of miR let-7, was upregulated in EC cells and was involved in EC carcinogenesis [34]. However, the relationship between miR-195 and HMGA2 in EC has not been reported. In our study, dual-luciferase assay demonstrated that there existed a signiﬁcant decrease of the relative luciferase activity for the reporter vector pMIR-REPORT containing wild-type 3′-UTR of HMGA2 but no obvious change in the mutant. The level of HMGA2 protein was inversely correlated with miR-195 expression. Moreover, cell proliferation inhibition and apoptosis of EC cells induced by overexpression of miR-195 were alleviated by overexpression of HMGA2. These ﬁndings indicated that HMGA2 was a direct target of miR-195, and miR-195 played the potential tumor- suppressive role in EC cells via targeting HMGA2. Yong Li and Dapeng Wu designed the experiments. Pei Wang and Xiaohui Li performed the experiments and acquired the data. Gongning Shi prepared the manuscript. Yong Li and Dapeng Wu contributed equally to this work. 5. Conclusion [12] Y. Cai, T. He, L. Liang, X. Zhang, and H. Yuan, “Upregulation of microRNA-337 promotes the proliferation of endometrial carcinoma cells via targeting PTEN,” Molecular Medicine Reports, vol. 13, no. 6, pp. 4827–4834, 2016. In summary, our ﬁndings demonstrated that miR-195 expression was downregulated in EC cells and inversely correlated with HMGA2 levels. Overexpression of miR-195 evidently restrained proliferation and promoted apoptosis of EC cells through the mTOR/p70s6k signaling pathway by targeting HMGA2. Therefore, miR-195 may function as a tumor suppressor and be developed as the potential diagnostic and therapeutic target for EC. [13] Z. Wang, Q. Qiao, M. Chen et al., “miR-625 down- regulation promotes proliferation and invasion in esophageal cancer by targeting Sox2,” FEBS Letters, vol. 588, no. 6, pp. 915–921, 2014. [14] Z. Li, H. Wang, Z. Wang, and H. 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It is reported that more than 30% of human gene expression are related to miRs [6]. Numerous studies have suggested that miRs can regulate carcinogenesis and progression of malig- nant cancers by target binding to the 3′-UTR of mRNAs [29]. It has been found that miR-195 has a crucial role in the progression of diﬀerent cancers. For instance, miR-195 expression was signiﬁcantly decreased in cervical cancer cell lines and overexpression of miR-195 exerted a signiﬁcant inhibitory impact on the proliferation of cervical cancer cells and induced apoptosis in vitro by targeting cyclin D1 [16]. miR-195 inhibited the tumor angiogenesis and growth by HMGA2 was found upregulated in various cancers, such as lung cancer [31], breast cancer [32], and oral squamous cell carcinomas [33]. Liu et al. reported that HMGA2, a target Disease Markers 8 of miR let-7, was upregulated in EC cells and was involved in EC carcinogenesis [34]. However, the relationship between miR-195 and HMGA2 in EC has not been reported. In our study, dual-luciferase assay demonstrated that there existed a signiﬁcant decrease of the relative luciferase activity for the reporter vector pMIR-REPORT containing wild-type 3′-UTR of HMGA2 but no obvious change in the mutant. The level of HMGA2 protein was inversely correlated with miR-195 expression. Moreover, cell proliferation inhibition and apoptosis of EC cells induced by overexpression of miR-195 were alleviated by overexpression of HMGA2. These ﬁndings indicated that HMGA2 was a direct target of miR-195, and miR-195 played the potential tumor- suppressive role in EC cells via targeting HMGA2. Conflicts of Interest Torbenson, “mTOR and P70 S6 kinase expression in pri- mary liver neoplasms,” Clinical Cancer Research, vol. 10, no. 24, pp. 8421–8425, 2004. [21] H. Heinonen, A. Nieminen, M. 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https://openalex.org/W4392787508	https://vinculategica.uanl.mx/index.php/v/article/download/463/686	es		Políticas Públicas como estrategia de Fiscalización de la Obligación Predial en el Municipio de La Barca, Jalisco	Vinculategica efan	2,024	cc-by	6,118	Políticas Públicas como estrategia de Fiscalización de la Obligación Predial en el Municipio de La Barca, Jalisco (Public Politics as an Inspection Strategy of Property Obligation in the Municipality of La Barca, Jalisco) Luz María Galán-Briseño1; Miguel Castro-Sánchez2 y Leticia Villarruel-Rodríguez3 Universidad de Guadalajara – Centro Universitario de la Ciénega (México), luz.galan@academicos.udg.mx, https://orcid.org/0000-0002-9803-4406 2 Universidad de Guadalajara – Centro Universitario de la Ciénega (México), m.castro@academicos.udg.mx, https://orcid.org/0000-0002-3604-3729 3 Universidad de Guadalajara – Escuela Preparatoria Regional de Jamay (México), leticia.villaruel@sems.udg.mx, https://orcid.org/0009-0005-4664-2142 1 Información revisada por pares Fecha de recepción: 9 de Abril del 2023 Fecha de aceptación: 18 de Mayo del 2023 Fecha de publicación en línea: 31 de Enero del 2024 DOI: https://doi.org/10.29105/vtga10.1-463 Resumen La Hacienda Municipal requiere desempeñar con eficiencia los procesos de recaudación para dar respuesta a las necesidades locales, para ello deben implementar acciones que impulsen a lograr estos propósitos. El objetivo busca proponer políticas públicas como estrategia para eficientizar los procesos de recaudación del impuesto predial, el trabajo presenta un enfoque de metodología mixta y un diseño que contempla la aplicación de los métodos inductivo, analítico, deductivo y síntesis, además de las técnicas de observación, análisis documental, entrevista y encuesta, ahora bien, es de tipo no experimental, de campo y longitudinal, se apoyó en el cuestionario como material y el el muestreo por conveniencia. Los resultados destacan que los funcionarios reconocen un rezago en los cobros del predial, la necesidad de incorporar las tecnologías para eficientizar el padrón y pagos catastrales, por su parte, los contribuyentes expresan las deficiencias administrativas para el pago del predial, el desacuerdo en los programas de apoyo en que se ejerce la contribución. Para concluir, la ley de ingresos municipales establece la cantidad esperada de recaudación del impuesto predial, vistos los resultados del pago de la contribución, constatamos la urgencia de innovar los procesos de recolección del impuesto a través de la aplicación de políticas públicas. Abstract The Municipal Treasury requires efficient performance of collection processes to respond to local needs, for this they must implement actions that promote achieving these purposes. The objective seeks to propose public policies as a strategy to streamline property tax collection processes, the work presents a mixed methodology approach and a design that contemplates the application of inductive, analytical, deductive and synthesis methods, in addition to observation techniques, documentary analysis, interview and survey, however, it is of a non-experimental, field and longitudinal type, it is supported by the questionnaire as material and the convenience sampling. The results highlight that officials recognize a lag in property tax collections, the need to incorporate technologies to streamline the register and cadastral payments, for their part, taxpayers express administrative deficiencies for property tax payments, disagreement in the programs of support in which the contribution is made. To conclude, the municipal income law establishes the expected amount of property tax collection, given the results of the payment of the contribution, we verify the urgency of innovating the tax collection processes through the application of public policies. Palabras clave: Políticas Públicas, Recaudación, Impuesto Predial, Municipio. Códigos JEL: M15, M42, M48. Key words: Public Policies, Collection, Property Tax, Municipality. JEL Codes: M15, M42, M48. e-ISSN: 2448-5101 VinculaTégica EFAN https://vinculategica.uanl.mx/ Vol. 10. Núm. 1 Enero-Febrero 2024 106 Introducción Tanto los gobiernos estatales y federales, así como los municipios manifiestan dificultades en los procesos de recaudación fiscal, las deficiencias en las políticas de fiscalización impiden a estos gobernantes hacerse llegar de los recursos para atender su propósito sustancial, proporcionar servicios a la ciudadanía. Este trabajo se centra en la obligación predial, que compete a los municipios, de ahí que, en el municipio de La Barca, Jalisco, la carencia de una base de datos actualizada del registro predial y catastral, tiene consecuencias en la disminución en la recaudación del impuesto, por consiguiente, impide el crecimiento en el desarrollo de infraestructura como pavimentación y alumbrado público, de la misma manera frena la calidad de los servicios como el aseo público, el manteamiento de parques y jardines, por citar algunos. El impuesto predial de competencia municipal que grava los bienes inmuebles registrados en la oficina de catastro de las localidades es cobrado por la autoridad municipal y pagado por el ciudadano que tiene registrado un bien inmueble, por lo que este tributo es relevante para el municipio en virtud de que con el ingreso de estos recursos el ayuntamiento solventa las necesidades básicas de la ciudadanía lo cual contribuye al desarrollo económico y social. El objetivo del trabajo busca proponer a la autoridad recaudatoria la aplicación de políticas públicas como estrategia de fiscalización para una efectiva recaudación que le permita a la autoridad disponer de los recursos económicos de la mayoría de estos adeudos atrasados y vigentes para hacer frente a las necesidades del municipio. La hipótesis describe que la falta de actualización y adaptación de los procesos administrativos de fiscalización del impuesto predial al entorno hoy vivido – en virtud de que en algunos municipios de Jalisco ya tienen implementadas formas de recaudación digital de este precepto, procedimiento que en la actualidad en La Barca no se ha contemplado–, es causante de la acumulación en los diversos periodos de gobierno de las cuentas por cobrar por este precepto el cual obstaculiza brindar a la ciudadanía programas y servicios de manera óptima. Por otra parte, la implementación de políticas públicas incentiva a los obligados a estar al actualizados en el cumplimiento de su obligación y contribuir en la mejora de los servicios públicos. Por consiguiente, este trabajo es producto de una investigación profunda y arroja resultados para una mejor comprensión de la importancia de la implementación de las políticas públicas como medio para resolución de problemáticas de las organizaciones, en este terreno la implementación brinda la oportunidad al gobierno municipal de recuperar y evitar el rezago de la deuda de este precepto y así cumplir con la encomienda de brindar a los ciudadanos servicios de altura, mediante e-ISSN: 2448-5101 VinculaTégica EFAN https://vinculategica.uanl.mx/ Vol. 10. Núm. 1 Enero-Febrero 2024 107 la propuesta de la adopción efectiva de las políticas públicas. Políticas Públicas como Agentes de Cambio Social (Ruiz y Cadenas, 2019) examinan las políticas públicas como medio de transformación social en el que la convección de los sistemas gobierno y ciudadanía converge en beneficio de los propósitos de estos sectores, ahora se mencionan los elementos más sobresalientes. Las políticas públicas hacen alusión al ámbito privilegiado de realizar “pactos” entre Estado y sociedad, priorizando el ejercicio óptimo de las atribuciones del Estado. Dentro de las prioridades de las políticas públicas se debe considerar la atención de las necesidades de la demarcación geográfica de que se trate. Las políticas públicas comprenden los lineamientos que debe respetar la autoridad con el propósito de cumplir con su función, siempre pugnando por la mejora de las condiciones de vida de los ciudadanos. Las medidas desarrolladas para recaudar de forma adecuada el impuesto predial, surgen del lineamiento constitucional de México, seguido de lo que establezca el fundamento legal del estado de Jalisco, a continuación, se atiende el lineamiento que establezca la administración municipal y por último, la Ley de Catastro del Estado de Jalisco. De modo que, el fundamento legal constitucional lo encontramos en el artículo 115 fracción I y fracción IV párrafo a, mientras que en la Constitución de Jalisco se encuentra en el 88 y 89, por otro lado, en la Ley de Hacienda el fundamento se dicta en el artículo 1º y 23, para posteriormente revisar la Ley de Catastro de Jalisco en los artículos 1º al 9º, 11º, 24º y 25º. Todo lo anterior hace referencia a la legislación federal y estatal, mientras que las leyes de ingresos municipales delimitan los importes que se recaudará derivados de impuestos y demás contribuciones correspondientes en el año fiscal de cada período. Es por esto que, en el presente estudio, fue necesario revisar lo descrito en la Ley de Ingresos del Municipio de La Barca, Jalisco en los ejercicios fiscales 2017, 2018, 2019 y 2020. El Impuesto Predial en Relación con la Constitución Política de los Estados Unidos Mexicanos En la Constitución Política de los Mexicanos, el gobierno refiere que los municipios denominados ayuntamientos están conformados por un Presidente o Presidenta municipal, regidores y síndicos. Por otro lado, se faculta a los ayuntamientos para el ejercicio de los preceptos que describen libremente las acciones de fiscalización, que se conforman de los rendimientos de los bienes propiedad del municipio, de la misma manera faculta a los municipios para la recaudación de las contribuciones sobre los bienes inmobiliarios. e-ISSN: 2448-5101 VinculaTégica EFAN https://vinculategica.uanl.mx/ Vol. 10. Núm. 1 Enero-Febrero 2024 108 Para darle fundamento a lo anterior, es necesario apegarse a lo establecido en Constitución Política de los Mexicanos (1917) que en su artículo 115, fracciones I y IV plantean: En primera instancia el apartado de la fracción I plantea que los municipios deberán ser gobernados por los Ayuntamientos de elección popular directa, integrados por el Presidente o Presidenta Municipal, así también por el número de regidurías y sindicaturas que la ley específica, en atención al principio de paridad. Las atribuciones que este mandato Constitucional otorga a los gobiernos municipales que deberán ser ejercidos por los Ayuntamientos con exclusividad y bajo ninguna circunstancia intervendrá autoridad intermedia entre éste y el gobierno del Estado. A su vez, lo señalado en la fracción IV, describe que los municipios administrarán libremente sus contribuciones y el ejercicio de las mismas, las cuales se formarán de los rendimientos de los bienes que les pertenezcan, así como de las contribuciones y otros ingresos que las legislaturas establezcan a su favor. El Impuesto Predial en Relación con la Constitución Política para el Estado de Jalisco De la misma manera que la carta magna faculta a los ayuntamientos para ejercer sus facultades libremente y por medio de ella recaudar el impuesto predial, (la Constitución Política del Estado de Jalisco fechada el 10 de abril de 2014), también faculta a los municipios para gestionar su ejercicio tributario que comprende la recaudación de las contribuciones de propiedad inmobiliaria, y esto encuentra su fundamento en el artículo 88º y adicionalmente el artículo 89º, dicta la autoridad facultada para visar las normativas de los ingresos de los municipios. El Municipio, Ayuntamiento y Hacienda Pública Municipal, Medios de Instrumentación de la Función Pública El H. Congreso del Estado de Jalisco en la Ley de Gobierno y Administración Pública Municipal del Estado de Jalisco (2019), en su artículo 2 refiere que el municipio representa un nivel de gobierno, en el artículo 3 señala que será gobernado por un Ayuntamiento electo de forma popular y en el artículo 10 menciona la forma como estará conformado el Ayuntamiento de los municipios: Presidente Municipal, Síndico y los regidores que marca la Ley Estatal electoral. El fundamento de la Hacienda Pública Municipal se describe en la Ley de Hacienda Municipal del Estado de Jalisco (2019) en el artículo 1º y en el artículo 23º se mencionan las funciones del titular de Hacienda Pública Municipal. La Figura de Catastro Para abordar a la dependencia de Catastro es necesario analizar lo que está plasmado en la Ley de Catastro dictada por el Congreso del Estado de Jalisco (2019), para su estudio, es necesario separar e-ISSN: 2448-5101 VinculaTégica EFAN https://vinculategica.uanl.mx/ Vol. 10. Núm. 1 Enero-Febrero 2024 109 los fragmentos aplicables que aporta dicha Ley. A continuación se describen los artículos más relevantes. Para el caso del artículo 2º, la citada Ley infiere que el término catastro distingue los elementos cualitativos y cuantitativos de las propiedades, que son considerados como inmuebles y se encuentran en la demarcación geográfica de los municipios. Es importante mantener información de dichas propiedades para su valoración en caso de que sean parte de procesos fiscales realizados por las autoridades municipales. Para el caso del artículo 3º, las personas que sean propietarias de los inmuebles considerados en el artículo 2º serán responsable de cumplir los lineamientos establecidos en la Ley respectiva y se ubiquen en el Estado. En el artículo 7º se considera toda la documentación que correspondan a los inmuebles objeto de la Ley y se encuentren ubicados en el territorio del municipio. Contribuciones y Delimitación del Impuesto Predial En atención a lo establecido en la Ley de Hacienda Municipal del Estado de Jalisco (2019) dictada por el Congreso del Estado, en el artículo 2º describe a los contribuyentes, en el artículo 3º a los impuestos, en el artículo 92º el objeto del impuesto predial y en el artículo 93º los sujetos obligados al pago de dicho impuesto. En el artículo 92º, se describe el objeto del impuesto predial, que considera las distintas formas de propiedad que se tengan de un inmueble, así como las construcciones realizadas sobre los inmuebles que son base de referencia. El Impuesto Predial en La Barca, Jalisco El estudio de este apartado tiene su origen en la Ley de Ingresos del Municipio de La Barca, Jalisco, para el propósito la oficina de transparencia municipal proporcionó las estadísticas de recaudación predial correspondiente a los ejercicios fiscales 2017 al 2020, la cual se tomó de base para la comparación de los ingresos que se esperaban recaudar y lo recaudado, por lo anterior, se determinó el rezago en las cuentas por cobrar de este precepto impositivo situación que justifica el objetivo de esta investigación. El Municipio de La Barca, Jalisco Según datos generados por el Instituto de Información Estadística y Geográfica de Jalisco (IIEG, 2019), mediante la revisión del municipio en marzo de 2019, la ciudad de La Barca pertenece a la región Ciénega, en el 2015 el censo poblacional confirmó 65,055 personas de las cuales el 48.4 por ciento son hombres y el 51.6 por ciento mujeres, lo cual podemos observar en la tabla 1. e-ISSN: 2448-5101 VinculaTégica EFAN https://vinculategica.uanl.mx/ Vol. 10. Núm. 1 Enero-Febrero 2024 110 Clave Tabla 1. Habitantes por género en el municipio de la Barca, Jalisco 2010- 2015. La Barca, Jalisco Población 2025 No. Municipio/localidad Población Total % en el Hombres Mujeres total 2010 municipio 018 LA BARCA 64,269 65,055 100 31,487 33,568 Fuente: IIEG, Instituto de Información Estadística y Geográfica del Estado de Jalisco con base en INEGI, Censo de Población y vivienda 2010. Encuesta Intercensal 2015. La estimación para el año 2020 es que la población aumentaría a 71,268 habitantes, 34,501 masculinos y 36,766 femeninos. Tanto en los datos del año 2015 como en la proyección al 2020, la presencia de las mujeres es ligeramente superior. Según la Enciclopedia de los Municipios y Demarcaciones Territoriales de México (Secretaría de Gobernación, 1988) el municipio de La Barca, Jalisco surge por la necesidad de traslado de los habitantes de Guadalajara hacia las áreas de comercio y diversa proveeduría en la ciudad de México, pues los usuarios solicitaron a la nueva Audiencia del Reino de Nueva Galicia, un acuerdo confiable y explícito para el cruce de los poderosos ríos Santiago y Lerma. De modo que, la Audiencia dio respuesta estableciendo una barca –canoa– para el cruce del río Lerma, el nombre del lugar en que ocurría tal cruce y que se convirtió en un interesante centro agrícola, fue la antigua Santa Mónica de La Barca, que al inicio fue simplemente un medio de paso, pero después se convirtió en un espacio de descanso muy importante en el sitio llamado por los arrieros e indios de la comarca. Metodología En atención al objetivo e hipótesis, el trabajo se realizó con un enfoque de investigación mixta, así mismo, para el diseño se utilizó el método lógico de la ciencia que combinó los métodos inductivo permitió evaluar los ingresos de la Hacienda Pública Municipal por concepto de impuesto predial, analítico ayudó a recolectar información por separado en forma individual de las variables, las cuales fueron las políticas públicas y la recaudación del impuesto predial, deductivo contribuyó a revisar la información recabada en el método analítico y síntesis el procedimiento mental permitió integrar los elementos previamente analizados. Además, el diseño se complementó con la aplicación de las técnicas de observación de datos, sucesos y fenómenos sobre el estudio, análisis documental ayudó a describir la problemática e identificar soluciones, entrevista permitió obtener información de los servidores públicos relacionada con las acciones que realiza la Hacienda Pública del municipio para la fiscalización del impuesto predial y encuesta ayudó para conocer el testimonio de los contribuyentes sobre el procedimiento de recaudación de esta obligación fiscal. Ahora bien, este trabajo es de tipo no experimental sin manipulación de las variables, de campo e-ISSN: 2448-5101 VinculaTégica EFAN https://vinculategica.uanl.mx/ Vol. 10. Núm. 1 Enero-Febrero 2024 111 que permitió la obtención de testimonios, longitudinal para el estudio de los datos en diferentes tiempos y descriptivo contribuyó en la descripción de las políticas públicas que proporcionen aportación a la solución de la problemática. Cabe resaltar que, por la lógica de intervención del estudio, la entrevista fue aplicada únicamente a dos funcionarios que tienen relación directa con la fiscalización del Impuesto Predial: Director del Catastro Municipal y Presidente de la Comisión de Catastro. Para la aplicación de la encuesta, el tamaño y muestreo de la muestra fue por conveniencia, para ello, se aplicaron encuestas en un lapso de 90 días hábiles a 100 personas que acudieron a pagar el importe de su impuesto predial en la presidencia municipal de La Barca Jalisco. Resultados En este apartado se muestran cifras sobre el nivel de cumplimiento de los contribuyentes del municipio, respecto al pago de la cuenta predial de los años 2017-2020. En la Tabla 2 se muestra concentrado de los contribuyentes con cuenta de la contribución predial, así como también de aquellos que efectivamente han cumplido con su responsabilidad. En cuanto al número de contribuyentes obligados, durante los años 2017, 2018 y 2019 la base se mantuvo en alrededor de 23,500, mientras que en el año 2020 la base creció hasta 25,565; el total de obligados en 2020 creció un 8.7% con respecto al promedio de los años pasados. La estadística de obligados al corriente en la liquidación del impuesto durante los años 2017 al 2019 estuvo cerca del 49% en promedio, mientras que en 2020 los contribuyentes cumplidos representaron sólo el 43%. En 2020 la base creció con respecto a los tres años anteriores y los contribuyentes cumplidos bajaron con respecto a los mismos periodos. Tomando en consideración las cifras incluidas en la Tabla 2 y lo ocurrido en el año 2020, es necesario atender las áreas de mejora relacionadas, particularmente en lo referente al destino de los recursos relacionados con el ingreso de las aportaciones prediales y al cumplimiento de responsabilidades imputadas al gobierno, sobre todo en lo relacionado con el fomento de la actividad económica. Tabla 2 Concentrado de cuentas del impuesto predial en el municipio de La Barca, Jalisco de los períodos 2017, 2018, 2019 y 2020. Contribuyentes 2017 2018 2019 2020 Obligados 23,488 23,788 23,352 25,565 Al corriente 11,033 11,205 11,838 10,981 Faltantes 12,455 12,673 11,514 14,584 Fuente: elaboración propia con información proporcionada por la oficina de Catastro del municipio de La Barca, Jalisco. Adicionalmente se realizó el estudio sobre la recaudación estimada y la recaudada, con e-ISSN: 2448-5101 VinculaTégica EFAN https://vinculategica.uanl.mx/ Vol. 10. Núm. 1 Enero-Febrero 2024 112 información de la Ley de Ingresos municipal, además de los datos proporcionados por la oficina de Catastro, la información se puede observar en la Tabla 3. La diferencia en los años 2017 y 2018 representó un faltante en la recaudación, pues representó el 23.5% y 26% respectivamente. El comparativo en los años 2019 y 2020 no se pudo determinar debido a que en el año 2019 se recaudaron $9,903,077.47, pero en este año se presentó el inconveniente de que en la Ley de Ingresos no se refleja la cantidad estimada de recaudación por rubro de aportación predial, mientras que para el 2020 tampoco fue posible conocer la cantidad recaudada por concepto de predial, ya que aún no se tenía el cálculo exacto al momento de la investigación. Tabla 3 Concentrado de recaudación (Miles de pesos) del impuesto predial en el municipio de La Barca, Jalisco de los años 2017, 2018, 2019 y 2020. Cantidad 2017 2018 2019 2020 Estimada 11,500 12,075 8,191 Recaudada 8,788 8,940 9,903 Diferencia 2,712 3,135 Fuente: elaboración propia con información proporcionada por la oficina de Catastro del municipio de La Barca, Jalisco, además de la obtenida de la Ley de Ingresos correspondiente. En la información incluida en las Tablas 2 y 3 hay situaciones que se deben observar: • La cantidad de contribuyentes que no están al día en cubrir las cuotas de fiscalización predial es alta, de 2017 a 2019 osciló en el 51%, mientras que en 2020 se incrementó a 57%. • La diferencia entre el impuesto predial estimado y recaudado sigue mostrando una situación desfavorable, que pasó de 23.5% a 26% en los años 2017 y 2018. En caso de haber contado con toda la información de los años 2019 y 2020, seguramente se hubiera observado una tendencia similar, tal y como se observó en el concentrado de contribuyentes. Considerando la información obtenida de los dos funcionarios con mayor injerencia en el proceso de tributación de la obligación predial en el Ayuntamiento de La Barca, Jalisco, es necesario hacer las siguientes precisiones: • Su formación académica se aleja del perfil deseable para desempeñar los nombramientos públicos que ostentan, lo recomendable sería una formación en Administración Pública, Finanzas Públicas o Contaduría. La determinación adecuada de las contribuciones conjuntamente con la administración efectiva de las mismas, son funciones que deberían desempeñar de forma efectiva. e-ISSN: 2448-5101 VinculaTégica EFAN https://vinculategica.uanl.mx/ Vol. 10. Núm. 1 Enero-Febrero 2024 113 • La antigüedad que tienen en sus nombramientos guarda una relación directa con los ejercicios de su nombramiento en las dinámicas municipales, en uno de los casos es evidente la mención y en el otro caso la “intermitencia” se asocia a distintos momentos o periodos de gobierno en los cuales desempeñó las actividades correspondientes. • Con respecto a la determinación del impuesto, sólo uno de los funcionarios mostró amplio conocimiento. • Sobre el procedimiento para la recaudación del impuesto, mostraron conocimiento suficiente y destacaron las acciones concretas que realizan los contribuyentes. • Estuvieron de acuerdo en que la implementación de políticas públicas puede mejorar la recaudación e incluso hicieron recomendaciones específicas. Sugirieron informar a los contribuyentes sobre sus adeudos, solicitar su opinión para la aplicación del impuesto en partidas específicas y otorgar, en caso de que sea posible, la aplicación de descuentos. También se sugirió la actualización de datos y el ejercicio del Procedimiento Administrativo de Ejecución (PAE). La disposición mostrada por los funcionarios respecto a la implementación de políticas públicas que mejoren la recaudación guarda un enfoque directo con el objetivo general de esta investigación. Una vez concluido el análisis de los resultados de las técnicas aplicadas a los contribuyentes seleccionados fue posible identificar las siguientes fortalezas: • El 84% está de acuerdo con el importe a cubrir por el precepto de la obligación inmobiliaria, lo cual demuestra la concientización de los contribuyentes para atender dicha responsabilidad y la equidad y de la contribución. • El 68% está de acuerdo con los medios de pago que actualmente se tienen implementados, aunque debería promoverse una diversificación para elevar el porcentaje. • El 63% de los sujetos de estudio considera que es eficiente el proceso de la fiscalización del impuesto a los predios. • El 100% está de acuerdo con la atención que recibe de la oficina de Catastro cuando acude a pagar su impuesto predial. • El 88% considera que el tiempo de espera que debe invertir en el pago de su contribución es adecuado. • El 100% manifestó no tener problema con los errores que se llegan a generar en el proceso de impresión de los recibos, señal de que son mínimos o inexistentes. • El 100% de los contribuyentes estuvo de acuerdo en los apoyos y estímulos que otorgó el gobierno durante la pandemia COVID-19. e-ISSN: 2448-5101 VinculaTégica EFAN https://vinculategica.uanl.mx/ Vol. 10. Núm. 1 Enero-Febrero 2024 114 Por otro lado, también fue posible identificar las principales áreas de mejora que se deben considerar: • El 92% está en desacuerdo con los servicios que brinda el municipio, pues consideran que no son atendidas de apropiadamente las demandas de la ciudadanía. • El 56% de los contribuyentes considera que el recurso recaudado del impuesto predial se debe asignar para mejorar la pavimentación de las calles y el 44% considera que se deben apoyar proyectos que generen empleo. • El 90% criticó la infraestructura que se tiene en el municipio, particularmente la situación actual de los medios de comunicación. • El 95% desaprobó los trabajos ejecutados por el Ayuntamiento municipal como apoyo en el regreso a clases, particularmente en lo relativo a servicios de seguridad pública y otorgamiento de apoyos en insumos para los estudiantes. • Únicamente el 1% de los contribuyentes estuvo a favor sobre el apoyo que otorga el gobierno municipal a las actividades económicas. • El 60% de los sujetos de estudio aprueba la instalación de una practicaja para diversificar los medios de pago del impuesto predial. Las áreas de mejora deben ser atendidas para que sea posible convertirlas en factores internos favorables. Las fortalezas se deben mantener e incrementar mediante la adecuada implementación de políticas públicas. Propuesta de Política Pública como Estrategia de Fiscalización de la Contribución de los Predios Tomando como referencia el ejercicio 2020 y según datos mostrados en la Tabla 2, hubo 25,565 contribuyentes con cuentas pendientes de cubrir por contribución predial de los cuales tan sólo 10,981 pagaron su recaudación, es decir que solo el 43%, acudió a pagar su contribución, por lo tanto hay un 57% no cumplió; dicho faltante en la recaudación trajo como resultado que no se contara con recursos suficientes para el otorgamiento servicios públicos y el desarrollo de obras que permitieran contribuir significativamente en la situación de bienestar de la ciudadanía, lo cual provocó una pobre valoración de los servicios que proporciona el municipio en voz de los habitantes. Para conocer la viabilidad de la aplicación de esta política pública es necesario definir los objetivos que se busca cumplir: • Otorgar a los contribuyentes la oportunidad de integrarse en la elaboración de programas relativos a la aplicación de los ingresos obtenidos motivo del pago de sus impuestos. • Incrementar el número de contribuyentes al corriente y de la misma manera disminuir el índice de contribuyentes morosos. e-ISSN: 2448-5101 VinculaTégica EFAN https://vinculategica.uanl.mx/ Vol. 10. Núm. 1 Enero-Febrero 2024 115 Facilitar el acceso a la información sobre destino de los recursos motivados de la fiscalización • de predios, así como a las cuentas catastrales de los obligados. Contribuir a cumplir con los protocolos de sanidad y evitar las aglomeraciones. • Formulación de Políticas Pública Con el objetivo de aminorar la problemática expresada en apartados anteriores, se propone la integración y la correspondiente aplicación de una política pública consistente en el diseño de una plataforma digital que permita mantener actualizada la base de contribuyentes sujetos a la obligación del precepto predial, así también de los que están al corriente en sus pagos y los que aún faltan por cumplir; dicha plataforma busca eficientizar el proceso de recaudación. Concretamente incluye los siguientes pasos: Paso 1. Presentación y aprobación en sesión del H. Ayuntamiento de la política pública, pues requiere el visto bueno del pleno para continuar su proceso en los filtros sucesivos hasta su ejecución. Paso 2. Una vez que el H. Ayuntamiento aprobó la aplicación de la política pública pueden presentarse dos alternativas de decisión: • Migrar el total de las cuentas catastrales de forma automática, considerando que existe compatibilidad entre los registros actuales y la plataforma propuesta. • Si no es posible migrar en masa las cuentas catastrales a la plataforma, se deberá conformar un equipo de capturistas, que trabajen paralelamente con el otorgamiento de servicios públicos de atención al contribuyente, es decir que se deberán seguir trabajando con normalidad haciendo los registros en los medios existentes y posteriormente compartir dicha información con los capturistas para alimentar la plataforma. Los capturistas serán preferentemente trabajadores de otras áreas que estén disponibles en tiempo parcial o total. La inclusión de personal de otras áreas en la oficina de Catastro, es con el propósito de evitar las cargas excesivas de trabajo del personal actual y priorizar la adecuada atención a los contribuyentes. También se busca evitar el incremento no justificado de la plantilla laboral y, por ende, el costo de la nómina. Según los datos mostrados en la tabla 2, en el año 2020 hubo 25,565 contribuyentes en total, por lo cual se estiman de 2 a 4 semanas para culminar con la captura. Para una eficiente ejecución de la política pública, es necesario que al momento de capturar las cuentas se elaboren 2 listados, haciendo la separación de los tipos de contribuyentes de la siguiente forma: contribuyentes al corriente y contribuyentes con adeudo. Adicionalmente el listado debe estar separado por colonias y comunidades, con la finalidad de elaborar rutas de notificación de los e-ISSN: 2448-5101 VinculaTégica EFAN https://vinculategica.uanl.mx/ Vol. 10. Núm. 1 Enero-Febrero 2024 116 deudores morosos. Paso 3. Una vez que se apruebe la política pública, debe ser comunicada mediante redes oficiales del Gobierno Municipal, periódicos, radio y de ser posible televisión, para que la propaganda de la política pública se concentre en muy pocos puntos y explotarlos de forma adecuada, hasta que todos los contribuyentes del predial, así como la ciudadanía en general, comprendan y sepan cómo se llevará a cabo la implementación de la política pública, abriendo la puerta para transitar a un mayor índice de políticas públicas aplicadas, para el mejoramiento de otros servicios y obras. Paso 4. Una vez concluida la captura con el mismo cuerpo de capturistas, comenzar a notificar a los contribuyentes morosos, haciéndoles saber de su adeudo, la forma en que puede consultar su cuenta y hacer pagos correspondientes de manera digital, de la misma manera comunicarles los descuentos y opciones de pago, así mismo indicarles que si desean revisar su situación personalmente para llegar algún tipo de acuerdo, lo pueden realizar asistiendo a la Oficina de Catastro del municipio de La Barca. Un ejemplo de este acuerdo podría ser si él o la contribuyente tienen un adeudo de una suma grande de dinero, (Definidas por el H. Ayuntamiento, a consideración del contador del municipio) puede ser la opción de abonar a la deuda por concepto de impuesto predial, desde una mínima cantidad, que también en su caso sería definida por el H. Ayuntamiento. Paso 5. Una vez que las personas acudan a pagar el predial y de la misma manera si hacen su pago de forma digital, las y los servidores públicos encargados de atención al contribuyente le harán una pregunta, para saber su opinión acerca de su preferencia en cuanto a la mejora de servicios u obras. En la plataforma digital, al finalizar el pago se realizaría la misma pregunta a la persona que paga la contribución, es importante aclarar que cualquier persona puede pagar el predial de un bien inmueble, mientras conozca el folio, la dirección y el nombre del propietario. La Previsión Por la cuestión de poderes, conflictos de intereses políticos y personales, lo más viable es que esta política se utilice al principio de mandato, y esto en razón de los siguientes dos escenarios plenamente argumentados: • En el principio del mandato quienes fueron candidatos y ganadores pasan por un momento de euforia, eso pone al presidente municipal y sus regidores de bancada, en sintonía, esto da como resultado que aprueben muchas iniciativas y decisiones. • Por otro lado, las y los regidores de oposición, los grupos y personas que estén en contra del partido que encabezado el gobierno o simplemente que formen parte del sector de la e-ISSN: 2448-5101 VinculaTégica EFAN https://vinculategica.uanl.mx/ Vol. 10. Núm. 1 Enero-Febrero 2024 117 población detractor de cualquier gobierno, pueden hacer una interpretación errónea de esta política pública, argumentando que el gobierno o presidente quiere cobrar más impuestos a la ciudadanía y crear desinformación que es muy nociva para la ciudadanía. Por otra parte, la mejora de algún servicio u obra pública, realizada con el porcentaje recaudado en un periodo de 3 años, o incluso de 1 año, y lo más importante comunicando de una forma efectiva la conclusión de la política pública, así como la implementación de la transparencia, abre la posibilidad a retomar la confianza en el gobierno, por ende, motivar a más contribuyentes a pagar sus impuestos, ya que se les hace partícipe de las acciones y el destino de su impuesto. Administrar mediante una plataforma específica el proceso de recaudación del impuesto predial busca lo siguiente: mantener registros actualizados, facilitar el proceso de notificación de adeudos, facilitar el pago de los contribuyentes, tomar en consideración la opinión de los usuarios con respecto al uso de los recursos y finalmente, mejorar la percepción que tiene la población de los servicios que presta el municipio. Conclusiones La obligación catastral es una de las acciones más importantes que deben recaudar los Ayuntamientos para una mejor gestión del progreso de la población, pues entre mayor importe sea recaudado, más acciones de gobierno se pueden realizar. En la Ley de Ingresos municipales de La Barca, Jalisco se señala la cantidad esperada de recaudación del impuesto predial, pero existen deficiencias en el proceso pues se observó que un número importante de contribuyentes no cumplieron con su obligación. Es necesario innovar el proceso mediante el cual se recolecta este impuesto, sobre todo utilizando herramientas digitales que están disponibles en el mercado. En el municipio objeto de estudio los servicios públicos no cumplen con las expectativas que tienen sus habitantes, se observan serias deficiencias en las siguientes áreas: alumbrado público, recolección de basura, mantenimiento de parques, así como la pavimentación en calles de la ciudad, que por mucho es el mayor problema que se tiene. El predial es un impuesto cobrado directamente por los municipios sobre la propiedad de bienes inmuebles y se puede decir que es una fuente de ingresos estable para la demarcación. El contar con un proceso de recaudación eficiente permitirá brindar mejores servicios públicos, beneficiando a la población en general e incrementando la plusvalía de sus propiedades. La política pública propuesta en la presente investigación involucra la actualización de las bases de datos de los contribuyentes del impuesto predial, así como los conocimientos del personal que trabaja en la Oficina de Catastro, también se busca la incorporación de herramientas tecnológicas e-ISSN: 2448-5101 VinculaTégica EFAN https://vinculategica.uanl.mx/ Vol. 10. Núm. 1 Enero-Febrero 2024 118 y el otorgamiento de incentivos a los contribuyentes para lograr una mayor recaudación. La diversificación de los medios de pago también se está considerando, en particular la realización de pagos en línea para facilitar el cumplimiento. Municipios como Zapopan, Guadalajara, Tlajomulco de Zúñiga y Tepatitlán de Morelos, ya cuentan con la modalidad de pago electrónico, donde además los contribuyentes pueden consultar lo que deben. El uso de medios electrónicos de pago evita aglomeraciones, previniendo contagios asociados a distintas enfermedades. En caso de que la propuesta de política pública que se propone en la presente investigación hubiera estado en funcionamiento durante el año 2020, los estragos en la recaudación del impuesto que generó la pandemia COVID-19 hubieran sido menores y se habría contado con un mayor volumen de recursos para otorgar los servicios públicos a la población. De la misma forma se hubieran aprovechado los estímulos por el pago puntual de la contribución en los primeros meses del año. Referencias Cámara de Diputados del Congreso del Estado de Jalisco. (10 de abril de 2014). Constitución Política del Estado de Jalisco. https://www.jalisco.gob.mx/sites/default/files/constituci_n_politica_del_estado_de_jalisco.pd f Congreso del Estado de Jalisco. (2020). Ley de Ingresos del municipio de La Barca, Jalisco año 2020. https://periodicooficial.jalisco.gob.mx/sites/periodicooficial.jalisco.gob.mx/files/12-24-19lxxiii_0.pdf Congreso del Estado de Jalisco. (2019). Ley de Catastro Municipal del Estado de Jalisco. https://info.jalisco.gob.mx/sites/default/files/leyes/Ley_Catastro_Municipal_Estado_Jalisco_ 0.pdf Congreso del Estado de Jalisco. (2019). Ley de Gobierno y la Administración Pública Municipal. https://transparencia.info.jalisco.gob.mx/sites/default/files/Ley%20del%20Gobierno%20y%2 0la%20Administraci%C3%B3n%20P%C3%BAblica%20Municipal%20del%20Estado%20d e%20Jalisco_1.pdf Congreso del Estado de Jalisco. (2019). Ley de Hacienda Municipal del Estado de Jalisco. https://info.jalisco.gob.mx/sites/default/files/leyes/Ley_Hacienda_Municipal_Jalisco.pdf Congreso del Estado de Jalisco. (2019). Ley de Ingresos del municipio de La Barca, Jalisco, para el ejercicio fiscal del año 2019. https://periodicooficial.jalisco.gob.mx/sites/periodicooficial.jalisco.gob.mx/files/la_barca_2.p df Congreso del Estado de Jalisco. (2018). Ley de Ingresos del municipio de La Barca, Jalisco, para el ejercicio fiscal del año 2018. https://periodicooficial.jalisco.gob.mx/sites/periodicooficial.jalisco.gob.mx/files/la_barca_1.p df Congreso del Estado de Jalisco. (2017). Ley de Ingresos del municipio de La Barca, Jalisco, para el ejercicio fiscal del año 2017. https://periodicooficial.jalisco.gob.mx/sites/periodicooficial.jalisco.gob.mx/files/la_barca_0.p df IIEG. (2019). La Barca, diagnóstico del municipio marzo de 2019. https://iieg.gob.mx/ns/wpcontent/uploads/2019/06/La-Barca.pdf INEGI. (2015). Encuesta Intercensal 2015. https://www.inegi.org.mx/programas/intercensal/2015/ e-ISSN: 2448-5101 VinculaTégica EFAN https://vinculategica.uanl.mx/ Vol. 10. Núm. 1 Enero-Febrero 2024 119 Jalisco. (s. f.). La Barca. https://www.jalisco.gob.mx/es/jalisco/municipios/la-barca Ruíz López, D. & Cadenas Ayala, C. E. (2019). ¿Qué es una política pública? https://es.scribd.com/document/449676105/QUE-ES-UNA-POLITICA-PUBLICA-porDOMINGO-RUIZ-LOPEZ-Y-CARLOS-EDUARDO-CADENAS-AYALA Secretaría de Gobernación. (1988). Colección: Enciclopedia de los Municipios y Demarcaciones Territoriales de México. http://www.snim.rami.gob.mx/enciclopedia_v2/PruebaPropuesta.php?id=586&tipo=m&e=14 &m=18 e-ISSN: 2448-5101 VinculaTégica EFAN https://vinculategica.uanl.mx/ Vol. 10. Núm. 1 Enero-Febrero 2024
https://openalex.org/W1977511249	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0013444&type=printable	English	null	Betacellulin Induces Increased Retinal Vascular Permeability in Mice	PloS one	2,010	cc-by	5,565	Abstract Funding: This work was supported by US National Institutes of Health EY016490, CA106415, EY015638, Research to Prevent Blindness (RPB) Challenge Grant and RPB Lew Wasserman award. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. Abstract Background: Diabetic maculopathy, the leading cause of vision loss in patients with type 2 diabetes, is characterized by hyper-permeability of retinal blood vessels with subsequent formation of macular edema and hard exudates. The degree of hyperglycemia and duration of diabetes have been suggested to be good predictors of retinal complications. Intervention studies have determined that while intensive treatment of diabetes reduced the development of proliferative diabetic retinopathy it was associated with a two to three-fold increased risk of severe hypoglycemia. Thus we hypothesized the need to identify downstream glycemic targets, which induce retinal vascular permeability that could be targeted therapeutically without the additional risks associated with intensive treatment of the hyperglycemia. Betacellulin is a 32 kD member of the epidermal growth factor family with mitogenic properties for the retinal pigment epithelial cells. This led us to hypothesize a role for betacellulin in the retinal vascular complications associated with diabetes. Methods and Findings: In this study, using a mouse model of diabetes, we demonstrate that diabetic mice have accentuated retinal vascular permeability with a concomitant increased expression of a cleaved soluble form of betacellulin (s-Btc) in the retina. Intravitreal injection of soluble betacellulin induced retinal vascular permeability in normoglycemic and hyperglycemic mice. Western blot analysis of retinas from patients with diabetic retinopathy showed an increase in the active soluble form of betacellulin. In addition, an increase in the levels of A disintegrin and metalloproteinase (ADAM)-10 which plays a role in the cleavage of betacellulin was seen in the retinas of diabetic mice and humans. Conclusions: These results suggest that excessive amounts of betacellulin in the retina may contribute to the pathogenesis of diabetic macular edema. Citation: Anand-Apte B, Ebrahem Q, Cutler A, Farage E, Sugimoto M, et al. (2010) Betacellulin Induces Increased Retinal Vascular ONE 5(10): e13444. doi:10.1371/journal.pone.0013444 Ebrahem Q, Cutler A, Farage E, Sugimoto M, et al. (2010) Betacellulin Induces Increased Retinal Vascular Permeability in Mice. PLoS 0.1371/journal.pone.0013444 Editor: Pieter H. Reitsma, Leiden University Medical Center, Netherlands Editor: Pieter H. Reitsma, Leiden University Medical Center, Netherlands Received April 30, 2010; Accepted September 11, 2010; Published October 18, 2010 Received April 30, 2010; Accepted September 11, 2010; Published October 18, 2010 Copyright: 2010 Anand-Apte et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Bela Anand-Apte1,2, Quteba Ebrahem1, Alecia Cutler1, Eric Farage1, Masahiko Sugimoto1, Joe Hollyfield1,2, Judah Folkman3 1 Department of Ophthalmology, Cole Eye Institute, Cleveland Clinic Lerner College of Medicine, Cleveland, Ohio, United States of America, 2 Department of Molecular Medicine, Lerner Research Institute, Cleveland Clinic Lerner College of Medicine, Cleveland, Ohio, United States of America, 3 Vascular Biology, Children’s Hospital, Boston, Massachusetts, United States of America PLoS ONE \| www.plosone.org Bela Anand-Apte1,2, Quteba Ebrahem1, Alecia Cutler1, Eric Farage1, Masahiko Sugimoto1, Joe Hollyfield1,2 Judah Folkman3 pte1,2, Quteba Ebrahem1, Alecia Cutler1, Eric Farage1, Masahiko Sugimoto1, Joe d h lk 3 -Apte1,2, Quteba Ebrahem1, Alecia Cutler1, Eric Farage1, Masahiko Sugimoto1, Joe , Judah Folkman3 Introduction disease [6]. Laser photocoagulation as well as anti-VEGF therapies have shown significant promise in the treatment of proliferating vessels in proliferative diabetic retinopathy. However, DME appears to be more resistant to these treatment approaches, suggesting that other factors might contribute to this phenotype. These observations led us to hypothesize the presence of a novel factor that contributes to the increased retinal vascular perme- ability and/or retinopathy seen in diabetes. The incidence of diabetes world-wide, is expected to reach epidemic proportions by 2025. Progression of diabetic retinopathy often results in diabetic macular edema, which is a consequence of the breakdown of the blood-retinal barrier, increased retinal vascular permeability and leakage of plasma from small blood vessels in the macula leading to loss of central vision. While epidemiological studies have suggested that glycemic control plays a major role in the development of vascular complications of diabetes[1], current insulin therapies for control of glucose metabolism have not been successful in the prevention of long- term complications[2,3]. Intervention studies have determined that while intensive treatment of diabetes reduced the develop- ment of proliferative diabetic retinopathy [4], it was associated with a two to three-fold increased risk of severe hypoglycemia[5] as well as an increased risk of mortality from cardiovascular Betacellulin (BTC) is a 32 kD member of the epidermal growth factor (EGF) family that was originally isolated from the conditioned medium of a mouse pancreatic b-cell tumor line[7]. BTC is a glycosylated (N- and O-linked) protein with an apparent molecular weight of 32 kDa, generated by cleavage of a 178- amino acid membrane anchored precursor protein pro-BTC. BTC binds and activates the EGF receptor (EGFR/erbB-1) and erbB-4[8,9,10]. BTC has been shown to be expressed in the human pancreas by pancreatic b cells [11,12,13] as well in other PLoS ONE \| www.plosone.org 1 October 2010 \| Volume 5 \| Issue 10 \| e13444 Betacellulin and Diabetes organs[7], induce differentiation of a pancreatic exocrine cell line into insulin secreting cells[13,14,15]and induce pancreatic b-cell neogenesis in diabetic mice[16]. Because of the proliferative effects of BTC on retinal pigment epithelial cells we hypothesized that it may contribute to increased retinal vascular permeability and the pathogenesis of diabetic macular edema. We have compelling evidence that betacellulin can increase retinal vascular permeabil- ity and may play a role in the pathogenesis of retinal vascular leakage in diabetes. gray-level threshold set between 230–255. Experimental Diabetes 6–8 week old mice were rendered diabetic with 3 consecutive daily intraperitoneal injections of STZ (55 mg/Kg) freshly dissolved in citrate buffer (pH 4.5). Control mice were given injections of buffer alone. Development of diabetes (defined by blood glucose greater than 250 mg/dl) was confirmed 1 week after the initial injection (One TouchHltraHTest Strips and One TouchHUltraMiniTMglucometer). Introduction Intensely stained vessels protruding from binarized leakage areas were removed using successive passes of morphological erosion and dilation filters; areas of processed, segmented regions were then exported to Excel. In original, unprocessed images, a threshold value obtained from an automated search for histogram minima was used to segment the entire retina from background for percent leakage calculations (leakage area divided by total retinal tissue area). Finally, boundaries of binarized leakage regions were extracted using a Sobel filter, pseudo-colored, and then superimposed upon original images to allow visualization of segmented vascular leakage regions. Increased soluble betacellulin in the retinas of diabetic mice with hyper-permeable retinal vessels Steptozotocin-induced diabetic mice with hyperglycemia (fast- ing blood sugar $200 mg/ml) were examined for increases in retinal vascular permeability using an Evans Blue leakage assay. As described previously [17], we observed a modest increase in dye leakage from the retinal vessels of diabetic mice (Fig. 1b,c) when compared with control normoglycemic mice (Fig. 1a,c). We examined the expression of betacellulin in the pancreas and retinas of control and diabetic mice. It was interesting to note that in the pancreas the majority of betacellulin was present as the cleaved soluble form (s-BTC) compared with the abundance of the higher molecular weight membrane bound pro-form (Pro-BTC) in normal mouse retinas (Fig. 1e). There appeared to be a trend towards a slight increase in the pro-BTC in the pancreas of diabetic mice (Fig. 1d). In the mouse retina the cleaved low- molecular weight form of active soluble betacellulin (s-BTC) was significantly increased in hyperglycemic mice (Fig. 1e,f) when examined by western blot analysis. Increase in retinal soluble betacellulin causes increased retinal vascular permeability in diabetic mice To determine if the increase in soluble active betacellulin seen in diabetic mice could contribute to increased vascular permeability we examined whether increased concentration of betacellulin in the retina could induce retinal vascular leakage in normal mice. Intravitreal injections of increasing doses of recombinant soluble betacellulin (0–300 ng) resulted in increased retinal hemorrhage (Fig. 2a upper panel,arrowheads) and increased retinal vascular permeability (Fig. 2b lower panel) in a dose dependent manner, similar to that induced by vascular endothelial growth factor (VEGF). Animals 6–8 week old C57BL6 mice (Jackson Laboratories, Maine) were used in this study. The animals were cared for in accordance with the ARVO statement for the Use of Animals in Ophthalmic and Vision research. All experimental procedures used aseptic sterile techniques and were approved by the Animal Care and Use Committee of the Cleveland Clinic (ARC08792). Materials Recombinant human VEGF was a kind gift from Genentech, CA and recombinant betacellulin was purchased from R&D. Antibodies: polyclonal anti-mouse and anti-human betacellulin antibody (R&D), monoclonal anti-ADAM-10 (Calbiochem) and HRP-conjugated anti-rabbit, anti-mouse IgG antibodies (Amer- sham Pharmacia Biotech) and anti-goat IgG (Santa Cruz). Post mortem human eyes were obtained from he National Disease Research Interchange (NDRI), Philadelphia. Postmortem time ranged from 3–10 hours. Immunoblotting Lysates from mouse or human retina prepared using sonication on ice with RIPA buffer (Boston Bioproducts) containing EDTA free protease inhibitors (Roche) were subjected to SDS-PAGE. Proteins were probed with antibody and detected with either a HRP-conjugated anti-rabbit or anti mouse IgG antibody (Amer- sham Pharmacia Biotech, Piscataway, NJ) followed by ECL. The blots were restripped with RestoreTM solution (Thermo Scientific) for 30 minutes and reprobed as indicated. PLoS ONE \| www.plosone.org Blood retinal barrier quantitation Leakage of Evans Blue dye from mouse retinal vessels in representative a) normoglycemic and b) hyperglycemic mice was quantitate whole mount retinas one hr after perfusion with the dye(n = 8). (c). Representative Western blot analysis of betacellulin in pancreas (d) and retina from control and streptozotocin-induced diabetic mice. (f) densitometric quantitation of the ratio of sBTC to ProBTC in mouse retina from con (n 12) and diabetic (n 12) mo se e es *p 0 008 (ANOVA) Figure 1. Diabetic mice show augmented retinal vascular permeability and increased expression of cleaved betacellulin in the retina. Leakage of Evans Blue dye from mouse retinal vessels in representative a) normoglycemic and b) hyperglycemic mice was quantitated in whole mount retinas one hr after perfusion with the dye(n = 8). (c). Representative Western blot analysis of betacellulin in pancreas (d) and retinas (e) from control and streptozotocin-induced diabetic mice. (f) densitometric quantitation of the ratio of sBTC to ProBTC in mouse retina from control (n = 12) and diabetic (n = 12) mouse eyes. p = 0.008 (ANOVA). doi:10.1371/journal.pone.0013444.g001 to pro-BTC was significantly increased in the retinas of patients with diabetes (Fig. 3c). dependent. No increase in VEGF was observed in mouse retinas injected with betacellulin (Fig. S1) indicating that betacellulin increased retinal vascular permeability via a VEGF-independent pathway. Blood retinal barrier quantitation Vascular permeability in the retina was quantitated after 3–4 weeks of diabetes using an Evans blue quantitation technique as described previously [17]. Briefly 3–4 weeks after the onset of diabetes or 24 hours after intravitreal injections, mice were anesthetized with isofluorane and injected intravenously (via the tail vein) with 200 ml of Evans Blue dye (Sigma) at a concentration of 45 mg/Kg. Quantitation of the retinal barrier breakdown was assessed as described previously [18]. Additionally, retinas from Evans Blue injected mice were examined by fluorescence microscopy (Nikon). For quantitative analysis of vascular leakage, retinal images were batch processed using a customized macro generated in Image-Pro Plus 6.1 (Media Cybernetics, Silver Spring, MD). In each image, leakage areas were segmented using a Since all patients with diabetes do not develop macular edema, we postulated that a combination of hyperglycemia and increased intravitreous betacellulin might lead to a more severe phenotype. Injection of 200 ng of recombinant betacellulin intravitreally in hyperglycemic mice induced a marked accentuation of retinal vascular leakage as detected by extravasation of intravascular Evans Blue dye (Figs. 2f,g) when compared with leakage induced in normoglycemic mice (Figs. 2d,g). Since VEGF has been proposed to be a critical factor in the increase in retinal vascular permeability in proliferative diabetic retinopathy (PDR) and diabetic macular edema (DME), we wanted to determine if the effect of betacellulin was VEGF- PLoS ONE \| www.plosone.org October 2010 \| Volume 5 \| Issue 10 \| e13444 October 2010 \| Volume 5 \| Issue 10 \| e13444 2 Betacellulin and Diabetes Figure 1. Diabetic mice show augmented retinal vascular permeability and increased expression of cleaved betacellulin in the retina. Leakage of Evans Blue dye from mouse retinal vessels in representative a) normoglycemic and b) hyperglycemic mice was quantitated in whole mount retinas one hr after perfusion with the dye(n = 8). (c). Representative Western blot analysis of betacellulin in pancreas (d) and retinas (e) from control and streptozotocin-induced diabetic mice. (f) densitometric quantitation of the ratio of sBTC to ProBTC in mouse retina from control (n = 12) and diabetic (n = 12) mouse eyes. *p = 0.008 (ANOVA). doi:10.1371/journal.pone.0013444.g001 Figure 1. Diabetic mice show augmented retinal vascular permeability and increased expression of cleaved betacellulin in retina. Increased expression of soluble betacellulin in human diabetic retinas Recent studies have determined that ADAM-10 (a disintegrin and metalloproteinase-10) can mediate the constitutive and activated shedding of pro-betacellulin[20,21]. In order to deter- mine if ADAM-10 plays a role in the increased expression of the soluble form of betacellulin in the retinas of diabetic mice we performed western blot analyses using an ADAM-10 antibody. We found that ADAM-10 (62 kD mature form) was increased in the retinas of streptozotocin-induced hyperglycemic mice (Fig. 4a) but not in mice injected intravitreally with recombinant VEGF (Fig. 4b). Immunoblot analysis on retinas from diabetic patients with without a clinical diagnosis of retinopathy determined an increase in mature ADAM 10 when compared with retinas from healthy controls (Fig. 4c,d). While the exact mechanism by which ADAM-10 is increased in the retinas of diabetics remains to be determined, these results indicate that ADAM-10 may contribute to the cleavage of membrane bound betacellulin that causes Betacellulin is expressed as a membrane anchored precursor protein (pro-betacellulin) containing an extracellular N-terminal ectodomain, a transmembrane domain and a cytoplasmic domain[13,19]. Like other members of the EGF family, the ectodomain of betacellulin can be proteolytically cleaved to release a soluble form of the protein. Protein levels of the pro-betacellulin as well as its cleaved soluble form were examined by western blot analysis in post-mortem retina tissue from healthy donors, patients with diabetes with no clinical diagnoses of retinopathy and patients with diabetic retinopathy. Diabetic retinas showed evidence of increased pro-BTC (38 kD) as well as the cleaved s-BTC (19 kD) compared with retinas from controls (Fig. 3a). Both the membrane anchored and soluble forms of betacellulin are believed to be biologically active, binding to ErbB receptors. The ratio of s-BTC PLoS ONE \| www.plosone.org October 2010 \| Volume 5 \| Issue 10 \| e13444 3 PLoS ONE \| www.plosone.org Betacellulin and Diabetes Figure 2. Intravitreal injection of betacellulin induces increased retinal haemorrhage and augmented retinal vascular permeability in normal and diabetic retinas. Dose response of the effect of intravitreal injection of betacellulin (50–300 ng), VEGF (200 ng) and PBS in normoglycemic mice on a) retinal haemorrhage as seen in a bright field image of dissected posterior pole of mouse eyes. Arrowheads indicate areas of hemorrhage. For BTC-300, inset is magnification of boxed area b) Evans Blue dye leakage in flat mounted retinas. Increased expression of soluble betacellulin in human diabetic retinas Leakage of Evans blue dye from retinal vessels of normoglycemic (c,d) and hyperglycemic (e,f) mice injected intravitreally with PBS(c,e) or 200 ng recombinant mouse betacellulin (d,f). Results are quantitated (g). p,0.009. doi:10.1371/journal.pone.0013444.g002 Figure 2. Intravitreal injection of betacellulin induces increased retinal haemorrhage and augmented retinal vascular permeability in normal and diabetic retinas. Dose response of the effect of intravitreal injection of betacellulin (50–300 ng), VEGF (200 ng) and PBS in normoglycemic mice on a) retinal haemorrhage as seen in a bright field image of dissected posterior pole of mouse eyes. Arrowheads indicate areas of hemorrhage. For BTC-300, inset is magnification of boxed area b) Evans Blue dye leakage in flat mounted retinas. Leakage of Evans blue dye from retinal vessels of normoglycemic (c,d) and hyperglycemic (e,f) mice injected intravitreally with PBS(c,e) or 200 ng recombinant mouse betacellulin (d,f). Results are quantitated (g). * p,0.009. doi:10.1371/journal.pone.0013444.g002 Figure 2. Intravitreal injection of betacellulin induces increased retinal haemorrhage and augmented retinal vascular permeability in normal and diabetic retinas. Dose response of the effect of intravitreal injection of betacellulin (50–300 ng), VEGF (200 ng) and PBS in normoglycemic mice on a) retinal haemorrhage as seen in a bright field image of dissected posterior pole of mouse eyes. Arrowheads indicate areas of hemorrhage. For BTC-300, inset is magnification of boxed area b) Evans Blue dye leakage in flat mounted retinas. Leakage of Evans blue dye from retinal vessels of normoglycemic (c,d) and hyperglycemic (e,f) mice injected intravitreally with PBS(c,e) or 200 ng recombinant mouse betacellulin (d,f). Results are quantitated (g). * p,0.009. doi:10.1371/journal.pone.0013444.g002 increased retinal vascular permeability seen as a complication of the disease. and proliferative diabetic retinopathy (PDR). In non-proliferative diabetic retinopathy, the earliest visible sign is the formation of microaneurysms. Increased capillary permeability, results in the leaking of fluid into the surrounding retinal tissue, pooling around the macula causing macular edema and loss of visual acuity. It is believed that the retinal neovascularization in PDR results from ischemia and the consequent release of local hypoxic and pro- angiogenic factors such as VEGF[27], hypoxia-inducible fac- tors[28] and erythropoietin[29]. These factors may stimulate abnormal vessel growth extending into the vitreous of the eye, leading to hemorrhage and visual loss. Laser photocoagulation as well as anti-VEGF therapies have shown significant promise in the treatment of proliferating vessels in PDR. Increased expression of soluble betacellulin in human diabetic retinas While PDR appears to be extremely responsive to treatment with VEGF inhibitors, early studies indicate that the response to these drugs in retinal edema is often partial and requires higher doses[30]. However, DME appears to be more resistant to these treatment approaches, suggesting that other factors might contribute to the retinal vascular leakage. These results and other recent studies have Discussion Diabetes mellitus is a global disease with considerable morbidity and mortality. The total number of people with diabetes is expected to reach 300 million by the year 2025, with the majority of increased cases predicted to be in developing countries. The disease is classified into type I diabetes (previously referred to as ‘‘insulin dependent’’ or ‘‘juvenile onset’’), characterized by an absolute insulin deficiency as a consequence of destruction of the insulin secretory pancreatic b cells of the islets of Langerhans; and type II diabetes (‘‘non-insulin dependent’’ adult-onset’’ which is characterized by an insulin secretory defect of the b-cell and insulin resistance in peripheral tissues. Diabetic retinopathy is a common complication of both type I (50% incidence) and type II (30% incidence)[22,23,24,25,26] diabetes. Diabetic retinopathy can be broadly divided into two clinical stages: non-proliferative PLoS ONE \| www.plosone.org October 2010 \| Volume 5 \| Issue 10 \| e13444 4 Betacellulin and Diabetes Figure 3. Increased expression of betacellulin in retinas of humans with diabetes. Western blot analysis of (a) BTC (b) GAPDH in retina tissue from human donors with no diabetes, diabetes or diabetes with diabetic retinopathy. (c) Densitometric quantitation of the ration of sBTC to full length BTC using GAPDH as a reference. ** p = 0.01 * p = 0.03. doi:10.1371/journal.pone.0013444.g003 Figure 3. Increased expression of betacellulin in retinas of humans with diabetes. Western blot analysis of (a) BTC (b) GAPDH in retina tissue from human donors with no diabetes, diabetes or diabetes with diabetic retinopathy. (c) Densitometric quantitation of the ration of sBTC to full length BTC using GAPDH as a reference. ** p = 0.01 * p = 0.03. doi:10.1371/journal.pone.0013444.g003 Recent studies have suggested Neurod-Btc gene therapy to be a promising approach to induce islet regeneration for the treatment of insulin-dependent diabetes[40]. Our data suggest that we should proceed with caution in this regard as there is a likelihood of inducing or worsening the ocular complications of macular edema. We observed that the magnitude of the effect of intravitreal betacellulin on retinal vascular permeability was equivalent to that of VEGF. While much attention has been focused on the role of VEGF in the pathophysiology of PDR and DME, it is becoming increasingly evident that other vasoperme- ability factors are involved [32,41] and more attention should be focused on targeting these factors as therapeutics. Discussion Recent findings have also suggested the need to re-evaluate the current diagnostic criteria for diabetes, and the risk of complications based on fasting plasma glucose levels, as neither macrovascular nor microvascular complications seem to respect a strict glycemic threshold [3]. Perhaps glycemia in combination with additional vasopermeability factors might be a more reliable prognostic indicator. In summary, our data suggests that betacellulin plays an important role in the development of increased retinal vascular permeability in diabetes and inhibitors of ADAM-10 or betacellulin signaling might be clinically useful in preventing the development and progression of macular edema in this disease. suggested that VEGF-independent mechanism(s) such as carbonic anhydrase[31,32] and erythropoietin[33,34], play a role in diabetic retinal edema. Our results suggest that cleaved betacellu- lin contributes to increased retinal vascular leakage in diabetes. suggested that VEGF-independent mechanism(s) such as carbonic anhydrase[31,32] and erythropoietin[33,34], play a role in diabetic retinal edema. Our results suggest that cleaved betacellu- lin contributes to increased retinal vascular leakage in diabetes. ADAMs and ADAM-10 in particular have been recently reported to be novel regulators of vascular permeability[7,35,36] and our data indicates that this might occur via increased expression of betacellulin. Cleavage of the membrane-anchored forms of BTC to release a secreted form occurs principally by ADAM-10 (a disintegrin and metalloprotease-10)[20,21,37,38] as well as endothelin-1[39]. ADAM-10 has recently been shown to induce endothelial cell permeability via regulation of VE- cadherin-dependent endothelial cell functions[36]. Whether cleav- age of betacellulin is necessary for the increased vascular permeability by ADAM-10 occurs in the retina or elsewhere remains to be determined. ADAMs and ADAM-10 in particular have been recently reported to be novel regulators of vascular permeability[7,35,36] and our data indicates that this might occur via increased expression of betacellulin. Cleavage of the membrane-anchored forms of BTC to release a secreted form occurs principally by ADAM-10 (a disintegrin and metalloprotease-10)[20,21,37,38] as well as endothelin-1[39]. ADAM-10 has recently been shown to induce endothelial cell permeability via regulation of VE- cadherin-dependent endothelial cell functions[36]. Whether cleav- age of betacellulin is necessary for the increased vascular permeability by ADAM-10 occurs in the retina or elsewhere remains to be determined. We have examined betacellulin levels in the plasma and found no significant increase in betacellulin in the circulating plasma of patients with PDR (n = 23) (Fig. S2). PLoS ONE \| www.plosone.org Discussion While this suggests that the betacellulin might be synthesized or processed in the retina we cannot exclude the possibility that the ELISA used to measure betacellulin may lack the sensitivity to detect small changes in circulating levels. PLoS ONE \| www.plosone.org October 2010 \| Volume 5 \| Issue 10 \| e13444 October 2010 \| Volume 5 \| Issue 10 \| e13444 5 Supporting Information Figure S1 Betacellulin induces retinal vascular leakage indepen- dent of VEGF. Retinas from mice injected intravitreally with PBS or betacellulin (200 ng) or no injection (control) were evaluated for VEGF protein 24 hours post injection. Found at: doi:10.1371/journal.pone.0013444.s001 (0.74 MB TIF) Figure S2 No significant increase in circulating plasma beta- cellulin in patients with proliferative diabetic retinopathy. Plasma was prepared from whole blood from patients with no diabetes, Figure 4. Increased expression of ADAM-10 in diabetic retinas. Western blot analysis of ADAM-10 in retina tissue from (a) control and STZ induced diabetic mice (b) control and intravitreal VEGF injected mice and (c) human retinas from patients with no diabetes, diabetes or diabetes with diabetic retinopathy(d) Densitometric quantitation of mature ADAM-10 (mADAM-10) relative to actin on the same blot. p#0.03. doi:10.1371/journal.pone.0013444.g004 Betacellulin and Diabetes Betacellulin and Diabetes Figure 4. Increased expression of ADAM-10 in diabetic retinas. Western blot analysis of ADAM-10 in retina tissue from (a) control and STZ induced diabetic mice (b) control and intravitreal VEGF injected mice and (c) human retinas from patients with no diabetes, diabetes or diabetes with diabetic retinopathy(d) Densitometric quantitation of mature ADAM-10 (mADAM-10) relative to actin on the same blot. p#0.03. doi:10.1371/journal.pone.0013444.g004 Supporting Information Found at: doi:10.1371/journal.pone.0013444.s001 (0.74 MB TIF) References 21. 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TDCaCTR (1993) The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. The Diabetes Control and Complications Trial Research Group. N Engl J Med 329: 977–986. 25. Klein R, Klein BE, Moss SE, Davis MD, DeMets DL (1984) The Wisconsin epidemiologic study of diabetic retinopathy. III. Prevalence and risk of diabetic retinopathy when age at diagnosis is 30 or more years. Arch Ophthalmol 102: 527–532. 6. Ismail-Beigi F, Craven T, Banerji MA, Basile J, Calles J, et al. References Effect of intensive treatment of hyperglycaemia on microvascular outcomes in type 2 diabetes: an analysis of the ACCORD randomised trial. Lancet 376: 419–430. 26. Orchard TJ, Dorman JS, Maser RE, Becker DJ, Drash AL, et al. (1990) Prevalence of complications in IDDM by sex and duration. Pittsburgh Epidemiology of Diabetes Complications Study II. Diabetes 39: 1116–1124. y 7. Shing Y, Christofori G, Hanahan D, Ono Y, Sasada R, et al. (1993) Betacellulin: a mitogen from pancreatic beta cell tumors. Science 259: 1604–1607. 7. Shing Y, Christofori G, Hanahan D, Ono Y, Sasada R, et al. (1993 27. Funatsu H, Yamashita H, Ikeda T, Mimura T, Eguchi S, et al. (2003) Vitreous levels of interleukin-6 and vascular endothelial growth factor are related to diabetic macular edema. Ophthalmology 110: 1690–1696. 8. Riese DJ, 2nd, Bermingham Y, van Raaij TM, Buckley S, Plowman GD, et al. (1996) Betacellulin activates the epidermal growth factor receptor and erbB-4, and induces cellular response patterns distinct from those stimulated by epidermal growth factor or neuregulin-beta. Oncogene 12: 345–353. p gy 28. Arjamaa O, Nikinmaa M (2006) Oxygen-dependent diseases in the retina: role of hypoxia-inducible factors. Exp Eye Res 83: 473–483. 9. Riese DJ, Kim ED, Elenius K, Buckley S, Klagsbrun M, et al. (1996) The epidermal growth factor receptor couples transforming growth factor-alpha, heparin-binding epidermal growth factor-like factor, and amphiregulin to Neu, ErbB-3, and ErbB-4. J Biol Chem 271: 20047–20052. 29. Manzoni P, Maestri A, Gomirato G, Takagi H, Watanabe D, et al. (2005) Erythropoietin as a retinal angiogenic factor. N Engl J Med 353: 2190–2191; author reply 2190-2191. 30. Aiello LP (2008) Targeting intraocular neovascularization and edema–one drop at a time. N Engl J Med 359: 967–969. 10. Watanabe T, Shintani A, Nakata M, Shing Y, Folkman J, et al. (1994) Recombinant human betacellulin. Molecular structure, biological activities, and receptor interaction. J Biol Chem 269: 9966–9973. 31. Gao BB, Chen X, Timothy N, Aiello LP, Feener EP (2008) Characterization of the vitreous proteome in diabetes without diabetic retinopathy and diabetes with proliferative diabetic retinopathy. J Proteome Res 7: 2516–2525. p J 11. Li L, Seno M, Yamada H, Kojima I (2003) Betacellulin improves glucose metabolism by promoting conversion of intraislet precursor cells to beta-cells in streptozotocin-treated mice. Am J Physiol Endocrinol Metab 285: E577–583. p p y 32. Gao BB, Clermont A, Rook S, Fonda SJ, Srinivasan VJ, et al. References (2007) Extracellular carbonic anhydrase mediates hemorrhagic retinal and cerebral vascular permeability through prekallikrein activation. Nat Med 13: 181–188. 12. Li L, Yi Z, Seno M, Kojima I (2004) Activin A and betacellulin: effect on regeneration of pancreatic beta-cells in neonatal streptozotocin-treated rats. Diabetes 53: 608–615. 33. Tong Z, Yang Z, Patel S, Chen H, Gibbs D, et al. (2008) Promoter polymorphism of the erythropoietin gene in severe diabetic eye and kidney complications. Proc Natl Acad Sci U S A 105: 6998–7003. 13. Seno M, Tada H, Kosaka M, Sasada R, Igarashi K, et al. (1996) Human betacellulin, a member of the EGF family dominantly expressed in pancreas and small intestine, is fully active in a monomeric form. Growth Factors 13: 181–191. 34. Watanabe D, Suzuma K, Matsui S, Kurimoto M, Kiryu J, et al. (2005) Erythropoietin as a retinal angiogenic factor in proliferative diabetic retinopathy. N Engl J Med 353: 782–792. 14. Mashima H, Ohnishi H, Wakabayashi K, Mine T, Miyagawa J, et al. (1996) Betacellulin and activin A coordinately convert amylase-secreting pancreatic AR42J cells into insulin-secreting cells. J Clin Invest 97: 1647–1654. g 35. Ponnuchamy B, Khalil RA (2008) Role of ADAMs in endothelial cell permeability: cadherin shedding and leukocyte rolling. Circ Res 102: 1139–1142. 15. Miyagawa J, Hanafusa O, Sasada R, Yamamoto K, Igarashi K, et al. (1999) Immunohistochemical localization of betacellulin, a new member of the EGF family, in normal human pancreas and islet tumor cells. Endocr J 46: 755–764. 36. Schulz B, Pruessmeyer J, Maretzky T, Ludwig A, Blobel CP, et al. (2008) ADAM10 regulates endothelial permeability and T-Cell transmigration by proteolysis of vascular endothelial cadherin. Circ Res 102: 1192–1201. 16. Yamamoto K, Miyagawa J, Waguri M, Sasada R, Igarashi K, et al. (2000) Recombinant human betacellulin promotes the neogenesis of beta-cells and ameliorates glucose intolerance in mice with diabetes induced by selective alloxan perfusion. Diabetes 49: 2021–2027. 37. Blobel CP (2005) ADAMs: key components in EGFR signalling and development. Nat Rev Mol Cell Biol 6: 32–43. p 38. Sahin U, Blobel CP (2007) Ectodomain shedding of the EGF-receptor ligand epigen is mediated by ADAM17. FEBS Lett 581: 41–44. 17. Xu Q, Qaum T, Adamis AP (2001) Sensitive blood-retinal barrier breakdown quantitation using Evans blue. Invest Ophthalmol Vis Sci 42: 789–794. p g y 39. Sanderson MP, Abbott CA, Tada H, Seno M, Dempsey PJ, et al. Found at: doi:10.1371/journal.pone.0013444.s001 (0.74 MB TIF) Figure S1 Betacellulin induces retinal vascular leakage indepen- dent of VEGF. Retinas from mice injected intravitreally with PBS or betacellulin (200 ng) or no injection (control) were evaluated for VEGF protein 24 hours post injection. Figure S2 No significant increase in circulating plasma beta- cellulin in patients with proliferative diabetic retinopathy. Plasma was prepared from whole blood from patients with no diabetes, PLoS ONE \| www.plosone.org October 2010 \| Volume 5 \| Issue 10 \| e13444 October 2010 \| Volume 5 \| Issue 10 \| e13444 6 Betacellulin and Diabetes diabetes with no evidence of retinopathy and proliferative diabetic retinopathy and analyzed by ELISA for betacellulin. diabetes with no evidence of retinopathy and proliferative diabetic retinopathy and analyzed by ELISA for betacellulin. Author Contributions Conceived and designed the experiments: BAA JF. Performed the experiments: QE AC EF MS. Analyzed the data: BAA QE JF. Contributed reagents/materials/analysis tools: JH. Wrote the paper: BAA. Found at: doi:10.1371/journal.pone.0013444.s002 (0.54 MB TIF) Found at: doi:10.1371/journal.pone.0013444.s002 (0.54 MB TIF) References (2006) Hydrogen peroxide and endothelin-1 are novel activators of betacellulin ectodomain shedding. J Cell Biochem 99: 609–623. 18. Qaum T, Xu Q, Joussen AM, Clemens MW, Qin W, et al. (2001) VEGF- initiated blood-retinal barrier breakdown in early diabetes. Invest Ophthalmol Vis Sci 42: 2408–2413. 40. Kojima H, Fujimiya M, Matsumura K, Younan P, Imaeda H, et al. (2003) NeuroD-betacellulin gene therapy induces islet neogenesis in the liver and reverses diabetes in mice. Nat Med 9: 596–603. 19. Holbro T, Hynes NE (2004) ErbB receptors: directing key signaling networks throughout life. Annu Rev Pharmacol Toxicol 44: 195–217. 41. Antonetti DA, Barber AJ, Bronson SK, Freeman WM, Gardner TW, et al. (2006) Diabetic retinopathy: seeing beyond glucose-induced microvascular disease. Diabetes 55: 2401–2411. 20. Sahin U, Weskamp G, Kelly K, Zhou HM, Higashiyama S, et al. (2004) Distinct roles for ADAM10 and ADAM17 in ectodomain shedding of six EGFR ligands. J Cell Biol 164: 769–779. PLoS ONE \| www.plosone.org October 2010 \| Volume 5 \| Issue 10 \| e13444 7
https://openalex.org/W4281667738	https://publications.aston.ac.uk/id/eprint/43857/1/unac007.pdf	English	null	Exporting the Luxembourg Rail Protocol to the Convention on International Interests in Mobile Equipment to Africa	Uniform law review	2,022	cc-by	16,996	Abstract This article recommends the Cape Town Convention on International Interests in Mobile Equipment and appeals to African countries to accede to the Luxembourg Protocol on Railway Rolling Stock (Rail Protocol). The discussion in this article focuses on the extent to which Article XXII of the Rail Protocol can be applied with respect to Africa. The article critically analyses the competence of the African Economic Community (AEC) and its sub-regional organizations to accede to the Cape Town Convention and Rail Protocol. The author provides a detailed argumentative analysis of the legal mandate of the AEC and the inﬂuence of African Union (AU) laws on the competence of the AEC’s sub-regional organizations to make declarations under the Cape Town Convention and Rail Protocol. The analysis conﬁrms that competence to accede to the Convention and Rail Protocol resides with sovereign States in Africa and not with the AU, the AEC, the Organisation pour l’harmonisation en Afrique du Droit des Affaires, or any existing sub-regional organization in Africa. Exporting the Luxembourg Rail Protocol to the Convention on International Interests in Mobile Equipment to Africa Iyare Otabor-Olubor* * Iyare Otabor-Olubor, Aston University, Birmingham, United Kingdom. Email: i.otabor- olubor@aston.ac.uk. The author would like to thank Professor Anna Veneziano and Professor Ignacio Tirado for their continuous support, advice, and the opportunity to carry out this research at UNIDROIT. This article was supported by the Aston University Global Challenges Research Fund. The author declares that there is no conflict of interest. I. Introduction The 1991 Treaty Establishing the African Economic Community (AEC Treaty) outlines the importance of transnational trade and uniform laws and policies among AEC countries.1 One objective of the AEC Treaty is to harmonize regula- tions to further develop African rail infrastructure and easy access to rail * Iyare Otabor-Olubor, Aston University, Birmingham, United Kingdom. Email: i.otabor- olubor@aston.ac.uk. The author would like to thank Professor Anna Veneziano and Professor Ignacio Tirado for their continuous support, advice, and the opportunity to carry out this research at UNIDROIT. This article was supported by the Aston University Global Challenges Research Fund. The author declares that there is no conflict of interest. 1 Treaty Establishing the African Economic Community 1991 (AEC Treaty), Art 4(1)(d) and Art 4(2)(d). 1 Treaty Establishing the African Economic Community 1991 (AEC Treaty), Art 4(1)(d) and Art 4(2)(d). V C The Author(s) (2022). Published by Oxford University Press on behalf of UNIDROIT. V C The Author(s) (2022). Published by Oxford University Press on behalf of UNIDROIT. y y This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Unif. L. Rev., 2022, 1–27 https://doi.org/10.1093/ulr/unac007 Unif. L. Rev., 2022, 1–27 https://doi.org/10.1093/ulr/unac007 Iyare Otabor-Olubor 2 finance.2 This objective aligns with those of the Agreement Establishing the African Continental Free Trade Area (AfCFTA),3 the 2001 Convention on International Interests in Mobile Equipment (Cape Town Convention),4 and the Protocol on Matters Specific to Railway Rolling Stock (Rail Protocol).5 The AfCFTA is designated to create a single market for the free movement of per- sons and easy carriage of freight across Africa.6 The AfCFTA covers a market of 1.2 billion people in Africa and a gross domestic product of US $2.5 trillion.7 Participating countries include all 55 Member States of the African Union (AU).8 In terms of participating countries, the AfCFTA is the world’s largest free trade area since the formation of the World Trade Organization in 1995.9 A single interconnected market with modern infrastructure, such as access to rail, would provide businesses with economies of scale and business mobility while enhancing Africa’s competitiveness.10 The success of the AfCFTA to facilitate the transportation of goods hinges on adequate financing of the transportation sector. Financial investment in Africa’s rail transport sector has grown over the years. 2 I.N. Kessides, Regionalising Infrastructure for Deepening Market Integration: The Case of East Africa (2012) 4 Journal of Infrastructure Development 115; See also A. Jerome and D. Nabena, ‘Infrastructure and Regional Integration in Africa’ in D. H. Levine and D. Nagar (eds) Region- building in Africa: Political and Economic Challenges (Springer 2016). 3 Agreement Establishing the African Continental Free Trade Area 2018. 4 4 UNIDROIT, Convention on International Interests in Mobile Equipment (Cape Town, 16 November 2001). I. Introduction At the first United Kingdom (UK)–Africa Investment Summit held in London on 20 January 2020, UK Prime Minister Boris Johnson declared that the UK had established itself as a ‘partner of choice’ for Africa in trade and infrastruc- ture investment.11 Successful private sector financing of monorail trains in Cairo, Egypt, supported by UK Export Finance, was cited as an example, with an assurance that similar arrangements will materialize in other African coun- tries.12 Furthermore, the availability of modern rail equipment in Africa will give impetus to African countries through a virtuous cycle to adopt the 1980 4 UNIDROIT, Convention on International Interests in Mobile Equipment (Cape Town, 16 November 2001). 5 UNIDROIT, Luxembourg Protocol to the Convention on International Interests in Mobile Equipment on Matters Specific to Railway Rolling Stock (Luxembourg, 23 February 2007). 6 Assessing Regional Integration in Africa VIII (UN Economic Commission for Africa 2017) 11. 7 7 D. Luke, ‘Making the Case for the African Continental Free Trade Area’ in D. Luke and J. Macleod (eds) Inclusive Trade in Africa the African Continental Free Trade Area in Comparative Perspective (Routledge 2019) 5. 8 The AU is an international organization formed in 2001 to accelerate the socio-economic and political integration of the African continent, see African Union Handbook 2021 (African Union Commission 2021). 9 Ibid. 10 L. Abrego, Maria A. Amado, Tunc Gursoy, Garth P. Nicholls, and Hector Perez-Saiz, The African Continental Free Trade Agreement: Welfare Gains Estimates from a General Equilibrium Model (IMF Working Paper WP/19/124, 2019) 4. 11 S. Kedem, ‘UK-Africa Investment Summit Hints at Post-Brexit Future’ (African Business, February 2020) 29. 12 ‘PM Africa Investment Summit Speech’ (International, 20 January 2020); ‘UK–Egypt Cooperation on Transportation Projects’ (Trade and Investment, 25 May 2021). Unif. L. Rev., 2022, 1–27 Luxembourg Rail Protocol in Africa 3 3 Convention on International Carriage by Rail (COTIF),13 the Uniform Rules Concerning the Contract for International Carriage of Passengers and Luggage by Rail,14 and the Uniform Rules Concerning the Contract for International Carriage of Goods by Rail.15 The potential adoption of these laws can advance the development of transnational contract law of carriage by rail in Africa. 13 Convention on International Carriage by Rail (Convention Relative aux Transports Internationaux Ferroviaires, ‘COTIF’), adopted in Bern, 9 May 1980, later revised in the Protocol of Modification of 3 June 1999 (in force 1 July 2006). Presently, Algeria, Tunisia and Morocco are the only African OTIF member states and contracting parties to COTIF. 16 The European Green Deal is a nascent growth initiative that seeks to transform the EU into a fair and prosperous society through sustainable railway transportation, see European Commission, ‘Decision (EU) 2020/2228 of the European Parliament and of the Council of 23 December 2020 on a European Year of Rail 2021 (OJ L 437, 28.12.2020, 108). 17 R. Goode, Convention on International Interests in Mobile Equipment and Luxembourg Protocol Thereto on Matters Specific to Railway Rolling Stock: Official Commentary (2nd edn, UNIDROIT 2014) para 3.1. 13 Convention on International Carriage by Rail (Convention Relative aux Transports Internationaux Ferroviaires, ‘COTIF’), adopted in Bern, 9 May 1980, later revised in the Protocol of Modification of 3 June 1999 (in force 1 July 2006). Presently, Algeria, Tunisia and Morocco are the only African OTIF member states and contracting parties to COTIF. 14 In force from 1 July 2006. 15 In force from 1 July 2006. 16 The European Green Deal is a nascent growth initiative that seeks to transform the EU into a fair and prosperous society through sustainable railway transportation, see European Commission, ‘Decision (EU) 2020/2228 of the European Parliament and of the Council of 23 December 2020 on a European Year of Rail 2021 (OJ L 437, 28.12.2020, 108). 17 R. Goode, Convention on International Interests in Mobile Equipment and Luxembourg Protocol Thereto on Matters Specific to Railway Rolling Stock: Official Commentary (2nd edn, UNIDROIT 2014) para 3.1. I. Introduction In addition, the inherent capacity of railway rolling stock to move volumes of freight or passengers in an environmentally friendly and energy-efficient way makes it an attractive and sustainable means of transportation.16 Acknowledging the socio-economic value of access to modern rail equipment, this article offers a call for reform directed to all African countries to accede to the Cape Town Convention and the Rail Protocol. The Rail Protocol will en- hance the availability of modern rail equipment in Africa while providing a glo- bal regime for recognizing, registering, and enforcing international security interests held by creditors and lessors of railway rolling stock. Creditors and les- sors require security to ensure that credit advanced through loans or leases will be repaid. Protection of property rights in rail equipment and easy and quick re- possession of collateral in the event of default or insolvency of the debtor—even when the collateral moves from one country to another—are often required for secured financing.17 The Rail Protocol will bring uniformity to rail private sec- tor financing laws both in Africa and globally, which can act as the needed cata- lyst for the modernization of rail infrastructure, thus leading to an efficient transportation system. The main discussion in this article focuses on the legal reasons why Regional Economic Integration Organizations (REIOs)—that is, sub-regional organiza- tions—in Africa cannot accede to the Cape Town Convention and the Rail Protocol. Following the provisions of Article 6 of the 1986 Vienna Convention on the Law of Treaties between States and International Organizations or be- tween International Organizations (1986 VCLT), the founding treaties of sub- regional organizations in Africa do not equip REIOs with the competence to legislate over matters in the Cape Town Convention and the Rail Protocol. Additionally, this article concludes that the AU and the AEC lack the compe- tence to make a declaration on behalf of their Member States subject to Article XXII(2) of the Rail Protocol. Rev. dr. unif., 2022, 1–27 4 Iyare Otabor-Olubor 4 Iyare Otabor-Olubor The essence of the Cape Town Convention and the Rail Protocol will first be explained, followed by an examination of the mechanism behind the European Union (EU) accession to the Cape Town Convention and the Rail Protocol. Next, a summary of the AU’s sub-structure and its inter-relationship with the AEC will be discussed. 18 UNIDROIT, ‘Diplomatic Conference to Adopt a Mobile Equipment Convention and an Aircraft Protocol’ (Cape Town, South Africa, 29 October—16 November 2001). 19 Pursuant to Article 49(1) of the Convention, it entered into force on 1 March 2006. 20 R. Goode, ‘Transcending the Boundaries of Earth and Space: The Preliminary Draft UNIDROIT Convention on International Interests in Mobile Equipment’ (1998) 3 Uniform Law Review 52. 21 M. Lehmann, ‘The State of Development of Uniform Law in the Fields of European and International Civil and Commercial Law’ (2008) 7 European Legal Forum 266, 269. 22 R. Goode and H. Rosen, ‘Explanatory Report of the Draft Protocol on Matters Specific to Railway Rolling Stock’ (UNIDROIT/OTIF 2006 DCME-RP—Doc. 4) 1. I. Introduction It demonstrates why the author concedes that the AU and AEC sub-re- gional organizations lack exclusive competence to accede to the Cape Town Convention and the Rail Protocol. A detailed account of the emergence and legal responsibilities of the AU, the AEC, and the Organisation pour l’harmonisation en Afrique du Droit des Affaires (OHADA) will also be undertaken to shed light on why they do not enjoy exclusive competence over matters governed by the Rail Protocol. Finally, the article expounds that any REIO/sub-regional organization in Africa that attempts to accede to the Cape Town Convention or the Rail Protocol on behalf of their Member States will be contravening Article 6 of the 1986 VCLT, the AEC Treaty, and other AU laws. Conclusively, the author holds that the direc- tion taken by African countries that have ratified or acceded to the Cape Town Convention and the Protocol on Matters Specific to Aircraft Equipment (Aircraft Protocol) is the justifiable approach that should be maintained when ratifying or acceding to the Rail Protocol. 18 UNIDROIT, ‘Diplomatic Conference to Adopt a Mobile Equipment Convention and an Aircraft Protocol’ (Cape Town, South Africa, 29 October—16 November 2001). II. Background to the Cape Town Convention The Cape Town Convention and Aircraft Protocol were jointly adopted on 16 November 2001 at a Diplomatic Conference hosted in Cape Town, following the invitation of the government of South Africa.18 The conference was coordi- nated under the joint aegis of the International Institute for the Unification of Private Law (UNIDROIT) and the International Civil Aviation Organization.19 The Cape Town Convention establishes a uniform law for secured financing of high-value mobile equipment that frequently crosses international borders.20 The lack of global uniformity in recognizing security interests in mobile equip- ment promoted its adoption. Mobile equipment may be attached unexpectedly or, in the worst case, perfected in another country with an unpredictable prop- erty system.21 There is also the risk of pluralism among national laws, inconsis- tencies in legal application and judicial interpretation, and inefficient collateral registries. The inoperability of the lex rei sitae rule governing proprietary rights, which can apply to the law of the jurisdiction where the mobile asset is located, presents another challenge.22 The Cape Town Convention and Aircraft Protocol were jointly adopted on 16 November 2001 at a Diplomatic Conference hosted in Cape Town, following the invitation of the government of South Africa.18 The conference was coordi- nated under the joint aegis of the International Institute for the Unification of Private Law (UNIDROIT) and the International Civil Aviation Organization.19 Unif. L. 23 R. Goode, ‘From Acorn to Oak Tree: The Development of the Cape Town Convention and Protocols’ (2012) 17 Uniform Law Review 599; see also Convention, Art 2(2). 31 S. Gopalan, ‘Harmonization of Commercial Law: Lessons from the Cape Town Convention on International Interests in Mobile Equipment’ (2003) 9 Law and Business Review of the Americas 255, 263. 24 UNIDROIT, Luxembourg Protocol to the Convention on International Interests in Mobile Equipment on Matters Specific to Railway Rolling Stock (Luxembourg, 23 February 2007). It will enter into force in accordance with Art XXIII. 29 B. von Bodungen and H. Rosen, ‘From the Luxembourg Rail Protocol to the Draft MAC Protocol’ (2018) 23 Uniform Law Review 190, 191. 25 UNIDROIT, Protocol to the Convention on International Interests in Mobile Equipment on Matters Specific to Space Assets (Berlin, 9 March 2012). It will enter into force pursuant to Art XXXVIII. 26 UNIDROIT, Protocol to the Convention on International Interests in Mobile Equipment on Matters Specific to Mining, Agriculture and Construction Equipment’ (Cape Town, 22 October 2019). It will enter into force pursuant to Art XXV. II. Background to the Cape Town Convention Rev., 2022, 1–27 Luxembourg Rail Protocol in Africa Luxembourg Rail Protocol in Africa Luxembourg Rail Protocol in Africa 5 5 The Cape Town Convention regulates the creation, registration, priority, and enforcement of international interests in high-value and uniquely identifiable mobile equipment held by a conditional seller under a retention of title con- tract, a lessor’s proprietary right under a leasing agreement, and a chargee’s se- curity interest.23 Using the original two-pronged structure (the convention and a protocol), the Cape Town Convention under Article 2(3) contemplates two other accompanying protocols (not yet in force): the Luxembourg Protocol for Railway Rolling Stock (Rail Protocol), adopted in 2007,24 and the Berlin Space Protocol for Space Equipment (Space Protocol), adopted in 2012.25 Article 51(1) of the Cape Town Convention acknowledges the creation of future proto- cols, which has culminated in the adoption of the Protocol for Mining, Agricultural and Construction Equipment (MAC Protocol) in 2019.26 Though these protocols operate separately under the Cape Town Convention; the Convention and each protocol should be read and interpreted together as one instrument.27 In the event of inconsistency between the Convention and a protocol, that protocol will prevail.28 These protocols are intended to supple- ment and amend the Convention’s provisions, thus being responsive to the modern requirements that arise with a particular category of equipment, includ- ing related industry practices and expectations.29 Matters within the Cape Town Convention’s scope include default remedies and an international registration system that governs the effects of an inter- national interest against competing claimants, insolvency matters, assignments of associated rights, and rights of subrogation. When concluding the agreement that creates the international interest in favour of the creditor, the debtor must be situated in a Contracting State, irrespective of whether the creditor is located in a non-Contracting State.30 The interpretative meaning given to ‘international interests’ under the Convention does not rely on any national law definition.31 Likewise, the motive of the Convention is not to unify national private law and 26 UNIDROIT, Protocol to the Convention on International Interests in Mobile Equipment on Matters Specific to Mining, Agriculture and Construction Equipment’ (Cape Town, 22 October 2019). It will enter into force pursuant to Art XXV. 27 Convention, Art 6(1). 28 Convention, Art 6(2). 29 B. von Bodungen and H. Rosen, ‘From the Luxembourg Rail Protocol to the Draft MAC Protocol’ (2018) 23 Uniform Law Review 190, 191. 30 Convention, Art 2(2). 31 S. 32 N. Orkun Akseli, ‘The Interpretation Philosophy of Secured Transaction Law Conventions’ (2013) 21 ERPL 1299; C. Bourbon-Seclet, ‘Cross-border Security Interests in Movable Property: An Attempt at Rationalising the International Patchwork—Part 2’ (2005) 20 JIBLR 501, 506. II. Background to the Cape Town Convention Gopalan, ‘Harmonization of Commercial Law: Lessons from the Cape Town Convention on International Interests in Mobile Equipment’ (2003) 9 Law and Business Review of the Americas 255, 263. Rev. dr. unif., 2022, 1–27 Iyare Otabor-Olubor 6 substantive rules on personal property security in a domestic context but, ra- ther, to create an optional and harmonized transnational secured transactions legal instrument specific to high-value mobile assets.32 Thus, by developing a sui generis global regime, the Convention and accompanying protocols do not interfere with existing domestic substantive rules on personal property security, with the intent of avoiding, as far as possible, the need to depend on conflict of law rules.33 At its irreducible core, the Cape Town Convention seeks to promote cross- border commercial transactions between debtors and creditors; second, it pro- motes certainty in making equipment financing reasonably accessible while ensuring that creditors have expeditious remedies to enforce their rights in identifiable assets; and, third, it cuts down on transactional costs and other costs involved with monitoring the collateral and enforcing the rights of creditors.34 Simply put, according to Sir Roy Goode, QC, the Convention and the protocols are governed by five underlying principles—namely, practicality, party auton- omy, predictability, transparency, and sensitivity to the local laws of national jurisdictions.35 36 European Commission, ‘Council Decision of 6 April 2009 on the accession of the European Community to the Convention on International Interests in Mobile Equipment and its Protocol on Matters Specific to Aircraft Equipment, adopted jointly in Cape Town on 16 November 2001’ (2009/370/EC, 15 May 2009). 33 K.F. Kreuzer, ‘Jurisdiction and Choice of Law Under the Cape Town Convention and the Protocols thereto’ (2013) 2 Cape Town Convention Journal 149. 37 Aircraft Protocol, Art XXVIII. The Luxembourg Rail Protocol requires four instruments of rati- fication, Art XXIII; the Berlin Space Protocol requires ten instruments of ratification, Art XXXVIII; the MAC Protocol requires five instruments of ratification, Art XXV. 34 I. Davies, ‘The New Lex Mercatoria: International Interests in Mobile Equipment’ (2003) 52 ICLQ 151, 153–4. 39 The eight countries were Ethiopia, Ireland, Malaysia, Nigeria, Oman, Panama, Pakistan, and USA. 35 R. Goode, Official Commentary n 17 above, para 2.13–2.18. 1. The Cape Town Convention The Cape Town Convention remains one of the most successful private inter- national law treaties today. Eighty-three countries, including the EU, are Contracting States.36 In total, 27 African countries have either ratified or acceded to the Convention. Today, the Aircraft Protocol is the only protocol in force, having already met the requirement of eight instruments of ratification, acceptance, approval or accession.37 The Convention enters into force only in the Contracting States to which the respective protocol applies.38 Two African countries—Ethiopia and Nigeria—were among the first eight countries that deposited their instrument of ratification, which brought the Cape Town Convention and the Aircraft Protocol into force on 1 March 2006.39 The Unif. L. Rev., 2022, 1–27 Luxembourg Rail Protocol in Africa 7 Convention is open for acceptance, approval, or accession by a sovereign State or an REIO, which may also be constituted by sovereign States that have trans- ferred competence to the REIO.40 However, the Convention does not specify the legal personality of a REIO. Nor does Article 48 of the Convention explain the degree of competence that a REIO should possess before legislating over matters governed by the Convention. The author supports the view that African REIOs are responsible for meeting their existing erga omnes obligations following the 2008 Protocol on Relations between the African Union and the Regional Economic Communities (Protocol on Relations). Article 28 of the Protocol on Relations obliges REIOs to realize their regional integration objectives by entering into cooperation agreements with international organizations or with third countries, provided that such agreements do not conflict with the objectives of the AEC Treaty. On the basis of this article, the competence of REIOs to legislate in specific matters on behalf of its Member States in Africa will be analysed. This view has been taken because of the gradual unification of law through the emerging AU legal framework, subject to Article 6 of the 1986 VCLT.41 This emerging framework recognizes cooperation and integration between REIOs and sovereign States in Africa on various policy levels such as human rights, economic, political, or other, indica- tive of the existence of legal personality in REIOs.42 The geographical delimita- tion of an REIO cannot be used as a determining factor because a country can belong to several REIOs.43 There are 54 countries in Africa, and the continent is fragmented into many political jurisdictions and international boundaries. 40 Convention, Art 47–8. Any State that has not signed the Convention at the time of adoption may accede to the Convention at a later date—see Art 47(3). 41 M. Olivier, ‘Conceptualizing AU Law within the Constitutional Framework of the AU’ in O. Amao, M. Olivier, and K.D. Magliveras (eds), The Emergent African Union Law: Conceptualization, Delimitation, and Application (OUP 2021) 11. 42 43 J.T. Gathii, ‘African Regional Trade Agreements as Flexible Legal Regime’ (2010) North Carolina Journal of International Law 572, 648. 43 J.T. Gathii, ‘African Regional Trade Agreements as Flexible Legal Regime’ (2010) North Carolina Journal of International Law 572, 648. 44 P. Collier, ‘Building an African Infrastructure’ (2011) 48 IMF Finance and Development 18, 19. 45 R. Tavares & V. Tang, ‘Regional Economic Integration in Africa: Impediments to Progress?’ (2011) 18 South African Journal of International Affairs 217, 223. 46 Financing the Development of Aviation Infrastructure, ‘Declaration and Framework for a Plan of Action for Development of Aviation Infrastructure in Africa’ (3rd International Civil Aviation Organization (ICAO) World Aviation Forum, 20–2 November 2017, Abuja, Nigeria). 46 Financing the Development of Aviation Infrastructure, ‘Declaration and Framework for a Plan of Action for Development of Aviation Infrastructure in Africa’ (3rd International Civil Aviation Organization (ICAO) World Aviation Forum, 20–2 November 2017, Abuja, Nigeria). 45 R. Tavares & V. Tang, ‘Regional Economic Integration in Africa: Impediments to Progress?’ (2011) 18 South African Journal of International Affairs 217, 223. 44 P. Collier, ‘Building an African Infrastructure’ (2011) 48 IMF Finance and Development 18, 19. 40 Convention, Art 47–8. Any State that has not signed the Convention at the time of adoption may accede to the Convention at a later date—see Art 47(3). 41 M. Olivier, ‘Conceptualizing AU Law within the Constitutional Framework of the AU’ in O. Amao, M. Olivier, and K.D. Magliveras (eds), The Emergent African Union Law: Conceptualization, Delimitation, and Application (OUP 2021) 11. 42 Ibid. 43 J.T. Gathii, ‘African Regional Trade Agreements as Flexible Legal Regime’ (2010) North Carolina Journal of International Law 572, 648. 44 P. Collier, ‘Building an African Infrastructure’ (2011) 48 IMF Finance and Development 18, 19. 45 R. Tavares & V. Tang, ‘Regional Economic Integration in Africa: Impediments to Progress?’ (2011) 18 South African Journal of International Affairs 217, 223. 46 Financing the Development of Aviation Infrastructure, ‘Declaration and Framework for a Plan of Action for Development of Aviation Infrastructure in Africa’ (3rd International Civil Aviation Organization (ICAO) World Aviation Forum, 20–2 November 2017, Abuja, Nigeria). 48 UNIDROIT, ‘Summary Record of the Meetings of the Conference Plenum—First Meeting’ in Diplomatic Conference to Adopt a Mobile Equipment Convention and an Aircraft Protocol: Acts and Proceedings (UNIDROIT, Monday 29 October 2001) 733, statement by Minister of Transport of South Africa ‘Permit me also to express a special word of welcome to the delegations from fellow African States. In the context of the newly created African Union and developments in the Aviation sector on our continent, your presence is precious to South Africa as we seek together to promote the interests of our continent’. 51 Report on Committee of Governmental Experts for the Preparation of a Draft Protocol to the Draft UNIDROIT Convention on International Interests in Mobile Equipment on Matters Specific to Railway Rolling Stock (First Joint Session, OTIF/JGR/3 - UNIDROIT 2001 Study LXXIIH— Doc. 5, Berne, 15–16 March 2001). The primary working paper for the Committee was the ‘Preliminary Draft Protocol to the Draft UNIDROIT Convention on International Interests in Mobile Equipment on Matters specific to Railway Rolling Stock’ (OTIF/JGR/2—UNIDROIT 2000 Study LXXIIH—DOC. 4). This preliminary draft Rail Protocol was produced by the Rail Working Group (RWG), co-ordinated and chaired by Mr Howard Rosen, an expert on inter- national railway financing. During the discussions, OTIF advised that relying entirely on railway financing models such as sovereign guarantees and taxpayers funding were unsustainable, but rather an asset-based financing approach should be considered—see H. Kronke, ‘The Luxembourg Protocol to the Cape Town Convention: A Pillar for the Bridge to the Future of Rail Transportation Secured Rail Financing’ (2007) 12 Uniform Law Review 420. 47 African States’ Position on the Draft Convention on International Interests in Mobile Equipment and The Draft Protocol on Matters Specific to Aircraft (Presented by the African States) DCME Doc No. 25, 19 October 2001. 1. The Cape Town Convention For many African countries to access global markets, they would inevitably depend on poor transport infrastructure and underdeveloped routes through other countries.44 This is an endemic problem in Africa because of the proliferation of several competing sub-regional organizations where countries belong to mul- tiple REIOs, resulting in duplication and overlapping economic activities.45 African governments’ declarations complement the Cape Town Convention’s application in Africa.46 The position of African governments on the Convention Rev. dr. unif., 2022, 1–27 Iyare Otabor-Olubor 8 and the Aircraft Protocol was presented at the Diplomatic Conference adopting these instruments.47 This fact was also supported in the former South African Minister of Transport’s opening statement at the First Meeting of the Diplomatic Conference adopting the Cape Town Convention and the Aircraft Protocol in 2001, directed towards African government delegates.48 African governments rec- ognize the socio-economic and financial benefits of the Convention, which in- clude the affordable acquisition of modern equipment, reduced travel fares, improved repayment interest rates, longer or slower repayment terms, increased level of financing, access to letters of credit, and reduced pressure on African gov- ernments to finance mobile equipment.49 Accomplishing these benefits would allow African governments to divert scarce public funds to other sectors of their economy. 50 Unform Law Instruments, ‘Luxembourg Protocol to the Convention on International Interests in Mobile Equipment on Matters Specific to Railway Rolling Stock’ (2007) 12 Uniform Law Review 585. 52 It has been signed by France, Gabon, Germany, Italy, Luxembourg, Mozambique, South Africa, Spain, Sweden, Switzerland, and United Kingdom. f 49 Dean N. Gerber and David R. Walton, ‘De-registration and Export Remedies under the Cape Town Convention’ (2014) 3 Cape Town Convention Journal 49. 2. The Rail Protocol Following the success of the Cape Town Convention and the Aircraft Protocol, the Rail Protocol was negotiated by UNIDROIT and the Intergovernmental Organization for International Carriage by Rail (OTIF).50 The negotiations began in 2001 with a joint UNIDROIT–OTIF Committee session of governmental experts attended by 38 participants of 20 States and six observers from four international organizations.51 On 23 February 2007, the Rail Protocol was offi- cially opened for signature. The Rail Protocol is not yet in force,52 but it has 49 Dean N. Gerber and David R. Walton, ‘De-registration and Export Remedies under the Cape Town Convention’ (2014) 3 Cape Town Convention Journal 49. 51 Report on Committee of Governmental Experts for the Preparation of a Draft Protocol to the Draft UNIDROIT Convention on International Interests in Mobile Equipment on Matters Specific to Railway Rolling Stock (First Joint Session, OTIF/JGR/3 - UNIDROIT 2001 Study LXXIIH— Doc. 5, Berne, 15–16 March 2001). The primary working paper for the Committee was the ‘Preliminary Draft Protocol to the Draft UNIDROIT Convention on International Interests in Mobile Equipment on Matters specific to Railway Rolling Stock’ (OTIF/JGR/2—UNIDROIT 2000 Study LXXIIH—DOC. 4). This preliminary draft Rail Protocol was produced by the Rail Working Group (RWG), co-ordinated and chaired by Mr Howard Rosen, an expert on inter- national railway financing. During the discussions, OTIF advised that relying entirely on railway financing models such as sovereign guarantees and taxpayers funding were unsustainable, but rather an asset-based financing approach should be considered—see H. Kronke, ‘The Luxembourg Protocol to the Cape Town Convention: A Pillar for the Bridge to the Future of Rail Transportation Secured Rail Financing’ (2007) 12 Uniform Law Review 420. Unif. L. Rev., 2022, 1–27 Luxembourg Rail Protocol in Africa 9 9 been signed by three African countries: Gabon, Mozambique, and South Africa. 59 R. Goode, Official Commentary n 17 above, para 2.4. 2. The Rail Protocol The Rail Protocol requires four instruments of either ratification, acceptance, approval, or accession, among other requirements, before it enters into force.53 So far, the EU and three countries (including Gabon) have submitted the required instruments.54 Many African countries have either ratified or acceded to the Cape Town Convention, compared to similar conventions such as COTIF55 and the 1988 UNIDROIT Convention on International Financial Leasing (Ottawa Convention).56 The Cape Town Convention will prevail over them to the extent of any inconsistency between it and the Ottawa Convention or COTIF regarding rail equipment leasing and financing.57 The Rail Protocol’s objectives are to facilitate private sector financing for roll- ing stock worldwide, eliminate bottlenecks that impede industry from partici- pating in rail sector development, and resolve cross-border legal issues in rail financing.58 An international legal framework to protect the rights of secured creditors and lessors who supply rolling stock under leasing agreements and re- tention of title contracts is a fundamental step in boosting access to finance.59 The Rail Protocol applies to all types of rolling stock such as movable vehicles The Rail Protocol applies to all types of rolling stock, such as movable vehicles on a fixed railway track or on, above, or below a guideway, along with parts installed on such vehicles and all manuals, records, and other data.60 Generally, railway systems globally have different methods of identifying roll- ing stock.61 Due to the inconsistent identification systems with rolling stock, different registers for recording security interests, and many manufacturers operating worldwide, there was no mechanism to validate these different sys- tems on which an international interest could attach successfully.62 The Rail Protocol considered three solutions to identifying rolling stock: practically, a unique identifier designated by the international registry;63 a national or region- al numbering system utilized by a Contracting State;64 and a manufacturer’s 57 Rail Protocol, Art XIX and Art XX. 58 H. Rosen, M. Fleetwood and B. von Bodungen, ‘The Luxembourg Rail Protocol—Extending Cape Town Benefits to the Rail Industry’ (2012) 17 Uniform Law Review 609. 58 H. Rosen, M. Fleetwood and B. von Bodungen, ‘The Luxembourg Rail Protocol—Extending Cape Town Benefits to the Rail Industry’ (2012) 17 Uniform Law Review 609. 59 R. Goode, Official Commentary n 17 above, para 2.4. 60 Rail Protocol, Art I(2)(e). 61 M.J. 53 Rail Protocol, Art XXIII. 54 Gabon submitted an acceptance on 4 April 2017. Luxembourg ratified on 31 January 2012. Sweden ratified on 2 July 2018. 55 Al i M d T i i th th Af i C t ti St t t COTIF 56 Ghana, Guinea, Morocco, Nigeria, and Tanzania are the African signatories to the Ottawa Convention. Only Nigeria has ratified it. 64 Rail Protocol, Art XIV (2–4). 61 M.J. Fleetwood, ‘All Tracks Lead to Luxembourg: the Luxembourg Rail Protocol’ (2007) 6 JIBFL 318; See also Rail Working Group, ‘What Equipment is Covered by the Luxembourg Protocol?’ (Briefing Paper). 53 Rail Protocol, Art XXIII. 54 Gabon submitted an acceptance on 4 April 2017. Luxembourg ratified on 31 January 2012. Sweden ratified on 2 July 2018. 55 Algeria, Morocco and Tunisia are the three African Contracting States to COTIF. 56 Ghana, Guinea, Morocco, Nigeria, and Tanzania are the African signatories to the Ottawa Convention. Only Nigeria has ratified it. 57 Rail Protocol, Art XIX and Art XX. 58 H. Rosen, M. Fleetwood and B. von Bodungen, ‘The Luxembourg Rail Protocol—Extending Cape Town Benefits to the Rail Industry’ (2012) 17 Uniform Law Review 609. 59 R. Goode, Official Commentary n 17 above, para 2.4. 60 Rail Protocol, Art I(2)(e). 61 M.J. Fleetwood, ‘All Tracks Lead to Luxembourg: the Luxembourg Rail Protocol’ (2007) 6 JIBFL 318; See also Rail Working Group, ‘What Equipment is Covered by the Luxembourg Protocol?’ (Briefing Paper). 62 B. von Bodungen and H. Rosen, ‘From the Luxembourg Rail Protocol to the draft MAC Protocol’ (2018) 23 Uniform Law Review 190, 199. 63 Rail Protocol, Art XIV (1) states that the Registrar shall allocate a unique number to an item of rolling stock which may either be affixed or associated with the existing manufacturer’s or na- tional and regional identification number. 64 Rail Protocol, Art XIV (2–4). 54 Gabon submitted an acceptance on 4 April 2017. Luxembourg ratified on 31 January 2012. Sweden ratified on 2 July 2018. 55 Algeria, Morocco and Tunisia are the three African Contracting States to COTIF. 56 Ghana, Guinea, Morocco, Nigeria, and Tanzania are the African signatories to the Ottawa Convention. Only Nigeria has ratified it. 57 Rail Protocol Art XIX and Art XX 2. The Rail Protocol Fleetwood, ‘All Tracks Lead to Luxembourg: the Luxembourg Rail Protocol’ (2007) 6 JIBFL 318; See also Rail Working Group, ‘What Equipment is Covered by the Luxembourg Protocol?’ (Briefing Paper). 61 M.J. Fleetwood, ‘All Tracks Lead to Luxembourg: the Luxembourg Rail Protocol’ (2007) 6 JIBFL 318; See also Rail Working Group, ‘What Equipment is Covered by the Luxembourg Protocol?’ (Briefing Paper). 62 B. von Bodungen and H. Rosen, ‘From the Luxembourg Rail Protocol to the draft MAC Protocol’ (2018) 23 Uniform Law Review 190, 199. 62 B. von Bodungen and H. Rosen, ‘From the Luxembourg Rail Protocol to the draft MAC Protocol’ (2018) 23 Uniform Law Review 190, 199. 63 Rail Protocol, Art XIV (1) states that the Registrar shall allocate a unique number to an item of rolling stock which may either be affixed or associated with the existing manufacturer’s or na- tional and regional identification number. 63 Rail Protocol, Art XIV (1) states that the Registrar shall allocate a unique number to an item of rolling stock which may either be affixed or associated with the existing manufacturer’s or na- tional and regional identification number. 64 Rail Protocol, Art XIV (2–4). Rev. dr. unif., 2022, 1–27 Iyare Otabor-Olubor 10 serial number.65 However, operating the international registry with three differ- ent identifiers can become cumbersome and expensive. 66 E. Hirst and N. Gavage, ‘The International Rail Registry and The Luxembourg Rail Protocol to The Cape Town Convention—Global Registration of Mobile Assets’ (2015) 46 UCCLJ 359, 368. URVIS was developed upon the completion of the Luxembourg Diplomatic Conference adopt- ing the Rail Protocol. The Rail Working Group led an industry task force to develop URVIS, consulting with representatives from the Association of the European Rail Industry (UNIFE), Community of European Railway and Infrastructure Companies (CER), and the European Commission. 67 B. M. Goodstein and H. Rosen, ‘Financing Rolling Stock: Luxembourg Rail Protocol Steams Ahead’ (2017) 257 New York Law Journal 1, 2. 69 S. Saidova, Security Interests under the Cape Town Convention on International Interests in Mobile Equipment (Hart 2018) 238; R. Goode, Official Commentary n 17 above, para 3.30. 71 Rail Protocol, Art XXV(3). Rail Protocol Article XXV(4) enables a Contracting State to disapply the rules enshrined in Art XXV(2) and (3) with regards to enforcement of an international interest in the rolling stock. 70 B. von Bodungen and K. Schott, ‘The Public Service Exemption under the Luxembourg Rail Protocol: A German Perspective’ (2007) Uniform Law Review 573, 577. 65 Rail Protocol, Art XIV (1). y p Mobile Equipment (Hart 2018) 238; R. Goode, Official Commentary n 17 above, para 3.30. 70 B. von Bodungen and K. Schott, ‘The Public Service Exemption under the Luxembourg Rail Protocol: A German Perspective’ (2007) Uniform Law Review 573, 577. 71 Rail Protocol, Art XXV(3). Rail Protocol Article XXV(4) enables a Contracting State to disapply 68 Rail Protocol, Art XXV. 69 S. Saidova, Security Interests under the Cape Town Convention on International Interests in Mobile Equipment (Hart 2018) 238; R. Goode, Official Commentary n 17 above, para 3.30. 70 B. von Bodungen and K. Schott, ‘The Public Service Exemption under the Luxembourg Rail Protocol: A German Perspective’ (2007) Uniform Law Review 573, 577. 71 Rail Protocol, Art XXV(3). Rail Protocol Article XXV(4) enables a Contracting State to disapply the rules enshrined in Art XXV(2) and (3) with regards to enforcement of an international interest in the rolling stock. 65 Rail Protocol, Art XIV (1). 66 E. Hirst and N. Gavage, ‘The International Rail Registry and The Luxembourg Rail Protocol to The Cape Town Convention—Global Registration of Mobile Assets’ (2015) 46 UCCLJ 359, 368. URVIS was developed upon the completion of the Luxembourg Diplomatic Conference adopt- ing the Rail Protocol. The Rail Working Group led an industry task force to develop URVIS, consulting with representatives from the Association of the European Rail Industry (UNIFE), Community of European Railway and Infrastructure Companies (CER), and the European Commission. 67 B. M. Goodstein and H. Rosen, ‘Financing Rolling Stock: Luxembourg Rail Protocol Steams Ahead’ (2017) 257 New York Law Journal 1, 2. 68 Rail Protocol, Art XXV. 69 S. Saidova, Security Interests under the Cape Town Convention on International Interests in Mobile Equipment (Hart 2018) 238; R. Goode, Official Commentary n 17 above, para 3.30. 70 B. von Bodungen and K. Schott, ‘The Public Service Exemption under the Luxembourg Rail Protocol: A German Perspective’ (2007) Uniform Law Review 573, 577. 71 Rail Protocol, Art XXV(3). Rail Protocol Article XXV(4) enables a Contracting State to disapply the rules enshrined in Art XXV(2) and (3) with regards to enforcement of an international interest in the rolling stock. 2. The Rail Protocol A viable solution was to use a unique identifier—a 12-digit number, known as the Unique Rail Vehicle Identification System (URVIS), allocated by the registrar on demand and applied to a specific item of rolling stock.66 Considering that not all countries have a dedicated registry for recording interests in rolling stock, having a harmonized recording system through URVIS, the Rail Protocol will make financing more secure for creditors while promoting transparency in ascertain- ing rights in rolling stock.67 g g g An apparent feature of the Rail Protocol is that a Contracting State, subject to a formal declaration, can prioritize public transport accessibility, whereby the rolling stock is ‘habitually used for the purpose of providing services of public importance’, either for the purpose of carriage of freights or passengers.68 Under this exemption, a Contracting State can declare that it will continue to apply its national rules of law and public regulations, which may preclude, sus- pend, or govern the Cape Town Convention and Rail Protocol’s enforcement remedies so long as the rolling stock is habitually used to offer public transport service.69 Effectively, the creditor may not be able to enforce the international interest over every item of railway rolling stock. Furthermore, enforcement may be unavailable because a Contracting State government may have policies dic- tated by constitutional matters, such as inter-city transportation, that the Rail Protocol may affect if the creditor of the rolling stock is allowed to foreclose on the rolling stock.70 The effects of this legal quagmire can potentially cripple the public rail transport system and, ultimately, the economy. In certain circum- stances, this enables the State to prevent the creditor from asserting super- priority rights against the collateral, so long as any person, including the State or a public authority retaining control, compensates the creditor of an amount equal to the greater of the rolling stock lease rental and the amount as agreed under national law.71 The Rail Protocol stipulates that a Contracting State Unif. L. 76 The regulation of railway rolling stock in Africa stretches back to the Geneva Convention on the International Regime of Railways 1923, following the old British Empire’s ratification, see Convention and Statute on the International Regime of Railways and Protocol of Signature (signed 9 December 1923, ratified 29 August 1924) UK Treaty Series No. 23 1925 (Cmd. 2418). See also J.G. de Matons, A Review of International Legal Instruments: Facilitation of Transport and Trade in Africa (2nd edn, SSATP 2014) 50. 74 S. Kozuka, ‘The Cape Town Convention and Its Implementation in Domestic Law: Between Tradition and Innovation’ in Souichirou Kozuka (eds), Implementing the Cape Town Convention and the Domestic Laws on Secured Transactions (Springer 2017) 25. 2. The Rail Protocol Rev., 2022, 1–27 Luxembourg Rail Protocol in Africa 11 should ensure that the relevant public establishments in the State swiftly cooper- ate with and support the creditor to the extent necessary to enforce the foreclos- ure order,72 unless the Contracting State declares that it will continue to apply the rules of law in force in its territory.73 The Cape Town Convention binds the courts of Contracting States to apply the provisions of the Convention and the Rail Protocol.74 This may happen when, for instance, an African State becomes a Contracting State. The court in that State must apply the legal rules when the debtor is situated in a Contracting State at the time of the conclusion of the se- curity agreement (for example, a lease agreement) that creates or provides for the international interest in the rolling stock. The location of the creditor, whether in a Contracting State or not, is irrelevant.75 77 PIDA, Progress Report 2019 Summary Update: Positioning Africa to deliver on Agenda 2063 and Economic Integration through Multi-sectoral Approaches to Infrastructure Development (25 November 2019). 72 Rail Protocol, Art VII(5). 73 Rail Protocol, Art XXV. 79 Rail Infrastructure in Africa: Financing Policy Options (African Development Bank 2015). See also, Towards an Enhanced Africa-EU Cooperation on Transport and Connectivity: Report by the Task Force on Transport and Connectivity (European Commission, February 2020). 78 Towards the African Integrated High-Speed Railway Network (AIHSRN) Development (African Union, March 2017) 2. 72 Rail Protocol, Art VII(5). 73 Rail Protocol, Art XXV. 74 S. Kozuka, ‘The Cape Town Convention and Its Implementation in Domestic Law: Between Tradition and Innovation’ in Souichirou Kozuka (eds), Implementing the Cape Town Convention and the Domestic Laws on Secured Transactions (Springer 2017) 25. 75 Convention, Art 3. 76 The regulation of railway rolling stock in Africa stretches back to the Geneva Convention on the International Regime of Railways 1923, following the old British Empire’s ratification, see Convention and Statute on the International Regime of Railways and Protocol of Signature (signed 9 December 1923, ratified 29 August 1924) UK Treaty Series No. 23 1925 (Cmd. 2418). See also J.G. de Matons, A Review of International Legal Instruments: Facilitation of Transport and Trade in Africa (2nd edn, SSATP 2014) 50. 77 PIDA, Progress Report 2019 Summary Update: Positioning Africa to deliver on Agenda 2063 and Economic Integration through Multi-sectoral Approaches to Infrastructure Development (25 November 2019). 78 Towards the African Integrated High-Speed Railway Network (AIHSRN) Development (African Union, March 2017) 2. 79 Rail Infrastructure in Africa: Financing Policy Options (African Development Bank 2015). See also, Towards an Enhanced Africa-EU Cooperation on Transport and Connectivity: Report by the Task Force on Transport and Connectivity (European Commission, February 2020). p g 75 Convention, Art 3. 3. Rolling stock regulation and policy in Africa The institutional mechanisms that have attempted to transform rolling stock in- frastructure in Africa since the dawn of the 20th century have led to an ava- lanche of initiatives that, for the most part, did not gain the necessary traction as anticipated.76 Since the 1970s, African railway companies have been managed as public enterprises with government oversight. By the mid-1980s, there were gradual changes to facilitate privatization and eliminate bureaucratic bottle- necks and enhance the financial sustainability of African railways.77 Many African railways reformed their legal status to accommodate liberalization and commercialization, encouraging private sector participation, with concessions concluded in some countries.78 However, in many cases, privatization did not promote positive financial results as envisaged. Since then, railway traffic has gradually declined because of resource mismanagement, inadequate infrastruc- ture, and outdated rolling stock equipment.79 One of the most significant of these initiatives is the Protocol on Transport, Communications and Tourism, annexed to the AEC Treaty, that confirms the 77 PIDA, Progress Report 2019 Summary Update: Positioning Africa to deliver on Agenda 2063 and Economic Integration through Multi-sectoral Approaches to Infrastructure Development (25 November 2019). 78 Towards the African Integrated High-Speed Railway Network (AIHSRN) Development (African Union, March 2017) 2. 79 Rail Infrastructure in Africa: Financing Policy Options (African Development Bank 2015). See also, Towards an Enhanced Africa-EU Cooperation on Transport and Connectivity: Report by the Task Force on Transport and Connectivity (European Commission, February 2020). Rev. dr. unif., 2022, 1–27 Iyare Otabor-Olubor 12 intention of African governments to modernize their rail infrastructure. 81 The conference was held from 13–14 April 2006 in Brazzaville, Republic of Congo and it was attended by several national government representatives. The Brazzaville Declaration was adopted by the Ministers from twenty-seven African States participating at the conference—see First African Union Conference of Ministers responsible for Railway Transport (International Union of Railways, 28 April 2006). 85 African Union, Developing Smart Infrastructure to Boost Africa’s Continental Transformation and Integration (Second Ordinary Session of the African Union Specialised Technical Committee on Transport, Transcontinental and Interregional Infrastructure, Energy and Tourism) STC-TTIIET/Min/Final/Rpt(II) (14–18 April 2019 Cairo, Egypt) 7. 83 Financing Railway Rolling Stock: A New Solution for Africa—Issue Paper (Thirty-eighth Meeting) UNECA E/ECA/COE/38/15 (Marrakech, Morocco, 20–2 March 2019). 84 Ibid, 1. 80 AEC Treaty, Art 61(1)(c), (2)(b) and (2)(f). 82 See AIHSRN, n 78, 2. 3. Rolling stock regulation and policy in Africa Article 61 of the AEC Treaty encourages AU Member States to harmonize transporta- tion rules and regulations and prepare plans to improve and harmonize rail net- works across the AEC Member States to support inter-connectivity and harmonize rail transport policies.80 Additionally, the Brazzaville Declaration and Plan of Action on African Railways was adopted in 2006 by the first meeting of the African Ministers responsible for railway transport, reaffirming African governments’ commitment to modernizing their railway system to promote de- velopment and economic integration in Africa.81 This event was followed by the Railway Professional Conference on Interconnection, Interoperability and Complementarity of African Railway Networks hosted in Johannesburg in November 2007, which considered the strategies for harmonizing standards for rolling stock and operational procedures for African railways.82 Further, the United Nations Economic Commission for Africa’s (UNECA) Committee of Experts of the Conference of African Ministers of Finance, Planning and Economic Development, at its 38th meeting in Marrakech, explored a holistic approach to reforming railway rolling stock financing in Africa.83 The Committee raised questions at this conference, such as the socio- economic benefits of the Rail Protocol and the extent of legal protection avail- able to creditors who finance rolling stock through leases, secured credit, and conditional sale contracts.84 Afterwards, the AU Commission took up the man- date to further explore the benefits of the Rail Protocol. Subsequently, it recom- mended the adoption of the Rail Protocol by all AU Member States: Recommendations: A resolution be considered to support the adoption of the Luxembourg Protocol to the Cape Town Convention on International Interests in Mobile Equipment on Matters Specific to Railway Rolling Stock by African Member States. The Rail Protocol will make it easier and cheaper for private rail operators to finance new procurements of rolling stock using private capital.85 This AU mandate reflects a welcome approach to accept the Rail Protocol as an international secured transactions law solution in Africa to modernize and fi- nance rail networks in Africa. Furthermore, the Resolution supports the reform initiatives calling upon African organizations such as UNECA to raise public Unif. L. 3. Rolling stock regulation and policy in Africa Rev., 2022, 1–27 Luxembourg Rail Protocol in Africa 13 awareness of the need for African countries to adopt the Rail Protocol and sen- sitize private and public sector stakeholders in regard to the benefits of adopting the Rail Protocol.86 The analysis shows that African countries have remained at- tentive to the work of UNIDROIT on the Rail Protocol. Also, delegates from the AU were present at the first session of the Economic Commission for Europe Working Party on Rail Transport in Geneva, thus reinforcing the commitment of African governments to adopt the Rail Protocol.87 In the next phase of this article, the EU’s accession to the Rail Protocol in the capacity of a REIO will be discussed before embarking on the challenges surrounding the AU and African REIOs’ competence to accede to the Rail Protocol. 87 Report of the Group of Experts on Permanent Identification of Railway Rolling Stock on its first session (Economic Commission for Europe Inland Transport Committee, Working Party on Rail Transport) ECE/TRANS/SC.2/PIRRS/2020/2 (Geneva, 2–4 September 2020). 86 Report of the Conference of Ministers on the work of its fifty-second session (Economic Commission for Africa Conference of African Ministers of Finance, Planning and Economic Development) E/ECA/CM/52/2 965(LII) (Marrakech, Morocco, 25–6 March 2019) 18–19. 93 H. Kronke, ‘UNIDROIT 75th Anniversary Congress on Worldwide Harmonisation of Private Law and Regional Economic Integration: Hypotheses, Certainties and Open Questions’ (2003) 8 Uniform Law Review 10, 22. 92 R. Goode, Official Commentary n 17 above, para 4.310–4.312. 89 R. Goode, Official Commentary n 17 above, para 4.310; See R. Goode and H. Rosen, ‘Explanatory Report’ n 22, 14. 88 The Convention applies in relation to international interests in railway rolling stock as provided by the terms and provisions of the Rail Protocol, see Rail Protocol, Art II (1); See also Convention, Art 48. 91 R. Goode and H. Rosen, ‘Explanatory Report’ n 22, 68. III. The EU accession to the Rail Protocol A REIO, constituted by sovereign States, can sign, accept, approve, or accede to the Cape Town Convention and the Rail Protocol,88 so long as the REIO has competence over matters covered by the Rail Protocol.89 Upon accession to the Rail Protocol, the REIO will be furnished with rights and obligations like a Contracting State to the extent that it has competence over matters governed by the Rail Protocol.90 Before acceding to the Convention, the EU (then, the European Community) issued two regulations: Council Regulation 1346/2000 on Insolvency Proceedings (Insolvency Regulation) and the Brussel Regulation on Jurisdiction and Judgements (Brussels I), which relates to matters under the Convention.91 It was concluded that the EU, and not its Member States, could conclude ‘international agreements’ that affect these two EU regulations.92 Article 48 of the Convention grants less flexibility to the EU (and REIOs broad- ly) in implementing a protocol than sovereign countries since EU law does not harmonize the legislative environment for the rights and obligations of debtors and creditors under substantive secured transactions law.93 Under Article XXII, the Rail Protocol deemed it necessary to permit the EU and other competent REIOs to become a Contracting State. As applicable to all Contracting States 88 The Convention applies in relation to international interests in railway rolling stock as provided by the terms and provisions of the Rail Protocol, see Rail Protocol, Art II (1); See also Convention, Art 48. 89 R. Goode, Official Commentary n 17 above, para 4.310; See R. Goode and H. Rosen, ‘Explanatory Report’ n 22, 14. Rev. dr. III. The EU accession to the Rail Protocol unif., 2022, 1–27 Iyare Otabor-Olubor 14 (inclusive of a REIO), a State can only effectively become a contracting party to the Rail Protocol when it has ratified or acceded to the Convention, and the Convention must be in force in that State.94 When acceding to the Rail Protocol, the REIO must declare competence by making a declaration under the Rail Protocol specifying the matters regarding which competence has been transferred to it from its Member States.95 The REIO must also notify the depository—that is UNIDROIT—in the event of changes in its competence over such matters under the Rail Protocol.96 For ex- ample, the EU deposited an instrument of approval on the basis of the: Declaration to be made pursuant to Article XXII(2) concerning the competence of the European Union over matters governed by the Protocol to the Convention on International Interests in Mobile Equipment on Matters Specific to Railway Rolling Stock (the ‘Rail Protocol’), adopted in Luxembourg on 23 February 2007, in respect of which the Member States have transferred their competence to the Union.97 This Declaration closely follows the European Council’s previous Decision to accede to the Aircraft Protocol in conjunction with adhering to the Cape Town Convention on 28 April 2009.98 In its proposal to accede to the Rail Protocol, it was stated that the EU has exclusive competence over some of the matters gov- erned by the Rail Protocol,99 such as jurisdiction and the recognition and en- forcement of judgments in civil and commercial matters,100 insolvency proceedings (other than substantive insolvency law),101 and law applicable to contractual obligations (Rome I).102 In contrast, EU Member States have com- petence over other matters governed by the law. This strategy wittingly lends to the narrative based on an agreed bilateral compromise; the Convention can be a 94 Vienna Convention on the Law of Treaties, Art 2(1)(g); See also R. Goode, Official Commentary n 17, para 5.74-5.75. 95 Rail Protocol, Art XXII(2). 96 Rail Protocol, Art XXII(2). See also Seminar—The European Community and the Cape Town Convention: Summary Report (UNIDROIT: DC9/DEP—Doc. 8, Rome 26 November 2009) 6. 97 Declarations Lodged by the European Union under the Luxembourg Protocol at the time of the deposit of its Instrument of Approval (UNIDROIT). 94 Vienna Convention on the Law of Treaties, Art 2(1)(g); See also R. Goode, Official Commentary n 17, para 5.74-5.75. III. The EU accession to the Rail Protocol 98 European Commission, ‘Council Decision of 6 April 2009 on the Accession of the European Community to the Convention on International Interests in Mobile Equipment and its Protocol on Matters Specific to Aircraft Equipment, adopted jointly in Cape Town on 16 November 2001’ (2009/370/EC, OJ L 121, 15 May 2009). 99 European Commission, ‘Proposal for a Council Decision on the Approval, on behalf of the European Union, of the Luxembourg Protocol to the Convention on International Interests in Mobile Equipment on Matters specific to Railway Rolling Stock adopted in Luxembourg on 23 February 2007’ (COM 349 Final 2013/0184 (NLE) Brussels, 11 June 2013) 8. 100 Regulation (EU) No 1215/2012 of the European Parliament and of the Council of 12 December 2012 on Jurisdiction and the Recognition and Enforcement of Judgments in Civil and Commercial Matters (OJ L 351, 20 December 2012). 101 Council Regulation (EC) No 1346/2000 of 29 May 2000 on Insolvency Proceedings (OJ L 160, 30 June 2000). 102 Regulation (EC) No 593/2008 of the European Parliament and of the Council of 17 June 2008 on the Law Applicable to Contractual Obligations (Rome I) (OJ L 177, 4 July 2008). Unif. L. Rev., 2022, 1–27 Luxembourg Rail Protocol in Africa 15 split competence instrument whereby some competence can be vested with a REIO, while other competencies can be vested in an REIO Member State.103 The EU accession to the Cape Town Convention and Rail Protocol was pos- sible because the Convention accords a REIO constituted by sovereign States— where Member States have transferred competence in some matters covered by the Convention to the REIO.104 The effect of a REIO exercising the right to ac- cede to the Convention is that it assumes all the rights and obligations of a Contracting State to the extent that such a REIO has the competence over mat- ters governed by the Rail Protocol.105 While the EU’s accession to the Convention and the Rail Protocol unmasks the scope for other REIO adoption, existing sub-regional organizations in Africa arguably possess REIO status. Still, they lack the competence to accede to the Convention and the Rail Protocol. The forthcoming analysis will address two main questions—that is, legal per- sonality of REIOs and their competence over matters governed by the Rail Protocol. 104 Convention, Art 48(1), Rail Protocol. Art XXII (1). 105 Convention, Art 48(1), Rail Protocol. Art XXII (1). See also R. Goode, ‘Transnational Commercial Law and the Influence of the Cape Town Convention and Aircraft Protocol’ (2011) 50 Canadian Business Law Journal 186, 207; R. Goode, ‘Private Commercial Law Conventions and Public and Private International Law: The Radical Approach of the Cape Town Convention 2001 and Its Protocols’ (2016) 65 ICLQ 523, 538. III. The EU accession to the Rail Protocol The extent to which these sub-regional organizations lack the compe- tence to legislate on matters under Rail Protocol will be scrutinized in this art- icle. The forthcoming analysis will initially discuss legal reception in Africa and, subsequently, the role of the AU in African legal reform, and, afterwards, a study of the AU’s interrelationship with the AEC’s sub-regional organizations. 103 H. Rosen, ‘The Luxembourg Rail Protocol to the Cape Town Convention: Some Practical Differences from the Aviation Protocol’ in S. Kozuka (eds), Implementing the Cape Town Convention and the Domestic Laws on Secured Transactions (Springer 2017) 364. 107 African economies have been categorised as either underdeveloped or developing, see World Economic Situation and Prospects as of Mid-2020 (United Nations New York 2020). 106 R. Goode, Official Commentary n 17. 104 Convention, Art 48(1), Rail Protocol. Art XXII (1). IV. Legal reception in Africa The EU’s accession to the Cape Town Convention and the Rail Protocol paves the way for other REIOs, including REIOs comprising developing and under- developed countries, to consider adopting these international instruments. The Convention has been designed to bring significant economic benefits to coun- tries, particularly to developing countries, by allowing them to easily access secured finance for mobile equipment that was once costly and unavailable.106 Globally, no African country is currently ranked as a developed country.107 Today, a fundamental issue with Africa’s relentless pursuit for development hinges on its economic integration policy, which is often dependent on the complicated relationship between the AU and the regional economic commun- ities (RECs) in Africa. Untangling this complex relationship contributes to the Rev. dr. unif., 2022, 1–27 Iyare Otabor-Olubor 16 critical discussion of whether the AU and RECs can accede to the Convention and the Rail Protocol as competent REIOs under Article XXII. Achieving uniformity in the interpretation of the Cape Town Convention and the Rail Protocol in an AU context is a challenge that is distinct from merely realiz- ing uniformity in implementation. Transnational legal rules do not exist in a vac- uum but must be incorporated into extant regional and domestic law systems.108 The Convention and the Rail Protocol encompass many discrete areas commonly under the jurisdiction of domestic and regional rules sometimes nested within do- mestic legal systems, such as collateral registration, priority rules against competing claimants, insolvency, enforcement remedies, and public service access to transport. Thus, there is an additional layer of complexity when considering the reception of treaties in a REIO. For this reason, it is of significance to understand the nature of the AU in recognizing international laws and whether the AU and AEC sub-region- al organizations possess the legal personality to accede to an international treaty that may bind its Member States. Achieving uniformity in the interpretation of the Cape Town Convention and the Rail Protocol in an AU context is a challenge that is distinct from merely realiz- ing uniformity in implementation. Transnational legal rules do not exist in a vac- uum but must be incorporated into extant regional and domestic law systems.108 There has never been a uniform approach to adopting any private inter- national law convention through the AU in Africa. 114 M. Killander & H. Adjolohoun, ‘International Law and Domestic Human Rights Litigation in Africa: An introduction’ in M. Killander (eds) International Law and Domestic Human Rights Litigation in Africa (Pretoria University Law Press 2010) 4. 108 B.F. Havel & J.Q. Mulligan, ‘The Cape Town Convention and The Risk of Renationalization: A Comment in Reply to Jeffrey Wool & Andrej Jonovic’ (2014) 3 Cape Town Convention Journal 81, 83. 109 M. Ssenyonjo, ‘The Influence of the International Covenant on Economic, Social and Cultural Rights in Africa’ (2017) 64 NILR 259, 273. 111 J.G. Starke, ‘Monism and Dualism in the Theory of International Law’ (1936) 17 British Yearbook of International Law 66, 74. 112 J. Turley, ‘Dualistic Values in the Age of International Legisprudence’ (1993) 44 Hastings Law Journal 185. M. Ssenyonjo, The Influence of the International Covenant on Economic, Social and Cultural Rights in Africa’ (2017) 64 NILR 259, 273. 110 Ibid. IV. Legal reception in Africa African States individually apply the ‘dualist’ or ‘monist’ approach to conventions, following the previous practices in the domestication of treaties used by colonial powers: mainly, Britain, France, and Portugal.109 However, many African State constitutions now embody both ‘dualist’ or ‘monist’ elements.110 Monism aligns with the ideology that international and domestic law encompasses a particular legal order or intertwined legal orders that produce a consistent and uniform re- sult.111 Municipalities may draw upon international law as a rule of decision where necessary. Dualism expounds that national governments may choose to incorporate international law elements into local domestic law.112 Still, national governments are generally free to reject other aspects of international law if they so choose.113 Traditionally, African common law countries have been seen as dualists and civil law countries as monists.114 Provisions of francophone African constitutions are modelled on the civil law monist doctrine, thus reflecting the spirit of Article 55 of the Constitution of France: ‘Treaties or agreements duly ratified or approved shall, upon publication, prevail over Acts of Parliament, 11 J.G. Starke, ‘Monism and Dualism in the Theory of International Law’ (1936) 17 British Yearbook of International Law 66, 74. 112 J. Turley, ‘Dualistic Values in the Age of International Legisprudence’ (1993) 44 Hastings Law Journal 185. Unif. L. Rev., 2022, 1–27 Luxembourg Rail Protocol in Africa 1 17 subject, with respect to each agreement or treaty, to its application by the other party.’115 Upon ratification or accession at an international level and subsequent publication at the municipal level, an international convention can be inte- grated into a civil law country’s domestic law. In a conflict between existing do- mestic law and the international convention, the constitution will prioritize the international convention, and domestic law will take a subordinate position.116 An opposite position has been taken by dualist common law African countries, ensuring that an international convention is first enacted into law by order of le- gislation before it can attract the force of national law.117 Based on the analysis above, international law cannot be directly transplanted into Africa through an international organization such as the AU. AU institu- tions and organs do not possess the authority to accede to international laws on behalf of AU Member States. 115 See the Francophone constitutional provisions of Benin Republic 1990, Article 147; Burkina Faso 1991, Article 151; Burundi 2018, Article 279; Cameroon 1972, Article 45; Central African Republic 2016, Article 94; Chad 2018, Article 225; Congo 2015, Article 223; Coˆte d’Ivoire 2016, Article 123; Democratic Republic of the Congo 2005, Article 215; Guinea 2010, Article 151; Mali 1992, Article 116; Mauritania 1991, Article 80; Niger Republic 2010, Article 171; Rwanda 2003, Article 168; Senegal 2001, Article 98; and Togo 1992, Article 140. IV. Legal reception in Africa This is because the relationship between the AU and its Member States is built upon voluntary accession to the AU, which is fundamentally different from the relationship between an African Sovereign State and individuals at the national level.118 The AU supports these findings under Article 4 of the AU Constitutive Act, which states that it is the AU’s func- tion to maintain sovereign equality and interdependence among the Member States and respect Member States’ independence.119 Thus, the AU recognizes the status of respective national laws as the applicable law in AU Member States, thereby superseding the law of the AU in the Member States. The AU also rec- ognizes democratic principles, human rights, the rule of law, and good govern- ance among its Member States.120 The AU coordinates and harmonizes the policies between existing and future sub-regional organizations of the AEC—that is, RECs.121 The AEC Treaty led to the creation of sub-regional organizations in Africa to harmonize the laws and policies in Africa.122 The Protocol on Relations defines a REC as ‘a regional 115 See the Francophone constitutional provisions of Benin Republic 1990, Article 147; Burkina Faso 1991, Article 151; Burundi 2018, Article 279; Cameroon 1972, Article 45; Central African Republic 2016, Article 94; Chad 2018, Article 225; Congo 2015, Article 223; Coˆte d’Ivoire 2016, Article 123; Democratic Republic of the Congo 2005, Article 215; Guinea 2010, Article 151; Mali 1992, Article 116; Mauritania 1991, Article 80; Niger Republic 2010, Article 171; Rwanda 2003, Article 168; Senegal 2001, Article 98; and Togo 1992, Article 140. 116 M. Killander & H. Adjolohoun, n 114, 5. 116 M. Killander & H. Adjolohoun, n 114, 5. 117 See the dualist constitutional provisions of Malawi 1994, section 211(1); Nigeria 1999, section 12(1); and Zimbabwe 2013, section 327(2). 118 M. Barnard, ‘Legal Reception in the AU against the Backdrop of the Monist/Dualist Dichotomy’ (2015) 48 CILJSA 144, 145. 119 AU Constitutive Act, Art 4. 119 AU Constitutive Act, Art 4. 120 AU Constitutive Act, Art 4. 120 AU Constitutive Act, Art 4. 121 AU Constitutive Act, Art 3(l), Art 6. 121 AU Constitutive Act, Art 3(l), Art 6. 122 There are eight sub-regional organizations—Common Market for Eastern and Southern Africa (COMESA), the Economic Community of Central and Eastern African States (ECCAS), the Southern African Development Community (SADC), the Community of Sahel-Saharan States (CEN-SAD), the Economic Community of West African States (ECOWAS), the Inter- Governmental Authority on Development (IGAD), the Arab Maghreb Union (AMU) and the East African Community (EAC). 123 Protocol on Relations, Art. 1. Signatories to the Protocol on Relations are the AU and seven RECs namely COMESA, ECCAS, SADC, ECOWAS, IGAD, CEN-SAD, and EAC. It was signed on 27 January 2008. 130 J.G. de Matons, ‘Facilitation of Transport and Trade in Sub-Saharan Africa: A Review of International Legal Instruments Treaties, Conventions, Protocols, Decisions, Directives’ (SSA Transport Policy Working Paper No. 73, January 2003) 61. 128 R.F. Oppong, ‘Redefining the Relations between the African Union and Regional Economic Communities in Africa’ (2009) 9 Monitoring Regional Integration in Southern Africa Yearbook 5, 6. IV. Legal reception in Africa 122 There are eight sub-regional organizations—Common Market for Eastern and Southern Africa (COMESA), the Economic Community of Central and Eastern African States (ECCAS), the Southern African Development Community (SADC), the Community of Sahel-Saharan States (CEN-SAD), the Economic Community of West African States (ECOWAS), the Inter- Governmental Authority on Development (IGAD), the Arab Maghreb Union (AMU) and the East African Community (EAC). Rev. dr. unif., 2022, 1–27 Iyare Otabor-Olubor 18 grouping of African States organised into a legal entity by treaty, with economic and social integration as main objectives’.123 The AEC Treaty declares that sig- natories and sub-regional organizations should observe the AEC’s legal system when implementing its objectives.124 The AEC supports the harmonization of African national policies to promote intercontinental activities such as transpor- tation (for example, rail and air)125 while gradually removing barriers affecting the free movement of persons, goods, and services.126 Article 61 of the AEC Treaty promotes the harmonization of transportation rules and regulations. It outlines the responsibilities of the AEC Treaty signatories to expand and mod- ernize transport infrastructures by mobilizing the necessary technological and financial resources to enable the Member States to be linked.127 The AEC Treaty ostensibly denotes the AEC as an umbrella body that allows the sub-regional organizations to operate.128 However, the sub-regional organizations’ activities must be geared towards realizing a primary objective—namely, creating an AEC.129 What currently exists in Africa is a complex web of economic integra- tion initiatives consisting of several geo-economic legal structures (that is, sub- regional organizations) and a non-supranational umbrella regime (the AEC), all working towards a uniform and singular treaty-mandated framework (the AU Constitutive Act). Despite Article 5 of the AEC Treaty stating that the AEC’s primary aim is developing sub-regional communities, there is a growing concern that sub-re- gional organizations tend to create parochial policies, thereby losing sight of the main objective, which is the establishment of a common market.130 The AEC’s close relationship with its sub-regional organizations is re-echoed under the Protocol on Relations. 131 D. Nagar and F. Nganje, ‘The AU and Africa’s Regional Economic Communities’ (African Union Report: Regional and Global Challenges, Centre for Conflict Resolution, 2016) 26. IV. Legal reception in Africa While the Protocol on Relations was created to provide horizontal and vertical cooperation and harmonization of economic integration programs and agendas between the AU and the REC, national governments have not conferred supranational rights on sub-regional organizations.131 Nevertheless, the signatories to the Protocol on Relations have agreed to con- tinue cooperating with national governments to harmonize their policies and Nevertheless, the signatories to the Protocol on Relations have agreed to con- tinue cooperating with national governments to harmonize their policies and 123 Protocol on Relations, Art. 1. Signatories to the Protocol on Relations are the AU and seven RECs namely COMESA, ECCAS, SADC, ECOWAS, IGAD, CEN-SAD, and EAC. It was signed on 27 January 2008. 124 AEC Treaty, Art 3(e). 125 AEC Treaty, Art 4 (2)(e). 126 AEC Treaty, Art 4 (2)(i). 127 AEC Treaty, Art 61 (2). 128 R.F. Oppong, ‘Redefining the Relations between the African Union and Regional Economic Communities in Africa’ (2009) 9 Monitoring Regional Integration in Southern Africa Yearbook 5, 6. 129 Ibid. 130 J.G. de Matons, ‘Facilitation of Transport and Trade in Sub-Saharan Africa: A Review of International Legal Instruments Treaties, Conventions, Protocols, Decisions, Directives’ (SSA Transport Policy Working Paper No. 73, January 2003) 61. 131 D. Nagar and F. Nganje, ‘The AU and Africa’s Regional Economic Communities’ (African Union Report: Regional and Global Challenges, Centre for Conflict Resolution, 2016) 26. 130 J.G. de Matons, ‘Facilitation of Transport and Trade in Sub-Saharan Africa: A Review of International Legal Instruments Treaties, Conventions, Protocols, Decisions, Directives’ (SSA Transport Policy Working Paper No. 73, January 2003) 61. 131 D. Nagar and F. Nganje, ‘The AU and Africa’s Regional Economic Communities’ (African Union Report: Regional and Global Challenges, Centre for Conflict Resolution, 2016) 26. Unif. L. Rev., 2022, 1–27 Luxembourg Rail Protocol in Africa 19 programs while enabling the implementation of the AEC Treaty and AU Constitutive Act’s objectives.132 The next section of this article will analyse the legal personality of the AU and the AEC and its sub-regional organizations, including their limitations in acceding to the Rail Protocol. 135 See examples in ECOWAS Treaty, Art 88(1); COMESA Treaty, Art 186(1); EAC Treaty, Art 138(1); SADC Treaty, Art 3(1). 132 Protocol on Relations, Art 4(a). 133 B. Møller, The Pros and Cons of Subsidiarity: The Role of African Regional and Subregional Organisations in Ensuring Peace and Security in Africa (Danish Institute for International Studies (DIIS) Working Paper, Copenhagen 2005) 3. 134 B. Fagbayibo, ‘Looking Back, Thinking Forward: Understanding the Feasibility of Normative Supranationalism in the African Union’ (2013) 20 South African Journal of International Affairs 411, 418. 132 Protocol on Relations, Art 4(a). 133 B. Møller, The Pros and Cons of Subsidiarity: The Role of African Regional and Subregional Organisations in Ensuring Peace and Security in Africa (Danish Institute for International Studies (DIIS) Working Paper, Copenhagen 2005) 3. 134 B. Fagbayibo, ‘Looking Back, Thinking Forward: Understanding the Feasibility of Normative Supranationalism in the African Union’ (2013) 20 South African Journal of International Affairs 411, 418. 135 See examples in ECOWAS Treaty, Art 88(1); COMESA Treaty, Art 186(1); EAC Treaty, Art 138(1); SADC Treaty, Art 3(1). 1. Legal personality: the status of the AU and AEC sub-regional organizations To the extent that it is relevant to this discussion, the legal status of the RECs, the AEC, and the AU is perhaps one of the most perplexing dilemmas in regard to Africa’s economic integration story. This legal dogma can be expounded by first explaining the principles of subsidiarity and proportionality within the AU context. The principle of subsidiarity establishes an international organization’s complementary role by prescribing that only matters that cannot be effectively dealt with at the regional level should be acted upon by the relevant internation- al organization.133 Thus, a community of a higher order (for example, the AU or the AEC sub-regional community) should not interfere in a matter within the jurisdiction of a sovereign State, thereby depriving the sovereign State of its objectives. Instead, they should be supported to coordinate their activities with the activities of the AU to meet the overarching goals of the AU and the AEC. The proportionality principle considers whether the actions employed by the higher order are suitable and necessary for achieving the objectives in their founding treaties.134 The above discussion feeds into this normative analysis—whether sub-region- al organizations or the AU possess the appropriate legal capacity to accede to the Cape Town Convention and the Rail Protocol as a REIO and whether they have competence in certain matters under Article XXII of the Rail Protocol. As a starting point, sub-regional organizations in Africa possess legal personality, with evidence of legal personality etched in their founding treaties.135 Likewise, implicitly, it can be inferred from Article 98(2) of the AEC Treaty that the AEC possesses legal personality. As the AEC’s legal representative, the provision explains that the AU Secretary-General may, on behalf of the Community, enter into contracts and be a party to judicial and other legal proceedings. Yet, curi- ously and rather critically, the AU Constitutive Act is silent on the international legal personality of the AU. A sensible explanation for this unfortunate omission can be traced back to the 1965 General Convention on Privileges and Immunities of the Organization of African Unity. Article 1 of the 1965 Rev. dr. unif., 2022, 1–27 20 Iyare Otabor-Olubor Convention states that the OAU (now the AU) possesses a ‘juridical personality’ and can enter into contracts and institute legal proceedings. 141 N.J. Udombana, ‘The Institutional Structure of the African Union: A Legal Analysis’ (2002) 33 California Western International Law Journal 69, 82. 136 The OAU Charter 1963, adopted in Addis Ababa, is also silent about the legal personality of the OAU. 137 K.D. Magliveras and G.J. Naldi, The African Union: History, Institutions, and Activities (Wolters Kluwer 2018) 105. 138 Ibid. 139 AU Constitutive Act, Art 4. 140 AU Constitutive Act, Preamble. 141 N.J. Udombana, ‘The Institutional Structure of the African Union: A Legal Analysis’ (2002) 33 California Western International Law Journal 69, 82. 142 African Court on Human and Peoples’ Rights, Application No. 001/2011. 143 AEC Treaty, Art 98(1). 144 AU Constitutive Act, Art 33(2). 142 African Court on Human and Peoples’ Rights, Application No. 001/2011. 137 K.D. Magliveras and G.J. Naldi, The African Union: History, Institutions, and Activities (Wolters Kluwer 2018) 105. 136 The OAU Charter 1963, adopted in Addis Ababa, is also silent about the legal personality of the OAU. 1. Legal personality: the status of the AU and AEC sub-regional organizations Regardless, this lack of clarity surrounding the AU’s legal personality represents a missed oppor- tunity for the AU in its Constitutive Act to clarify the status of its existence as a legal person and its capacity to enter into international agreements and ratify conventions.136 It is noteworthy that the OAU’s objectives were not aligned with those of the AU because the OAU was formed almost exclusively to tackle colonial issues.137 In contrast, the AU was later formed to advance African sovereign States’ eco- nomic integration and socio-economic development while promoting demo- cratic principles and institutions.138 The AU has supranational authority over its Member States,139 although the AU was brought into existence by its Member States.140 The AU has been described as a body ‘endless in size and eternal in time, allowing for the admission of other members at any time after the entry into force of the [Constitutive] Act’.141 The AU Constitutive Act itself does not confer international legal personality on the AU. Instead, it was recog- nized in the decision of Femi Falana v African Union142 that, as an international organization, the AU has its distinct legal personality separate from its Member States with the legal capacity to become a party to an international treaty. This conferred status of ‘international organization’ on the AU makes the AU more responsive to the international law-making process. It has also been stated that the AEC is an integral part of the AU,143 and the AU Constitutive Act will supersede and take precedence over the AEC Treaty where there are inconsistencies or contrary provisions in its application.144 This is because the AU represents a political umbrella organization for the independ- ent African States championing a social, cultural, and political agenda. At the same time, the AEC is a part of the AU’s plan for economic integration. However, the same cannot be agreed in regard to sub-regional organizations as dependent on the AU because of their legal personality status. Surprisingly, the AEC Treaty is silent about the legal status of sub-regional organizations within the AEC and whether they are subject to the AEC Treaty regarding inconsisten- cies between their founding treaties and the AEC Treaty. 142 African Court on Human and Peoples’ Rights, Application No. 001/2011. 143 AEC Treaty, Art 98(1). 144 AU Constitutive Act, Art 33(2). Unif. L. 1. Legal personality: the status of the AU and AEC sub-regional organizations Rev., 2022, 1–27 Luxembourg Rail Protocol in Africa 21 There is no express indication under the AEC Treaty about whether sub-re- gional organizations (that is, REIOs) are organs, members, or agents of the AEC. The AEC Treaty does not specify any membership criteria. However, it provides a benchmark stating that contracting parties to the AEC Treaty are members of the AEC.145 Membership of the AEC comprises African sovereign States. Thus, it can be argued that sub-regional organizations are not organs of the AEC.146 To take a purposive and holistic approach, it is conclusive that sub- regional organizations are neither members nor organs of the AEC. Since they are neither organs nor members of the AEC, in consideration of their objectives, sub-regional organizations are agents of the AEC with a mandate to realize the AEC’s objectives, strengthen their relationship with the AU, and align their poli- cies with those of the AU.147 As agents of the AEC with legal personality, REIOs in Africa can enter into contractual obligations under treaty law. The position of an REIO as a body with legal personality was further reinforced under Article 1 of the Protocol on Relations, where it was stated that ‘Regional Economic Community’ means a corporate legal entity established by its Treaty (AEC Treaty) whose objective is to promote economic integration towards the estab- lishment of the AEC. 145 AEC Treaty, Art 2. 146 AEC Treaty, Art 7(1) states that the organs of the AEC are Assembly of Heads of State and Government, Council of Ministers, Pan-African Parliament, Economic and Social Commission, Court of Justice, General Secretariat and Specialised Technical Committees. 147 Protocol on Relations, Art 5. y 146 AEC Treaty, Art 7(1) states that the organs of the AEC are Assembly of Heads of State and Government, Council of Ministers, Pan-African Parliament, Economic and Social Commission, Court of Justice, General Secretariat and Specialised Technical Committees. 2. Competence to accede to the Cape Town Convention and the Rail Protocol: challenges and opportunities An example of a legal foundation for applying the Cape Town Convention and the Rail Protocol is substantive law rules on insolv- ency law.151 The rights of competing claimants in the enforcement of security interests over personal property are not currently within the competence of sub-regional organizations in Africa. Also, the priority of competing security rights and enforcement of secured and unsecured security rights falls exclusively within national law. Similarly, remedies on insolvency regarding the three alter- natives under Article IX of the Rail Protocol fall under national law jurisdiction to implement its provisions. The ideology of harmonization is discussed inconsistently across the sub-re- gional organizations’ founding treaties. There is no clear guidance on how sub- regional organizations will implement legal harmonization in the Member States, which institutions will be responsible, or which instruments (for ex- ample, directives, protocols, conventions, and so on) will be adopted by Member States. Harmonization of law in sub-regional organizations requires the existence of strong institutions to facilitate this vital objective. Such institu- tions should possess the authority to create, enforce, and interpret harmonized laws.149 No single institution is explicitly assigned the responsibility of harmo- nizing private international law within African sub-regional organizations.150 Harmonization and unification of law within a sub-regional organization de- pend on the availability of a legal foundation. This legal foundation necessitates an imposition on its Member States to adhere to the legal obligations stemming from such legal foundation. An example of a legal foundation for applying the Cape Town Convention and the Rail Protocol is substantive law rules on insolv- ency law.151 The rights of competing claimants in the enforcement of security interests over personal property are not currently within the competence of sub-regional organizations in Africa. Also, the priority of competing security rights and enforcement of secured and unsecured security rights falls exclusively within national law. Similarly, remedies on insolvency regarding the three alter- natives under Article IX of the Rail Protocol fall under national law jurisdiction to implement its provisions. Also, there are no uniform sub-regional transport policies for rolling stock regarding maintaining public service access. In Africa, sub-regional organiza- tions cannot make declarations relating to public services under the Rail Protocol because they do not have exclusive competence over rail transport ser- vice laws. 149 R.F. Oppong, ‘Regional Integration and Fundamentals of Legal Harmonisation’ in J. Do¨veling and H.I. Majamba (eds), Harmonisation of Laws in the East African Community: The State of Affiars with Comparartive Insights from the European Union and other Regional Economic Communities (TGCL Series 5, 2018) 125. 2. Competence to accede to the Cape Town Convention and the Rail Protocol: challenges and opportunities It has been realized that sub-regional organizations in Africa have legal personality and the capacity to enter into contractual obligations. Still, the extent to which they can achieve this over sovereign States is questionable. The effect of private inter- national laws needs to be discussed here because the Cape Town Convention and the Rail Protocol require contracting parties—in this case, REIOs—to make neces- sary declarations regarding competence that directly affect their Member States. A modern private international law can provide legal certainty for transnational secured transactions over mobile equipment that may cross national borders of sov- ereign States in Africa while simultaneously ensuring that substantive domestic laws are not fundamentally altered. Notwithstanding, there has been little success in developing a corpus juris on private international law in Africa. However, there have been some superficial attempts to harmonize private international law in the sub-regions. For example, Article 57(1) of the Economic Community of West African States (ECOWAS) Treaty states that the Member States should ‘cooperate in judicial and legal matters with a view to harmonizing their judicial and legal sys- tems’. Also, Article 126 of the EAC Treaty requires Member States to ‘encourage the standardisation of judgements of court within the community’. Likewise, Article 86(2) of the Common Market for Eastern and Southern Africa (COMESA) Rev. dr. unif., 2022, 1–27 Iyare Otabor-Olubor 22 Treaty expects Member States to ‘harmonise or approximate their laws to the extent required for the proper functioning of the Common Market’.148 The ideology of harmonization is discussed inconsistently across the sub-re- gional organizations’ founding treaties. There is no clear guidance on how sub- regional organizations will implement legal harmonization in the Member States, which institutions will be responsible, or which instruments (for ex- ample, directives, protocols, conventions, and so on) will be adopted by Member States. Harmonization of law in sub-regional organizations requires the existence of strong institutions to facilitate this vital objective. Such institu- tions should possess the authority to create, enforce, and interpret harmonized laws.149 No single institution is explicitly assigned the responsibility of harmo- nizing private international law within African sub-regional organizations.150 Harmonization and unification of law within a sub-regional organization de- pend on the availability of a legal foundation. This legal foundation necessitates an imposition on its Member States to adhere to the legal obligations stemming from such legal foundation. 152 Public Services Charter, Art 30(1). The nineteen countries are Algeria, Benin Republic, Burkina Faso, Burundi, Cameroon, Co^te d’Ivoire, Comoros, Congo, Kenya, Mali, Malawi, Mozambique, Mauritius, Namibia, Rwanda, South Africa, Sao Tome and Principe, Tanzania, and Zambia. 148 The COMESA Treaty enjoins its member states to co-operate in trade liberalisation, transpor- tation, and a host of other socio-economic areas of importance, see Art 82(6)(c). 151 Convention, Art 29. 150 Ibid, 126. 2. Competence to accede to the Cape Town Convention and the Rail Protocol: challenges and opportunities The 2011 African Charter on Values and Principles of Public Services and Administration (Public Services Charter) strengthens cooperation among Contracting States and the AU to improve public services, such as transporta- tion, in each Contracting State. The Public Services Charter is now in force, hav- ing received 38 signatures and been ratified by 19 African countries.152 The Contracting States to the Public Services Charter are required to implement its Unif. L. Rev., 2022, 1–27 Luxembourg Rail Protocol in Africa 23 provisions by ensuring the continuity of public services ‘under all circumstan- ces’, while protecting the rights of public transport users.153 The Public Services Charter instructs sub-regional organizations to encourage their Member States to ratify or accede to its Charter and implement it.154 There is no indication whatsoever that the Public Service Charter is open to signature from the sub-re- gional organizations. The Public Service Charter has been designed for adoption at the national level and not at a sub-regional level. While it can be argued that sub-regional organizations possess legal personal- ity, they do not have the competence to accede to an international convention, such as the Cape Town Convention and the Rail Protocol, which could become community law to bind the Member States.155 Their founding treaties do not empower them to conclude treaties and conventions with respect to Article 6 of the 1986 VCLT. However, sub-regional organizations may encourage Member States and cooperate with them to become Contracting Parties to international conventions that align with the objectives of the sub-regional organizations and the AU. In this respect, the 2014 Convention on Cross-Border Cooperation (Niamey Convention)156 facilitates cross-border cooperation at local and sub- regional levels.157 It commits contracting parties to promote cross-border co- operation in areas such as industry and transportation.158 Regional cooperation can be the facilitator of choice of law rules on secured transactions over mobile equipment in the sub-regions while ensuring adherence to uniform standards outlined under the respective treaties of the sub-regional organizations. The rec- ognition and enforcement of foreign judgments in sovereign States that have ratified the Convention and the Protocol would ensure the effectiveness of judi- cial decisions, thus aiding harmonized transnational settlement of disputes in sovereign States. Regional cooperation between national courts would furnish sub-regional organizations with greater insight and influence that may impact Africa’s future rolling stock legal policies, leading to uniform interpretation and consistent application of the Convention and the Protocol. 153 Public Services Charter, Art 3. 154 Public Services Charter, Art 23(ii). 155 R.F. Oppong, Legal Aspects of Economic Integration in Africa (CUP 2011) 69–72. 156 The Niamey Convention has been signed by seventeen African states. It has received five ratifi- cations. It will come into force after the fifteenth instrument of ratification has been received by the AU Commission. 157 Niamey Convention, Art 2(1). 158 Niamey Convention, Art 3(2). 157 Niamey Convention, Art 2(1). 2. Competence to accede to the Cape Town Convention and the Rail Protocol: challenges and opportunities While it is conclusive that sub-regional organizations lack exclusive compe- tence over private law matters under the Cape Town Convention and the Rail Protocol, other contemporary legal frameworks have made attempts to reform private laws in Africa by harmonizing substantive business law. These frame- works are influenced by historical legal traditions and past affiliations with for- eign administration and colonialists. A prime example is the OHADA and its collection of legal instruments (uniform acts) designed to harmonize business Rev. dr. unif., 2022, 1–27 Iyare Otabor-Olubor 24 laws of participating African States.159 It represents an ambitious effort in reforming investment and business law.160 However, OHADA has been set up with a particular structure and purpose. This article will subsequently consider the mandate of OHADA and whether OHADA can exercise competence to le- gislate over matters under the Convention and the Rail Protocol. 159 R. Beauchard and M.J. Vital Kodo, ‘Can OHADA Increase Legal Certainty in Africa?’ (Justice and Development Working Papers 65989, 2011) 7. 160 Ibid. 161 The OHADA Member States are Benin, Burkina Faso, Cameroon, Central African Republic, Chad, Comoros, Coˆte d’Ivoire, Democratic Republic of Congo, Equatorial Guinea, Gabon, Guinea, Guinea-Bissau, Mali, Niger, Republic of the Congo, Senegal, Togo. See also M. Frilet, ‘Legal Innovation for Development: The OHADA Experience’ (2013) 4 World Bank Legal Review 335. 167 Article 53 of the OHADA Treaty welcomes non-OHADA member countries to sign its Treaty, including non-Member States of the OAU (i.e., now AU); See B. Fagbayibo, ‘Towards the Harmonisation of Laws in Africa: is OHADA the way to go?’ (2009) 42 Comparative and International Law Journal of Southern Africa 309. 162 OHADA Treaty, Art 46. 163 C.M. Dickerson, ‘The OHADA Common Court of Justice and Arbitration: Exogenous Forces Contributing to Its Influence’ (2016) 79 Law & Contemporary Problems 63. 165 J. J. Milingo Ellong, ‘OHADA and the Intra-African Regulation of Commercial Exchanges: Critical Study’ (2015) International Business Law Journal 517, 528. 64 The UAS was adopted on 17 April 1997. It came into force on 1 January 1998, and subsequent- ly revised and adopted on 15 December 2010 in Lome, Togo. 3. Is OHADA the answer? Rev., 2022, 1–27 Luxembourg Rail Protocol in Africa 25 Africa, there is a proliferation of several other competing organizations (that is, sub-regional organizations), resulting in occasional duplication and overlapping economic activities.168 Such proliferation makes the task of challenging eco- nomic integration due to overlapping membership, thereby leading to irrational configurations and inconsistencies in harmonization and coordination of trade liberalization and facilitation. Africa, there is a proliferation of several other competing organizations (that is, sub-regional organizations), resulting in occasional duplication and overlapping economic activities.168 Such proliferation makes the task of challenging eco- nomic integration due to overlapping membership, thereby leading to irrational configurations and inconsistencies in harmonization and coordination of trade liberalization and facilitation. On the other hand, some OHADA Member States belong to one or more sub- regional organizations in Africa, thus presenting a challenge for OHADA to be considered an REIO under Article XXII of the Rail Protocol. For instance, Senegal and Co^te d’Ivoire are Member States of ECOWAS and the Community of Sahel-Saharan States—two different sub-regional organizations with separate regional economic objectives. Yet, these two countries are also OHADA Member States. While OHADA promotes trade and investment in predomin- antly OHADA civil law countries regardless of their sub-regional affiliations, sub-regional organizations take a different approach, enhancing economic inte- gration within their respective communities.169 In the event of overlapping membership, transfer of competence from one REIO to another is unprecedented. A comprehensive legal framework would need to be agreed upon by Member States at both the continental and sub-re- gional levels to transfer competence or move REIOs towards supranational gov- ernance. To do this, certain requirements need to be met. First, normative instruments will need to be adopted and ratified. Currently, ratifying regional standards is a passive and non-binding process that leads to delays in the opera- tionalization and effectiveness of regional laws in Africa.170 Second, there needs to be a concerted effort to transfer power to REIOs. Striking a balance between State sovereignty and supranationalism should be carefully considered. The AU Commission on International Law is responsible for the codification and pro- gressive development of international law in African.171 However, a framework that details the gradual transfer of competence to institutions responsible for monitoring governance standards will need to be established by the AU Commission on International Law. OHADA was created to increase investment and trade participation in civil law countries in Africa. 168 R. Tavares & V. Tang, ‘Regional Economic Integration in Africa: Impediments to Progress?’ (2011) 18 South African Journal of International Affairs 217, 223. 169 3. Is OHADA the answer? The OHADA Treaty was enacted to facilitate business activities and foreign in- vestment in 17 francophone African countries.161 OHADA possesses an inter- national legal personality and may enter into contracts to fulfil its objectives.162 The OHADA Treaty creates a single supranational court—the Common Court of Justice and Arbitration—to hear disputes on OHADA law.163 OHADA’s Uniform Act Organising Securities (UAS) regulates substantive commercial law rules over real security and personal security in OHADA countries.164 The type of securities encompasses legal mortgages, pledge of debt, pledge of documen- tary intangibles, reservation of title agreements, assignment of debts, liens, and personal guarantees.165 OHADA’s status as an international organization with a sub-regional man- date that can exercise competence under the Cape Town Convention and the Rail Protocol is disputable. First, the Protocol on Relations defines a sub-region- al organization as a group of African States with ‘economic and social integra- tion’ as a primary objective.166 Article 28 of the AEC Treaty requires sub-regional organizations to coordinate and harmonize their activities in ‘all fields or sectors’. These definitions do not align with the mandate of OHADA, which is to facilitate a single, cross-border regime of uniform business law in civil law, French-speaking African States.167 Second, although the AU has aligned its policies with the AEC, tasked to further the economic integration of 162 OHADA Treaty, Art 46. 163 C.M. Dickerson, ‘The OHADA Common Court of Justice and Arbitration: Exogenous For Contributing to Its Influence’ (2016) 79 Law & Contemporary Problems 63. 164 The UAS was adopted on 17 April 1997. It came into force on 1 January 1998, and subsequent- ly revised and adopted on 15 December 2010 in Lome, Togo. 165 J. J. Milingo Ellong, ‘OHADA and the Intra-African Regulation of Commercial Exchanges: Critical Study’ (2015) International Business Law Journal 517, 528. 166 Protocol on Relations, Art 1. 167 Article 53 of the OHADA Treaty welcomes non-OHADA member countries to sign its Treaty, including non-Member States of the OAU (i.e., now AU); See B. Fagbayibo, ‘Towards the Harmonisation of Laws in Africa: is OHADA the way to go?’ (2009) 42 Comparative and International Law Journal of Southern Africa 309. Unif. L. 170 B. Fagbayibo, ‘Enhancing Good Governance in Africa: The Imperative of a Supranational Approach’ (2012) 14 U Botswana LJ 135, 143. 171 Statute of the AU Commission on International Law 2009 (‘AUCIL Statute’), Art. 4(a). 169 Protocol on Relations, Art 3(b). 173 There are five different categories of Declarations: Opt-in, Opt-out, Declarations relating to State’s Own Laws, Mandatory Declarations, Others. The Declaration under Article 54(2) of the Convention is required to be made at the time of the Contracting Party’s deposit of its instru- ment of ratification, acceptance, approval of, or accession to, the Rail Protocol. All other Declarations under the Convention as they pertain to rolling stock may be made by Contracting Parties at the time or at a later time—see R. Goode, Official Commentary n 17 above, Appendix X. 3. Is OHADA the answer? Signatories to the Cape Town Convention and the Rail Protocol and the Member States of sub-regional organizations are not delineated based on the legal system of their participating countries. The Convention and the Rail Protocol are open to accession by any country, Rev. dr. unif., 2022, 1–27 Iyare Otabor-Olubor 26 regardless of the legal system—whether common law, Shariah law, civil law, and so on.172 Several rules under the Convention and the Rail Protocol enable con- tracting parties to make certain declarations about the applicability or scope of their legal provisions or the approach they wish to take in its implementation.173 This step was necessary to accommodate the diversity of legal systems world- wide regarding the recognition and implementation of the Convention’s provi- sions (for instance, some countries may need a particular approach to exercise extra-judicial remedies). The declarations are open to both Contracting States and REIOs, with the intention that all Contracting States make the declarations fully compliant with the Convention and the Rail Protocol’s obligations. Though OHADA possesses supranational authority over its Member States, it cannot be classified as an REIO due to a lack of a primary economic integration mandate. Thus, OHADA cannot claim competence to legislate over matters under Article 48 of the Convention and Article XXII of the Rail Protocol. regardless of the legal system—whether common law, Shariah law, civil law, and so on.172 Several rules under the Convention and the Rail Protocol enable con- tracting parties to make certain declarations about the applicability or scope of their legal provisions or the approach they wish to take in its implementation.173 172 Contracting States may make declarations under the Convention and Rail Protocol Declarations arranged by Article Deposited Under the Cape Town Convention On International Interests In Mobile Equipment. 174 V. Foster and C. Brice~no-Garmendia, Africa’s Infrastructure A Time for Transformation (World Bank Group, Paper 52102, 2010) 113. V. Conclusion This article concludes the argument surrounding the competence of the sub-re- gional organizations in Africa to accede to the Cape Town Convention and the Rail Protocol under Article XXII of the Rail Protocol as REIOs. According to the AU Constitutive Act and the AEC Treaty provisions, sub-regional organiza- tions cannot accede to the Convention and the Rail Protocol. It has been con- cluded that the AU and the AEC’s legal structures and economic objectives do not furnish sub-regional organizations with the exercise of exclusive compe- tence on matters under the Convention and the Rail Protocol. African States can ratify the Convention and the Rail Protocol in their sovereign capacities. In total, 27 African States have either ratified or acceded to the Convention; two have signed the Rail Protocol, while only Gabon has accepted the Rail Protocol. Acceding to the Rail Protocol will have several impacts for African States. Railway rolling stock is crucial to support economic growth and sustainable de- velopment in Africa. This infrastructure deficit is a significant hindrance to intra-African trade.174 Africa lost 25 per cent of its expected growth in the last 20 years due to inadequate infrastructure, thus leading to the high cost of Unif. L. Rev., 2022, 1–27 27 Luxembourg Rail Protocol in Africa 27 trade.175 Improving infrastructure by acceding to the Cape Town Convention and Rail Protocol will enable African countries to engage fully in intra-African trade and reap socio-economic development benefits. Accession will open up multinational private sector financing from investment entities such as leasing companies, banks, and private equity funds, rather than national public funding. The vast size of Africa and its landlocked countries encourages the develop- ment and use of high-speed, high-capacity rail rolling stock. The availability of modern rail rolling stock can also accelerate membership of OTIF in African States, thus leading to reform of railway contract law in Africa. Such reform could be achieved by adopting COTIF,176 the Uniform Rules concerning the Contract for International Carriage of Passengers and Luggage by Rail,177 and the Uniform Rules Concerning the Contract for International Carriage of Goods by Rail.178 With the launch of the AfCFTA, the realization of the 2030 Agenda for Sustainable Development and Agenda 2063 (‘The Africa We Want’) is achievable.179 Interregional connectivity and efficient logistic services through better rail rolling stock are at the core of this agenda. V. Conclusion Rail transport is a safe and cost-effective way to transport large freights and valuable commodities such as crude oil, mined natural resources, and agricultural goods while ensuring greater energy efficiency and lower greenhouse gas emissions.180 The AfCFTA has created a single interconnected market to provide African businesses with economies of scale and enhanced product mobility, thereby creating enormous markets. African countries accession to the Rail Protocol can bring long-term economic growth and sustainable development to the continent. -article/doi/10.1093/ulr/unac007/6601423 by guest on 07 June 2022 175 Assessing the Status of Regional Integration in Africa (Thirty-eighth Meeting) UNECA E/ECA/ COE/38/3/Rev.1 (Marrakech, Morocco, 20-22 March 2019) 9-10. 176 ‘Accession to the [COTIF] Convention shall be open to any State on the territory of which rail- way infrastructure is operated’, COTIF, Art 37. 177 Applicable from 1 July 2006. 178 Applicable from 1 July 2006. 179 L. Abrego, M.A. Amado, T. Gursoy, G.P. Nicholls, and H. Perez-Saiz, The African Continental Free Trade Agreement: Welfare Gains Estimates from a General Equilibrium Model (IMF Working Paper WP/19/124, 2019) 4. 180 Vera Songwe and Howard Rosen, ‘The Luxembourg Rail Protocol: Making the Railways Work for Africa’ (Rail Working Group). Rev. dr. unif., 2022, 1–27
https://openalex.org/W188051537	https://europepmc.org/articles/pmc2961023?pdf=render	English	null	5-Amino-3-methyl-1-phenyl-1<i>H</i>-1,2,4-triazole	Acta crystallographica. Section E	2,008	cc-by	3,189	5-Amino-3-methyl-1-phenyl-1H-1,2,4- triazole H atoms treated by a mixture of independent and constrained reﬁnement max = 0.19 e A˚ 3 min = 0.17 e A˚ 3 organic compounds Data collection Bruker SMART CCD area-detector diffractometer Absorption correction: multi-scan (SADABS; Bruker, 1998) Tmin = 0.984, Tmax = 0.997 16028 measured reﬂections 3882 independent reﬂections 1997 reﬂections with I > 2(I) Rint = 0.047 Reﬁnement R[F 2 > 2(F 2)] = 0.050 wR(F 2) = 0.148 S = 0.94 3882 reﬂections 124 parameters 3 restraints H atoms treated by a mixture of independent and constrained reﬁnement max = 0.19 e A˚ 3 min = 0.17 e A˚ 3 Data collection Bruker SMART CCD area-detector diffractometer Absorption correction: multi-scan (SADABS; Bruker, 1998) Tmin = 0.984, Tmax = 0.997 Reﬁnement R[F 2 > 2(F 2)] = 0.050 wR(F 2) = 0.148 S = 0.94 3882 reﬂections 124 parameters 3 restraints Acta Crystallographica Section E Structure Reports Online ISSN 1600-5368 16028 measured reﬂections 3882 independent reﬂections 1997 reﬂections with I > 2(I) Rint = 0.047 Related literature Supplementary data and ﬁgures for this paper are available from the IUCr electronic archives (Reference: DN2319). For related literature, see: Altman & Solomost (1993); Genady & Gabel (2003); Kanazawa et al. (1988); Karanik et al. (2003); Hashimoto et al. (1990); Allouch et al. (2004). For a discussion of hydrogen-bond patterns, see: Bernstein et al. (1995); Etter et al. (1990). Fatma Allouch,a Fatma Zouari,b* Fakher Chabchouba and Mansour Salema aLaboratoire de Chimie Applique´e: He´te´rocycles, Corps gras et Polyme`res, Faculte´ des Sciences de Sfax, BP 802, 3018 Sfax, Tunisia, and bLaboratoire de Sciences de Mate´riaux et d’Environnement, Faculte´ des Sciences de SFAX, BP 802, 3018 SFAX, Tunisia Table 1 Hydrogen-bond geometry (A˚ , ). D—H A D—H H A D A D—H A N4—H4A N3i 0.886 (8) 2.110 (8) 2.9923 (13) 173.1 (12) N4—H4B N2ii 0.917 (8) 2.210 (10) 3.0415 (14) 150.4 (10) Symmetry codes: (i) x þ 1; y; z þ 2; (ii) x; y þ 1 2; z þ 1 2. Table 1 Hydrogen-bond geometry (A˚ , ). D—H A D—H H A D A D—H A N4—H4A N3i 0.886 (8) 2.110 (8) 2.9923 (13) 173.1 (12) N4—H4B N2ii 0.917 (8) 2.210 (10) 3.0415 (14) 150.4 (10) Symmetry codes: (i) x þ 1; y; z þ 2; (ii) x; y þ 1 2; z þ 1 2. Correspondence e-mail: fatmazouari2003@yahoo.fr Received 16 February 2008; accepted 1 March 2008 Key indicators: single-crystal X-ray study; T = 296 K; mean (C–C) = 0.002 A˚; R factor = 0.051; wR factor = 0.148; data-to-parameter ratio = 31.3. Data collection: SMART (Bruker, 1998); cell reﬁnement: SAINT (Bruker, 1998); data reduction: SAINT; program(s) used to solve structure: SHELXS97 (Sheldrick, 2008); program(s) used to reﬁne structure: SHELXL97 (Sheldrick, 2008); molecular graphics: ORTEPIII (Burnett & Johnson, 1996), ORTEP-3 for Windows (Farrugia, 1997) and PLATON (Spek, 2003); software used to prepare material for publication: WinGX (Farrugia, 1999). In the title compound, C9H10N4, the phenyl and triazole rings make a dihedral angle of 38.80 (2). N—H N hydrogen bonds link the molecules, forming centrosymmetric R2 2(8) rings; these rings are interconnected through a C(5) chain, building up a zigzag layer parallel to the (100) plane. In the title compound, C9H10N4, the phenyl and triazole rings make a dihedral angle of 38.80 (2). N—H N hydrogen bonds link the molecules, forming centrosymmetric R2 2(8) rings; these rings are interconnected through a C(5) chain, building up a zigzag layer parallel to the (100) plane. References Allouch, F., Chabchoub, F., Ben Hassine, B. & Salem, M. (2004). Heterocycl. Commun. 10, 63–66 . Altman, A. & Solomost, T. (1993). Hortic. Sci. 28, 201–203. Bernstein, J., Davis, R. E., Shimoni, L. & Chang, N.-L. (1995). Angew. Chem. Int. Ed. Engl. 34, 1555–1573. Bruker (1998). SMART, SAINT and SADABS. Bruker AXS Inc., Madison, Wisconsin, USA. , Burnett, M. N. & Johnson, C. K. (1996). ORTEPIII. Report ORNL-6895. Oak Burnett, M. N. & Johnson, C. K. (1996). ORTEPIII. Report ORNL 6895. Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA. Ridge National Laboratory, Oak Ridge, Tennessee, USA. Etter, M. C., MacDonald, J. C. & Bernstein, J. (1990). Acta Cryst. B46, 256–262. Farrugia L J (1997) J Appl Cryst 30 565 Etter, M. C., MacDonald, J. C. & Bernstein, J. (1990). Acta Cryst. B46, 256–262. Farrugia, L. J. (1997). J. Appl. Cryst. 30, 565. , , , , ( ) y , Farrugia, L. J. (1997). J. Appl. Cryst. 30, 565. Farrugia, L. J. (1997). J. Appl. Cryst. 30, 565. Farrugia, L. J. (1999). J. Appl. Cryst. 32, 837–838. Genady, A. & Gabel, D. (2003). Tetrahedron Lett. 44, 2915–2917. Experimental Experimental Experimental Crystal data C9H10N4 Mr = 174.21 Monoclinic, P21=c a = 8.5110 (5) A˚ b = 11.2490 (8) A˚ c = 10.1048 (7) A˚ = 101.866 (4) V = 946.76 (11) A˚ 3 Z = 4 Mo K radiation = 0.08 mm1 T = 296 (7) K 0.49 0.14 0.08 mm Hashimoto, F., Sugimoto, C. & Hayashi, H. (1990). Chem. Pharm. Bull. 38, 2532–2536. Kanazawa, S., Driscoll, M. & Struhl, K. (1988). Mol. Cell. Biol. 8, 644–673. V = 946.76 (11) A˚ 3 Z = 4 Mo K radiation = 0.08 mm1 T = 296 (7) K 0.49 0.14 0.08 mm Karanik, M., Paetzel, M. & Liebscher, J. (2003). Synthesis, 8, 1201–1208. Karanik, M., Paetzel, M. & Liebscher, J. (2003). Synth Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122. Spek, A. L. (2003). J. Appl. Cryst. 36, 7–13. o684 Allouch et al. Acta Cryst. (2008). E64, o684 doi:10.1107/S1600536808005801 F. Allouch, F. Zouari, F. Chabchoub and M. Salem F. Allouch, F. Zouari, F. Chabchoub and M. Salem F. Allouch, F. Zouari, F. Chabchoub and M. Salem Experimental A mixture containing 3.56 g (0.02 mol) of N-phenyl ethyl acetydrazonateand 0.88 g (0.021 mol) of cyanamidein 20 ml of methanol was stirred and heated to reflux for 12 h. The solvent was removed under rotary evaporation. The crude product was washed with ether then recrystallized from methanol to give analytically pure crystals. Comment The aminotriazoles are crucial heterocyclic substances which have a great interest thanks to their biological and pharmaco- logical activity (Kanazawa et al., 1988; Hashimoto et al., 1990) such as antitumoral and inhibition of cholesterol activity. In addition they have many applications in agriculture domain (Altman et al., 1993). Aminotriazole are useful binucleophil- ic agents that lead to polycondensed heterocycles (Genady et al., 2003; Karanik et al., 2003). However, the studies that deal with N1-phenyl-aminotriazole are very limited (Allouch et al., 2004). Until now only a few reactions were reported concerning the addition-cyclizations of bielectrophile compounds with N1-phenyl-aminotriazoles. In fact, these later are not well identified, this can be explicable by the existence of the tautomer equilibrium. That is why we have undertaken a crystallographic study. In this paper, we report the synthesis of 5-amino-3-méthyl N1-phényl-1,2,4-triazole. The reaction of N-phenyl ethyl acet- ydrazonate with cyanamidegave aminotriazole could lead to structure (I) or its isomer (II) (Scheme). The structure elucid- ation was achieved by X-ray diffraction, and proved that the reaction occurs cleanly to form 5-amino-3-méthyl N1-phényl- 1,2,4-triazole (I). In the title compound, the phenyl and the triazole rings remain planar with mean deviations from planarity of 0.0072 and 0.0049Å respectively. However, the two rings are twisted with respect to each other making a dihedral angle of 38.80 (2)° (Fig. 1). The occurrence of N—H···N hydrogen bonds links the molecules through inversion centre to form R22(8) ring (Etter et al., 1990; Bernstein et al., 1995) and these rings are interconnected through C(5) chain to build up a like zigzag layer developping along the (1 0 0) plane (Table 1, Fig. 2) Acta Cryst. (2008). E64, o684 [ doi:10.1107/S1600536808005801 ] 5-Amino-3-methyl-1-phenyl-1H-1,2,4-triazole supplementary materials supplementary materials supplementary materials Acta Cryst. (2008). E64, o684 [ doi:10.1107/S1600536808005801 ] Acta Cryst. (2008). E64, o684 [ doi:10.1107/S1600536808005801 ] Refinement The molecular structure of the title compound with the atom-labelling scheme. Dis- placement ellipsoids are drawn at the 30% probability level. H atoms are represented as small spheres of arbitrary radii. Fig. 2. Partial packing view showing the formation of the R22(8) ring and C(5) chains through N—H···N hydrogen bonds drawn as dashed lines. H atoms not involved in hydrogen bondings have been omitted for clarity. [Symmetry codes: (i) −x + 1, −y, −z + 2; (ii) x, −y + 1/2, z + 1/2] Fig. 3. The tautomeric forms of the title compound. Fig. 1. The molecular structure of the title compound with the atom-labelling scheme. Dis- placement ellipsoids are drawn at the 30% probability level. H atoms are represented as small spheres of arbitrary radii. Fig. 2. Partial packing view showing the formation of the R22(8) ring and C(5) chains through N—H···N hydrogen bonds drawn as dashed lines. H atoms not involved in hydrogen bondings have been omitted for clarity. [Symmetry codes: (i) −x + 1, −y, −z + 2; (ii) x, −y + 1/2, z + 1/2] Fig. 2. Partial packing view showing the formation of the R22(8) ring and C(5) chains through N—H···N hydrogen bonds drawn as dashed lines. H atoms not involved in hydrogen bondings have been omitted for clarity. [Symmetry codes: (i) −x + 1, −y, −z + 2; (ii) x, −y + 1/2, z + 1/2] Fig. 3. The tautomeric forms of the title compound. Refinement All H atoms attached to C atoms were fixed geometrically and treated as riding with C—H = 0.96 Å (methyl) and 0.93 Å (aromatic) with Uiso(H) = 1.2Ueq(Phenyl) or Uiso(H) = 1.5Ueq(methyl). The methyl was found to be statistically disordered over two positions. H atoms attached to nitrogen were located in difference Fourier maps and included in the subsequent refinement using soft restraints (N—H= 0.90 (1)Å and H···H= 1.59 (2) Å) with Uiso(H) = 1.2Ueq(N). sup-1 supplementary materials Figures Fig. 1. The molecular structure of the title compound with the atom-labelling scheme. Dis- placement ellipsoids are drawn at the 30% probability level. H atoms are represented as smal spheres of arbitrary radii. Fig. 2. Partial packing view showing the formation of the R22(8) ring and C(5) chains through N—H···N hydrogen bonds drawn as dashed lines. H atoms not involved in hydrogen bonding have been omitted for clarity. [Symmetry codes: (i) −x + 1, −y, −z + 2; (ii) x, −y + 1/2, z + 1/2 Fig. 3. The tautomeric forms of the title compound. 5-Amino-3-methyl-1-phenyl-1H-1,2,4-triazole Crystal data C9H10N4 F000 = 368 Mr = 174.21 Dx = 1.222 Mg m−3 Monoclinic, P21/c Mo Kα radiation λ = 0.71073 Å Hall symbol: -P 2ybc Cell parameters from 2898 reflections a = 8.5110 (5) Å θ = 2.5–23.3º b = 11.2490 (8) Å µ = 0.08 mm−1 c = 10.1048 (7) Å T = 296 (7) K β = 101.866 (4)º Prism, colourless V = 946.76 (11) Å3 0.49 × 0.14 × 0.08 mm Z = 4 Data collection Bruker SMART CCD area-detector diffractometer 3882 independent reflections Radiation source: sealed tube 1997 reflections with I > 2σ(I) Monochromator: graphite Rint = 0.047 T = 296(2) K θmax = 34.4º φ and ω scans θmin = 3.0º supplementary materials Figures Fig. 1. The molecular structure of the title compound with the atom-labelling scheme. Dis- placement ellipsoids are drawn at the 30% probability level. H atoms are represented as small spheres of arbitrary radii. Fig. 2. Partial packing view showing the formation of the R22(8) ring and C(5) chains through N—H···N hydrogen bonds drawn as dashed lines. H atoms not involved in hydrogen bondings have been omitted for clarity. [Symmetry codes: (i) −x + 1, −y, −z + 2; (ii) x, −y + 1/2, z + 1/2] Fig. 3. The tautomeric forms of the title compound. Figures Fig. 1. supplementary materials Absorption correction: multi-scan (SADABS; Bruker, 1998) h = −11→13 Tmin = 0.984, Tmax = 0.997 k = −17→17 16028 measured reflections l = −16→16 Absorption correction: multi-scan (SADABS; Bruker, 1998) Tmin = 0.984, Tmax = 0.997 16028 measured reflections h = −11→13 k = −17→17 l = −16→16 Secondary atom site location: difference Fourier map Hydrogen site location: inferred from neighbouring sites H atoms treated by a mixture of independent and constrained refinement H atoms treated by a mixture of independent and constrained refinement w = 1/[σ2(Fo 2) + (0.0699P)2] where P = (Fo 2 + 2Fc 2)/3 (Δ/σ)max < 0.001 Δρmax = 0.19 e Å−3 Δρmin = −0.16 e Å−3 w = 1/[σ2(Fo 2) + (0.0699P)2] where P = (Fo 2 + 2Fc 2)/3 (Δ/σ)max < 0.001 Δρmax = 0.19 e Å−3 Δρmin = −0.16 e Å−3 Extinction correction: none Refinement 5-Amino-3-methyl-1-phenyl-1H-1,2,4-triazole Crystal data C9H10N4 F000 = 368 Mr = 174.21 Dx = 1.222 Mg m−3 Monoclinic, P21/c Mo Kα radiation λ = 0.71073 Å Hall symbol: -P 2ybc Cell parameters from 2898 reflections a = 8.5110 (5) Å θ = 2.5–23.3º b = 11.2490 (8) Å µ = 0.08 mm−1 c = 10.1048 (7) Å T = 296 (7) K β = 101.866 (4)º Prism, colourless V = 946.76 (11) Å3 0.49 × 0.14 × 0.08 mm Z = 4 Data collection Bruker SMART CCD area-detector diffractometer 3882 independent reflections Radiation source: sealed tube 1997 reflections with I > 2σ(I) Monochromator: graphite Rint = 0.047 T = 296(2) K θmax = 34.4º φ and ω scans θmin = 3.0º Crystal data C9H10N4 F000 = 368 Mr = 174.21 Dx = 1.222 Mg m−3 Monoclinic, P21/c Mo Kα radiation λ = 0.71073 Å Hall symbol: -P 2ybc Cell parameters from 2898 reflections a = 8.5110 (5) Å θ = 2.5–23.3º b = 11.2490 (8) Å µ = 0.08 mm−1 c = 10.1048 (7) Å T = 296 (7) K β = 101.866 (4)º Prism, colourless V = 946.76 (11) Å3 0.49 × 0.14 × 0.08 mm Z = 4 Data collection Bruker SMART CCD area-detector diffractometer 3882 independent reflections Radiation source: sealed tube 1997 reflections with I > 2σ(I) Monochromator: graphite Rint = 0.047 T = 296(2) K θmax = 34.4º φ and ω scans θmin = 3.0º sup-2 supplementary materials Special details Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes) are estimated using the full covariance mat- rix. The cell e.s.d.'s are taken into account individually in the estimation of e.s.d.'s in distances, angles and torsion angles; correlations between e.s.d.'s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s. planes. Refinement. Refinement of F2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F2, convention- al R-factors R are based on F, with F set to zero for negative F2. The threshold expression of F2 > 2sigma(F2) is used only for calculat- ing R-factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F2 are statistically about twice as large as those based on F, and R– factors based on ALL data will be even larger. Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) x y z Uiso/Ueq Occ. (<1) N1 0.22326 (10) 0.20008 (8) 0.83687 (9) 0.0445 (2) N2 0.28380 (11) 0.22738 (9) 0.72310 (10) 0.0530 (3) N3 0.43302 (11) 0.08529 (9) 0.84645 (9) 0.0500 (3) N4 0.29094 (12) 0.06921 (9) 1.02612 (10) 0.0528 (3) H4A 0.3772 (12) 0.0290 (10) 1.0662 (12) 0.063 H4B 0.2503 (14) 0.1219 (9) 1.0797 (11) 0.063* C4 0.08173 (12) 0.25677 (10) 0.85950 (12) 0.0454 (3) C3 0.31616 (12) 0.11612 (9) 0.90899 (11) 0.0424 (3) C2 0.40669 (14) 0.15538 (11) 0.73467 (12) 0.0535 (3) C9 −0.03152 (14) 0.19287 (12) 0.90944 (15) 0.0606 (3) H9 −0.0159 0.1124 0.9286 0.073* C5 0.05851 (16) 0.37569 (12) 0.82934 (14) 0.0663 (4) sup-3 supplementary materials supplementary materials supplementary materials H5 0.1354 0.4193 0.7968 0.080* C1 0.51079 (17) 0.15076 (16) 0.63304 (15) 0.0792 (5) H1A 0.5936 0.0925 0.6601 0.119* 0.50 H1B 0.5588 0.2273 0.6270 0.119* 0.50 H1C 0.4471 0.1295 0.5464 0.119* 0.50 H1D 0.4727 0.2070 0.5623 0.119* 0.50 H1E 0.5076 0.0722 0.5953 0.119* 0.50 H1F 0.6192 0.1700 0.6760 0.119* 0.50 C7 −0.19460 (19) 0.36600 (17) 0.89769 (18) 0.0921 (6) H7 −0.2890 0.4028 0.9085 0.111* C6 −0.0814 (2) 0.42885 (14) 0.84843 (18) 0.0877 (5) H6 −0.0991 0.5089 0.8274 0.105* C8 −0.16821 (16) 0.24917 (14) 0.93076 (18) 0.0797 (5) H8 −0.2428 0.2072 0.9679 0.096* Atomic displacement parameters (Å2) U11 U22 U33 U12 U13 U23 N1 0.0431 (4) 0.0488 (5) 0.0457 (5) 0.0065 (4) 0.0189 (4) 0.0059 (4) N2 0.0492 (5) 0.0649 (6) 0.0499 (6) 0.0084 (4) 0.0218 (5) 0.0126 (5) N3 0.0474 (5) 0.0574 (6) 0.0487 (6) 0.0104 (4) 0.0181 (4) 0.0014 (4) N4 0.0584 (6) 0.0544 (6) 0.0507 (6) 0.0157 (5) 0.0228 (5) 0.0088 (5) C4 0.0414 (5) 0.0509 (6) 0.0457 (6) 0.0077 (4) 0.0130 (5) 0.0019 (5) C3 0.0439 (5) 0.0418 (6) 0.0437 (6) 0.0034 (4) 0.0140 (5) −0.0003 (5) C2 0.0480 (6) 0.0669 (7) 0.0500 (7) 0.0048 (5) 0.0200 (5) 0.0064 (6) C9 0.0476 (6) 0.0612 (8) 0.0777 (9) 0.0036 (5) 0.0243 (6) 0.0070 (7) C5 0.0711 (8) 0.0611 (8) 0.0754 (10) 0.0174 (6) 0.0353 (7) 0.0164 (7) C1 0.0679 (8) 0.1133 (12) 0.0673 (10) 0.0214 (8) 0.0392 (7) 0.0151 (8) C7 0.0709 (9) 0.1057 (13) 0.1108 (14) 0.0396 (9) 0.0445 (9) 0.0214 (10) C6 0.0926 (11) 0.0767 (10) 0.1056 (13) 0.0420 (8) 0.0481 (10) 0.0290 (9) C8 0.0534 (8) 0.0923 (11) 0.1032 (12) 0.0100 (7) 0.0385 (8) 0.0146 (9) Geometric parameters (Å, °) N1—C3 1.3459 (14) C5—C6 1.3811 (18) N1—N2 1.3874 (11) C5—H5 0.9300 N1—C4 1.4224 (12) C1—H1A 0.9600 N2—C2 1.3090 (14) C1—H1B 0.9600 N3—C3 1.3294 (12) C1—H1C 0.9600 N3—C2 1.3577 (15) C1—H1D 0.9600 N4—C3 1.3528 (14) C1—H1E 0.9600 N4—H4A 0.886 (8) C1—H1F 0.9600 N4—H4B 0.917 (8) C7—C8 1.363 (2) C4—C5 1.3773 (17) C7—C6 1.369 (2) C4—C9 1.3785 (15) C7—H7 0.9300 C2—C1 1.4888 (15) C6—H6 0.9300 C9—C8 1.3799 (17) C8—H8 0.9300 C9—H9 0.9300 Atomic displacement parameters (Å2) sup-4 supplementary materials C3—N1—N2 109.09 (7) H1A—C1—H1C 109.5 C3—N1—C4 130.56 (8) H1B—C1—H1C 109.5 N2—N1—C4 120.33 (9) C2—C1—H1D 109.5 C2—N2—N1 102.42 (9) H1A—C1—H1D 141.1 C3—N3—C2 103.43 (9) H1B—C1—H1D 56.3 C3—N4—H4A 109.5 (8) H1C—C1—H1D 56.3 C3—N4—H4B 114.3 (8) C2—C1—H1E 109.5 H4A—N4—H4B 115.9 (11) H1A—C1—H1E 56.3 C5—C4—C9 120.55 (10) H1B—C1—H1E 141.1 C5—C4—N1 119.18 (9) H1C—C1—H1E 56.3 C9—C4—N1 120.27 (10) H1D—C1—H1E 109.5 N3—C3—N1 109.84 (9) C2—C1—H1F 109.5 N3—C3—N4 125.66 (10) H1A—C1—H1F 56.3 N1—C3—N4 124.49 (9) H1B—C1—H1F 56.3 N2—C2—N3 115.20 (9) H1C—C1—H1F 141.1 N2—C2—C1 122.54 (11) H1D—C1—H1F 109.5 N3—C2—C1 122.26 (11) H1E—C1—H1F 109.5 C4—C9—C8 119.55 (13) C8—C7—C6 119.63 (12) C4—C9—H9 120.2 C8—C7—H7 120.2 C8—C9—H9 120.2 C6—C7—H7 120.2 C4—C5—C6 118.57 (12) C7—C6—C5 121.25 (14) C4—C5—H5 120.7 C7—C6—H6 119.4 C6—C5—H5 120.7 C5—C6—H6 119.4 C2—C1—H1A 109.5 C7—C8—C9 120.39 (13) C2—C1—H1B 109.5 C7—C8—H8 119.8 H1A—C1—H1B 109.5 C9—C8—H8 119.8 C2—C1—H1C 109.5 Hydrogen-bond geometry (Å, °) D—H···A D—H H···A D···A D—H···A N4—H4A···N3i 0.886 (8) 2.110 (8) 2.9923 (13) 173.1 (12) N4—H4B···N2ii 0.917 (8) 2.210 (10) 3.0415 (14) 150.4 (10) Symmetry codes: (i) −x+1, −y, −z+2; (ii) x, −y+1/2, z+1/2. sup-5 supplementary materials Fig. 1 Fig. 1 sup-6 supplementary materials Fig. 2 sup-7 supplementary materials Fig. 3 supplementary materials Fig. 3 Fig. 3 Fig. 3 sup-8
https://openalex.org/W3198929262	https://www.researchsquare.com/article/rs-895693/latest.pdf	English	null	Impact of myocardial scars on left ventricular deformation in type 2 diabetes mellitus after myocardial infarction by contrast-enhanced cardiac magnetic resonance	Cardiovascular diabetology	2,021	cc-by	6,965	Impact of Myocardial Scars on Left Ventricular Deformation in Type 2 Diabetes Mellitus After Myocardial Infarction by Contrast- Enhanced Cardiac Magnetic Resonance g Yue Gao Sichuan University West China Hospital Hua-yan Xu West China Women\'s and Children\'s Hospital: Sichuan University West China Second University Hospital Ying-kun Guo West China Women\'s and Children\'s Hospital: Sichuan University West China Second University Hospital Xiao-ling Wen Sichuan University West China fourth University Hospital Rui Shi Sichuan University West China Hospital Yuan Li Sichuan University West China Hospital Zhi-Gang Yang (  yangzg666@163.com ) West China Hospital, Sichuan University https://orcid.org/0000-0001-9341-7697 Research Article Keywords: Diabetic cardiomyopathy, Myocardial Infarction, Myocardium, Cardiac Magnetic Resonance Posted Date: September 21st, 2021 DOI: https://doi.org/10.21203/rs.3.rs-895693/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Conclusions The myocardial scarring size in T2DM patients after MI is negatively correlated with LV global PS and PSSR, especially in the circumferential direction. Additionally, different MI regions have different effects on the reduction of LV deformation, and relevant clinical evaluations should be strengthened. Background Type 2 diabetes mellitus (T2DM) is a major risk factor for coronary artery disease and myocardial infarction (MI). The interaction of diabetic cardiomyopathy and MI scars on myocardial deformation in T2DM patients is unclear. Therefore, we aimed to evaluated myocardial deformation using cardiac magnetic resonance (CMR) in T2DM patients with previous MI and investigated the influence of MI on left ventricular (LV) deformation Results There was a decrease in LV function and LV global PS, PSSR, and PDSR in the T2DM(MI+) group compared with those of the other groups. Furthermore, reduced LV deformation (p < 0.017) was observed in the T2DM(MI+) group with anterior wall infarction. The increased total LV infarct extent and infarct mass of LV were related to decreased LV global PS (radial, circumferential, and longitudinal directions; p < 0.01) and LV global PSSR (radial and circumferential directions, p < 0.02). Multivariate analysis demonstrated that NYHA functional class and total LV infarct extent were independently associated with LV global radial PS (β = −0.400 and β = −0.446, respectively, all p < 0.01; model R2 = 0.37) and circumferential PS (β = −0.339 and β = −0.530, respectively, all p < 0.01; model R2 = 0.41), while LV anterior wall infarction was independently associated with LV global longitudinal PS (β = −0.398, p = 0.006). Methods Two hundred and two T2DM patients, including 46 with MI (T2DM(MI+)) and 156 without MI (T2DM(MI−)), and 59 normal controls who underwent CMR scans were included. Myocardial scars were assessed by late gadolinium enhancement. LV function and deformation, including LV global function index, LV global peak strain (PS), peak systolic strain rate (PSSR), and peak diastolic strain rate (PSDR), were compared among these groups. Correlation analysis and multivariate linear regression analyses were used to investigate the relationship between myocardial scars and LV deformation. Research Article License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/17 Introduction Diabetic cardiomyopathy (DCM) is defined as myocardial dysfunction that is independent of coronary artery disease and hypertension and can lead to heart failure1,2.Diastolic dysfunction is one of the important indicators of early left ventricular (LV) dysfunction prior to reduced LV ejection fraction in DCM, and impaired global longitudinal strain was associated with cardiovascular events in type 2 diabetes mellitus (T2DM) patients3–5. As DCM develops, myocardial microvascular dysfunction, remodeling of the extracellular matrix are also related in contractile dysfunction in DCM6. Coronary artery disease and myocardial infarction (MI) are major causes of global morbidity and mortality, and T2DM is considered a major risk factor for coronary artery disease, T2DM patients are at high risk of MI and have a poor prognosis7,8. Previous studies have pointed out that the MI size and transmurally in MI patients has an important effect on the prognosis and survival. In patients with previous MI, myocardial scarring causes weakened or even contradictory LV wall movement, fibrotic repair of the infarcted area, and compensatory cardiomyocyte hypertrophy in remote infarcted areas, all of which have an impact on the LV myocardial deformation9. At present, few studies have been carried out on the effect of myocardial scar on myocardial deformation after MI in T2DM patients with DCM. Cardiac magnetic resonance (CMR) imaging, which has been commonly used in the last decades in clinical practice, provides information on various characteristics of cardiac structure, function, and myocardial tissue10–12. CMR feature tracking has been used to measure myocardial deformation13. The late gadolinium enhancement (LGE) sequence detects and assesses myocardial scar location with high spatial resolution and quantifies the area of the MI14. Therefore, this study aimed to evaluate myocardial deformation using CMR in T2DM patients with previous MI and to investigate the influence of MI scar on LV deformation. Study population The study protocol was approved by Biomedical Research Ethics Committee of our hospital. Initially, we retrospective enrolled 698 patients who were diagnosed with T2DM according to the World Health Organization standards, between January 2015 and May 2021, and all the patients have completed CMR examinations15. Exclusion criteria were as follow: (1) known cardiomyopathy, congenital heart disease, or valvular heart disease (confirmed by echocardiography, electrocardiogram, or coronary computed tomographic angiography); (2) uncontrollable hypertension (systolic blood pressure > 160 mmHg); and (3) severe renal failure (estimated glomerular filtration rate, eGFR < 30 ml/min). Following these criteria, a total of 227 T2DM patients were included in this study, of which 156 T2DM patients (48 males and 106 females; mean age 55.55 ± 11.8 years) had no previous MI or coronary revascularization, 25 T2DM patients had acute or subacute MI during CMR examination, and 46 TDM patients (28 males and 18 females; mean age 61.54 ± 9.11 years) had previous acute MI (> 6 months). Final, we exclude the 27 T2DM patients had acute or subacute MI. In this study, the MI was defined according to the European Society of Cardiology/American College of Cardiology/American Heart Association committee criteria16. In addition, age-, sex-, and body mass index-matched healthy volunteers were recruited in the controls group. Exclusion criteria for the control group were as follows: (1) DM or impaired glucose tolerance; (2) known acute or chronic disease such as hypertension; (3) presence of dyspnea, chest pain, palpitation or other cardiovascular disease-related symptoms; (4) electrocardiogram abnormalities; and (5) cardiovascular abnormalities detected by CMR (perfusion defect, local, or diffuse myocardial late-gadolinium enhancement, abnormal ventricular motion, valvular stenosis, etc.). Hence, 59healthy controls (27 males and 32 females; mean age 58.53 ± 8.23 years) were included in this study. A detailed flow chart of the present study is provided by Fig. 1. Clinical characteristics, family history, surgery history, medication, and serum biochemical indexes of all patients and healthy controls were collected. Blood sampling for serum biochemical indexes was performed within one week of the CMR scan without changing the subject's medication regimen. Methods And Materials Page 2/17 Page 2/17 CMR scanning protocol All subjects underwent CMR scan using a 3.0-T whole-body scanner (Skrya; Siemens Medical Solutions, Erlangen, Germany) in the supine position. A standard ECG-triggering device was used and end-inspiratory breath holding were performed. Following a survey scan, cine images such as long-axis four-chamber views and short-axis two-chamber views were acquired using a steady-state free-precession sequence (temporal time = 39.34 ms, echo time = 1.22 ms, flip angle = 40°, slice thickness = 8 mm, field of view = 360 × 300 mm2, matrix size = 256×166). Gadobenate dimeglumine (MultiHance; Bracco, Milan, Italy) was intravenously injected at a dose of 0.2 ml/kg body weight at an injection rate of 2.5–3.0 ml/s, followed by a 20 ml saline flush at a rate of 3.0 ml/s. Late gadolinium enhancement (LGE) images were acquired in the corresponding slice position as the cine imaging 10–15 min after contrast injection. The images were obtained using a phase-sensitive inversion recovery sequence with the follow parameters: temporal time = 300ms, echo time = 1.44 ms, flip angle = 40°, slice thickness = 8 mm, field of view = 275 × 400 mm2, matrix size = 256×184. CMR data analysis All CMR images data to an offline workstation using a semi-automated software (Cvi42; Circle Cardiovascular Imaging, Inc., Calgary, Canada). Endocardial and epicardial traces were delineated manually by two experienced radiologists in the serial short-axis slices during the end-diastolic and end-systolic phases(figure.2.). Papillary muscles were considered as part of the ventricular cavity, and epicardial fat was excluded. Subsequently, LV functional parameters and LV mass were automatically determined. LV remodeling was characterized by the ratio of LV mass to LVEDV (LVMVR). The LV global function index (LVGFI) was calculated using the following formula: Comparison of the LV function and deformation among th The CMR imaging results for LV function and deformation are summarized in Table 2. LVESVi was higher in the T2DM(MI-) group than in the control group. LVESVi, and LV mass were higher (all p < 0.005) in the T2DM(MI+) group compared with the T2DM(MI-) and normal control groups. Meanwhile, LVSVi (T2DM(MI+) vs. T2DM(MI-): 39.33(31.96–46.71) vs. 48.17(41.04–54.36); T2DM(MI+) vs. control: 39.33(31.96–46.71) vs. 47.52(42.13–53.67), p < 0.001) and LVGFI (T2DM(MI+) vs. T2DM(MI-): 27.37(18.12– 40.02) vs. 48.99(44.38–54.33); T2DM(MI+) vs. control: 27.37(18.12–40.02) vs. 51.56(48.03–56.06)), p < 0.001) were lower in the T2DM(MI+) group compared with the T2DM(MI-) and control groups. Regarding LV deformation, the global radial PS, global circumferential PS, and global longitudinal PS (all p < 0.001) were lower in the T2DM(MI+) group than in the T2DM(MI-) and control groups. The global circumferential PS (p < 0.017) and global longitudinal PS (p = 0.001) were lower in the T2DM(MI-) group than in the control group. There was no statistically significant difference in global radial PS between the T2DM(MI-) and control group. The global radial, circumferential and longitudinal PSSR and PDSR (all p < 0.001) were significantly lower in the T2DM(MI+) group than those in the T2DM(MI-) and control groups. except for longitudinal PDSR in the T2DM(MI+) group compared with the T2DM(MI-) group. For the T2DM(MI+) group, the circumferential and longitudinal PDSR were lower than those in the control group (p < 0.017). LVGFI = {LVSV / [(LVEDV + LVESV) / 2 + (LV mass/1.05)]} × 100 For analysis of LV myocardial strain, long-axis 2-chamber, 4-chamber, and short-axis slices were loaded into the 3-dimensional tissue tracking module, and the LV global myocardial strain parameters were acquired automatically, including radial, circumferential, and longitudinal peak strain (PS), peak systolic strain rate (PSSR), and peak diastolic strain rate (PDSR). For LGE imaging analysis, LV segment–based analysis was performed in accordance with the 16-segment model of the American Heart Association. The hyper- enhanced myocardium area was defined as myocardial scar on the LGE short-axis images when the signal intensity was five standard deviations above the mean intensity of the normal myocardium (Figure.2)17. We assessed the extent of the LGE regions involving the LV wall, dividing it into the interventricular septum, anterior wall, inferior wall and lateral wall using the 16-segment model. Two radiologists evaluated the images separately, and if the results were inconsistent, they discussed and agreed on the result. g p y, Variability analysis Variability analysis Page 3/17 Page 3/17 To determine intra-observer variability, LV deformation and LGE parameters in 70 random cases that included 50 T2DM patients and 20 normal controls were measured twice within a week interval by a radiologist. Then, a second radiologist, who was blinded to the first investigator’s results reperformed the measurements to assess the inter-observer variability. l l To determine intra-observer variability, LV deformation and LGE parameters in 70 random cases that included 50 T2DM patients and 20 normal controls were measured twice within a week interval by a radiologist. Then, a second radiologist, who was blinded to the first investigator’s results reperformed the measurements to assess the inter-observer variability. Statistical analysis Statistical analyses were performed with commercially available SPSS (version 21.0 for windows; SPSS, Inc., Chicago, IL, USA). Continuous variables are presented as the mean ± standard deviation, or as median values and interquartile ranges. One-way analysis of variance test was performed to evaluate the differences among the following groups: T2DM with MI, T2DM without MI and normal control. Based on Bonferroni’s correction for multigroup comparisons, p-values of < 0.017 were considered as statistically significant. Spearman’s and Pearson’s correlation analysis were conducted to identify the relationship between infarction and cardiac deformation. Moreover, multivariable stepwise linear regression analysis was employed to identify the relationship between infarction parameters and cardiac dysfunction. Variables with a prob- ability value of 0.1 in the univariable analyses were then included in a stepwise multivariable analysis based on a linear regression model. A p-value of < 0.05 was considered statistically significant. Patient characteristics Of the 202 T2DM patients, 156 were in T2DM(MI-) group (48[30.7%]) males, mean age 55.55±11.80 years) and 46 in T2DM(MI+) group (28[60.8%]) males, mean age 61.54±9.11 years). Table 1 presents their baseline characteristics, cardiovascular risk factors, metabolic parameters, and medications. The results showed that there were no statistically significant differences in the baseline characteristics among these groups, except that there were more males in the T2DM(MI+) group than that in T2DM(MI-) group. As for cardiovascular risk factors, more patients were previous or current smoker (50.5% vs. 28.3%) in T2DM(MI+) group than that in T2DM(MI-) group, but there was no difference in hyperlipidemia or family history of T2DM between the groups. HbA1c and GLU showed no statistically significant differences between the T2DM(MI+) and T2DM(MI-) groups. In the T2DM(MI+) group, 20 patients received percutaneous coronary intervention, and one patient received coronary artery bypass grafting surgery. A total of 34 patients were identified with culprit vessels, among which 14 (30.43%) were the left anterior descending coronary artery (LAD), 6 (13.0%) were the left circumflex coronary artery (LCx) and 14 (30.43%) were the right coronary artery (RCA). Association between LV deformation and LGE area in T2D Regarding infarction-involve regions in the T2DM(MI+) group, 28 patients had LGE areas involving the interventricular septum, 20 involving the LV inferior wall, 13 involving the LV lateral wall, and 12 involving the LV anterior wall. There were no LGE areas detected in the T2DM(MI-) group. For the LGE area in different regions of the LV wall, patients with anterior wall infarction had lower LV global radial PS (anterior vs. non- anterior: 11.91 ± 1.924 vs. 18.45 ± 1.671, p = 0.037), circumferential PS (anterior vs. non-anterior: -8.447 ± 0.7525 vs. -13.24 ± 0.8778, p = 0.034. figure.3) and longitudinal PS (anterior vs. non-anterior: -5.289 ± 0.4827 vs. -8.351 ± 0.6052 p = 0.006). Patients with interventricular septum infarction have lower LV global longitudinal PS (interventricular septum vs. non- interventricular septum: -6.515 ± 0.708 vs. -8.503 ± 0.6702, p = 0.043) (Figure.4). In addition, LV anterior wall infarction was independently associated with global longitudinal PS (β = − 0.398; p = 0.006, model R2 = 0.16; Table 4). Association between LV deformation and LGE size in T2DM patients with MI Page 4/17 Page 4/17 In this study, the range of infarct size of LV and total LV infarct extent were 17.96(11.07-25.26) and 17.12(9.58-27.53), respectively. As shown in Table 3, for T2DM(MI+) patients, there was a negative correlation between increased infarction size and decreased LVGFI (all p <0.001) and LV global PS in all three directions (all p <0.01). LV global radial and circumferential PSSR were inversely correlated with total LV infarct extent (r = − 0.353, p = 0.016; r = 0.533, p <0.001, respectively), enhanced mass of LV (r = − 0.302, p = 0.042; r = 0.525, p <0.001, respectively), and enhanced area of LV (r = − 0.297, p = 0.045; r = 0.528, p <0.001, respectively) in T2DM patients with chronic MI. However, LV global longitudinal PSSR and global PDSR in the three directions showed no significant relationship with infarction size (all p >0.05). In this study, the range of infarct size of LV and total LV infarct extent were 17.96(11.07-25.26) and 17.12(9.58-27.53), respectively. As shown in Table 3, for T2DM(MI+) patients, there was a negative correlation between increased infarction size and decreased LVGFI (all p <0.001) and LV global PS in all three directions (all p <0.01). LV global radial and circumferential PSSR were inversely correlated with total LV infarct extent (r = − 0.353, p = 0.016; r = 0.533, p <0.001, respectively), enhanced mass of LV (r = − 0.302, p = 0.042; r = 0.525, p <0.001, respectively), and enhanced area of LV (r = − 0.297, p = 0.045; r = 0.528, p <0.001, respectively) in T2DM patients with chronic MI. However, LV global longitudinal PSSR and global PDSR in the three directions showed no significant relationship with infarction size (all p >0.05). Multivariate linear regression analysis demonstrated that NYHA functional class and total LV infarct extent were independently associated with global radial PS (β = -0.400, p = 0.002; and β = -0.446, p = 0.001, respectively; model R2 = 0.37) and global circumferential PS (β = -0.339, p = 0.006; and β = -0.530, p = 0.001, respectively; model R2 = 0.41). Inter‑ and Intraobserver variability Table 5 summarizes the inter- and Intraobserver variability for LV deformation and LGE analysis. The ICCs for intra- and interobserver variability were 0.828–0.959 and 0.777–0.955, respectively, for LV deformation, and 0.827–0.895 and 0.882–0.893 respectively, for LGE parameters, which suggesting that both techniques are in agreement. Discussion Scarring extent and size are strictly related not only to adverse cardiac remodeling but also to cardiovascular events28. Scar recognition is also a potential predictor for arrhythmia substrates, Histological examination has shown that isolated bundles of surviving myocytes are interwoven within strands of fibrous tissue. These viable cells can form reentry circuits within fibrous tissue, thus contributing to ventricular tachycardia29. LGE-CMR is considered the best available technique for noninvasive assessment of myocardial scar tissue following MI, and it quantitatively evaluates the myocardial scar, discriminates the infarction transmurally, and defines the infarct territory of the LV ventricular wall27. Scarring extent and size are strictly related not only to adverse cardiac remodeling but also to cardiovascular events28. Scar recognition is also a potential predictor for arrhythmia substrates, Histological examination has shown that isolated bundles of surviving myocytes are interwoven within strands of fibrous tissue. These viable cells can form reentry circuits within fibrous tissue, thus contributing to ventricular tachycardia29. Previous studies have shown that there is a correlation between the extent of MI and LV deformation in acute MI patients9,30. However, after acute MI, with the absorption of myocardial edema and inflammation, or infarction core fiber gliosis, the delayed enhancement area of LGE imaging changes. We found that on the basis of DCM, which inherently exists myocardial deformation and damage, myocardial scarring after MI has a significant negative correlation with LV radial, circumferential, and longitudinal PS and PSSR. This indicates that after acute MI, the recovery of LV myocardial systolic function and deformation, is affected by the extent and quality of MI, especially in the circumferential direction. Furthermore, we also found that T2DM (MI+) patients had reduced LV global PDSR in the three directions compared to T2DM (MI-)patients, but there was no correlation between LV global PDSR and the extent of myocardial scarring. The decreasing in PDSR does not seem to be directly related to the local myocardial scarring, but may be secondary to reduced LV systolic function. Decreased LV function may further lead to myocardial metabolic disorder, and hyperglycemia may have an additive effect on diastolic dysfunction, which further leads to reduced LV PDSR. These speculations need to be verified by further studies. At present, a few studies have explored the relationship between MI territory (involving the interventricular septum, anterior wall, lateral wall or inferior wall) and LV global deformation and peak strain rate. Conclusions The study found that systolic dysfunction is more likely to occur in T2DM patients after MI, with a significant reduction in LV global circumferential deformation. The size of myocardial scarring is negatively correlated with the LV global PS and PSSR. In addition, MI in different regions has different effects on the reduction of LV deformation. Therefore, more attention should be paid to MI scarring and involved scope in T2DM patients in clinical evaluations. Limitations There are several limitations to our study. First, this study was a single-center study, so there may be some biases influencing the results. Second, the medication and the NT-proBNP of the control group was not measured. However, the detailed history and the physical examination report were carefully checked to ensure that inclusion criteria were met. Thirdly, since this was a retrospective study, the long- term development in T2DM patients after myocardial infarction should be further investigated in follow-up in the future. Discussion The results in this study show that if MI involved in the LV anterior wall, the LV global PS in all three directions is significantly affected, and anterior wall involvement is an independent factor for the decrease in LV global longitudinal PS. Most of the Culprit vessel in the infarction of the LV anterior wall was LAD, while the right bundle branch and left anterior fascicular are slender, and the LAD is the only blood supply. When LAD stenosis and the blood supply is blocked, there might be an effect on LV deformation. More studies have focused on the stratification effects of MI size on prognosis and survival risk, and these studies have shown that MI size is an important predicator for the quality of life of patients31,32. However, the MI involved different areas of the LV wall may result in different prognoses, implying that more attention should be paid to LV infarction involvement in future clinical evaluation. Abbreviations DM: diabetes mellitus; T2DM: Type 2 diabetes mellitus; DCM: diabetic cardiomyopathy; CMR: cardiac magnetic resonance; LGE: late gadolinium enhancement; LV: left ventricular; LVGFI: left ventricular global function index; MI: myocardial infarction; PDSR: peak diastolic strain rate; PS: peak strain; PSSR: peak systolic strain rate; LAD: left anterior descending coronary artery; EDVi: End‑diastolic volume index; ESVi: End systolic volume index; SVi: Stroke volume index; LVEF: Left ventricle ejection fraction; ICC: Intraclass correlation coefficient. Discussion T2DM is a chronic metabolic disease involving multiple organs, and the main reason for the increasing mortality of patients with T2DM is DCM. In addition, T2DM is a high-risk factor for cardiovascular events such as coronary heart disease and MI18,19. In this study, the following principal findings were obtained: 1) T2DM (MI-) patients have reduced LV longitudinal deformation, while T2DM (MI+) patients have reduced deformation in all three directions, especially circumferential deformation; 2) in T2DM (MI+) patients, LV global PS, and PSSR are decreased, which is related to the extent of myocardial scarring; and 3) anterior wall infarction, and then ventricular septal infarction, are more likely to lead to decreased LV deformation in T2DM (MI+) patients. The pathogenesis of DCM is complex and multifactorial, and several causative mechanisms, include metabolic effects on myocytes, myocardial steatosis, myocardial fibrosis, microangiopathy, and autonomic nervous dysfunction20–22. Microvascular ischemia of DCM is most likely to occur in the sub-endocardium, where the longitudinal myocardial fibers are predominantly located; therefore, a reduction in longitudinal LV deformation occurs in the early stages of DCM. As in previous studies, our results show reduced longitudinal PS but preserved circumferential and radial PS in T2DM (MI-) patients compared to control subjects23. However, for T2DM patients after MI, deformation in all three directions of LV global PS were decreased, and were especially reduced in the circumferential rather than the longitudinal direction. We speculate that after MI and scar formation in T2DM (MI-) patients, the myocardial cells in the scar area are replaced by the fibrous matrix, and local ventricular wall movement is reduced or impaired to varying degrees. Different from the longitudinal myocardial fibers, circumferential myocardial fibers are mainly located subepicardial, resulting in significantly reduced circumferential LV deformation24. Additionally, reduced longitudinal and circumferential PDSR suggests that diastolic dysfunction begins in the initial stage of DCM, which we have shown in our previous research2526. LV enlargement, especially LVESV, and reduced LVEF, are common cardiac morphological changes after MI, which was more significant than the early diastolic functional damage of DCM. Therefore, in our study, T2DM(MI+) patients had not only decreased PDSR, but also decreased PSSR in all three directions. Page 5/17 Page 5/17 LGE-CMR is considered the best available technique for noninvasive assessment of myocardial scar tissue following MI, and it quantitatively evaluates the myocardial scar, discriminates the infarction transmurally, and defines the infarct territory of the LV ventricular wall27. Declarations Page 6/17 Funding This work was supported by the National Natural Science Foundation of China (821201080, 81971586, 81771887, 81471722), 1·3·5 project for disciplines of excellence, West China Hospital, Sichuan University (ZYGD18013). Consent for publication Not applicable. Authors’ Contributions YG and ZGY designed the study. YG performed the experiments, and wrote the manuscript. YL participated in the study design, analyzed the data, drafted the manuscript and editing and review of the manuscript. YZG supervised the overall study and contributed to study design, editing and review of the manuscript. YG and HYX performed the experiments and review the manuscript. YG, XLW and RS performed the experiments and was responsible for collecting, sorting and statistical data. ZGY is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Acknowledgements Not applicable Author details 1 Department of Radiology, West China Hospital, Sichuan University, 37# Guo Xue Xiang, Chengdu, Sichuan 610041, China. 2 Department of Radiology, Key Laboratory of Birth Defects and Related Diseases of Women and Children of Ministry of Education, West China Second University Hospital, Sichuan University, Chengdu, Sichuan, China. 3 Department of Radiology, West China Fourth Hospital, Sichuan University, Chengdu, Sichuan, China. Availability of data and materials The datasets used and analyzed during the current study are available from the corresponding author on reasonable request The authors declare that there are no conflicts of interest. The authors declare that there are no conflicts of interest. Page 6/17 The study complied with the Declaration of Helsinki and was approved by the West‑China hospital of Sichuan University biomedical research ethics committee (Chengdu, Sichuan, China; No. 2016‑24). 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Eur Heart J Cardiovasc Imaging. 2020;21:632–9. 15. Alberti KG, Zimmet PZ. Definition, diagnosis and classification of diabetes mellitus and its complications. Part 1: diagnosis and classification of diabetes mellitus provisional report of a WHO consultation. Diabet Med. 1998;15:539–53. 15. Alberti KG, Zimmet PZ. Definition, diagnosis and classification of diabetes mellitus and its complications. Part 1: diagnosis and classification of diabetes mellitus provisional report of a WHO consultation. Diabet Med. 1998;15:539–53. 6. Thygesen K, et al. Fourth Universal Definition of Myocardial Infarction (2018). J Am Coll Cardiol. 17. Bondarenko O, et al. Standardizing the definition of hyperenhancement in the quantitative assessment of infarct size and myocardial viability using delayed contrast-enhanced CMR. J Cardiovasc Magn Reson Off J Soc Cardiovasc Magn Reson. 2005;7:481–5. 17. Bondarenko O, et al. Standardizing the definition of hyperenhancement in the quantitative assessment of infarct size and myocardial viability using delayed contrast-enhanced CMR. J Cardiovasc Magn Reson Off J Soc Cardiovasc Magn Reson. 2005;7:481–5. 18. Seferovic JP, et al. Retinopathy, Neuropathy, and Subsequent Cardiovascular Events in Patients with Type 2 Diabetes and Acute Coronary Syndrome in the ELIXA: The Importance of Disease Duration. J. Diabetes Res. 2018, 1631263 (2018). 18. Seferovic JP, et al. Retinopathy, Neuropathy, and Subsequent Cardiovascular Events in Patients with Type 2 Diabetes and Acute Coronary Syndrome in the ELIXA: The Importance of Disease Duration. J. Diabetes Res. 2018, 1631263 (2018). 19. Engelen SE, et al. Incidence of cardiovascular events and vascular interventions in patients with type 2 diabetes. Int J Cardiol. 2017;248:301–7. 19. Engelen SE, et al. Incidence of cardiovascular events and vascular interventions in patients with type 2 diabetes. Int J Cardiol. 2017;248:301–7. 20. Cohen CD, et al. Diastolic dysfunction in a pre-clinical model of diabetes is associated with changes in the cardiac non-myocyte cellular composition. Cardiovasc Diabetol. 2021;20:116. 20. Cohen CD, et al. References JACC Cardiovasc Imaging. 2017;10:230–40. 28. Bulluck H, Hammond-Haley M, Weinmann S, Martinez-Macias R, Hausenloy DJ. Myocardial Infarct Size by CMR in Clinical Cardioprotection Studies: Insights From Randomized Controlled Trials. JACC Cardiovasc Imaging. 2017;10:230–40. 29. Wong DTL, et al. Electro-mechanical characteristics of myocardial infarction border zones and ventricular arrhythmic risk: Novel insights from grid-tagged cardiac magnetic resonance imaging. Eur Radiol. 2012;22:1651–8. 29. Wong DTL, et al. Electro-mechanical characteristics of myocardial infarction border zones and ventricular arrhythmic risk: Novel insights from grid-tagged cardiac magnetic resonance imaging. Eur Radiol. 2012;22:1651–8. 30. Lustosa RP, et al. Changes in Global Left Ventricular Myocardial Work Indices and Stunning Detection 3 Months After ST-Segment Elevation Myocardial Infarction. Am J Cardiol. 2021. doi:10.1016/j.amjcard.2021.07.012. 30. Lustosa RP, et al. Changes in Global Left Ventricular Myocardial Work Indices and Stunning Detection 3 Months After ST-Segment Elevation Myocardial Infarction. Am J Cardiol. 2021. doi:10.1016/j.amjcard.2021.07.012. Page 8/17 Page 8/17 Page 8/17 31. Sandoval Y, Jaffe AS. Type 2 Myocardial Infarction: JACC Review Topic of the Week. J Am Coll Cardiol. 2019;73:1846–60. 32. Amier RP, et al. Long-Term Prognostic Implications of Previous Silent Myocardial Infarction in Patients Presenting With Acute Myocardial Infarction. JACC Cardiovasc Imaging. 2018;11:1773–81. Tables Page 9/17 Normal (n=59) T2DM (M I -) (n=156) T2DM (MI +) (n=46) Baseline characteristics Age, years 58.53±8.23 55.55±11.8 61.54±9.11 Male, n (%) 27(48.2%) 48(30.7%) 28(60.8%) § BMI, kg/m² 23.17±3.14 23.67±3.06 25.32±3.35 Systolic blood pressure, mmHg 120.19±8.85 127.96±13.11 123.67±19.27 Diastolic blood pressure, mmHg 75.73±10.36 79.88±9.88 77.89±12.76 Heart rate, bpm 73.28±10.85 72.67±15.42 73.80±14.60 Cardiovascular risk factors Previous/current smoker, n (%) 9(15.2%) 44(28.3%) 23(50.5%) § Hyperlipidemia 0 29(18.5%) 8(17.4%) Family history of DM 0 32(20.5%) 11(23.9%) Pervious PCI, n (%) 0 0 20(43.5) % Previous CABG, n (%) 0 0 1(2.2%) NYHA functional class, n I - - 3(6.5%) II - - 27(58.7%) III - - 15(32.6%) IV - - 1(2.2%) Culprit vessel n (%) LM - - 0 LAD - - 14(30.43%) LCx - - 6(13.0%) RCA - - 14(30.43%) Metabolic characteristics HbA1c, % 5.46±0.34 7.82±0.87 8.10±1.32 GLU, mmol/L 5.08±0.45 8.28±1.23 8.78±2.34 eGFR, mL/min/1.73m2 107.23±8.45 95.84 ± 9.32 79.60±10.45 NT-proBNP - - 874(204.75-2032.75) Medication, n（%） Aspirin, n (%) - 17(6.5%) 28(60.8%) ß-blockers, n (%) - 0 11(19.6%) ACEI/ARB, n (%) - 5(3.2%) 16(34.7%) Diuretics - 1(0.06%) 13(28.2%) Calcium-channel blocker - 8(5.1%) 5(10.8%) Page 10/17 T2DM: type 2 diabetes mellitus; MI: myocardial infarct; BMI: body mass index; PCI: Percutaneous Transluminal Coronary Intervention; CAGB: coronary artery bypass grafting; NYHA: New York Heart Association; HbA1c: glycated hemoglobin; Glu: glucose, eGFR estimated glomerular filtration rate; ACEI: angiotensin converting enzyme inhibitor。 * p < 0.017 versus normal group (Bonferroni’s) Table.2 Page 11/17 Normal (n=59) T2DM (M I (-)) (n=156) T2DM (MI (+)) (n=46) LVEDVi, ml/m2 70.75(63.97-80.18) 76.49(67.45-84.18) 114.56(80.18-137.75) * LVESVi, ml/m2 23.64(19.16-28.39) 26.46(20.92-32.32) * 72.52(39.62-102,18) § LVSVi, ml/m2 47.52(42.13-53.67) 48.17(41.04-54.36) 39.33(31.96-46.71) § LVCO, l/min 5.54(5.00-6,63) 5.85(4.56-7.00) 5.11(3.95-6.25) LVEF, % 66.14(62.85-70.86)) 64.74(59.32-69.69) 38.26(24.75-55.68) § LV mass, g/m2 77.61(68.40-88.32) 91.86.86(74.13-111.40) 127.80(103.68,156.91) § LVGFI, % 51.56(48.03-56.06) 48.99(44.38-54.33) 27.37(18.12-40.02) § PS, % Radial 35.32(32.38-40.72) 32.11(25.73-38.83) 14.71(9.29-22.83) § Circumferential -20.92(-22.67-(-18.84)) -19.56(-21.47-(-17.64)) -11.11(-16.10 -(-8.68)) § Longitudinal -15.03(-16.68-(-12.31)) -12.24(-14.67-(-10.08)) * -7.41(-9.95-(-4.75)) § PSSR, 1/s Radial 2.05(1.77-2.48) 1.745(1.41-2.26) 0.83(0.61-1.44) § Circumferential -1.02(-1.15-(-0.92)) -0.98(-1.13-(-0.86)) -0.61(-0.81-(-0.45)) § Longitudinal -0.75(-0.87-(-0.67)) -0.69(-0.86-(-0.55)) -0.38(-0.56-(0.20)) § PDSR, 1/s Radial -2.80(-3.22-(-2.12)) -2.05(-2.65-(-1.58)) -0.83(-1.24-(-0.51)) § Circumferential 1.32(1.19-1.53) 1.12(0.97-1.36) 0.65(0.50-0.93) § Longitudinal 0.93(0.70-1.11) 0.79(0.60-0.97) 0.49(0.33-0.62) * Infarct size, g - - 20.69(11.69-32.12) Infarct size, g % of LV - - 17.96(11.07-25.26) Total LV infarct extent (%) - - 17.12(9.58-27.53) Infarct territory, n (%) - Interventricular septum - - 22(47.8%) Inferior - - 20(43.4%) Lateral - - 13(28.2%) Anterior - - 12(26.09%) Notes: Data are presented as median (25th, 75th percentile). Table.2 LVEDVi, left ventricular end diastolic volume index; LVESVi, left ventricular end systolic volume index; LVSVi, left ventricular stroke volume index; LVEF, left ventricular ejection fraction; LVGFI: left ventricular global function index; MI: myocardial infarction; PDSR: peak diastolic strain rate; PS: peak strain; PSSR: peak systolic strain rate * p < 0.017 versus normal group (Bonferroni’s) § p < 0.017 versus T2DM patients without MI (Bonferroni’s) Page 12/17 Table.3 Correlation analysis of LV global strain parameters with the myocardial infarction parameters Correlation analysis of LV global strain parameters with the myocardial infarction parameters Correlation analysis of LV global strain parameters with the myocardial infarction parameters Total LV infarct extent (%) enhanced mass (g % of LV) enhanced area (ml %of LV) r P r P r P LVGFI, % -0.533 <0.001 -0.507 <0.001 -0.506 <0.001 PS, % Radial -0.455 0.001 -0.437 0.002 -0.435 0.001 Circumferential 0.538 <0.001 0.530 <0.0001 0.533 .000 Longitudinal 0.395 0.007 0.434 0.003 0.431 0.007 PSSR, % Radial -0.353* 0.016 -0.302* 0.042 -0.297* 0.045 Circumferential 0.533 <0.001 0.525 <0.001 0.528** <0.001 Longitudinal 0.097 0.520 0.106 0.484 0.110 0.465 PDSR, % Radial 0.160 0.287 0.163 0.280 0.172 0.254 Circumferential -0.235 0.116 -0.220 0.141 -0.218 0.146 Longitudinal -0.123 0.417 -0.083 0.586 -0.071 0.639 * p < 0.05 ** p < 0.01 Inter- and intra-observer variability of tissue tracking and LGE Page 13/17 Radial PS Circumferential PS Longitudinal PS Univariable Multivariable Univariable Multivariable Univariable Multivariable r P value r P value r P value r P value r P value r P value Age -0.107 0.480 0.057 0.707 -0.045 0.768 gender -0.262 0.079 0.269 0.071 0.262 0.079 NYHA functional class -0.400 0.006 -0.400 0.002 0.322 0.029 0.339 0.006 0.174 0.249 BMI 0.160 0.287 -0.112 0.458 -0.060 0.693 Hyperlipidemia 0.151 0.318 -0.200 0.182 -0.027 0.860 Smoking -0.252 0.091 0.310 0.026 0.336 0.022 Total LV infarct extent (%) -0.455 0.001 -0.446 0.001 0.538 <0.001 0.530 <0.001 0.395 0.007 Anterior infarct -0.317 0.032 0.433 0.003 0.410 0.005 0.398 0.006 Intra-observer (n = 70) 95% CI Inter-observer (n =70) 95% CI PS, % Radial PS (%) 0.959 0.901–0.982 0.955 0.905–0.979 Circumferential PS (%) 0.932 0.851–0.969 0.938 0.866–0.972 Longitudinal PS (%) 0.923 0.831–0.965 0.883 0.745–0.947 PSSR, 1/s Radial 0.938 0.899–0.977 0.920 0.833–0.962 Circumferential 0.866 0.862–0.968 0.915 0.823–0.960 Longitudinal 0.828 0.717–0.935 0.849 0.516–0.948 PDSR, 1/s Radial 0.952 0.871–0.970 0.925 0.843–0.964 Circumferential 0.934 0.720–0.936 0.847 0.596–0.934 Longitudinal 0.864 0.641–0.918 0.777 0.485–0.913 LGE Infarct size, g 0.895 0.790–0.898 0.893 0.782–0.947 Infarct size, g % of LV 0.891 0.780–0.896 0.885 0.667–0.923 Total LV infarct extent (%) 0.827 0.544–0.905 0.882 0.651–0.893 Figures Page 14/17 Figure 1 Flow chart of the study Page 15/17 Figure 1 Flow chart of the study Figure 1 Page 15/17 Figure 2 Measurement of LV global strain and enhanced area in LGE Cardiac magnetic resonance tissue tracking in short-axis and long-axis two- chamber and four-chamber cine images at end-diastole (A1-3) and end-systole (B1-3). LGE images for quantification of infarct size (A4, B4) the signal intensity of yellow region was five standard deviations above the mean intensity of the normal myocardium (bule circle) Figure 2 Measurement of LV global strain and enhanced area in LGE Cardiac magnetic resonance tissue tracking in short-axis and long-axis two- chamber and four-chamber cine images at end-diastole (A1-3) and end-systole (B1-3). LGE images for quantification of infarct size (A4, B4) the signal intensity of yellow region was five standard deviations above the mean intensity of the normal myocardium (bule circle) Figure 3 CMR-LGE images and 3D pseudo-color images of LV circumferential strain in T2DM(MI+) patients. A1-3: T2DM(MI+) patient with LV anterior wall and interventricular septum infarction, female, 58years old, left ventricular short axis (A1), end-diastolic 3D pseudo-color image(A2), end-systolic 3D pseudo-color image(A3). B1-3: T2DM(MI+) patient with LV inferior wall infarction, male, 69years old, left ventricular short axis (B1), end-diastolic 3D pseudo-color image(B2), end-systolic 3D pseudo-color image(B3). Figure 2 Measurement of LV global strain and enhanced area in LGE Cardiac magnetic resonance tissue tracking in short-axis and long-axis two- chamber and four-chamber cine images at end-diastole (A1-3) and end-systole (B1-3). LGE images for quantification of infarct size (A4, B4) the signal intensity of yellow region was five standard deviations above the mean intensity of the normal myocardium (bule circle) Figure 3 Figure 3 CMR-LGE images and 3D pseudo-color images of LV circumferential strain in T2DM(MI+) patients. A1-3: T2DM(MI+) patient with LV anterior wall and interventricular septum infarction, female, 58years old, left ventricular short axis (A1), end-diastolic 3D pseudo-color image(A2), end-systolic 3D pseudo-color image(A3). B1-3: T2DM(MI+) patient with LV inferior wall infarction, male, 69years old, left ventricular short axis (B1), end-diastolic 3D pseudo-color image(B2), end-systolic 3D pseudo-color image(B3). Page 16/17 Page 16/17 Figure 4 Left ventricular global PS in T2DM(MI+) patients with different area of myocardial infarct involved Figure 4 Left ventricular global PS in T2DM(MI+) patients with different area of myocardial infarct involved Figure 4 Left ventricular global PS in T2DM(MI+) patients with different area of myocardial infarct involved Page 17/17
https://openalex.org/W4287775118	https://zenodo.org/records/3859817/files/%D0%AE%D1%85%D0%B8%D0%BC%D0%B5%D0%BD%D0%BA%D0%BE.pdf	Ukrainian	null	Досвід презентації субкультури у Twitter на прикладі k-pop community	Zenodo (CERN European Organization for Nuclear Research)	2,020	cc-by	3,380	Дніпровський національний університет імені Олеся Гончара У статті закцентовано увагу на засобах презентації комунікації за інтересами в Twitter. На прикладі спільноти шанувальників корейської поп-культури описано феномен stan-сегмента Twitter. Використовуючи метод включеного спостереження через власний фан-акаунт, презентовані особливості комунікації k-pop community, схарактеризовані базові чинники її функціонування та поширення. Ключові слова: корейська культура; k-pop community; комунікація в Twitter; фан-спільно Масова комунікація у глобальному та національному вимірах. 2020. Вип. 13 Масова комунікація у глобальному та національному вимірах. 2020. Вип. 13 16. У зоні відчуження затверджено 21 маршрут для відвідувачів. URL: http://dazv.gov.ua/novini-ta- media/vsi-novyny/u-zoni-vidchuzhennya-zatverdzheno-21-marshrut-dlya- vidviduvachiv.html?fbclid=IwAR1TaB3GhijcJX6d4qHorgtHpPxuf6KldwNNfktBVeBO8VAD-qvH_hvh3Qo (дата звернення: 26.04.2020). 17. Поток туристов в Припять вырос после сериала «Чернобыль». URL: https://riafan.ru/1186191-potok- turistov-v-pripyat-vyros-posle-seriala-chernobyl (дата звернення: 26.04.2020). Chornobylskyi A., Kyrylova O. The transformation of the Chernobyl toponym into the precedent phenomenon The study analyzes media coverage of the Chernobyl disaster and related topics as well as their influence on the transformation of the Chernobyl toponym into the precedent name and precedent phenomenon. The article defines the metaphors that were formed behind the toponym as the result of media and social influence. It was found out that 4 played a key role in the process of toponym transformation, which are Soviet media coverage, tourism to the Chernobyl Exclusion Zone, popularity of the computer game S.T.A.L.K.E.R. and HBO “Chernobyl” series. To investigate the transformation of the toponym into the precedent phenomenon in chronological order the historical method was applied. Through the analysis and the comparative method, the characteristic features of media reception were found out. The novelty of the research is in its attempt to describe the way in which the precedent phenomenon appears and transforms due to media and social influence, as well as the way in which the precedent phenomenon is enriched with new metaphors and meanings. The results of the work are important for understanding of mass media impact on communication and development of language means. Keywords: Chernobyl disaster; Chernobyl toponym; precedent name; precedent phenomenon; ‘Chernobyl’ series. DOI: 1. Вступ Постановка проблеми. На сьогодні соціальні мережі активно використовують для міжособистісного спілкування, маркетингу, продажу товарів, просування артистів та Гудошник О. В., кандидат філологічних наук, доцент, електронна адреса: vgudoshnik@gmail.com, Юхименко Я. В., студентка, електронна адреса: yukhymenko_y@fszmk.dnulive.dp.uа, Дніпровський національний університет імені Олеся Гончара, вул. Наукова 13, Дніпро, 49050, Україна 143 Масова комунікація у глобальному та національному вимірах. 2020. Вип. 13 політичних діячів. Зокрема, Twitter використовується як інструмент для політичної комунікації між представниками влади і електоратом, канал швидкого поширення повідомлень про останні світові події та як платформа для обговорення різних сфер приватного життя. Згідно квартальному звіту самої компанії Q1 2019 Earnings Report, мережею користується понад 145 млн унікальних користувачів на день. Умовно простір Twitter самі користувачі поділяють на дві групи: local Twitter (для персонального спілкування) та stan Twitter (усе що пов’язано з хобі та фанатськими спільнотами). Науковці приділяють особливу увагу вивченню політичної комунікації, аналізу персонального іміджу представників влади та дослідженню комунікації в ситуаціях кризового характеру. Проте сегмент так званого приватного та позаполітичного спілкування в Twitter не достатньо вивчений. Зокрема, феномен stan Twitter – окремої великої спільноти користувачів соцмережі, які реалізують у Twitter власні хобі та фан-інтереси. Останнім часом цей напрямок південнокорейської масової культури набув неабиякого розвитку і шаленої популярності за межами країни, став основою особливої субкультури та чисельних клубів фанів, зі своєю комунікацією, стилем, сленгом та поведінковими стандартами. Отже, увагу зосереджено на вивченні особливостей комунікації в середовищі спільнот stan Twitter та шанувальників k-pop. Аналіз останніх досліджень та публікацій. Вплив Twitter на масову комунікацію описує американський соціолог D. Murthy у книзі «Twitter: social communication in the twitter age. (Digital Media and Society)» [1], де на основі досліджень різних країн надає характеристику платформи як інструменту для мікроблогінгу, виокремлює відмінності Twitter від інших соцмереж та висвітлює вплив сервісу на журналістику, громадський активізм, політику та поп-культуру. З активізацією політичної складової мережевої комунікації відбулося суттєве розширення простору наукових досліджень, у яких акцентується безпосередній вплив Twitter на побудову довіри між громадянами та органами влади [2]. Активний моніторинг Twitter сформував цілий напрямок досліджень, що інтерпретують «великі дані», які зібрані у мережі. Разом з тим, не можна не погодитися з учасниками конференції ASONAM '17 про необхідність традиційних експериментальних досліджень як опитування, експеримент, дистанційне спостереження [3]. На цей дискретний рівень аналізу звернули увагу передусім журналісти, адже окремі лайф-сторі і до сьогодні – найзатребуваніша тематика ЗМК. 1. Вступ Мета статті: дослідити особливості реалізації власних інтересів представниками k-pop community та виявити прояви хобі-комунікації між користувачами цієї соцмережі. Методи дослідження. Основою дослідження став метод включеного спостереження – зареєструвавшись в Twitter (було створено власний фан-акаунт), ми мали можливість дистанційно досліджувати внутрішні комунікаційні зв’язки, тематичні преференції учасників спільноти, зсередини спостерігати й оцінювати процеси, які відбуваються у цьому середовищі. Було використано також описовий метод – на прикладі процесів, які відбуваються у k-pop community, та тих подій, що впливають на спільноту, презентовано комунікаційні особливості досліджуваного сегмента соціальної мережі. 1. Вступ Поняття stan Twitter характеризується у зарубіжній пресі як онлайн-феномен, зокрема The Atlantic згадує його як одне із явищ, що існує на рівні з local Twitter, іншим проявом онлайн-простору (йдеться про мережу користувачів Twitter, які існують у межах локальних спільнот — основне коло їх інтересів базується навколо приватного життя та подій, що відбуваються у місцевості, де вони проживають) [4]. Оглядачка сайту Affinity China Tony категорична у характеристиці stan Twitter і називає його хуліганською спільнотою та джерелом формування типажу «садистського фанату» [5]. The Guardian звертається до мовних характеристик контенту у квізі «Did you solve it? Can you speak Twitter?» [6]. Спільнота k-pop з 2010 рр. активно досліджується. Напрямки наукової проблематики – від описового характеру представленості національних версій фандому в Австралії (S. Keith ), Спільнота k-pop з 2010 рр. активно досліджується. Напрямки наукової проблематики – від описового характеру представленості національних версій фандому в Австралії (S. Keith ), Канаді (K. Yoon), Ізраїлі та Палестині (N. Otmazgin, I. Lyan ), Австрії (S.Y. Sung), Сінгапурі (J.P. Williams ), Чехії (V. Mazaná ), Лімі, Перу (S. Ho), на Філіппінах (J Pacis ) до обговорення складних проблем функціонування сучасного молодіжного віртуального суспільства та нових технологій культури. Технологізована k-pop культура виступає як варіант 144 Масова комунікація у глобальному та національному вимірах. 2020. Вип. 13 загальноазійського співтовариства, розмикає кордони, активно співпрацює з державою і впливає на формування музичного мейнстриму настільки агресивно, що авторка Suk-Young Kim промовисто озаглавила своє дослідження «Катастрофічно креативний» [7]. Полікультуралізм фандому та формування транснаціональної ідентичності розглядаються як прояви глобалізації з комунікаційним вектором формування симетричних дискурсів [8]. Вивчаються лінгвістичні особливості, притаманні культурі. Наприклад, поняття Code mixing – змішування двох або більше мов разом [9]. Гібридизація мови досліджуваної спільноти стала формою протесту проти мовного пуристського абсолютизму і викликала дискусію у корейському суспільстві [10]. загальноазійського співтовариства, розмикає кордони, активно співпрацює з державою і впливає на формування музичного мейнстриму настільки агресивно, що авторка Suk-Young Kim промовисто озаглавила своє дослідження «Катастрофічно креативний» [7]. Полікультуралізм фандому та формування транснаціональної ідентичності розглядаються як прояви глобалізації з комунікаційним вектором формування симетричних дискурсів [8]. Вивчаються лінгвістичні особливості, притаманні культурі. Наприклад, поняття Code mixing – змішування двох або більше мов разом [9]. Гібридизація мови досліджуваної спільноти стала формою протесту проти мовного пуристського абсолютизму і викликала дискусію у корейському суспільстві [10]. Об’єкт дослідження – комунікативні особливості фан-спільноти у stan-сегменті Twitter. Об’єкт дослідження – комунікативні особливості фан-спільноти у stan-сегменті Twitter. Мета статті: дослідити особливості реалізації власних інтересів представниками k-pop community та виявити прояви хобі-комунікації між користувачами цієї соцмережі. Масова комунікація у глобальному та національному вимірах. 2020. Вип. 13 Масова комунікація у глобальному та національному вимірах. 2020. Вип. 13 Алгоритми, наприклад, YouTube спрямовані на підтримку інтересу до тематичних відео – зробивши однократний вибір, глядач отримує велику кількість схожих кліпів, а оскільки k- pop спирається на різний контент – одним відео чи кліпом обмежитися важко. Алгоритми, наприклад, YouTube спрямовані на підтримку інтересу до тематичних відео – зробивши однократний вибір, глядач отримує велику кількість схожих кліпів, а оскільки k- pop спирається на різний контент – одним відео чи кліпом обмежитися важко. Twitter допомагає налагодити зв’язок між фанатами та артистами. Проте не всі фанати вступають у офіційні фан-клуби, адже членство в них часто вимагає певних фінансових та інших зобов’язань. З k-pop спільнотою себе ідентифікують шанувальники південнокорейської розважальної індустрії. Отже, люди, які входять до цієї спільноти та шукають однодумців, реєструються в соцмережах, зокрема в Twitter. Зауважимо також, що представники k-pop community теж входять до stan Twitter. Виокремити користувачів, які належать до цієї спільноти, можна завдяки зовнішньому вигляду їх акаунту. На фото профілю – улюблений артист, а замість справжнього імені – нікнейм. Більшість з них не бажають повідомляти свій гендер, але жінок значно більше аніж чоловіків. Загалом це молодь віком від 17 до 25 років, проте є і старші прихильники. Для більшості користувачів приєднання до якогось фандому лише привід познайомитися з однодумцями. Вони ведуть персональні мікроблоги, поєднуючи захоплення корейською хвилею з повсякденним життям. Створюють окремі фан-акаунти, де публікують контент, який стосується тільки фандомного життя, тим самим набираючи аудиторію, з якою надалі можуть взаємодіяти та яка взаємодіє між собою. Іноді персональний акаунт може перетворитися на новинний ресурс. Наприклад, акаунт Twitter @OH_mes2 з аудиторією більше 300 тис. підписників, спершу був персональним, де публікували посилання на корейські новинні сайти і надавалися переклади подій. Тепер це один із головних джерел інформації про корейську розважальну індустрію в Twitter. Люди обирають саме Twitter через те, що тут не потрібно повідомляти справжнього імені і можна розпочати розмову з незнайомою людиною, просто відповівши на її допис. Серед позитивних сторін приєднання до певного фандому користувачі виділяють можливість знайти друзів, інтерес до вивчення іноземних мов і опанування креативних навичок та можливість створювати власний текстовий або візуальний продукт. Одна із основних складових життя будь-якої фанатської спільноти, а зокрема k-pop community – контент, який продукують самі фанати. Це можуть бути арти, фанфікшин або пісенні та денс- кавери на пісні. За допомогою Twitter влаштовуються онлайн-фестивалі фан-творчості. Наприклад, користувач @k-pop_aufest – координатор щорічного фестивалю пісенних текстів, який проводиться на базі Twitter з використанням стороннього ресурсу diary.ru. 2. Результати і обговорення Stan Twitter характеризується приналежністю користувачів до певної фанатської спільноти. Цей феномен бере початок з онлайнового явища stan culture (поєднання слів «stalker» і «fan» вперше використано у пісні Емінема «Stan» 2000 р.). Слово «stan», згідно з онлайн-словником Merriam Webster, означає «бути надзвичайно відданим і захопленим шанувальником когось чи чогось» [11]. Зазвичай, використовується у негативному значенні; так називають занадто захоплених або навіть божевільних фанатів. Виокремити представника stan Twitter можна завдяки їх унікальному сленгу. Наприклад, «ugly» (негарний) використовується не у значенні зовнішності, а як спосіб описати погану поведінку людини. Часто зустрічаються плітки, сварки, тролінг. Ці користувачі зазвичай мають велику аудиторію, публікують суперечливі твіти, щоб спровокувати емоційну реакцію на дописи у своєму або чужому фандомі. Зазвичай, це гумористичний тролінг з мемами, «реакційними» картинками або відео, які використовуються для вираження емоцій та кращого сприйняття іншими. Hallyu або Korean Wave (корейська хвиля) – термін на позначення швидкого розповсюдження корейської культури у всьому світі. Розповсюдження Hallyu почалося у кінці 1990-х рр., і спочатку цей феномен поширювався на інші країни Азії. З розвитком цифрових технологій, зокрема зі створенням соціальних мереж та YouTube, корейська масова культура прийшла і на західний ринок. За підрахунками The Korea Foundation кількість фан-клубів за межами Кореї, що пов’язані з Hallyu, станом на грудень 2018 р. складала 1,843 в 113 країнах світу. Кількість фанатів перевищувала 90 млн (у статистику увійшли лише офіційні фан-клуби) [12]. Індустрія k-pop – це добре продуманий масовий розважальний продукт, який дивує своєю організацією і змушує глибше розбиратися у ньому. Велику роль у просуванні Hallyu і корейської масової культури відіграли соцмережі. 145 Масова комунікація у глобальному та національному вимірах. 2020. Вип. 13 Масова комунікація у глобальному та національному вимірах. 2020. Вип. 13 було походження співачки, яке до того ж супроводжувалося расистськими закликами. Цікаво, що самі корейці спокійно поставилися до появи Лани в розважальній індустрії. Активно представники спільноти реагують і на світові події. Під час пандемії COVID-19 фанати використовують захоплення k-pop як спосіб пережити вимушену самоізоляцію, беруть активну участь у гуманітарних акціях. Наприклад, фанати BTS зібрали більше 300 тис. доларів та передали їх до Korea Disaster Relief Association (The Korea Herald. 03.03.2020). Також створюються фан-проєкти, де збирають благодійні внески як, наприклад, дописувачі One in an Army (@OneinanArmy), які щомісяця передають гроші на благодійність. Серед проектів виокремляються жартівливі 30-секундні ролики під мотиви улюблених пісень з інструкціями як, наприклад, правильно мити руки. Разом з тим, агресивна комунікація широко представлена й у фандомі в цілому, і у сегменті Twitter зокрема. Сваряться як з тими хто не «в темі», так і з іншими фандомами і навіть усередині спільноти. Найчастіше суперечки відбуваються між представниками k-pop спільноти та радикальними феміністками. Останні звинувачують фанатів в асоціальності, ескапізмі, інфантильності, називають їхні спроби створити власний художній продукт «грою в ляльки». Претензії зі сторони представників радикального фемінізму часто однотипні, але вони викликають негативну реакцію зі сторони фанатської спільноти. Іноді реакція фанатів на критику у свою адресу невиправдано агресивна. Так, фанати BTS «Army» відомі надто активною реакцією на будь-яку згадку про групу. Існують навіть жарти про те, що про них потрібно говорити обережно, тому що фанати можуть атакувати тебе, якщо їм щось не сподобається. Наприклад, після сюжету ТСН від 6 вересня 2019 р. «Стрімка популярність K-pop, або чому все більше дітей захоплюються корейською культурою» та сюжету «Сніданку з 1+1» від 19 вересня фанати створити петицію на ресурсі change.org «Петиція проти не- професіональних сюжетів ТСН щодо кей поп індустрії і БТС», де наголошували на непрофесіоналізмі журналістів «1+1». Зокрема, у «Сніданку з 1+1» журналісти переплутали два гурти,: під час розповіді про гурт BTS показували кадри з кліпу GOT7 «Just right». Також, фанати наголошували на однобічності висвітлення теми, зокрема, що це не тільки субкультура підлітків, а весь напрямок не може обмежуватися розповіддю про одну групу. Відомо, що петицію підписала лише 51 людина, але сюжет було видалено. 3. Висновки 3. Висновки Приналежність до певної спільноти має позитивні аспекти: освітні можливості, налагодження соціальних контактів з однодумцями, можливість реалізувати свій творчий потенціал та отримати фідбек. Існують різні форми презентації свого хобі та захоплення. Зокрема, фанатські спільноти, зосереджені навколо масової корейської культури, мету своєї діяльності вбачають в об’єднані людей зі схожими інтересами та можливостями. Учасники також продукують власний контент — текстовий, візуальний або перекладацький. Разом з тим анонімність та відсутність бар’єрів у спілкуванні часто призводить до виникнення конфліктної комунікації, а спільнота, яка за своєю природою об’єднує людей навколо спільного інтересу, часто стає токсичною навіть для своїх представників. 1. Murthy D. Twitter: social communication in the twitter age. Digital Media and Society. Cambridge : Polity Press, 2013. 193 p. Масова комунікація у глобальному та національному вимірах. 2020. Вип. 13 У рамках цієї події користувачі реалізують свій творчий потенціал та знаходять відгук у аудиторії. Опубліковані роботи обговорюються між учасниками протягом усього періоду проведення заходу. Учасники спільноти об’єднуються, незважаючи на мовний бар’єр, національність, расову приналежність, соціальний та фінансовий стан. Проте варто зазначити, що ці характеристики часто стають тригерами конфліктних ситуацій. Наприклад, дебют у Кореї співачки із Росії Лани (@Hicc_LANA) викликав бурю невдоволення серед користувачів з Європи та США. Вони звинувачували артистку в тому, що вона має «привілеї білих» через своє походження та зовнішність. Зокрема, хвилю ненависті викликала участь дівчини в китайському шоу на виживання Produce Camp 2020. Під її дописом у Twitter щодо участі в шоу з’явилися відповіді на кшталт you are WHITE, who let you in. Користувачі в коментарях звинувачували тих, хто підтримує дівчину, а одним із головних аргументів її противників 146 Список використаних джерел DOI: 10.1017/S0047404516000828. 11. Stan. Merriam-Webster. URL: https://www.merriam-webster.com/dictionary/stan (дата звернення: 26.04.2020). 11. Stan. Merriam-Webster. URL: https://www.merriam-webster.com/dictionary/stan (дата звернення: 26.04.2020). 12. Jin D. Y. New Korean Wave: Transnational Cultural Power in the Age of Social Media. University of Illinois Press, 2016. URL: www.jstor.org/stable/10.5406/j.ctt18j8wkv (дата звернення: 26.04.2020). 12. Jin D. Y. New Korean Wave: Transnational Cultural Power in the Age of Social Media. University of Illinois Press, 2016. URL: www.jstor.org/stable/10.5406/j.ctt18j8wkv (дата звернення: 26.04.2020). eywords: Korean culture; k-pop community; Twitter communication; fan community; hobby. 3. Falzon L., McCurrie C., Dunn J. Representation and Analysis of Twitter Activity: A Dynamic Network Perspective. ASONAM '17: Proceedings of the 2017 IEEE/ACM International Conference on Advances in Social Networks Analysis and Mining. Sydney, Australia, 2017. P. 1183–1190. DOI: 10.1145/3110025.3122118. 2. Park M. J., Kang D., Rho J. J. & Lee D. H. Policy Role of Social Media in Developing Public Trust: Twitter communication with government leaders. Public Management Review. 2016. Vol. 18. Issue 9. P. 1265–1288. DOI: 10.1080/14719037.2015.1066418. Список використаних джерел 1. Murthy D. Twitter: social communication in the twitter age. Digital Media and Society. Cambridge : Polity Press, 2013. 193 p. 147 Масова комунікація у глобальному та національному вимірах. 2020. Вип. 13 2. Park M. J., Kang D., Rho J. J. & Lee D. H. Policy Role of Social Media in Developing Public Trust: Twitter communication with government leaders. Public Management Review. 2016. Vol. 18. Issue 9. P. 1265–1288. DOI: 10.1080/14719037.2015.1066418. 3. Falzon L., McCurrie C., Dunn J. Representation and Analysis of Twitter Activity: A Dynamic Network Perspective. ASONAM '17: Proceedings of the 2017 IEEE/ACM International Conference on Advances in Social Networks Analysis and Mining. Sydney, Australia, 2017. P. 1183–1190. DOI: 10.1145/3110025.3122118. 4. How Twitter Became Home to the Teen Status Update. The Atlantic. July 3, 2018. URL: https://www.theatlantic.com/technology/archive/2018/07/how-twitter-became-home-to-the-teen-status- update/564404/ (дата звернення: 27.04.2020). 4. How Twitter Became Home to the Teen Status Update. The Atlantic. July 3, 2018. URL: https://www.theatlantic.com/technology/archive/2018/07/how-twitter-became-home-to-the-teen-status- update/564404/ (дата звернення: 27.04.2020). 5. How Stan Twitter is Turning Into a Community of Bullies. URL: http://affinitymagazine.us/ 2016/12/29/how-stan-twitter-is-turning-into-a-community-of-bullies/(дата звернення: 26.04.2020). 5. How Stan Twitter is Turning Into a Community of Bullies. URL: http://affinitymagazine.us/ 2016/12/29/how-stan-twitter-is-turning-into-a-community-of-bullies/(дата звернення: 26.04.2020). 6. Did you solve it? Can you speak Twitter? URL: https://www.theguardian.com/science/2018/dec/17/did- you-solve-it-can-you-speak-twitter (дата звернення: 26.04.2020). 6. Did you solve it? Can you speak Twitter? URL: https://www.theguardian.com/science/2018/dec/17/did- you-solve-it-can-you-speak-twitter (дата звернення: 26.04.2020). 7. Kim S.-Y. Disastrously Creative: K-pop, Virtual Nation, and the Rebirth of Culture Technology. TDR / The Drama Review. 2020. Vol. 64. Issue 1. Р. 22–35. DOI: https://doi.org/10.1162/dram_a_00894. 7. Kim S.-Y. Disastrously Creative: K-pop, Virtual Nation, and the Rebirth of Culture Technology. TDR / The Drama Review. 2020. Vol. 64. Issue 1. Р. 22–35. DOI: https://doi.org/10.1162/dram_a_00894. 8. Kwon E.J. Korean Wave: Discourse Analysis on Korean Popular Culture in US and UK Digital Newspapers. MA Thesis Arts and Culture. DOI: https://theses.ubn.ru.nl/handle/123456789/4898. 8. Kwon E.J. Korean Wave: Discourse Analysis on Korean Popular Culture in US and UK Digital Newspapers. MA Thesis Arts and Culture. DOI: https://theses.ubn.ru.nl/handle/123456789/4898. 9. Dwiyota S. The use of code mixing in tweets by kpop fans in Twitter. Lingua Litera. 2018. Vol.3. Issue 1. URL: https://www.semanticscholar.org/paper/THE-USE-OF-CODE-MIXING-IN-TWEETS-BY-KPOP-FANS-IN-Dwiyota/ 63ebd9ed17895fc583af57a2555144c624984417(дата звернення: 26.04.2020). 9. Dwiyota S. The use of code mixing in tweets by kpop fans in Twitter. Lingua Litera. 2018. Vol.3. Issue 1. URL: https://www.semanticscholar.org/paper/THE-USE-OF-CODE-MIXING-IN-TWEETS-BY-KPOP-FANS-IN-Dwiyota/ 63ebd9ed17895fc583af57a2555144c624984417(дата звернення: 26.04.2020). ( р ) 10. Chun E. How to drop a name: Hybridity, purity, and the K-pop fan. Language in Society. 2017. Vol. 46. Issue 1. Р. 57–76. Yukhymenko Y., Hudoshnyk O. Experience of subculture presentation on Twitter on the example of the k-pop community This article describes the ways of presenting leisure activity though communication on the example of Twitter social network. The community of the Korean pop music fans and their ways of communication with each other were chosen as the object of the study. With the help of the data collected from Twitter, it became possible to describe the phenomenon of stan twitter and the Korean pop music community that exist within this area. The research was based on the experimental method, which means that the author of the article created the personal fan account on Twitter to remotely explore internal communications links and thematic preferences of the community members. This procedure has contributed to the evaluation of the process that takes place in this community. In addition, a descriptive method was applied to characterize the communication features of this association. These actions promoted understanding of not only how the processes in this community effect their own members, but also those who are not a part of this phenomenon. The research showed that belonging to a particular community has numerous benefits; one of them is educational opportunities or establishing social contacts with people of similar interests. There are various forms of presentation of your leisure activity by producing your own content and receiving feedback from others. In particular, the point of activity within this examined community is attempt to unite the members for a specific purpose. It could be promotion of a favourite artist, as well as charitable activities. However, anonymity and lack of barriers to connect frequently lead to conflict communication. Furthermore, communities based on the common interests often show detrimental effects on their representatives. The scientific novelty of the research is that it is an attempt to study the processes that take place in non-political parts of the Twitter segment community. This article helps to understand why the examined community appears and how it is affected by their participants or in the world in general. Keywords: Korean culture; k-pop community; Twitter communication; fan community; hobby. 148
https://openalex.org/W4232908846	https://europepmc.org/articles/pmc2983359?pdf=render	Latin	null	Ranunculin	Acta crystallographica. Section E	2,010	cc-by	3,595	Related literature For background to ranunculin, see: Hill & van Heyningen (1951); Bai et al. (1996); Benn & Yelland (1968); Boll (1968); Camps et al. (1982); Fang et al. (1989). For chemical and spectrometric data for closely related, simple butenolides, see: Perry et al. (1996). For comparison bond distances, see: Allen et al. (1987). For puckering parameters, see: Cremer & Pople (1975). Supplementary data and ﬁgures for this paper are available from the IUCr electronic archives (Reference: FL2315). Received 17 August 2010; accepted 29 August 2010 Table 1 Hydrogen-bond geometry (A˚ , ). Table 1 Hydrogen-bond geometry (A˚ , ). D—H A D—H H A D A D—H A O5—H5O O8i 0.86 (3) 2.17 (3) 2.910 (2) 144 (3) O6—H6O O5ii 0.92 (3) 1.94 (3) 2.824 (2) 162 (3) O7—H7O O6ii 0.84 (3) 1.84 (3) 2.668 (2) 173 (3) O8—H8O O2iii 0.88 (3) 1.96 (3) 2.830 (2) 167 (3) O6—H6O O7 0.92 (3) 2.56 (3) 2.894 (2) 102 (2) Symmetry codes: (i) x 1; y 1; z; (ii) x; y þ 1 2; z þ 1; (iii) x þ 2; y þ 1 2; z þ 2. Key indicators: single-crystal X-ray study; T = 173 K; mean (C–C) = 0.003 A˚; R factor = 0.026; wR factor = 0.069; data-to-parameter ratio = 6.2. Key indicators: single-crystal X-ray study; T = 173 K; mean (C–C) = 0.003 A˚; R factor = 0.026; wR factor = 0.069; data-to-parameter ratio = 6.2. In the title molecule {systematic name: (5S)-5-[(-d-gluco- pyranosyloxy)methyl]furan-2(5H)-one}, C11H16O8, the ﬁve- membered ring is essentially planar, the maximum deviation being 0.0151 (13) A˚ for the O atom. The six-membered ring adopts a chair conformation with puckering parameters Q = 0.581 (2) A˚ , = 9.0 (2) and ’ = 39.7 (13), and with all of the substituents of the glucoside unit having normal equatorial orientations. The crystal structure is stabilized by extensive O—H O and C—H O hydrogen bonding, resulting in a three-dimensional network. In the title molecule {systematic name: (5S)-5-[(-d-gluco- pyranosyloxy)methyl]furan-2(5H)-one}, C11H16O8, the ﬁve- membered ring is essentially planar, the maximum deviation being 0.0151 (13) A˚ for the O atom. The six-membered ring adopts a chair conformation with puckering parameters Q = 0.581 (2) A˚ , = 9.0 (2) and ’ = 39.7 (13), and with all of the substituents of the glucoside unit having normal equatorial orientations. The crystal structure is stabilized by extensive O—H O and C—H O hydrogen bonding, resulting in a three-dimensional network. Data collection: COLLECT (Hooft, 1998); cell reﬁnement: DENZO (Otwinowski & Minor, 1997); data reduction: SCALE- PACK (Otwinowski & Minor, 1997); program(s) used to solve structure: SHELXS97 (Sheldrick, 2008); program(s) used to reﬁne structure: SHELXL97 (Sheldrick, 2008); molecular graphics: ORTEP-3 for Windows (Farrugia, 1997); software used to prepare material for publication: SHELXL97. organic compounds Acta Crystallographica Section E Structure Reports Online ISSN 1600-5368 Acta Crystallographica Section E Structure Reports Online ISSN 1600-5368 Data collection Nonius KappaCCD diffractometer with APEXII CCD Absorption correction: multi-scan (SORTAV; Blessing, 1997) Tmin = 0.961, Tmax = 0.997 1926 measured reﬂections 1133 independent reﬂections 1112 reﬂections with I > 2(I) Rint = 0.018 References Allen, F. H., Kennard, O., Watson, D. G., Brammer, L., Orpen, A. G. & Taylor, R. (1987). J. Chem. Soc. Perkin Trans. 2, pp. S1–19. ( ) Bai, Y., Benn, M. H., Majak, W. & McDiarmid, R. (1996). J. Agric. Food Chem. 44, 2235–2238. Experimental Crystal data C11H16O8 Mr = 276.24 Monoclinic, P21 a = 5.7944 (4) A˚ b = 6.9359 (3) A˚ c = 15.0491 (10) A˚ = 97.895 (2) V = 599.08 (6) A˚ 3 Z = 2 Mo K radiation = 0.13 mm1 T = 173 K 0.30 0.24 0.02 mm Benn, M. H. & Yelland, L. J. (1968). Can. J. Chem. 46, 729–732. Blessing, R. H. (1997). J. Appl. Cryst. 30, 421–426. Boll, P. M. (1968). Acta Chem. Scand. 22, 3245–3250. Camps, P., Cardellach, J., Font, J., Ortuno, R. M. & Ponsati, O. (1982). Tetrahedron, 38, 2395–2402. remer, D. & Pople, J. A. (1975). J. Am. Chem. Soc. 97, 1354–1 Cremer, D. & Pople, J. A. (1975). J. Am. Chem. Soc. 97, 1354–1358. Fang, Z., Zhou, J. & Huang, L. (1989). Yaoxue Xuebao, 24, 182–188. p ( ) Fang, Z., Zhou, J. & Huang, L. (1989). Yaoxue Xuebao, 24, 182–188. Farrugia, L. J. (1997). J. Appl. Cryst. 30, 565. Hill, R. & van Heyningen, R. (1951). Biochem. J. 49, 332–335 Ranunculin H atoms treated by a mixture of independent and constrained reﬁnement max = 0.18 e A˚ 3 min = 0.16 e A˚ 3 Michael Benn, Lois Jean Yelland and Masood Parvez* Department of Chemistry, The University of Calgary, 2500 University Drive NW, Calgary, Alberta, Canada T2N 1N4 Correspondence e-mail: parvez@ucalgary.ca Received 17 August 2010; accepted 29 August 2010 Received 17 August 2010; accepted 29 August 2010 Experimental y g ( ) Hooft, R. (1998). COLLECT. Nonius BV, Delft, The Netherlands. Otwinowski, Z. & Minor, W. (1997). Methods in Enzymology, Vol. 276, Macromolecular Crystallography Part A edited by C W Carter Jr & R M Otwinowski, Z. & Minor, W. (1997). Methods in Enzymology, Vol. 276, Macromolecular Crystallography, Part A, edited by C. W. Carter Jr & R. M. Sweet, pp. 307–326. New York: Academic Press. Otwinowski, Z. & Minor, W. (1997). Methods in Enzym Macromolecular Crystallography, Part A, edited by C. W. Sweet, pp. 307–326. New York: Academic Press. Macromolecular Crystallography, Part A, edited by C. W. Carter Jr & R. M. Sweet, pp. 307–326. New York: Academic Press. V = 599.08 (6) A˚ 3 Z = 2 Mo K radiation = 0.13 mm1 T = 173 K 0.30 0.24 0.02 mm y g y Sweet, pp. 307–326. New York: Academic Press. Perry, N. B., Benn, M. H., Forster, L. M., Routledge, A. & Weavers, R. T. (1996). Phytochemistry, 42, 453–459. y (1996). Phytochemistry, 42, 453–459. Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122. Sheldrick, G. M. (2008). Acta Cryst. A64 Benn et al. o2503 Acta Cryst. (2010). E66, o2503 doi:10.1107/S1600536810034847 Experimental The details of the isolation, and some of the physical properties, of our sample of (I) have been reported previously (Benn & Yelland 1968). Suitable crystals of the title compound for X-ray study were grown from a solution in aqueous ethanol (ca 1:20) in the form of plates. Ranunculin M. Benn, L. J. Yelland and M. Parvez M. Benn, L. J. Yelland and M. Parvez Acta Cryst. (2010). E66, o2503 [ doi:10.1107/S1600536810034847 ] Acta Cryst. (2010). E66, o2503 [ doi:10.1107/S1600536810034847 ] Comment Ranunculin (I) is the glycosidic precursor of the vesicant protoanemonin present in numerous species of Rannunculaceae and is especially associated with the burning sensation on chewing leaves of buttercup plants. It was first obtained in crystalline form by Hill and van Heyningen (1951) who established its gross structure and showed that it undergoes enzymatic cleavage by β-glucosidase to yield the aglucone, which underwent easy dehydration to protoanemonin. These processes were shown to occur readily under autolytic conditions (Bai et al., 1996). The S-stereochemistry of the dihydrofuranone ring was deduced by Benn and Yelland (1968), and Boll (1968), as shown in the schematic diagram, and later confirmed by synthesis (Camps et al., 1982; Fang et al., 1989). Our sample of (I) was a natural product, and as such had been biosynthesised in the plant (and not made in a laboratory). The only stereoisomer which occurs naturally is the D-isomer, and both that and the anomeric configuration of the glycoosidic bond in (I) were established by cleavage of the glycoside by β-D-glucosidases. The X-ray structure reported here provides the simplest unequivocal proof of that stereochemistry since the chirality follows from that of the D-glucopyranosyl moiety. The molecular structure of (I) is presented in Fig. 1. The five membered ring, O1/C2—C5, is essentially planar with the maximum deviation of any atom being 0.0151 (13) Å for O1. The six-membered ring adopts a chair conformation with puckering parameters (Cremer & Pople, 1975): Q = 0.581 (2) Å, θ = 9.0 (2)° and φ = 39.7 (13)°, with all of the substituents of the glucoside unit having normal equatorial orientations. The 5-membered ring folds back away from the six-membered ring as reflected by the torsion angle C6—O3—C1—C5 -162.86 (17)°. The bond distances and angles are as expected (Allen, 2002). The structure is stabilized by extensive O—H···O and C—H···O hydrogen bonding resulting in a three diemsional network (Table 1 & Fig. 2). There are two other, closely related, simple butenolides known, though their structures rest on chemical and spectro- metric data, i.e., without X-ray crystallographic support. They are the (5R,6R) and (5S,6R) steeroisomers of 5-([1-β-D- glucopyranosyloxy]ethyl)-2(5H)-furanone (Perry et al., 1996); the glycosidic precursor of (Z)-5-ethylidene-2(5H)-furanone, a homologue of protoanemonin in Halocarpus biformis juvenile foliage. supplementary materials supplementary materials Refinement An absolute structure could not be established reliably because of insufficient anomalous scattering effects. Therefore, 792 Friedel pairs were merged. The H-atoms were located from difference maps and were included in the refinements at geometrically idealized positions with C—H distances = 0.95, 0.99 and 1.00 Å for aryl, methylene and methine type H-atoms, sup-1 supplementary materials supplementary materials respectively; the positions of hydroxyl H-atoms were allowed to refine freely. The H-atoms were assigned Uiso = 1.2 and 1.5 × Ueq of the parent C and O-atoms, respectively. The final difference map was free of chemically significant features. Figures Fig. 1. ORTEP-3 (Farrugia, 1997) drawing of the title compound with displacement ellipsoid plotted at 50% probability level. Fig. 2. Unit cell packing of the title compound showing intermolecular hydrogen bonds of O—H···O; H-atoms not involved in hydrogen bonds have been excluded. (5S)-5-[(β-D-glucopyranosyloxy)methyl]furan-2(5H)-one Figures Figures Fig. 1. ORTEP-3 (Farrugia, 1997) drawing of the title compound with displacement ellipsoids plotted at 50% probability level. F O Fig. 2. Unit cell packing of the title compound showing intermolecular hydrogen bonds of O—H···O; H-atoms not involved in hydrogen bonds have been excluded. Fig. 2. Unit cell packing of the title compound showing intermolecular hydrogen bonds of O—H···O; H-atoms not involved in hydrogen bonds have been excluded. (5S)-5-[(β-D-glucopyranosyloxy)methyl]furan-2(5H)-one sup-2 supplementary materials φ & ω scans θmax = 25.0°, θmin = 3.2° Absorption correction: multi-scan (SORTAV; Blessing, 1997) h = −6→6 Tmin = 0.961, Tmax = 0.997 k = −6→8 1926 measured reflections l = −17→17 θmax = 25.0°, θmin = 3.2° θmax = 25.0°, θmin = 3.2° h = −6→6 k = −6→8 l = −17→17 Absorption correction: multi-scan (SORTAV; Blessing, 1997) Tmin = 0.961, Tmax = 0.997 1926 measured reflections Primary atom site location: structure-invariant direct methods Secondary atom site location: difference Fourier map Hydrogen site location: difference Fourier map H atoms treated by a mixture of independent and constrained refinement w = 1/[σ2(Fo 2) + (0.040P)2 + 0.1617P] where P = (Fo 2 + 2Fc 2)/3 (Δ/σ)max = 0.002 Δρmax = 0.18 e Å−3 Δρmin = −0.16 e Å−3 w = 1/[σ2(Fo 2) + (0.040P)2 + 0.1617P] where P = (Fo 2 + 2Fc 2)/3 (Δ/σ)max = 0.002 Δρmax = 0.18 e Å−3 Δρmin = −0.16 e Å−3 Special details Experimental. NMR data (400 MHz, 1H; 100 MHz 13C) for a solution in D2O containing sodium 3-trimethylsilylpropionate-2,3 - d4 as reference: δH (400 MHz) 7.77 (1H, dd, J = 1.5 and 5.8 Hz, H-4), 6.3 (1H, dd, J = 2.1 and 5.8 Hz, H-3), 5.47 (1H, m), 4.48 (1H, d, J = 7.9 Hz, H-1'), 4.30 (1H, dd, J = 3.2 and 12.2 Hz, H-6 A), 3.95 (1H, dd, J = 5.8 and 12.2 Hz, H-6B), 3.91 (1H, dd, J = 2.1 and 12.5 Hz, H-6A'), 3.72 (1H, dd, J = 5.8 and 12.5 Hz, H-6B'), 3.48 (1H, dd, dd, J = ca 9 Hz H-3'), 3.43 (1H, m, H-5'), 3.37 (1H, dd, J = ca 9 Hz, H=4'), and 3.25 (1H, dd, J = 7.9 and 9.2 Hz, H-2'); δC 179.2 s (C-2), 158.5 d (C-4), 124.7 d (C-3), 105.6 d (C-1'), 86.9 d (C-5), 78.7 d (C-5'), 78.3 d (C-3'), 75.6 d (C-2'), 72.2 d (C-4'), 71.7 t (C-6), 63.3 t (C-6'). Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes) are estimated using the full covariance mat- rix. The cell e.s.d.'s are taken into account individually in the estimation of e.s.d.'s in distances, angles and torsion angles; correlations between e.s.d.'s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s. planes. Refinement. Refinement of F2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F2, convention- al R-factors R are based on F, with F set to zero for negative F2. The threshold expression of F2 > σ(F2) is used only for calculating R- factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F2 are statistically about twice as large as those based on F, and R- factors based on ALL data will be even larger. Refinement. Refinement of F2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F2, convention- al R-factors R are based on F, with F set to zero for negative F2. The threshold expression of F2 > σ(F2) is used only for calculating R- factors(gt) etc. and is not relevant to the choice of reflections for refinement. supplementary materials supplementary materials O6 −0.0241 (2) 0.2692 (2) 0.53178 (10) 0.0208 (4) H6O −0.025 (5) 0.336 (5) 0.479 (2) 0.031* O7 0.3667 (3) 0.5126 (3) 0.50561 (9) 0.0215 (3) H7O 0.259 (5) 0.591 (5) 0.4895 (18) 0.032* O8 0.8805 (3) 0.6376 (3) 0.72096 (10) 0.0277 (4) H8O 0.893 (5) 0.563 (5) 0.769 (2) 0.042* C1 0.6798 (3) 0.0332 (4) 0.84401 (13) 0.0216 (5) H1A 0.7191 0.1601 0.8721 0.026* H1B 0.7947 0.0036 0.8030 0.026* C2 0.8918 (4) −0.0724 (3) 1.05518 (13) 0.0225 (5) C3 0.6434 (4) −0.0666 (4) 1.06406 (14) 0.0252 (5) H3 0.5794 −0.0486 1.1183 0.030* C4 0.5229 (4) −0.0912 (3) 0.98301 (14) 0.0243 (5) H4 0.3578 −0.0895 0.9699 0.029* C5 0.6862 (4) −0.1216 (3) 0.91570 (13) 0.0204 (5) H5 0.6609 −0.2517 0.8874 0.025* C6 0.4469 (4) 0.1343 (3) 0.71336 (13) 0.0178 (4) H6 0.5552 0.0694 0.6765 0.021* C7 0.2004 (3) 0.1338 (3) 0.66293 (13) 0.0176 (4) H7 0.0906 0.1980 0.6994 0.021* C8 0.2093 (3) 0.2433 (3) 0.57590 (13) 0.0167 (4) H8 0.2985 0.1652 0.5363 0.020* C9 0.3238 (3) 0.4413 (3) 0.59065 (13) 0.0172 (4) H9 0.2164 0.5310 0.6170 0.021* C10 0.5559 (3) 0.4305 (3) 0.65204 (13) 0.0180 (4) H10 0.6738 0.3622 0.6208 0.022* C11 0.6423 (4) 0.6302 (3) 0.67948 (14) 0.0218 (5) H11A 0.6248 0.7137 0.6256 0.026* H11B 0.5424 0.6840 0.7217 0.026* Atomic displacement parameters (Å2) U11 U22 U33 U12 U13 U23 O1 0.0203 (7) 0.0294 (9) 0.0169 (7) 0.0038 (6) −0.0002 (5) 0.0024 (6) O2 0.0327 (8) 0.0329 (10) 0.0233 (8) 0.0036 (8) −0.0095 (7) −0.0014 (8) O3 0.0212 (7) 0.0242 (8) 0.0137 (7) −0.0011 (7) −0.0005 (5) 0.0038 (6) O4 0.0226 (7) 0.0190 (8) 0.0143 (7) −0.0026 (6) 0.0005 (5) −0.0001 (6) O5 0.0317 (8) 0.0223 (8) 0.0172 (7) −0.0077 (7) −0.0007 (6) 0.0020 (7) O6 0.0177 (7) 0.0259 (8) 0.0174 (7) −0.0007 (6) −0.0027 (6) 0.0030 (7) O7 0.0218 (7) 0.0257 (8) 0.0172 (7) 0.0016 (7) 0.0036 (6) 0.0063 (7) O8 0.0262 (8) 0.0330 (9) 0.0227 (8) −0.0102 (8) −0.0010 (6) 0.0034 (8) C1 0.0200 (10) 0.0262 (12) 0.0175 (10) −0.0020 (10) −0.0010 (8) 0.0041 (9) C2 0.0302 (11) 0.0188 (10) 0.0175 (9) 0.0063 (10) 0.0000 (9) 0.0026 (9) C3 0.0306 (11) 0.0257 (11) 0.0201 (10) 0.0037 (10) 0.0064 (8) 0.0025 (10) C4 0.0235 (10) 0.0234 (12) 0.0264 (12) 0.0004 (10) 0.0044 (9) 0.0071 (10) C5 0.0217 (10) 0.0214 (11) 0.0168 (10) −0.0001 (9) −0.0023 (8) 0.0016 (9) C6 0.0216 (10) 0.0179 (10) 0.0138 (9) −0.0003 (9) 0.0020 (7) 0.0016 (9) C7 0.0190 (10) 0.0188 (10) 0.0151 (9) −0.0014 (9) 0.0025 (7) 0.0003 (9) Special details R-factors based on F2 are statistically about twice as large as those based on F, and R- factors based on ALL data will be even larger. Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) x y z Uiso/Ueq O1 0.9146 (3) −0.1112 (2) 0.96815 (9) 0.0225 (4) O2 1.0618 (3) −0.0493 (3) 1.11025 (10) 0.0309 (4) O3 0.4499 (2) 0.0377 (2) 0.79524 (9) 0.0200 (3) O4 0.5209 (2) 0.3269 (2) 0.73224 (9) 0.0188 (3) O5 0.1227 (3) −0.0568 (2) 0.63887 (10) 0.0241 (4) H5O 0.085 (5) −0.121 (5) 0.6833 (19) 0.036 Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) sup-3 supplementary materials Atomic displacement parameters (Å2) sup-4 supplementary materials supplementary materials supplementary mat C8 0.0152 (10) 0.0207 (10) 0.0135 (9) 0.0003 (9) −0.0001 (8) −0.0008 C9 0.0185 (10) 0.0191 (10) 0.0143 (9) 0.0010 (9) 0.0038 (7) 0.0010 C10 0.0180 (10) 0.0216 (10) 0.0147 (9) −0.0004 (10) 0.0038 (8) 0.0017 C11 0.0253 (11) 0.0206 (11) 0.0189 (10) −0.0016 (9) 0.0004 (8) 0.0006 Geometric parameters (Å, °) O1—C2 1.361 (2) C2—C3 1.464 (3) O1—C5 1.447 (2) C3—C4 1.331 (3) O2—C2 1.208 (3) C3—H3 0.9500 O3—C6 1.401 (2) C4—C5 1.493 (3) O3—C1 1.430 (2) C4—H4 0.9500 O4—C6 1.419 (3) C5—H5 1.0000 O4—C10 1.443 (2) C6—C7 1.523 (3) O5—C7 1.427 (3) C6—H6 1.0000 O5—H5O 0.86 (3) C7—C8 1.521 (3) O6—C8 1.434 (2) C7—H7 1.0000 O6—H6O 0.92 (3) C8—C9 1.528 (3) O7—C9 1.425 (2) C8—H8 1.0000 O7—H7O 0.84 (3) C9—C10 1.525 (2) O8—C11 1.435 (3) C9—H9 1.0000 O8—H8O 0.88 (3) C10—C11 1.511 (3) C1—C5 1.519 (3) C10—H10 1.0000 C1—H1A 0.9900 C11—H11A 0.9900 C1—H1B 0.9900 C11—H11B 0.9900 C2—O1—C5 109.40 (15) O4—C6—H6 109.9 C6—O3—C1 111.09 (15) C7—C6—H6 109.9 C6—O4—C10 112.00 (15) O5—C7—C8 106.92 (16) C7—O5—H5O 113 (2) O5—C7—C6 111.72 (17) C8—O6—H6O 110.7 (18) C8—C7—C6 106.69 (16) C9—O7—H7O 105.7 (19) O5—C7—H7 110.5 C11—O8—H8O 107 (2) C8—C7—H7 110.5 O3—C1—C5 108.08 (17) C6—C7—H7 110.5 O3—C1—H1A 110.1 O6—C8—C7 108.63 (16) C5—C1—H1A 110.1 O6—C8—C9 108.56 (17) O3—C1—H1B 110.1 C7—C8—C9 112.90 (16) C5—C1—H1B 110.1 O6—C8—H8 108.9 H1A—C1—H1B 108.4 C7—C8—H8 108.9 O2—C2—O1 120.56 (19) C9—C8—H8 108.9 O2—C2—C3 130.8 (2) O7—C9—C10 108.24 (15) O1—C2—C3 108.66 (17) O7—C9—C8 107.89 (16) C4—C3—C2 108.1 (2) C10—C9—C8 111.93 (17) C4—C3—H3 125.9 O7—C9—H9 109.6 C2—C3—H3 125.9 C10—C9—H9 109.6 C3—C4—C5 109.8 (2) C8—C9—H9 109.6 C3—C4—H4 125.1 O4—C10—C11 107.91 (16) C5—C4—H4 125.1 O4—C10—C9 108.49 (15) O1—C5—C4 103.91 (16) C11—C10—C9 110.63 (17) O1—C5—C1 106.48 (16) O4—C10—H10 109.9 sup-5 supplementary materials supplementary materials supplementary materials C4—C5—C1 115.19 (19) C11—C10—H10 109.9 O1—C5—H5 110.3 C9—C10—H10 109.9 C4—C5—H5 110.3 O8—C11—C10 114.47 (18) C1—C5—H5 110.3 O8—C11—H11A 108.6 O3—C6—O4 107.90 (15) C10—C11—H11A 108.6 O3—C6—C7 109.54 (16) O8—C11—H11B 108.6 O4—C6—C7 109.80 (16) C10—C11—H11B 108.6 O3—C6—H6 109.9 H11A—C11—H11B 107.6 C6—O3—C1—C5 −162.86 (17) O3—C6—C7—C8 179.11 (17) C5—O1—C2—O2 −176.8 (2) O4—C6—C7—C8 60.8 (2) C5—O1—C2—C3 3.2 (2) O5—C7—C8—O6 68.0 (2) O2—C2—C3—C4 176.8 (3) C6—C7—C8—O6 −172.30 (16) O1—C2—C3—C4 −3.3 (3) O5—C7—C8—C9 −171.50 (16) C2—C3—C4—C5 2.0 (3) C6—C7—C8—C9 −51.8 (2) C2—O1—C5—C4 −2.0 (2) O6—C8—C9—O7 −71.42 (19) C2—O1—C5—C1 120.06 (19) C7—C8—C9—O7 168.07 (16) C3—C4—C5—O1 −0.1 (3) O6—C8—C9—C10 169.60 (14) C3—C4—C5—C1 −116.2 (2) C7—C8—C9—C10 49.1 (2) O3—C1—C5—O1 −175.41 (16) C6—O4—C10—C11 −177.97 (16) O3—C1—C5—C4 −60.8 (2) C6—O4—C10—C9 62.1 (2) C1—O3—C6—O4 −61.2 (2) O7—C9—C10—O4 −169.84 (18) C1—O3—C6—C7 179.26 (17) C8—C9—C10—O4 −51.1 (2) C10—O4—C6—O3 171.64 (15) O7—C9—C10—C11 72.0 (2) C10—O4—C6—C7 −69.02 (19) C8—C9—C10—C11 −169.25 (17) O3—C6—C7—O5 −64.4 (2) O4—C10—C11—O8 74.2 (2) O4—C6—C7—O5 177.31 (17) C9—C10—C11—O8 −167.27 (16) Hydrogen-bond geometry (Å, °) D—H···A D—H H···A D···A D—H···A O5—H5O···O8i 0.86 (3) 2.17 (3) 2.910 (2) 144 (3) O6—H6O···O5ii 0.92 (3) 1.94 (3) 2.824 (2) 162 (3) O7—H7O···O6ii 0.84 (3) 1.84 (3) 2.668 (2) 173 (3) O8—H8O···O2iii 0.88 (3) 1.96 (3) 2.830 (2) 167 (3) C3—H3···O4iv 0.95 2.55 3.413 (3) 151 C4—H4···O1v 0.95 2.57 3.504 (3) 168 C8—H8···O7vi 1.00 2.37 3.306 (3) 155 C1—H1A···O2iii 0.99 2.38 3.289 (3) 153 C10—H10···O6vii 1.00 2.43 3.415 (3) 167 O6—H6O···O7 0.92 (3) 2.56 (3) 2.894 (2) 102 (2) C11—H11A···O7 0.99 2.59 2.986 (3) 104 Symmetry codes: (i) x−1, y−1, z; (ii) −x, y+1/2, −z+1; (iii) −x+2, y+1/2, −z+2; (iv) −x+1, y−1/2, −z+2; (v) x−1, y, z; (vi) −x+1, y−1/2, −z+1; (vii) x+1, y, z. supplementary materials C4—C5—C1 115.19 (19) C11—C10—H10 109.9 O1—C5—H5 110.3 C9—C10—H10 109.9 C4—C5—H5 110.3 O8—C11—C10 114.47 (18) C1—C5—H5 110.3 O8—C11—H11A 108.6 O3—C6—O4 107.90 (15) C10—C11—H11A 108.6 O3—C6—C7 109.54 (16) O8—C11—H11B 108.6 O4—C6—C7 109.80 (16) C10—C11—H11B 108.6 O3—C6—H6 109.9 H11A—C11—H11B 107.6 C6—O3—C1—C5 −162.86 (17) O3—C6—C7—C8 179.11 (17) C5—O1—C2—O2 −176.8 (2) O4—C6—C7—C8 60.8 (2) C5—O1—C2—C3 3.2 (2) O5—C7—C8—O6 68.0 (2) O2—C2—C3—C4 176.8 (3) C6—C7—C8—O6 −172.30 (16) O1—C2—C3—C4 −3.3 (3) O5—C7—C8—C9 −171.50 (16) C2—C3—C4—C5 2.0 (3) C6—C7—C8—C9 −51.8 (2) C2—O1—C5—C4 −2.0 (2) O6—C8—C9—O7 −71.42 (19) C2—O1—C5—C1 120.06 (19) C7—C8—C9—O7 168.07 (16) C3—C4—C5—O1 −0.1 (3) O6—C8—C9—C10 169.60 (14) C3—C4—C5—C1 −116.2 (2) C7—C8—C9—C10 49.1 (2) O3—C1—C5—O1 −175.41 (16) C6—O4—C10—C11 −177.97 (16) O3—C1—C5—C4 −60.8 (2) C6—O4—C10—C9 62.1 (2) C1—O3—C6—O4 −61.2 (2) O7—C9—C10—O4 −169.84 (18) C1—O3—C6—C7 179.26 (17) C8—C9—C10—O4 −51.1 (2) C10—O4—C6—O3 171.64 (15) O7—C9—C10—C11 72.0 (2) C10—O4—C6—C7 −69.02 (19) C8—C9—C10—C11 −169.25 (17) O3—C6—C7—O5 −64.4 (2) O4—C10—C11—O8 74.2 (2) O4—C6—C7—O5 177.31 (17) C9—C10—C11—O8 −167.27 (16) Hydrogen-bond geometry (Å, °) D—H···A D—H H···A D···A D—H···A O5—H5O···O8i 0.86 (3) 2.17 (3) 2.910 (2) 144 (3) O6—H6O···O5ii 0.92 (3) 1.94 (3) 2.824 (2) 162 (3) O7—H7O···O6ii 0.84 (3) 1.84 (3) 2.668 (2) 173 (3) O8—H8O···O2iii 0.88 (3) 1.96 (3) 2.830 (2) 167 (3) C3—H3···O4iv 0.95 2.55 3.413 (3) 151 C4—H4···O1v 0.95 2.57 3.504 (3) 168 C8—H8···O7vi 1.00 2.37 3.306 (3) 155 C1—H1A···O2iii 0.99 2.38 3.289 (3) 153 C10—H10···O6vii 1.00 2.43 3.415 (3) 167 O6—H6O···O7 0.92 (3) 2.56 (3) 2.894 (2) 102 (2) C11—H11A···O7 0.99 2.59 2.986 (3) 104 Symmetry codes: (i) x−1, y−1, z; (ii) −x, y+1/2, −z+1; (iii) −x+2, y+1/2, −z+2; (iv) −x+1, y−1/2, −z+2; (v) x−1, y, z; (vi) −x+1, y−1/2, −z+1; (vii) x+1, y, z. sup-6 supplementary materials Fig. 1 sup-7 supplementary materials supplementary materials Fig. 2 sup-8
https://openalex.org/W1447962669	https://masujournal.org/store_file/archive/37-1-1-7-10.pdf	Maltese	null	Fish manures and their importance to soil fertility	Madras Agricultural Journal	1,950	cc-by	2	https://doi.org/10.29321/MAJ.10.A04459 https://doi.org/10.29321/MAJ.10.A04459
https://openalex.org/W1976304296	https://link.springer.com/content/pdf/10.1007/s10238-013-0268-z.pdf	English	null	Guilty bystanders: nurse-like cells as a model of microenvironmental support for leukemic lymphocytes	Clinical and Experimental Medicine	2,013	cc-by	8,133	Guilty bystanders: nurse-like cells as a model of microenvironmental support for leukemic lymphocytes Agata A. Filip • Bogumiła Ciseł • Ewa Wa˛sik-Szczepanek Received: 14 June 2013 / Accepted: 29 November 2013 / Published online: 12 December 2013 The Author(s) 2013. This article is published with open access at Springerlink.com Abstract B-cell chronic lymphocytic leukemia (B-CLL) is one of the most common leukemias among the elderly and, despite many efforts, still stays incurable. Recent studies point to the microenvironment as the critical factor providing leukemic lymphocytes with pro-survival signals. Thus, the neighboring cells appear to be a perfect target for antileukemic therapy. Nurse-like cells (NLCs) largely contribute to CLL microenvironmental support. We developed the CLL lymphocyte/NLC co-culture model for the investigation of microenvironmental interactions. Via- bility and apoptosis were investigated in CLL lymphocytes treated with dexamethasone (DEX) and chlorambucil (CLB), with and without NLCs’ support. For the ﬁrst time, the capacity of DEX and CLB to affect NLCs viability was also evaluated. Apoptosis-associated gene expression pro- ﬁles of leukemic lymphocytes ex vivo and cultured with NLCs were assessed by expression arrays. CLL lympho- cytes escaped spontaneous apoptosis for several months when cultured with NLCs. The presence of NLCs signiﬁ- cantly reduced apoptosis induced with DEX and CLB (p \ 0.001; p = 0.012, respectively), and their protective effect was more evident than the effect of recombinant SDF1. Both DEX and CLB also decreased NLCs viability, but to a lesser extent (mean viability in DEX-treated cul- tures was 37.79 % in NLCs compared to 29.24 % in lymphocytes). NLCs induced the expression of important anti-apoptotic genes in cultured CLL lymphocytes; median expression of BCL2, SURVIVIN, BCL2A1, and XIAP was signiﬁcantly higher as compared to ex vivo status. The CLL lymphocyte/NLC co-culture makes up the convenient and close to the natural-state model for studying the rela- tionship between leukemic cells and the microenvironment. Direct cell-to-cell contact with NLCs increases the expression of anti-apoptotic genes in CLL lymphocytes, thus protecting them against induced apoptosis. As the effect of antileukemic drugs is not so apparent in NLCs, the combined therapy targeted at both lymphocytes and the microenvironment should be considered for CLL patients. Simultaneous aiming at the disruption of several different signaling pathways and/or anti-apoptotic proteins may further improve treatment efﬁciency. Electronic supplementary material The online version of this article (doi:10.1007/s10238-013-0268-z) contains supplementary material, which is available to authorized users. Keywords CLL Nurse-like cells Microenvironment Apoptosis Gene expression proﬁling A. A. Clin Exp Med (2015) 15:73–83 DOI 10.1007/s10238-013-0268-z Clin Exp Med (2015) 15:73–83 DOI 10.1007/s10238-013-0268-z ORIGINAL ARTICLE Guilty bystanders: nurse-like cells as a model of microenvironmental support for leukemic lymphocytes Filip (&) Department of Cancer Genetics, Medical University of Lublin, Radziwillowska 11, 20-080 Lublin, Poland e-mail: aaﬁlip@hotmail.com E. Wa˛sik-Szczepanek Department of Hematooncology and Bone Marrow Transplantation, Medical University of Lublin, Staszica 11, 20-081 Lublin, Poland Introduction B-cell chronic lymphocytic leukemia (B-CLL) is charac- terized by an accumulation of leukemic lymphocytes in peripheral blood, bone marrow, and lymphatic organs [1]. Increase in lymphocyte number is due both to decreased apoptosis and to slightly increased proliferation of B cells observed in proliferation centers [2]. Once isolated from circulation, leukemic cells die rapidly by apoptosis, which B. Ciseł Department of Oncologic Surgery, Medical University of Lublin, Staszica 11, 20-081 Lublin, Poland E. Wa˛sik-Szczepanek Department of Hematooncology and Bone Marrow Transplantation, Medical University of Lublin, Staszica 11, 20-081 Lublin, Poland 12 3 3 Clin Exp Med (2015) 15:73–83 74 ﬁrst to evaluate the sensitivity of NLCs to these antileu- kemic agents. Finally, we have analyzed gene expression pattern of CLL lymphocytes cultured with NLCs with special focus on anti-apoptotic genes and compared it to ex vivo status. ﬁrst to evaluate the sensitivity of NLCs to these antileu- kemic agents. Finally, we have analyzed gene expression pattern of CLL lymphocytes cultured with NLCs with special focus on anti-apoptotic genes and compared it to ex vivo status. suggests that not only their intrinsic properties contribute to the prolonged survival. Indeed, growing evidence conﬁrms the importance of microenvironmental signals for leukemic lymphocyte growth and resistance to the therapy [2]. CLL microenvironment is composed of cells of different origin, including activated T lymphocytes, dendritic cells, stromal cells, endothelial cells, and nurse-like cells (NLCs) [3]. The latter were named after thymic nurse cells, which were found to be necessary for proper maturation and differentiation of thymocytes [4]. CLL nurse-like cells were ﬁrst described by Burger et al. in 2000 [5]. They differentiate from peripheral blood monocytes of CLL patients in in vitro cultures, but were also found in vivo, within pseudo follicles present in tissue inﬁltrates [6]. In vitro, NLCs protect leukemic cells against spontaneous apoptosis by producing chemokines and interleukins, i.e., SDF1, IL8, CCL2, CXCL9, and by direct cell-to-cell contact [5, 7–9]. Recently, we characterized the gene expression pattern of NLCs and stated that they resemble tumor-associated macrophages (TAMs), which support growth of solid tumor cells and thus may inﬂuence the prognosis [9]. Patients With informed consent, in accordance with the Declaration of Helsinki and approval from the Medical University Bioethics Committee, peripheral blood was obtained from 35 previously untreated patients, hospitalized at the Department of Hematooncology and Bone Marrow Transplantation, Medical University of Lublin. Five patients were excluded because less than 15 NLCs/mm2 was obtained in the culture. Among the remaining 30, there were 10 women and 20 men, aged 36–80 years (median 68.5), diagnosed with B-CLL according to standard criteria [11]. According to Rai classiﬁcation, 5 patients were at stage 0 (16.6 %), 5 at stage I, 18 at stage II (60 %), 1 at stage III (0.3 %), and 1 at stage IV [12]. WBC ranged 10.6–530.0 9 109/l (median 77.7 9 109/l) (Table 1). Detailed patients’ clinical data are presented in Supple- mentary Table S1. The discovery of the role played by microenvironment in CLL development and course resulted in intensive research in this ﬁeld, mainly aimed at disrupting the pro- survival signalization pathways. Many models mimicking the interactions between CLL cells and their microenvi- ronment were proposed for this purpose. However, the cells used to date resemble CLL environment only in some aspects [10]. Here, we developed the natural model for investigation, which utilizes NLCs grown from peripheral blood of CLL patients, and we compared it with non-cell model of culture supplemented with SDF1, which is con- sidered as the most important NLC-derived chemokine [5]. We also assessed the viability of lymphocytes cultured as such after exposure to two drugs of different mechanisms of action: dexamethasone and chlorambucil. We were the Cell isolation, culture, and analyses PB mononuclear cells (PMBCs) were isolated by gradient density centrifugation (LymphoprepTM, AXIS-SHIELD) in glass tubes, as described elsewhere [13]. Immunopheno- type was assessed ex vivo by ﬂow cytometry with FAC- SCalibur apparatus equipped with CellQuest software (Becton–Dickinson Immunocytometry System) on 10,000 cells for each sample, after incubation with monoclonal Table 1 Summarized clinical characteristics of CLL patients n Mean Median Minimum Maximum SD Age 30 63.3667 68.5000 36.0000 80.0000 14.0430 WBC (9 109/l) 30 100.8900 77.7000 10.6000 530.0000 99.1000 Absolute lymphocyte count (9 109/l) 30 92.3823 68.1500 7.5700 491.0000 93.7696 Absolute monocyte count (9 109/l) 26 1.6126 0.6210 0.1160 14.4000 2.8841 Monocytes (%) 26 2.1719 1.0450 0.1000 10.9000 2.7014 B2 M (mg/l) 28 4.3700 3.6700 0.8000 17.6200 3.2457 LDH (IU/l) 28 364.4286 340.0000 172.0000 664.0000 123.5066 CD5/CD19 (%) 30 89.3480 91.0250 64.9100 97.1600 7.7009 CD19/ZAP70 (%) 27 21.9300 15.8400 2.6000 68.5800 18.7887 CD19/CD38 (%) 30 31.6283 23.1900 0.4400 88.4300 31.1744 SD standard deviation, WBC white blood cells count, B2 M beta-2-microglobulin, LDH lactate dehydrogenase Table 1 Summarized clinical characteristics of CLL patients 3 Clin Exp Med (2015) 15:73–83 75 10 9 and 20 9 high chromatic correction objectives and PASCAL 42SP1 imaging software. mouse antihuman CD5-PE, CD19-PE-Cy5, CD38-FITC, and ZAP70 antibodies along with appropriate isotype controls (all from BD PharMingen). Antibodies were applied at 1 lg/100 ll of cell suspension (1 9 106 cells in 1 % BSA/PBS), and samples were processed according to the manufacturer’s instructions. ZAP70 gene expression was conﬁrmed by RT-PCR using primers shown in sup- plementary Table S2 (DNA Gdansk). As median percent- age of CD5(?)/CD19(?) lymphocytes was 91 % (Table 1), we decided not to enrich leukemic cell sub- population for further analyses. Microarray analyses Gene expression proﬁling (GEP) was assessed in CLL lymphocytes isolated ex vivo and in CLL lymphocytes cul- tured with NLCs for 14 days by means of expression cDNA arrays (BD Atlas Human cDNA Expression Array—Human Apoptosis Array, Clontech), as described, according to the manufacturer’s protocol, utilizing 2–5 lg DNase digested RNA per reaction [14]. Probes were synthesized using 350 mCi/l of [a-32P]dATP, puriﬁed by column chromatog- raphy, and the radioactivity was checked by scintillation counting. After hybridization and washing, the membranes were incubated in a phosphorimager cassette with an MS Multisensitive Storage Phosphor Screen (PerkinElmer) for 5–17 h at RT, and the image was read with a Cyclon Phos- phor Imager (PerkinElmer). The results were analyzed with AtlasImageTM2.7 software (BD Biosciences, Clontech). All reactions were performed in duplicate. RNA was isolated by means of TRI reagent (Sigma), checked by spectrometry (Lambda 25 UV/VIS Spectrom- eter, PerkinElmer) and used for microarray analyses or for reverse transcription, followed by PCR. CLL PBMCs (3 9 106/ml) were cultured in culture ﬂasks for adherent cells (for analyses) or on Petri dishes with grid on the bottom (squares 1 9 1 mm; Sarstedt) for counting NLCs, in RPMI 1640 with L-glutamine, supple- mented with 15 % FCS and 1 9 antibiotics/antimycotics solution (all from Gibco) at 37 C, 5 % CO2. Culture medium was partially changed (1/3 vol/vol) three times a week. For statistical analyses, CLL lymphocyte suspension was decanted at day 14, and NLCs were stained with May- Gru¨nwald Giemsa (MGG) and counted (from 50 squares of 1 mm2) with an Olympus CX31 microscope equipped with PLAN C 40 9/0.65 objective. In order to verify the results, RNA isolated from the same samples was reverse-transcribed and PCR was carried out with primers on BCL2, SURVIVIN, and GAPDH genes (Supplementary Table S2). Both reactions were performed according to the manufacturer’s instructions (ImProm-IITM Reverse Transcription System, Promega and Taq PCR Core Kit, Qiagen) on 0.3 lg RNA digested with RNase- free DNase (BD PharMingen) per reaction. PCR products were assessed after gel electrophoresis with TotalLab version 1.11 Gel Analysis software. For apoptosis induction, three versions of culture were prepared: 1. CLL lymphocytes cultured alone in the stan- dard medium (L), 2. CLL lymphocytes co-cultured with NLCs in the standard medium (L/NLC), and 3. CLL lymphocytes cultured in the standard medium supple- mented with SDF1 (L/SDF1) (rhSDF1a, 100 ng/ml, UPSTATE) [5]. Statistics Statistical analyses were performed with a free accessible R statistical package (www.R-project.org) and Statistica 10.0 PL software. Apoptosis was induced by treatment with either dexa- methasone (DEX, 100nM, 30 patients) or chlorambucil (CLB, 35 lM, 8 patients) (both Sigma-Aldrich) for 24 h. The concentrations of DEX and CLB were adjusted empirically. For array data analysis, the genes were ﬁltered based on background threshold, according to the manufacturer’s instructions. The original data were then expressed in loga- rithmic scale and subjected to quantile normalization [15]. For comparison of pairs of groups, Welch’s corrected t-test was applied. The signiﬁcance of diversiﬁcation in the two groups was assessed by a variance analysis test (test F). For analyses, NLCs were carefully washed out from lymphocytes with fresh medium, harvested by incubation with trypsin/EDTA solution (Gibco) at 37 C for 5 min and gently scrapped with a cell-scraper (Sarstedt). Lymphocyte and NLCs’ viability was assessed using trypan blue exclusion (TBE) assay (Trypan Blue, Sigma) and by means of ﬂow cytometry, using ﬂuorescein diace- tate assay (FDA) (Fluorescein Diacetate, Sigma); apoptosis was examined with the Active Caspase-3 Mab Apoptosis Kit (BD PharMingen), according to the manufacturer’s protocol. For other analyses, the normality of data distribution was tested by means of the Shapiro–Wilk test. Descriptive statistical analysis was performed utilizing median, mini- mal, and maximal values. The signiﬁcance of differences between dependent samples was tested by means of the Wilcoxon matched pairs test and between independent samples by the Mann–Whitney U test. The strength of interdependency of two variables was expressed with Spearman’s rank correlation coefﬁcient (R). Live cell confocal microscopy was performed utilizing the Zeiss Axiovert 200 M inverted microscope with ﬂuo- rescence/phase or DIC (Nomarski) imaging, ﬁtted with 12 123 Clin Exp Med (2015) 15:73–83 76 The differences were considered statistically signiﬁcant with P values of less than 0.05. ﬂasks (Fig. 1). The number of NLCs increased up to the 7–8th day and then achieved a plateau. At day 14, it ranged 18–52 cells/mm2 (median 35) (Table 2, Supplementary Table S1). The number of NLCs positively correlated with absolute monocyte count, monocyte percentage, and b-2- microglobulin (B2 M) level (r = 0.45597, r = 0.476191, and r = 0.383003, respectively). No signiﬁcant correlation was observed for NLC count and the stage of the disease according to Rai, WBC, serum LDH, as well as the expression of ZAP70 and CD38. Results NLCs’ outgrowth and number After 3–4 days of CLL cell culture, adherent NLCs with long projections were observed at the bottom of the culture CLL lymphocytes’ viability in cultures with NLCs Fig. 1 Confocal live cell imaging of CLL PBMCs culture at day 14. Cells were cultured on 3-ml Petri dishes, in standard RPMI-1640 medium supplemented with 15 % FCS and antibiotics/antimycotics. Note, large NLCs with radial projections among small, translucent lymphocytes Median lymphocyte viability assessed by TBE assay in cultures with NLCs at day 14 was 93.88 % as compared with 96.09 % at day 0, and it signiﬁcantly positively cor- related with the number of NLCs (r = 0.845075, p \ 0.05) (Fig. 2a). While most of CLL lymphocytes died after 10 days in cultures depleted of NLCs, the CLL lympho- cyte/NLC co-cultures were successfully carried out up to 14 weeks (data not shown). Apoptosis induced by DEX and CLB in CLL lymphocytes y y yp ** Gene expression was assessed by RT-PCR and compared to GAPDH expression 2 yp sessed by RT-PCR and compared to GAPDH expression Apoptosis induced by DEX and CLB in CLL lymphocytes CLL lymphocytes were cultured for 14 days as described in ‘‘Methods.’’ CLL lymphocyte viability was evaluated by trypan blue exclusion (TBE) assay. NLCs number was assessed on Petri dishes by counting cells from 50 squares of 1 mm2. b Apoptosis induced in CLL lymphocytes cultures by DEX and CLB, assessed by ﬂow cytometry using active caspase-3 test. c The viability of CLL lymphocytes treated with DEX depending on culture conditions: L-CLL lymphocytes alone (control), L/NLC—CLL lymphocyte/NLC co-culture, L/SDF1—CLL lymphocytes supplemented with recombi- nant human SDF1. d The viability of CLL lymphocytes and NLCs treated with DEX. L ? DEX—CLL lymphocytes treated with DEX for 24 h, NLC ? DEX—NLCs treated with DEX for 24 h Fig. 2 a Scatter diagram of CLL lymphocytes viability against NLCs number. CLL lymphocytes were cultured for 14 days as described in ‘‘Methods.’’ CLL lymphocyte viability was evaluated by trypan blue exclusion (TBE) assay. NLCs number was assessed on Petri dishes by counting cells from 50 squares of 1 mm2. b Apoptosis induced in CLL lymphocytes cultures by DEX and CLB, assessed by ﬂow cytometry using active caspase-3 test. c The viability of CLL lymphocytes treated with DEX depending on culture conditions: L-CLL lymphocytes alone (control), L/NLC—CLL lymphocyte/NLC co-culture, L/SDF1—CLL lymphocytes supplemented with recombi- nant human SDF1. d The viability of CLL lymphocytes and NLCs treated with DEX. L ? DEX—CLL lymphocytes treated with DEX for 24 h, NLC ? DEX—NLCs treated with DEX for 24 h L/NLC ? DEX, p = 0.000002 and L ? DEX vs. L/SDF1 ? DEX, p = 0.000148) (Fig. 2c). Similarly, in CLB-treated cultures, median viability was 45.84 % for lymphocytes alone, 53.69 % for co-cultures with NLCs and 49.66 % for cultures with SDF1 (Supplementary Table S4). In our experiments, SDF1 alone did not improve lymphocyte viability as effectively as NLCs; median viability without drugs was 82.13 % for lymphocytes alone, 82.94 % for SDF1 cultures and 88.53 % for co-cultures with NLCs (Supplementary Table S3). The difference in lymphocyte viability between the latter two culture variants was statis- tically signiﬁcant (p = 0.000047). (Supplementary Tables S3 and S4). DEX at a concentration of 100 nM was more efﬁcient in inducing cell death as compared to 35 lM CLB (median viability 31.81 vs 45.84 %, respectively, p = 0.003923) (Fig. 2b). Apoptosis induced by DEX and CLB in CLL lymphocytes Treatment with either DEX or CLB induced apoptosis in leukemic cells as compared to control cultures (Fig. 3, Supplementary Table S3 and S4). Median lymphocyte viability assessed by FDA was lower in cultures treated with DEX or CLB than in untreated ones (31.81 vs 82.13 %, p \ 0.0001 and 45.84 vs 79.34 %, p \ 0.0001, respectively). Fig. 1 Confocal live cell imaging of CLL PBMCs culture at day 14. Cells were cultured on 3-ml Petri dishes, in standard RPMI-1640 medium supplemented with 15 % FCS and antibiotics/antimycotics. Note, large NLCs with radial projections among small, translucent lymphocytes Median apoptosis assessed by caspase-3 activity test was higher in DEX-treated cultures (27.12 vs 6.15 % positive cells, p \ 0.0001) and CLB-treated cultures (9.1 vs 6.3 %, p \ 0.0001) as compared to control cultures nd SURVIVIN genes’ expression in CLL lymphocytes ex vivo and cultured with NLCs, and NLCs count at day Table 2 Cell viability, BCL2, and SURVIVIN genes’ expression in CLL lymphocytes ex vivo and cultured with NLCs, and NLCs count at day 14 n Mean Median Minimum Maximum SD Viability* 0 (%) 30 96.0937 96.47000 89.20000 100.0000 2.60281 BCL2 0 30 93.9490 90.72500 46.15000 135.0000 18.68148 Survivin 0 30 20.5127 17.25500 0.00000 51.9200 15.19931 Viability 14 (%) 30 93.8843 95.60000 80.60000 100.0000 5.57868 BCL2 14 30 102.2823 98.34000 72.51000 143.8000 17.05437 Survivin 14 30 29.8413 30.94000 0.00000 69.4500 16.63554 NLC count* 30 36.2000 35.00000 18.00000 52.0000 9.51369 SD standard deviation * Viability was assessed by trypan blue exclusion test Gene expression was assessed by RT-PCR and compared to GAPDH expression Gene expression was assessed by RT-PCR and compared to GAPDH expression 2 12 Clin Exp Med (2015) 15:73–83 77 (Supplementary Tables S3 and S4). DEX at a concentration of 100 nM was more efﬁcient in inducing cell death as compared to 35 lM CLB (median viability 31.81 vs 45.84 %, respectively, p = 0.003923) (Fig. 2b). CLL lymphocytes cultured with NLCs were found to be L/NLC ? DEX, p = 0.000002 and L ? DEX vs. L/SDF1 ? DEX, p = 0.000148) (Fig. 2c). Similarly, in CLB-treated cultures, median viability was 45.84 % for lymphocytes alone, 53.69 % for co-cultures with NLCs and 49 66 % for cultures with SDF1 (Supplementary Table S4) Fig. 2 a Scatter diagram of CLL lymphocytes viability against NLCs number. Apoptosis induced by DEX and CLB in CLL lymphocytes CLL lymphocytes cultured with NLCs were found to be less sensitive to DEX and CLB, as compared with lympho- cytes alone and lymphocytes cultured with SDF1 (Figs. 2c, 3). Median lymphocyte viability in DEX-treated cultures was 31.81 % for lymphocytes alone (L ? DEX), 42.02 % for co-cultures with NLCs (L/NLC ? DEX), and 34.75 % for cultures with the addition of SDF1 (L/SDF1 ? DEX). The differences were signiﬁcant (L ? DEX vs. 12 3 Clin Exp Med (2015) 15:73–83 78 R1 R1 R1 R1 Fig. 3 Exemplary CLL lymphocytes viability assessed by FDA in DEX-treated cultures—different variants of culture (patient # 6). FDA— ﬂuorescein diacetate assay, L— untreated CLL lymphocytes (control), L ? DEX—CLL lymphocytes treated with dexamethasone for 24 h, L/NLC—CLL lymphocyte/NLC co-culture, L/NLC ? DEX— CLL lymphocyte/NLC co- culture treated with DEX for 24 h, L/SDF1—CLL lymphocytes supplemented with SDF1, L/SDF1 ? DEX—CLL lymphocytes supplemented with SDF1 treated with DEX for 24 h. (see ‘‘Methods’’ for details). Numbers at the upper right present the proportion of viable cells R1 R1 ary CLL ability assessed X-treated rent variants of # 6). FDA— cetate assay, L— lymphocytes DEX—CLL eated with for 24 h, lymphocyte/NLC LC ? DEX— te/NLC co- with DEX for —CLL upplemented with ? DEX—CLL upplemented with with DEX for thods’’ for ers at the upper he proportion of R1 R1 R1 R1 R1 R1 Apoptosis induced by DEX and CLB in NLCs Apoptosis induced by DEX and CLB in NLCs Genes down-regulated in cultured CLL lymphocytes included pro-apoptotic BCL2 family members: BAD, BNIP1, and BNIP3; genes coding for repair proteins: GADD45 and ERCC6; growth factor/chemokine receptors: IGFBP1, IGFBP2, and IGFBP6; genes for antioxidant enzymes: MGST12, GSTP1, and GPX1; and gene coding for death receptor FAS (Table 4). Apoptosis-inducing drugs induced decrease in viability of NLCs as well, but to a lesser extent than that of lymphocytes. Mean NLCs’ viability after DEX treatment was 37.79 %, as compared to 29.24 % of CLL lymphocytes’ viability, and the difference was statistically signiﬁcant (p = 0.0387) (Fig. 2d, Supplementary Table S5). CLB at concentration used was less toxic than DEX; mean viability of CLB-treated NLCs was 50.66 % as assessed by FDA test. The Spearman’s rank correlation test demonstrated that the expression of BCL2, SURVIVIN, and GAPDH genes assessed by microarrays and RT-PCR in the same samples was comparable (p \ 0.02). Gene expression proﬁling After data ﬁltering and normalization for both groups examined, the informative data concerning the expression level of 179 genes (out of 218) were obtained (Supple- mentary Table S6). Signiﬁcant differences between CLL lymphocytes ex vivo and CLL lymphocytes cultured with NLCs concerned 36 genes (20 %), 17 genes being up- regulated at day 14. Higher expression in cultured CLL lymphocytes was observed for genes encoding anti-apop- totic proteins: BCL2, BCL2A1, SURVIVIN, XIAP, and regulators of the cell cycle, differentiation and transcrip- tion: CDK4, E2F3, CDK9, CDK5R1, MAPK3, MAPK7, and CDKN1C (Table 3). Increased BCL2 and SURVIVIN expressions were conﬁrmed by means of RT-PCR (Table 2). Moreover, using RT-PCR, we have identiﬁed three alternatively spliced SURVIVIN transcript variants in CLL lymphocytes: SURVIVIN-wt, SURVIVIN-2B, and SURVIVIN-DEx3 (Supplementary Figure 1). Discussion 14* (%) Fold change p Apoptosis regulator BCL-2 M14475 88 100 1.13 0.0139 Cyclin-dependent kinase 4 (CDK4) M14505 68 80 1.17 0.0178 CASP8 and FADD-like apoptosis regulator, CFLAR AF010127 54 75 1.38 0.0448 E2F-3, E2F transcription factor 3 Y10479 53 74 1.39 0.0412 BCL-2-related protein A1 (BCL2A1) U29680 47 60 1.27 0.0320 Cell division protein kinase 9 (CDK9) L25676 42 60 1.42 0.0184 RBP2 retinoblastoma binding protein S66431 36 63 1.75 0.0223 G2/mitotic-speciﬁc cyclin G1 (CCNG1; CYCG1) U47413 33 58 1.75 0.0467 p53-associated mdm2 protein; MDM2 Z12020 28 45 1.61 0.0487 Extracellular signal-regulated kinase 5 (ERK5); MAPK7 U25278 27 50 1.85 0.0382 Receptor interacting serine/threonine protein kinase 2 (RIPK2) U25994 26 40 1.53 0.0275 Apoptosis inhibitor Survivin U75285 24 58 2.41 0.0046 TNF receptor-associated factor 5, TRAF5 U69108 22 42 1.9 0.0421 Inhibitor of apoptosis protein 3 (API3; IAP3); XIAP U45880 18 52 2.88 0.0116 Cyclin-dependent kinase 5 activator precursor (CDK5R1) X80343 16 49 3.06 0.0277 Extracellular signal-regulated kinase 3 (ERK3); MAP kinase 3, MAPK3 X80692 14 35 2.5 0.0323 Cyclin-dependent kinase inhibitor 1C (CDKN1C); p57-KIP2 U22398 11 33 3 0.0418 * Expression assessed by means of expression arrays CLL lymphocytes cultured with NLCs for 14 days compared to the ex vivo status (in descending order of mean Table 4 Genes down-regulated in CLL lymphocytes cultured with NLCs for 14 days compared to the ex vivo status (in descending order of mean expression values at day 0) Gene/description GeneBank accession # Exp. 0* (%) Exp. Discussion 14* (%) Fold change p Apoptosis regulator BCL-2 M14475 88 100 1.13 0.0139 Cyclin-dependent kinase 4 (CDK4) M14505 68 80 1.17 0.0178 CASP8 and FADD-like apoptosis regulator, CFLAR AF010127 54 75 1.38 0.0448 E2F-3, E2F transcription factor 3 Y10479 53 74 1.39 0.0412 BCL-2-related protein A1 (BCL2A1) U29680 47 60 1.27 0.0320 Cell division protein kinase 9 (CDK9) L25676 42 60 1.42 0.0184 RBP2 retinoblastoma binding protein S66431 36 63 1.75 0.0223 G2/mitotic-speciﬁc cyclin G1 (CCNG1; CYCG1) U47413 33 58 1.75 0.0467 p53-associated mdm2 protein; MDM2 Z12020 28 45 1.61 0.0487 Extracellular signal-regulated kinase 5 (ERK5); MAPK7 U25278 27 50 1.85 0.0382 Receptor interacting serine/threonine protein kinase 2 (RIPK2) U25994 26 40 1.53 0.0275 Apoptosis inhibitor Survivin U75285 24 58 2.41 0.0046 TNF receptor-associated factor 5, TRAF5 U69108 22 42 1.9 0.0421 Inhibitor of apoptosis protein 3 (API3; IAP3); XIAP U45880 18 52 2.88 0.0116 Cyclin-dependent kinase 5 activator precursor (CDK5R1) X80343 16 49 3.06 0.0277 Extracellular signal-regulated kinase 3 (ERK3); MAP kinase 3, MAPK3 X80692 14 35 2.5 0.0323 Cyclin-dependent kinase inhibitor 1C (CDKN1C); p57-KIP2 U22398 11 33 3 0.0418 * Expression assessed by means of expression arrays Table 3 Genes up-regulated in CLL lymphocytes cultured with NLCs for 14 days compared to the ex vivo status (in descending order of mean expression values at day 0) Gene/description GeneBank accession # Exp. 0* (%) Exp. * Expression assessed by means of expression arrays Discussion Historically, CLL has been considered as an accumulative disease of lymphocytes defective in apoptosis, and this particular mechanism, but not increased proliferation, was thought to contribute toward leukemogenesis. The dis- covery of high spontaneous apoptosis level of CLL lym- phocytes transferred to an ex vivo conditions has completely changed our conception of their intrinsic properties. In addition, telomere length studies together with proliferation assays based on deuterated water label- ing pointed to the role of proliferation in lymphocyte accumulation as well [16]. Since it was not evident in peripheral blood, the thorough investigations were carried out, which unraveled dividing cells within proliferation centers of pseudofollicles located in bone marrow, spleen, and lymph nodes of CLL patients [1]. Pseudofollicle 123 123 79 Clin Exp Med (2015) 15:73–83 Table 4 Genes down-regulated in CLL lymphocytes cultured with NLCs for 14 days compared to the ex vivo status (in descending order of mean expression values at day 0) Gene/description GeneBank accession # Exp. 0* (%) Exp. Discussion 14* (%) Fold change p Insulin-like growth factor binding protein 2 (IGFBP2) M35410 83 46 1.8 0.0421 Insulin-like growth factor binding protein 6 precursor (IGFPB6) M62402 83 34 2.44 0.0365 Microsomal glutathione S-transferase 12 (MGST12) J03746 72 53 1.35 0.0474 Growth arrest and DNA damage-inducible protein (GADD45) M60974 70 48 1.46 0.0238 Insulin-like growth factor 1 (somatomedin C), IGF1 M27544 67 13 5.15 0.0312 BCL2-associated agonist of cell death, BAD U66879 62 47 1.31 0.0121 Retinoic acid receptor epsilon (RAR-epsilon); RARB X07282 48 29 1.65 0.0452 Cyclin-dependent protein kinase 2 (CDK2) M68520 47 15 3.13 0.0339 Cell division cycle 25A, CDC25A M81933 44 21 2.09 0.0386 FAS soluble protein; APO1 Z70519 44 26 1.69 0.0211 Glutathione S-transferase pi (GSTP1) X15480 41 4 10.25 0.0439 BCL2/adenovirus E1B kDa interacting protein 1, BNIP1 U15172 39 7 5.27 0.0398 Cytochrome P450 reductase, POR S90469 38 3 12.6 0.0385 Defender against cell death 1 (DAD1) D15057 37 7 5.28 0.0486 BCL2/adenovirus E1B kDa interacting protein 3, BNIP3 U15174 33 2 16.5 0.0284 Glutathione peroxidase (GPX1) Y00433 31 6 5.16 0.0399 Excision repair protein ERCC6 L04791 30 17 1.76 0.0437 Insulin-like growth factor 2 (IGF2); somatomedin A M29645 27 14 1.92 0.0486 CSE1 chromosome segregation 1-like (yeast); cellular apoptosis susceptibility protein U33286 25 8 3.5 0.0436 * Expression assessed by means of expression arrays Table 3 Genes up-regulated in CLL lymphocytes cultured with NLCs for 14 days compared to the ex vivo status (in descending order of mean expression values at day 0) Gene/description GeneBank accession # Exp. 0* (%) Exp. The CLL lymphocyte/NLC co-culture model Previously, we characterized one of the components of CLL microenvironment, nurse-like cells (NLCs) [9]. Their number in vitro correlated with serum beta-2-microglobu- lin, absolute monocyte count, and monocyte percentage, but not with other clinical/hematological features of CLL patients, such as stage of the disease according to Rai, lymphocytosis or CD38 and ZAP70 expression. The aforementioned observations were conﬁrmed in the current study group. The relationship between CLL lymphocytes and NLCs is clearly mutual, as the former secrete CCL3 and CCL4, which recruit NLC precursors and T lymphocytes to CLL niches [18]. In our experiments, NLCs from the cultures depleted of lymphocytes survived several days (data not shown). As the network of mutual lymphocyte–NLC interactions is so complex, the animal models that mimic the natural CLL microenvironment are far inaccurate. NLCs considerably support the growth and survival of leukemic lymphocytes in vitro—in our laboratory, the co- cultures were successfully carried out for up to 14 weeks. NLCs also seem to protect leukemic cells in vivo, as we observed the tendency for longer overall survival in our patients producing less NLCs in vitro [9]. In the present study, we established the natural model for the investigation of mutual lymphocyte–environment interaction, utilizing NLCs grown from peripheral blood of CLL patients. It is easy to assemble and very convenient for studies on new therapies. Different cell culture systems mimicking CLL microenvironment have been proposed before, including murine ﬁbroblast cells (NIH3T3) expressing CD154, murine bone marrow stromal cells (M2-10B4 line), and others [5, 10, 19]. However, these cells only in part resemble the natural cells, which compose the microenvironment. Another issue important for the studies of new drugs is that NLC/lymphocyte model allows for simultaneous assessment of the effect of particular treatment both on lymphocytes and on NLCs as microenvironmental compounds. Apoptosis induced in the CLL lymphocyte/NLC co- cultures Although treatment for CLL patients has dramatically changed during the last decade with the introduction of monoclonal antibodies, glucocorticoids (GCs) still are part of the therapeutic regimen, especially in ﬂudarabine- refractory cases [24, 25]. One of the most important chemokines responsible for the enhanced survival of CLL lymphocytes is SDF1 (CXCL12), which acts via its receptor, CXCR4 [5]. SDF1 is normally produced by stromal cells within bone marrow, and it regulates B-cell development by retaining their precursors within the supportive hematopoietic microen- vironment, until they are mature enough to be released into the circulation [5]. Discussion 14* (%) Fold change p Insulin-like growth factor binding protein 2 (IGFBP2) M35410 83 46 1.8 0.0421 Insulin-like growth factor binding protein 6 precursor (IGFPB6) M62402 83 34 2.44 0.0365 Microsomal glutathione S-transferase 12 (MGST12) J03746 72 53 1.35 0.0474 Growth arrest and DNA damage-inducible protein (GADD45) M60974 70 48 1.46 0.0238 Insulin-like growth factor 1 (somatomedin C), IGF1 M27544 67 13 5.15 0.0312 BCL2-associated agonist of cell death, BAD U66879 62 47 1.31 0.0121 Retinoic acid receptor epsilon (RAR-epsilon); RARB X07282 48 29 1.65 0.0452 Cyclin-dependent protein kinase 2 (CDK2) M68520 47 15 3.13 0.0339 Cell division cycle 25A, CDC25A M81933 44 21 2.09 0.0386 FAS soluble protein; APO1 Z70519 44 26 1.69 0.0211 Glutathione S-transferase pi (GSTP1) X15480 41 4 10.25 0.0439 BCL2/adenovirus E1B kDa interacting protein 1, BNIP1 U15172 39 7 5.27 0.0398 Cytochrome P450 reductase, POR S90469 38 3 12.6 0.0385 Defender against cell death 1 (DAD1) D15057 37 7 5.28 0.0486 BCL2/adenovirus E1B kDa interacting protein 3, BNIP3 U15174 33 2 16.5 0.0284 Glutathione peroxidase (GPX1) Y00433 31 6 5.16 0.0399 Excision repair protein ERCC6 L04791 30 17 1.76 0.0437 Insulin-like growth factor 2 (IGF2); somatomedin A M29645 27 14 1.92 0.0486 CSE1 chromosome segregation 1-like (yeast); cellular apoptosis susceptibility protein U33286 25 8 3.5 0.0436 ated in CLL lymphocytes cultured with NLCs for 14 days compared to the ex vivo status (in descending order of day 0) 123 Clin Exp Med (2015) 15:73–83 80 NLCs were characterized by high expression of SDF1 mRNA, and synthetic SDF1 rescued CLL lymphocytes from spontaneous apoptosis. This is why we decided to use recombinant SDF1 as the control of our experiments. centers are composed of a mixture of small to medium and sporadically larger lymphocytes, prolymphocytes, and paraimmunoblasts [3]. They are surrounded and inﬁltrated by stromal cells, follicular dendritic cells, endothelial cells, and nurse-like cells. Expanded size of such proliferation centers seems to be associated with aggressive clinical course [17]. It was the second hint on the role of envi- ronment in CLL development. Recently, growing evidence conﬁrms the signiﬁcance of microenvironmental factors for CLL cell survival and proliferation. Hidden in their microenvironmental niches, leukemic lymphocytes are also resistant to therapy, which makes this issue even more important [18]. Nurse-like cells exert their support not only by secreting SDF1. They enhance CLL cells survival by releasing BAFF (B-cell activating factor, CD257) and proliferation- inducing ligand APRIL (CD256) [8]. Discussion Our studies showed that NLCs might protect lympho- cytes also by the secretion of IL8, the interleukin of many important functions [9]. IL8 was found to decrease CLL lymphocyte apoptosis, both spontaneous and induced by glucocorticoids [21]. Furthermore, gene expression proﬁl- ing demonstrated that NLCs strongly express genes for other molecules important for lymphocyte ‘‘nursing,’’ such as growth factors, chemokines, cell signalization mole- cules, and cell adhesion molecules (i.e., CCL2, CCL8, CCL19, CXCL5, CXCL9, CX3CL1, FLT3LG, IL10, MIC3, CD44H, CD105, ICAM1, ITGAM, ITGAX, ITGB2, and SELP) [9]. High expression of genes encoding cell adhesion molecules points to the importance of direct NLC/lymphocyte contact, which in turn enables CD31– CD38 interaction, resulting in the increase in leukemic cells proliferation and migration [19, 22, 23]. The CLL lymphocyte/NLC co-culture model The CLL lymphocyte/NLC co-culture model The CLL lymphocyte/NLC co-culture model CLL lymphocytes express high levels of CXCR4, and activation of this signal transduction pathway was found to reduce spontaneous apoptosis via AKT and ERK phosphorylation [20]. Burger et al. [5] stated that Dexamethasone (DEX) triggers apoptosis by either transactivation through the glucocorticoid response ele- ment (GRE), transrepression of NF-kappaB, phosphoryla- tion of intracytoplasmic tyrosine kinase RAFTK, or induction of BCL2L11 (BIM) gene [26]. Moreover, since 1940 s, when GCs were introduced as ﬁrst systemic ther- apy for CLL patients, it is known that they interfere with 123 123 Clin Exp Med (2015) 15:73–83 81 many years, and BCL2 has been considered as one of certain contributors to CLL development [29]. Its overexpression is linked to enhanced resistance to apoptosis observed in CLL lymphocytes. Deletion of 13q14, a common cytogenetic aberration in CLL, may partially account for BCL2 up-reg- ulation, because it involves miR-15a and miR-16 loci, both being negative BCL2 regulators [30]. BCL2 expression may be also regulated by environmental signals. Deaglio and coworkers discovered that the expression of BCL2 was increased in CLL lymphocytes located within proliferation centers [2]. Our results conﬁrm that the resistance of leuke- mic cells to apoptosis associated with high BCL2 expression is not solely their intrinsic feature, but is largely induced by the microenvironmental stimuli. leukemic lymphocytes homing and redistribution between blood and secondary lymphoid tissues [27]. This brings to mind an attractive idea of combining GCs with other cytotoxic agents, which will act at leukemic cells expelled from their supportive niches. Chlorambucil (CLB) is an alkylating agent, which impedes DNA replication and induces cellular apoptosis via the accumulation of cytosolic TP53 and subsequent activation of pro-apoptotic BAX gene. For many years, chlorambucil has been a standard ﬁrst-line chemothera- peutic agent for patients with CLL who required treatment [11]. As orally administered, well tolerated, and inexpen- sive drug, it still remains an appropriate option for elderly or unﬁt patients [28]. BCL2 itself is considered as very attractive target of the therapy in many types of cancer. First attempts with ob- limersen (BCL2 antisense phosphorothioate oligodeoxy- nucleotide G3139) and Oblataclax (BH3 mimetic BCL2 inhibitor GX15-070) were not very successful in CLL [26]. The results of the phase I study of Navitoclax (ABT-263, disruptor of BCL2–BCL-xL interactions with pro-apoptotic proteins) in patients with relapsed or refractory CLL were more promising, but it caused thrombocytopenia due to BCL-xL inhibition [31, 32]. The CLL lymphocyte/NLC co-culture model Treatment with another spe- ciﬁc BCL2 inhibitor, ABT-199, is expected to be associ- ated with less adverse effects [32]. However, such monotherapy still aims at only one out of many factors responsible for malignant potential of CLL cells. We have chosen dexamethasone and chlorambucil as drugs of different mechanisms of action to induce apop- tosis in CLL lymphocytes cultured in different conditions. We also wanted to evaluate whether DEX and CLB at used concentrations have any activity on NLCs. In our study, NLCs attenuated apoptosis induced in CLL lymphocytes by both DEX and CLB (Fig. 2c). Their indirect protective effect is much more evident than the effect of recombinant SDF1, which was also observed for spontaneous apoptosis (Supplementary Table S4) [5]. This again points to the complexity of the network of pathways activated between lymphocytes and microenvironment, as well as the role of direct cell-to-cell contact [22, 23]. For the ﬁrst time, the sensitivity of NLCs to antileuke- mic drugs was evaluated. Both DEX and CLB induced NLCs apoptosis, but the resulting decrease in their viability was not as obvious as that of lymphocytes (Fig. 2d). Given that the above observations concern the in vitro conditions, one may assume that in vivo NLCs may be even more resistant. It suggests that to achieve better response, the combined therapy, aimed both at lymphocytes and at their microenvironment, should deﬁnitely be considered. SURVIVIN/BIRC5 belongs to the inhibitor of apoptosis (IAP) family genes, which encode negative regulatory pro- teins that prevent apoptotic cell death. Its overexpression in cancer cells is an adverse prognostic factor, for it is associ- ated with chemotherapy resistance, increased tumor recur- rence, and shorter patient survival [33]. SURVIVIN expression in circulating CLL lymphocytes is low, and it was only found to be induced within proliferation centers [17]. By means of RT-PCR, we identiﬁed three alternatively spliced SURVIVIN transcript variants in CLL lymphocytes: SURVIVIN-wt, SURVIVIN-2B, and SURVIVIN-DEx3. The predominant one was SURVIVIN-wt, which was described also in ALL, AML, and B-cell lymphoma [33]. NLCs may induce SURVIVIN expression via activation of PI3 K/AKT pathway through BAFF and APRIL, and this up-regulation contributes to the enhanced survival of leukemic cells [8]. Conclusion 5. Burger JA, Tsukada N, Burger M, Zvaiﬂer NJ, Dell’Aquilla M, Kipps TJ (2000) Blood derived nurse cells protect leukemia B cells from spontaneous apoptosis through stromal-cell derived factor-1. Blood 96:2655–2663 Despite many efforts, as elegantly reviewed by Hallek and by Burger, CLL still stays an incurable disease [25, 28]. The network of factors inﬂuencing proliferation and sur- vival of leukemic cells is quite complex. Undisputedly, microenvironmental signals are crucial and should abso- lutely be considered in the design of new therapies. Our results proved that conventional antileukemic drugs affec- ted microenvironment only in a small degree. Thus, the combined therapy, targeted both on lymphocytes and on the cells composing the microenvironment, should be developed. However, we have shown that NLC-derived chemokine SDF1 alone was not as effective in the pro- tection of leukemic cells against induced apoptosis as was the presence of NLCs, which points to the involvement of other microenvironment-related factors. Hence, therapies combining conventional antileukemic drugs with disruptors of single signaling pathway, such as CXCR4 inhibitors or inhibitors of B-cell receptor-associated kinases, may not be sufﬁcient. Strategies aimed at several different targets simultaneously should be worked out to bring an evident progress in CLL treatment. The CLL lymphocyte/NLC co- culture, as entirely comprised of the human leukemic cells, seems to be the perfect model for preliminary studies. 6. Tsukada N, Burger AJ, Zvaiﬂer NJ, Kipps TJ (2002) Distinctive features of ‘‘nurselike’’ cells that differentiate in the context of chronic lymphocytic leukemia. Blood 99:1030–1037 7. Burger JA, Quiroga MP, Hartmann E, Bu¨rkle A, Wierda WG, Keating MJ et al (2009) High-level expression of the T-cell chemokines CCL3 and CCL4 by chronic lymphocytic leukemia B cells in nurselike cell co-cultures and after BCR stimulation. Blood 113:3050–3058 8. Cols M, Barra CM, He B, Puga I, Xu W, Chiu A, Tam W, Knowles DM, Dillon SR, Leonard JP, Furman RR, Chen K, Cerutti A (2012) Stromal endothelial cells establish a bidirec- tional crosstalk with chronic lymphocytic leukemia cells through the TNF-related factors BAFF, APRIL, and CD40L. J Immunol 188:6071–6083. doi:10.4049/jimmunol.1102066 9. Filip AA, Ciseł B, Koczkodaj D, Wa˛sik-Szczepanek E, Piersiak T, Dmoszyn´ska A (2013) Circulating microenvironment of CLL: are nurse-like cells related to tumor-associated macrophages? Blood Cells Mol Dis 50:263–270. doi:10.1016/j.bcmd.2012.12. 003 10. Natoni A, O’Dwyer M, Santocanale C (2013) A cell culture system that mimics chronic lymphocytic leukemia cells micro- environment for drug screening and characterization. References 1. Mu¨ller-Hermerink HK, Montserrat E, Catovsky D, Campo E, Harris NL, Stein H (2008) Chronic lymphocytic leukaemia/small lymphocytic leukaemia. In: Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H et al (eds) WHO classiﬁcation of tumours of haematopoietic and lymphoid tissues, 4th edn. IARC Press, Lyon, pp 180–182 2. Deaglio S, Malavasi F (2009) Chronic lymphocytic leukemia microenvironment: shifting the balance from apoptosis to pro- liferation. Haematologica 94:752–756 3. Ponzoni M, Doglioni C, Caligaris-Cappio F (2011) Chronic lymphocytic leukemia: the pathologist’s view of lymph node microenvironment. Semin Diagn Pathol 28:161–166 4. Hendrix TM, Chilukuri RV, Martinez M, Olushoga Z, Blake A, Brohi M, Walker C, Samms M, Guyden JC (2010) Thymic nurse cells exhibit epithelial progenitor phenotype and create unique extra-cytoplasmic membrane space for thymocyte selection. Cell Immunol 261:281–292. doi:10.1016/j.cellimm.2009.11.004 Conclusion Methods Mol Biol 986:217–226. doi:10.1007/978-1-62703-311-4_14 11. Hallek M, Cheson BD, Catovsky D, Caligaris-Cappio F, Dighiero G, Do¨hner H, Hillmen P, Keating MJ, Montserrat E, Rai KR, Kipps TJ, International Workshop on Chronic Lymphocytic Leukemia (2008) Guidelines for the diagnosis and treatment of chronic lymphocytic leukemia: a report from the International Workshop on chronic lymphocytic leukemia updating the National Cancer Institute—working Group 1996 guidelines. Blood 111:5446–5456. doi:10.1182/blood-2007-06-093906 Acknowledgments The authors are grateful to the patients who participated in this study. We would like to thank Barbara Kwiat- kowska-Drabik and Małgorzata Luterek for assistance with culture issues, Dr. Krystyna Ro´ _zyn´ska and Dr. Tomasz Kubiatowski for preparing and reading the arrays, Dr. Piotr Pokarowski for help with the statistical analyses, and Dr. Tomasz Piersiak for photographic documentation. This project was supported by the Medical University of Lublin, Poland (Grants PW 487/03-04-06 and DS225 to AAF). 12. Rai KR, Sawitsky A, Cronkite EP, Chanana AD, Levy RN, Pasternack BS (1975) Clinical staging of chronic lymphocytic leukemia. Blood 46:219–234 13. Bo¨yum A (1968) Separation of leukocytes from blood and bone marrow, introduction. Scand J Lab Clin Invest Suppl 97:7 Changes in gene expression proﬁles Changes in gene expression proﬁles CLL lymphocyte gene expression in NLC co-cultures was studied by Burger and colleagues [7]. However, in our studies, we have focused strictly on the expression of genes contributing to apoptosis (Human Apoptosis Array, Clon- tech, 218 genes) as the process that is known to be altered in CLL. We have also selected the cases where the number of NLCs was [ 15/mm2, to be sure that the potential gene expression changes were NLC-associated. Another phenomenon observed in CLL lymphocytes cul- tured with NLCs was down-regulation of genes BAD, BNIP1, and BNIP3. These are BCL2 family genes encoding pro- apoptotic proteins. The higher is the ratio of anti-apoptotic BCL2familyproteinstopro-apoptotic ones,the stronger isthe signal for survival. Decreased expression of BAD, BNIP1, and BNIP3 may contribute to the resistance to apoptosis. CLL lymphocytes cultured with NLCs demonstrated the up-regulation of genes encoding anti-apoptotic proteins, i.e., BCL2, BCL2A1, SURVIVIN (BIRC5), and XIAP (Table 3). The most important apoptosis-related gene in CLL is BCL2 (B-cell leukemia/lymphoma). We ﬁrst described in follicular lymphoma as translocated to Ig heavy chain gene locus, for Cultured CLL lymphocytes demonstrated also the decrease in the expression of genes coding for antioxidant 12 3 Clin Exp Med (2015) 15:73–83 82 enzymes, i.e., MGST12, GSTP1, and GPX1. Oxidative stress is well known to play an important role in solid tumors development and response to the therapy, but there were only a few studies performed in hematological malignancies [34]. Pasanen et al. [34] described the increased expression of oxidative stress markers and anti- oxidative enzymes in B-cell-derived lymphomas, and its positive correlation with aggressive clinical course. As some antioxidants may also act as growth factors by inhibiting apoptosis and activating a number of transcrip- tion factors (e.g., NF-jB), the observed up-regulation may also be important for prolonged CLL cells’ survival [35]. References Conﬂict of interest None. 14. 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https://openalex.org/W2969826381	http://pdf.blucher.com.br/chemicalengineeringproceedings/cobecic2019/EAT66.pdf	Portuguese	null	ANÁLISE DO CONFORTO TÉRMICO EM EDIFICAÇÕES: EFEITO DE DIFERENTES REVESTIMENTOS QUÍMICOS	Blucher Chemical Engineering Proceedings	2,019	cc-by	3,229	ANÁLISE DO CONFORTO TÉRMICO EM EDIFICAÇÕES: EFEITO DE DIFERENTES REVESTIMENTOS QUÍMICOS G. M. PALMA, A. C. L. HORTA e P. R. F. ROSA Universidade Federal de São Carlos, Departamento de Engenharia Química E-mail para contato: gui.mpalma@gmail.com RESUMO – O objetivo deste estudo é analisar as distribuições de temperatura, o conforto térmico e a contenção de ilhas de calor, proporcionados por diferentes produtos químicos, de forma a promover soluções simples e acessíveis para a redução do consumo de energia na refrigeração de ambientes. Para isso, foi realizado um estudo do isolamento térmico a partir de diferentes revestimentos aplicados: sem revestimento (RN), placas rígidas de poliuretano (PU) e dois tipos de resinas acrílicas (RA e RB) em edificações. A análise foi realizada a partir da exposição ao sol de quatro unidades experimentais revestidas com os produtos mencionados. Os resultados indicam que a aplicação de resinas acrílicas tem influência significativa na redução da temperatura de superfícies e, portanto, no abrandamento de ilhas de calor e que o uso de poliuretano é recomendável para o caso de isolamento de ambientes refrigerados. 1. INTRODUÇÃO Notoriamente, a sustentabilidade tem sido um dos temas mais discutidos em pautas internacionais nas últimas décadas. Tendo em vista os compromissos do Brasil em reduzir a emissão de gases do efeito estufa e aumentar sua eficiência energética, é imprescindível a utilização de produtos químicos e suas propriedades térmicas para a obtenção de melhores resultados energéticos. Devido à baixa condutividade térmica das placas de poliuretano (PU), elas podem exercer um importante papel como isolante térmico (Demharter, 1997). Já as resinas acrílicas brancas, correntemente denominadas resinas de “Cool Roof”, aumentam a refletividade da superfície revestida e, portanto, reduzem a temperatura e energia emitida por radiação (Suehrcke et al., 2008; Akbari et al., 2005). Dessa forma, é esperado que ambos permitam uma diminuição nas temperaturas internas e externas de ambientes. A diminuição de temperatura promovida pelos revestimentos de PU e resinas acrílicas é altamente desejável, visto que pode acarretar uma redução na temperatura média de centros urbanos, aumento da convecção natural em ambientes fechados e diminuição nos gastos com refrigeração. Assim, o objetivo desse estudo foi, através das propriedades térmicas destes produtos avaliados, mapear a efetividade de cada revestimento e compreender as possibilidades de sua utilização para a solução de problemas ambientais relacionados à eficiência energética. 2. METODOLOGIA Foram construídas quatro unidades experimentais distintas, utilizando tijolos e telhas de barro, conforme mostram a Tabela 1 e Figura 1. Tabela 1 – Produtos aplicados às unidades experimentais Unidade Experimental 1 Unidade Experimental 2 Unidade Experimental 3 Unidade Experimental 4 Revestimento Aplicado RN (sem revestimento) PU (placas de poliuretano) RA (Denversol Top Reflective) RB (Metalatex Eco Telha Térmica) Figura 1 – Unidades experimentais. Para a medição dos valores de temperatura interna e de telha, cada unidade continha um sensor dentro de seu espaço interno (DHT11 – faixa de medição 0 a 50°C) e um sensor no interior de sua telha (LM35 – faixa de medição -55 a 150°C), como mostra a Figura 2. Tabela 1 – Produtos aplicados às unidades experimentais Figura 1 – Unidades experimentais. Para a medição dos valores de temperatura interna e de telha, cada unidade continha um sensor dentro de seu espaço interno (DHT11 – faixa de medição 0 a 50°C) e um sensor no interior de sua telha (LM35 – faixa de medição -55 a 150°C), como mostra a Figura 2. Para a medição dos valores de temperatura interna e de telha, cada unidade continha um sensor dentro de seu espaço interno (DHT11 – faixa de medição 0 a 50°C) e um sensor no interior de sua telha (LM35 – faixa de medição -55 a 150°C), como mostra a Figura 2. Figura 2 – Sensores de temperatura. Figura 2 – Sensores de temperatura. Todos os dados medidos foram enviados a um Arduino UNO, programado para a obtenção, conversão e impressão dos valores. Esses valores foram então filtrados pelo método da média móvel, plotados em gráficos e analisados no programa MS Excel. As medições tiveram início às 5pm, os dados eram adquiridos a cada cinco minutos (vinte e quatro medições a cada duas horas) e foram obtidos entre os dias 01/11/2017 e 19/11/2017, estação de primavera no Brasil, no interior do campus da Universidade Federal de São Carlos. Todos os dados medidos foram enviados a um Arduino UNO, programado para a obtenção, conversão e impressão dos valores. Esses valores foram então filtrados pelo método da média móvel, plotados em gráficos e analisados no programa MS Excel. 2. METODOLOGIA As medições tiveram início às 5pm, os dados eram adquiridos a cada cinco minutos (vinte e quatro medições a cada duas horas) e foram obtidos entre os dias 01/11/2017 e 19/11/2017, estação de primavera no Brasil, no interior do campus da Universidade Federal de São Carlos. 3. RESULTADOS Os quatro dias iniciais apresentaram dados interessantes para análise. Os dois primeiros dias foram quentes e os dois seguintes amenos, com valores de radiação global em São Carlos de 56.521,7 e 49.190,9 KJ/m2 respectivamente (INMET, 2019). Assim, foi possível comparar o comportamento das unidades em diferentes condições de temperatura. Os dados brutos de temperatura de telha, sem tratamento pelo método da média móvel, foram plotados apenas para se destacar a suavização e importância da utilização do método para amenizar ruídos dos sensores. As análises a seguir, portanto, são baseadas nos dados das médias móveis. As distribuições de temperatura da unidade sem revestimento podem ser visualizadas na Figura 3. Figura 3 – Distribuição de Temperatura da Unidade 1: a) Dias 1 e 2; b) Dias 3 e 4. Figura 3 – Distribuição de Temperatura da Unidade 1: a) Dias 1 e 2; b) Dias 3 e 4. Na Figura 3.a, dias quentes, observa-se que os valores de temperatura interna foram inferiores aos de telha nos períodos quentes do dia (ΔT chega a 32°C às 2pm). Isso limita a ocorrência de convecção natural no interior da unidade, massa de ar quente acima da massa de ar frio, e gera desconforto térmico (Suehrcke et al., 2008). Os altos valores da temperatura de Figura 3 – Distribuição de Temperatura da Unidade 1: a) Dias 1 e 2; b) Dias 3 e 4. Figura 3 – Distribuição de Temperatura da Unidade 1: a) Dias 1 e 2; b) Dias 3 e 4 Na Figura 3.a, dias quentes, observa-se que os valores de temperatura interna foram inferiores aos de telha nos períodos quentes do dia (ΔT chega a 32°C às 2pm). Isso limita a ocorrência de convecção natural no interior da unidade, massa de ar quente acima da massa de ar frio, e gera desconforto térmico (Suehrcke et al., 2008). Os altos valores da temperatura de telha, que ultrapassam os 60°C, indicam também que a energia emitida pela superfície é elevada e ocasiona um grande aumento na temperatura externa. Na Figura 3.b, dias amenos, o desprendimento entre as temperaturas internas e de telha se manteve durante o dia todo. As distribuições de temperatura da unidade com PU podem ser visualizadas na Figura 4. Figura 4 – Distribuição de Temperatura da Unidade 2: a) Dias 1 e 2; b) Dias 3 e 4. 3. RESULTADOS Figura 4 – Distribuição de Temperatura da Unidade 2: a) Dias 1 e 2; b) Dias 3 e 4. Figura 4 – Distribuição de Temperatura da Unidade 2: a) Dias 1 e 2; b) Dias 3 e 4. Figura 4 – Distribuição de Temperatura da Unidade 2: a) Dias 1 e 2; b) Dias 3 e 4. Na Figura 4.a, novamente a linha de temperatura interna se localizou abaixo da temperatura de telha nos períodos mais quentes, porém, durante a noite a temperatura interna foi maior que a temperatura externa. Diferentemente das outras unidades, o PU promoveu temperaturas internas até 10°C superiores às temperaturas de telha no período noturno. Isso se dá pelo fato de o PU confinar a energia no interior da unidade. Apesar da temperatura de telha ser menor que a interna, nesse caso também não há o gradiente de temperatura para que ocorra convecção natural no interior da unidade (afinal a placa de PU estará promovendo o isolamento), ou seja, para o caso de climas tropicais o PU é altamente indesejável. Na Figura 4.b, dias amenos, a temperatura interna se manteve mais constante, sendo um pouco mais alta que a externa durante a noite e um pouco mais baixa durante o dia. As distribuições de temperatura da unidade revestida com RA podem ser visualizadas na Figura 5. Na Figura 4.a, novamente a linha de temperatura interna se localizou abaixo da temperatura de telha nos períodos mais quentes, porém, durante a noite a temperatura interna foi maior que a temperatura externa. Diferentemente das outras unidades, o PU promoveu temperaturas internas até 10°C superiores às temperaturas de telha no período noturno. Isso se dá pelo fato de o PU confinar a energia no interior da unidade. Apesar da temperatura de telha ser menor que a interna, nesse caso também não há o gradiente de temperatura para que ocorra convecção natural no interior da unidade (afinal a placa de PU estará promovendo o isolamento), ou seja, para o caso de climas tropicais o PU é altamente indesejável. Na Figura 4.b, dias amenos, a temperatura interna se manteve mais constante, sendo um pouco mais alta que a externa durante a noite e um pouco mais baixa durante o dia. As distribuições de temperatura da unidade revestida com RA podem ser visualizadas na Figura 5. 3. RESULTADOS Figura 5 – Distribuição de Temperatura da Unidade 3: a) Dias 1 e 2; b) Dias 3 e 4. Figura 5 – Distribuição de Temperatura da Unidade 3: a) Dias 1 e 2; b) Dias 3 e 4. Diferentemente dos casos anteriores, nas Figuras 5.a e 5.b não se observou um grande desprendimento entre as temperaturas interna e de telha, independente do horário do dia. Esse fato é consequência da menor absorção de energia solar pela telha recoberta com a resina RA. Nos dias amenos, por exemplo, em nenhum momento a temperatura de telha atinge 40°C. A temperatura mais baixa da superfície favorece tanto a redução da energia emitida por radiação (para evitar ilhas de calor) quanto a convecção natural no interior do ambiente por conta da formação de massas de ar frio acima de massas de ar quente no interior da unidade. As distribuições de temperatura da unidade revestida com RB podem ser visualizadas na Figura 6. Figura 5 – Distribuição de Temperatura da Unidade 3: a) Dias 1 e 2; b) Dias 3 e 4 Figura 5 Distribuição de Temperatura da Unidade 3: a) Dias 1 e 2; b) Dias 3 e 4. Diferentemente dos casos anteriores, nas Figuras 5.a e 5.b não se observou um grande desprendimento entre as temperaturas interna e de telha, independente do horário do dia. Esse fato é consequência da menor absorção de energia solar pela telha recoberta com a resina RA. Nos dias amenos, por exemplo, em nenhum momento a temperatura de telha atinge 40°C. A temperatura mais baixa da superfície favorece tanto a redução da energia emitida por radiação (para evitar ilhas de calor) quanto a convecção natural no interior do ambiente por conta da formação de massas de ar frio acima de massas de ar quente no interior da unidade. As distribuições de temperatura da unidade revestida com RB podem ser visualizadas na Figura 6 Diferentemente dos casos anteriores, nas Figuras 5.a e 5.b não se observou um grande desprendimento entre as temperaturas interna e de telha, independente do horário do dia. Esse fato é consequência da menor absorção de energia solar pela telha recoberta com a resina RA. Nos dias amenos, por exemplo, em nenhum momento a temperatura de telha atinge 40°C. 3. RESULTADOS A temperatura mais baixa da superfície favorece tanto a redução da energia emitida por radiação (para evitar ilhas de calor) quanto a convecção natural no interior do ambiente por conta da formação de massas de ar frio acima de massas de ar quente no interior da unidade. As distribuições de temperatura da unidade revestida com RB podem ser visualizadas na Figura 6 6. Figura 6 – Distribuição de Temperatura da Unidade 4: a) Dias 1 e 2; b) Dias 3 e 4. Apesar do recobrimento da unidade 4 com a resina RB, observa-se nas Figuras 6.a e 6.b uma tendência próxima às unidades experimentais sem tratamento de telha. Períodos quentes do dia apresentam temperaturas de telha frequentemente acima dos 50°C. Dessa forma, conclui-se que a RB não foi tão efetiva quanto a RA para amenização das temperaturas de superfície Vale citar que ambas foram utilizadas de acordo com as recomendações do Figura 6 – Distribuição de Temperatura da Unidade 4: a) Dias 1 e 2; b) Dias 3 e 4. Figura 6 – Distribuição de Temperatura da Unidade 4: a) Dias 1 e 2; b) Dias 3 e 4 Apesar do recobrimento da unidade 4 com a resina RB, observa-se nas Figuras 6.a e 6.b uma tendência próxima às unidades experimentais sem tratamento de telha. Períodos quentes do dia apresentam temperaturas de telha frequentemente acima dos 50°C. Dessa forma, conclui-se que a RB não foi tão efetiva quanto a RA para amenização das temperaturas de superfície. Vale citar que ambas foram utilizadas de acordo com as recomendações do fabricante. A análise das médias de todo conjunto de dados, relativos aos dezenove dias de experimento, reforça as tendências consideradas nos gráficos de temperatura em função do tempo. As médias, máximos e mínimos de temperatura são apresentadas na Tabela 2. Apesar do recobrimento da unidade 4 com a resina RB, observa-se nas Figuras 6.a e 6.b uma tendência próxima às unidades experimentais sem tratamento de telha. Períodos quentes do dia apresentam temperaturas de telha frequentemente acima dos 50°C. Dessa forma, conclui-se que a RB não foi tão efetiva quanto a RA para amenização das temperaturas de superfície. Vale citar que ambas foram utilizadas de acordo com as recomendações do fabricante. A análise das médias de todo conjunto de dados, relativos aos dezenove dias de experimento, reforça as tendências consideradas nos gráficos de temperatura em função do tempo. 3. RESULTADOS As médias, máximos e mínimos de temperatura são apresentadas na Tabela 2. Tabela 2 – Médias das temperaturas RN PU RA RB Média temperaturas de telha (°C) 25,59 26,21 22,53 25,98 Média temperaturas internas (°C) 19,74 23,93 20,47 20,45 Temperatura máxima (°C) 61,74 63,78 51,45 56,27 Temperatura mínima (°C) 9,88 8,74 8,16 11,33 Tendo em vista a média das temperaturas de telha, era esperado que as telhas sem resina, RN e PU, tivessem valores similares, e que RA e RB apresentassem temperaturas menores. Conforme já citado, a resina RB não impediu a absorção de energia (térmica) proveniente da luz solar. Observando agora as médias das temperaturas internas, fica evidente que a aplicação de resinas não reduziu significativamente a temperatura interna das unidades. Na verdade, a menor média foi observada na unidade sem nenhum tratamento. Esse fato provavelmente é consequência da existência de convecção mássica forçada que mistura o ar externo ao interno das unidades experimentais (que possuem aberturas), o mesmo que ocorre em residências quando se abrem janelas e portas. Pode-se considerar também a maior retenção de água pela telha sem resina (não impermeabilizada), que resfria mais rapidamente com o sereno noturno ao perder essa água de acordo com o conceito de temperatura de bulbo úmido. Contudo, essa segunda hipótese é menos provável, afinal RN não demonstrou temperatura de telha menor. O maior valor de média de temperatura interna foi atingido pela unidade com placas de PU, o que já era esperado por conta da característica isolante do material. externo ao interno das unidades experimentais (que possuem aberturas), o mesmo que ocorre em residências quando se abrem janelas e portas. Pode-se considerar também a maior retenção de água pela telha sem resina (não impermeabilizada), que resfria mais rapidamente com o sereno noturno ao perder essa água de acordo com o conceito de temperatura de bulbo úmido. Contudo, essa segunda hipótese é menos provável, afinal RN não demonstrou temperatura de telha menor. O maior valor de média de temperatura interna foi atingido pela unidade com placas de PU, o que já era esperado por conta da característica isolante do material. Comparando os resultados obtidos com valores da literatura, Suehrcke et al. (2008) apresenta dados de medição de temperatura máxima e mínima de telha. Evidentemente, a comparação de valores absolutos para diferentes regiões não é representativa. Os parâmetros e condições climáticas encontrados por Suehrcke et al. 3. RESULTADOS (2008) são relativas ao clima australiano, que notoriamente se diferem do brasileiro. Todavia, é possível analisar as variações entre unidades com e sem resina e comparar os resultados. No caso da literatura, a temperatura máxima obtida com utilização da resina, 35°C, equivale a 63,6% da temperatura máxima sem resina, 55°C, enquanto a maior diferença entre temperaturas de telha máximas encontradas neste trabalho foi entre PU e RA, com percentual de 80,7%. Visto que não há variação relevante entre a absorbância e emissividade da resina utilizada na literatura e RA, um fator que pode explicar a discrepância é a não consideração da condução de calor entre a telha e o sensor neste estudo. Com relação às temperaturas mínimas, Suehrcke et al. (2008) apresenta valor mínimo de 14°C com resina e 13°C sem resina. Como são sempre relativas a períodos noturnos, em que os fatores de absorbância e emissividade das resinas são irrelevantes, é natural que resultados de unidades com e sem resina sejam similares. 4. CONCLUSÃO Com relação às resinas de “Cool Roof”, a resina RA reduziu significativamente a temperatura de telha. A temperatura média de telha foi aproximadamente 3°C abaixo das outras unidades experimentais, por conta disso o fluxo de calor por convecção natural no interior da unidade tende a ser maior e o de radiação para o exterior menor, o que promove maior conforto térmico e redução de ilhas de calor. Todavia, a resina RB não obteve os mesmos resultados, o que indica que a qualidade do produto é fundamental para ganhos de eficiência relevantes. Com relação às temperaturas internas, independentemente da resina utilizada, não foram observados resultados satisfatórios. O isolamento térmico a partir de placas rígidas de PU se mostrou muito adequado em ocasiões onde há refrigeração interna na edificação, afinal, evita que grande parte da energia emitida por radiação das telhas seja transferida para o ambiente interno. SUEHRCKE, E. L.; PETERSON, E. L.; SELBY, N. Effect of roof solar reflectance on the building heat gain in a hot climate. 13p. Townsville, Australia, 2008. AKBARI, H.; LEVINSON, R.; RAINER, L. Monitoring the energy-use effects of cool roofs on Califronia commercial buildings. Berkeley, CA, 2005. DEMHARTER, A. Polyurethane rigid foam, a proven thermal insulatin material for applications between +130°C and -196°C. Uberlingen, Germany, 1997. SUEHRCKE, E. L.; PETERSON, E. L.; SELBY, N. Effect of roof solar reflectance on the building heat gain in a hot climate. 13p. Townsville, Australia, 2008. 5. REFERÊNCIAS AKBARI, H.; LEVINSON, R.; RAINER, L. Monitoring the energy-use effects of cool roofs on Califronia commercial buildings. Berkeley, CA, 2005. DEMHARTER, A. Polyurethane rigid foam, a proven thermal insulatin material for applications between +130°C and -196°C. Uberlingen, Germany, 1997. SUEHRCKE, E. L.; PETERSON, E. L.; SELBY, N. Effect of roof solar reflectance on the building heat gain in a hot climate. 13p. Townsville, Australia, 2008. SUEHRCKE, E. L.; PETERSON, E. L.; SELBY, N. Effect of roof solar reflectance on the building heat gain in a hot climate. 13p. Townsville, Australia, 2008.
https://openalex.org/W121556386	https://www.cifor.org/publications/pdf_files/Books/BLiswanti1202.pdf	Indonesian	null	Panduan praktis untuk survei mata pencaharian sosial-ekonomi dan hak dan kepemilikan lahan untuk digunakan dalam perencanaan penggunaan lahan kolaboratif yang berbasis ekosistem	null	2,012	cc-by	26,048	Panduan praktis untuk survei mata pencaharian Panduan praktis untuk survei mata pencaharian Nining Liswanti, Bayuni Shantiko, Emily Fripp, Esther Mwangi dan Yves Laumonier Panduan praktis untuk survei mata pencaharian sosial-ekonomi dan hak dan kepemilikan lahan untuk digunakan dalam perencanaan penggunaan lahan kolaboratif yang berbasis ekosistem Nining Liswanti, Bayuni Shantiko, Emily Fripp, Esther Mwangi dan Yves Laumonier Panduan praktis untuk survei mata pencaharian sosial-ekonomi dan hak dan kepemilikan lahan untuk digunakan dalam perencanaan penggunaan lahan kolaboratif yang berbasis ekosistem Nining Liswanti, Bayuni Shantiko, Emily Fripp, Esther Mwangi dan Yves Laumonier Panduan praktis untuk survei mata pencaharian sosial-ekonomi dan hak dan kepemilikan lahan untuk digunakan dalam perencanaan penggunaan lahan kolaboratif yang berbasis ekosistem Panduan praktis untuk survei mata pencaharian sosial-ekonomi dan hak dan kepemilikan lahan untuk digunakan dalam perencanaan penggunaan lahan kolaboratif yang berbasis ekosistem Nining Liswanti CIFOR Bayuni Shantiko CIFOR Emily Fripp EFECA Esther Mwangi CIFOR Yves Laumonier CIRAD-CIFOR Center for International Forestry Research Center for International Forestry Research © 2012 Center for International Forestry Research Hak cipta dilindungi oleh undang-undang ISBN 978-602-8693-91-2 Liswanti, N., Shantiko, B., Fripp, E., Mwangi, E. dan Laumonier, Y. 2012 Panduan praktis untuk survei mata pencaharian sosial-ekonomi dan hak dan kepemilikan lahan untuk digunakan dalam perencanaan penggunaan lahan kolaboratif yang berbasis ekosistem. CIFOR, Bogor, Indonesia. Diterjemahkan dari: Liswanti, N., Shantiko, B., Fripp, E., Mwangi, E. and Laumonier, Y. 2012 Practical guide for socio-economic livelihood, land tenure and rights surveys for use in collaborative ecosystem-based land use planning. CIFOR, Bogor, Indonesia. Ilustrasi oleh Pandu Dharma Wijaya (TELAPAK) Ilustrasi oleh Pandu Dharma Wijaya (TELAPAK) CIFOR Jl. CIFOR, Situ Gede Bogor Barat 16115 Indonesia T +62 (251) 8622-622 F +62 (251) 8622-100 E cifor@cgiar.org CIFOR Jl. CIFOR, Situ Gede Bogor Barat 16115 Indonesia T +62 (251) 8622-622 F +62 (251) 8622-100 E cifor@cgiar.org © 2012 Center for International Forestry Research Hak cipta dilindungi oleh undang-undang ISBN 978-602-8693-91-2 cifor.org Semua pendapat yang dinyatakan dalam publikasi ini berasal dari para penulis dan tidak serta merta mencerminkan pendapat CIFOR, lembaga tempat bernaung penulis atau penyandang dana publikasi ini. Daftar isi Bagian A: Pendekatan 1 Tujuan dari panduan 1 Apakah perencanaan penggunaan lahan kolaboratif itu? 2 Bagian B: Melaksanakan survei sosial-ekonomi 5 Tahap 1 – Desain survei, pengambilan sampel dan data yang dibutuhkan 5 Tahap 2 – Perencanaan dan pelatihan tim 8 Tahap 3 – Pelaksanaan 8 Bagian C: Menggunakan data yang dikoleksi 13 Tahap 1 – Analisis data 14 Tahap 2 – Menggunakan hasil 15 Catatan Pendukung 1 sampai 9 17 Catatan 1 – Definisi kunci 18 Catatan 2 – Teknik penarikan sampel 20 Catatan 3 – Memilih pertanyaan yang tepat 23 Catatan 4 – Melaksanakan survei 27 Catatan 5 – Panduan pertemuan dengan masyarakat 29 Catatan 6 – Teknik wawancara 31 Catatan 7 – Diskusi kelompok terfokus 33 Catatan 8 – Contoh kuesioner baku: Rumah tangga, desa, dan informan kunci 38 Catatan 9 – Pengalaman dari proyek CoLUPSIA 87 Rujukan 93 Situs web 94 Bagian A: Pendekatan 1 Tujuan dari panduan 1 Apakah perencanaan penggunaan lahan kolaboratif itu? 2 Bagian B: Melaksanakan survei sosial-ekonomi 5 Tahap 1 – Desain survei, pengambilan sampel dan data yang dibutuhkan 5 Tahap 2 – Perencanaan dan pelatihan tim 8 Tahap 3 – Pelaksanaan 8 Bagian C: Menggunakan data yang dikoleksi 13 Tahap 1 – Analisis data 14 Tahap 2 – Menggunakan hasil 15 Catatan Pendukung 1 sampai 9 17 Catatan 1 – Definisi kunci 18 Catatan 2 – Teknik penarikan sampel 20 Catatan 3 – Memilih pertanyaan yang tepat 23 Catatan 4 – Melaksanakan survei 27 Catatan 5 – Panduan pertemuan dengan masyarakat 29 Catatan 6 – Teknik wawancara 31 Catatan 7 – Diskusi kelompok terfokus 33 Catatan 8 – Contoh kuesioner baku: Rumah tangga, desa, dan informan kunci 38 Catatan 9 – Pengalaman dari proyek CoLUPSIA 87 Rujukan 93 Bagian A: Pendekatan 1 Tujuan dari panduan 1 Apakah perencanaan penggunaan lahan kolaboratif itu? 2 Catatan 6 – Teknik wawancara Catatan 7 – Diskusi kelompok terfokus Catatan 8 – Contoh kuesioner baku: Rumah tangga, desa, dan informan kunci Catatan 9 – Pengalaman dari proyek CoLUPSIA Bagian A: Pendekatan Bagian A: Pendekatan Memahami kondisi sosial-ekonomi, kepemilikan lahan dan hak-hak masyarakat merupakan aspek penting dari perencanaan penggunaan lahan kolaboratif. Daftar gambar, tabel dan kotak Gambar 1 Mengembangkan dan melaksanakan survei 5 2 Desa-desa penelitian di empat lokasi pilot di Kapuas Hulu, Kalimantan Barat 90 3 Desa-desa penelitian di lima lokasi pilot di Maluku Tengah (Pulau Seram), Maluku 90 Tabel 1 Keunggulan dan kelemahan pertanyaan terbuka 24 2 Keunggulan dan kelemahan pertanyaan semiterbuka 25 3 Keunggulan dan kelemahan pertanyaan tertutup 26 4 Contoh tema dan topik untuk bahan diskusi dalam FGD 34 5 Data sosial-ekonomi yang dibutuhkan untuk kolaboratif perencanaan penggunaan lahan 92 Kotak 1 Contoh catatan penjelasan untuk mengenalkan anggota tim survei di desa 30 2 Panduan wawancara 32 3 Peran fasilitator 36 Tujuan dari panduan Panduan ini bertujuan untuk memberikan langkah-langkah praktis bagi para praktisi di lapangan dalam melakukan survei sosial-ekonomi rumah tangga dan desa, termasuk diskusi kelompok terfokus dan wawancara informan kunci. Panduan ini disertai sembilan Catatan Pendukung, yang merupakan alat penting untuk mengumpulkan informasi sosial-ekonomi. Hasil informasi yang dikumpulkan dapat digunakan sebagai masukan untuk mengembangkan prosedur dan alat pengambilan keputusan tentang penggunaan lahan kolaboratif. Alat survei sosial-ekonomi dirancang untuk mengumpulkan informasi sebagai sarana meningkatkan pemahaman tentang sistem pengelolaan sumberdaya lokal, penggunaan sumberdaya dan kepentingan relatif masing-masing untuk rumah tangga dan desa. Survei juga memberikan informasi tentang interaksi antara sistem pengambilan keputusan oleh pemerintah dan tren persepsi masyarakat dan isu-isu prioritas. Pengetahuan tentang lembaga berbasis masyarakat, yang juga Nining Liswanti, Bayuni Shantiko, Emily Fripp, Esther Mwangi dan Yves Laumonier 2 2 diperoleh melalui survei, dan peran mereka dalam pemanfaatan berkelanjutan dan konservasi sumberdaya alam, membantu untuk melancarkan atau memperkuat konsensus tentang kepemilikan lahan dan hak-hak di daerah, sekarang dan di masa depan. Dengan demikian, survei juga berfungsi untuk menyoroti peluang yang memungkinkan untuk resolusi konflik antara para pemangku kepentingan. Konflik atas sumberdaya sering muncul akibat ketidakjelasan batas dan respek terhadap pengaturan kelembagaan untuk lahan. Apakah perencanaan penggunaan lahan kolaboratif itu? Perencanaan penggunaan lahan kolaboratif melibatkan kerja sama dengan semua pemangku kepentingan – pemerintah, masyarakat lokal, sektor swasta dan individu lain yang relevan – untuk memastikan bahwa lahan digunakan secara berkelanjutan, menghindari dampak negatif atau ancaman dari degradasi lingkungan dan hilangnya hutan sambil memastikan bahwa pertimbangan sosial dan ekonomi dari semua pengguna dipertimbangkan. Dalam kaitan khususnya dengan masyarakat, tujuan perencanaan penggunaan lahan kolaboratif adalah sebagai berikut: Untuk memastikan bahwa keputusan perencanaan penggunaan lahan diambil dengan mempertimbangkan pendapat masyarakat lokal, kebutuhan penggunaan lahan dan kondisi sosial-ekonomi (peluang dan kendala), termasuk hak akses ke lahan dan untuk menggunakannya Langkah pertama dalam proses ini adalah melibatkan masyarakat lokal. Hal ini dapat dicapai melalui penggunaan survei rumah tangga dan desa, dan diskusi kelompok terfokus. Langkah ini merupakan cara penting positif yang melibatkan para pemangku kepentingan lokal dalam proses perencanaan dan untuk memastikan bahwa suara-suara lokal didengar. Survei dan diskusi kelompok terfokus juga menyediakan cara untuk mendapatkan pemahaman menyeluruh tentang hubungan masyarakat lokal dengan sumberdaya yang relevan – ekonomi dan sosial – dan hak hukum mereka serta akses untuk menggunakan sumberdaya. Informasi ini sangat penting untuk perencanaan penggunaan lahan yang efektif, yaitu perencanaan yang akan dapat diterapkan secara praktis dan yang memenuhi kebutuhan lokal, sehingga berpotensi menghindari konflik atas sumberdaya di antara pihak-pihak yang berkepentingan. Istilah-istilah kunci didefinisikan dalam Catatan Pendukung 1. Panduan praktis untuk survei mata pencaharian sosial-ekonomi dan hak dan kepemilikan lahan 3 Bagian B: Melaksanakan survei sosial-ekonomi Melakukan survei rumah tangga dan desa dan diskusi kelompok terfokus untuk memahami mata pencaharian dan hak dan kepemilikan lahan Gambar 1. Mengembangkan dan melaksanakan survei Tahap 1 - Merancang survei • Desain, pengambilan sampel dan data yang dibutuhkan Tahap 2 - Perencanaan • Perencanaan dan pelatihan tim Tahap 3 - Pelaksanaan • Wawancara informan kunci • Survei rumah tangga • Diskusi kelompok terfokus Tahap 1 – Desain survei, pengambilan sampel dan data yang dibutuhkan Ketika melakukan survei, langkah pertama adalah menentukan maksud dan tujuan survei. Langkah ini menjelaskan kerangka kerja survei, termasuk muatan dan cakupan pekerjaan survei dan juga digunakan untuk membantu mengidentifikasi jenis pemangku kepentingan dan masyarakat yang harus disurvei. Bagian B: Melaksanakan survei sosial-ekonomi Melakukan survei rumah tangga dan desa dan diskusi kelompok terfokus untuk memahami mata pencaharian dan hak dan kepemilikan lahan Gambar 1. Mengembangkan dan melaksanakan survei Tahap 1 - Merancang survei • Desain, pengambilan sampel dan data yang dibutuhkan Tahap 2 - Perencanaan • Perencanaan dan pelatihan tim Tahap 3 - Pelaksanaan • Wawancara informan kunci • Survei rumah tangga • Diskusi kelompok terfokus Gambar 1. Mengembangkan dan melaksanakan survei Tahap 1 – Desain survei, pengambilan sampel dan data yang dibutuhkan Data yang dibutuhkan dan desain survei Data yang akan dikumpulkan melalui proses survei harus mencerminkan tujuan survei. Dalam mengembangkan survei, pertimbangan lainnya adalah lamanya waktu wawancara dan kebutuhan sumberdaya (keuangan, manusia) yang efektif melakukan survei, memasukkan data dan menganalisis hasilnya. Alat yang berbeda (survei, diskusi, wawancara) digunakan untuk mendapatkan berbagai jenis informasi dari berbagai kelompok informan. Misalnya, survei rumah tangga dapat digunakan untuk mengumpulkan informasi tentang umur, jenis kelamin, pendidikan, sumber pendapatan (pertanian, kehutanan dan Nining Liswanti, Bayuni Shantiko, Emily Fripp, Esther Mwangi dan Yves Laumonier 6 g y y pp g lapangan kerja lainnya), dan persepsi tentang perubahan penggunaan lahan dan akses ke sumberdaya hutan. Sebaliknya, pemahaman fungsi (pemerintahan dan kelembagaan) desa, pembangunan, isu-isu yang lebih luas tentang akses lahan, pertumbuhan penduduk, kondisi sosial dan kendala dapat diperoleh melalui diskusi kelompok terfokus dan wawancara dengan informan kunci. Metode survei dijelaskan dalam Langkah 3. lapangan kerja lainnya), dan persepsi tentang perubahan penggunaan lahan dan akses ke sumberdaya hutan. Sebaliknya, pemahaman fungsi (pemerintahan dan kelembagaan) desa, pembangunan, isu-isu yang lebih luas tentang akses lahan, pertumbuhan penduduk, kondisi sosial dan kendala dapat diperoleh melalui diskusi kelompok terfokus dan wawancara dengan informan kunci. Metode survei dijelaskan dalam Langkah 3. lapangan kerja lainnya), dan persepsi tentang perubahan penggunaan lahan dan akses ke sumberdaya hutan. Sebaliknya, pemahaman fungsi (pemerintahan dan kelembagaan) desa, pembangunan, isu-isu yang lebih luas tentang akses lahan, pertumbuhan penduduk, kondisi sosial dan kendala dapat diperoleh melalui diskusi kelompok terfokus dan wawancara dengan informan kunci. Metode survei dijelaskan dalam Langkah 3. Pengambilan sampel acak dan ukuran sampel Ukuran sampel harus cukup untuk memastikan bahwa hasil survei secara statistik akan relevan. Namun, ukuran sampel umumnya juga harus diimbangi sumberdaya yang tersedia – keuangan, manusia dan waktu. Pengambilan sampel acak digunakan untuk memastikan agar sampel yang dipilih mewakili daerah penelitian, menghindari hasil yang bias dan memastikan bahwa semua elemen dari suatu populasi memiliki kesempatan yang sama untuk diwawancarai. Ada beberapa pendekatan dalam menentukan sampel acak, misalnya sistematis, bertingkat dan cluster. Dalam kaitannya dengan perencanaan penggunaan lahan kolaboratif, pendekatan sampel acak sederhana sudah memadai (Catatan Pendukung 2). Siapa yang perlu dilibatkan dalam survei? Individu-individu dan masyarakat yang akan disurvei harus diputuskan saat mempertimbangkan maksud dan tujuan survei. Individu atau kelompok individu yang berbeda akan diwawancarai sesuai dengan data yang diperlukan. Untuk survei rumah tangga yang diwawancarai adalah kepala rumah tangga, namun dalam sebuah wawancara informan kunci maka yang diwawancarai adalah kepala desa atau kepala adat. Diskusi kelompok terfokus akan melibatkan anggota masyarakat, dengan kelompok-kelompok yang dibagi berdasarkan usia dan jenis kelamin yang berbeda. Panduan praktis untuk survei mata pencaharian sosial-ekonomi dan hak dan kepemilikan lahan nduan praktis untuk survei mata pencaharian sosial-ekonomi dan hak dan kepemilikan lahan 7 Tahap 2 – Perencanaan dan pelatihan tim Sebuah tim yang terlatih dan berpengalaman sangat penting bagi keberhasilan setiap survei sosial-ekonomi. Pengalaman praktis sebelumnya adalah aset besar untuk tim, misalnya dengan pengetahuan teknis yang relevan di bidang sosial- ekonomi, kehutanan dan sumberdaya alam. Semua anggota tim membutuhkan pelatihan secara lengkap dan rinci untuk semua jenis survei (rumah tangga, desa, diskusi kelompok terfokus, wawancara informan kunci) yang akan dilakukan. Semua anggota tim harus memiliki pemahaman yang menyeluruh tentang tujuan kegiatan dan makna setiap pertanyaan yang akan ditanyakan. Kombinasi pelatihan di kelas dan di lapangan akan memberikan pemahaman terbaik tentang prosedur dan metode survei. Selain itu, pelatihan lapangan sangat berguna untuk memeriksa pertanyaan survei dan maksud pengumpulan data, sehingga memberikan kesempatan untuk melakukan revisi yang diperlukan untuk survei, pertanyaan wawancara dan prosedur surveinya. Pembagian tugas antara anggota tim juga perlu dipertimbangkan – fasilitator, manajer sumberdaya, pengamat/perekam, dan pemimpin tim. Melalui persiapan yang matang (merancang pekerjaan survei, melatih tim, dll) dan penjadualan pekerjaan survei sangat penting untuk keberhasilan survei (Catatan Pendukung 4). Persiapan tim survei dan kesepakatan rencana kerja dan kerangka waktu untuk menyelesaikan survei harus ditetapkan lebih dahulu. Desa yang akan disurvei juga harus diberitahu terlebih dahulu sebelumnya dan izin untuk melakukan wawancara harus diperoleh dan dipastikan. Nining Liswanti, Bayuni Shantiko, Emily Fripp, Esther Mwangi dan Yves Laumonier Nining Liswanti, Bayuni Shantiko, Emily Fripp, Esther Mwangi dan Yves Laumonier 8 Pertanyaan tertutup, seperti pilihan ganda atau ya/tidak ada jawaban, digunakan ketika informasi kunci yang diperlukan bisa diperoleh tanpa perlu penjelasan lebih lanjut atau pemahaman mendalam tentang jawaban atau masalahnya. Pertanyaan- pertanyaan seperti ini dapat ditanyakan dan dianalisis dengan cepat, namun hasil jawabannya dapat menjadi kurang dalam dan jelas. Jenis pertanyaan Untuk semua jenis wawancara (untuk rumah tangga, diskusi kelompok terfokus dan informan kunci), pertanyaannya dapat terstruktur dan ditanyakan sebagai pertanyaan terbuka, tertutup atau semiterbuka. Jenis pertanyaan yang digunakan bergantung pada informasi yang sedang dikumpulkan. Setiap jenis pertanyaan ini ada keuntungan dan kerugiannya (Catatan Pendukung 3). Melalui pertanyaan terbuka, contohnya ‘Mengapa panen padi menurun tahun ini?”, pewawancara dapat mengungkap makna di balik jawaban, yang memungkinkan responden memberikan contoh dan menjelaskan jawaban mereka. Pertanyaan-pertanyaan ini lebih memakan waktu untuk dikelola dan dianalisis. Pertanyaan terbuka bisa sulit untuk ditanyakan dan untuk menafsirkan responnya, sehingga diperlukan pelatihan untuk melakukannya. Suatu pertanyaan semiterbuka sebagian, contohnya ‘Konsumsi produk hutan meningkat, karena...’ membutuhkan responden untuk menguraikan setiap jawaban yang diberikan. Keuntungan dari pertanyaan ini adalah mereka lebih cepat dan lebih mudah ditanyakan dan di analisis daripada pertanyaan terbuka, tetapi si pewawancara mungkin kehilangan beberapa informasi karena kurangnya kategori yang sesuai atau tingkat jawaban responden yang lebih rinci. Untuk menghindari masalah ini, jawaban responden harus dicatat secara penuh, dan si pewawancara harus mengulang pertanyaan apakah jawaban responden masih kurang lengkap. Tahap 3 – Pelaksanaan Pendekatan survei bervariasi. Pendekatan yang berbeda digunakan untuk tujuan yang berbeda. Untuk perencanaan penggunaan lahan kolaboratif, digunakan tiga metode survei, yaitu: •• Wawancara informan kunci, •• Survei rumah tangga, dan •• Diskusi kelompok terfokus Panduan praktis untuk survei mata pencaharian sosial-ekonomi dan hak dan kepemilikan lahan 9 Sebelum memulai setiap pekerjaan survei, sebaiknya dilakukan pertemuan dengan masyarakat. Sebelum memulai setiap pekerjaan survei, sebaiknya dilakukan pertemuan dengan masyarakat. Sebelum memulai setiap pekerjaan survei, sebaiknya dilakukan pertemuan dengan masyarakat. Nining Liswanti, Bayuni Shantiko, Emily Fripp, Esther Mwangi dan Yves Laumonier Nining Liswanti, Bayuni Shantiko, Emily Fripp, Esther Mwangi dan Yves Laumonier 10 Pertemuan dengan masyarakat Pertemuan dengan masyarakat merupakan cara yang berharga dan produktif bagi tim survei untuk bertemu dengan penduduk desa, menjelaskan pekerjaan survei – tujuan dan pendekatan – dan memastikan bahwa semua anggota masyarakat memahami hasil yang diharapkan dari survei (Catatan Pendukung 5). Keputusan melakukan pertemuan masyarakat harus diambil setelah bertemu dengan kepala masyarakat atau desa. Jika memungkinkan, kepala desa bisa membantu mengatur dan melakukan pertemuan dengan masyarakat. Survei desa dan rumah tangga Survei desa dan rumah tangga terutama digunakan untuk mengumpulkan data kuantitatif dan kualitatif, melalui wawancara terstruktur dengan kepala rumah tangga. Jenis pertanyaan yang digunakan bisa berupa pertanyaan tertutup dan terbuka. Formulir survei, baik untuk tingkat rumah tangga maupun desa, dirancang untuk mendapatkan informasi yang spesifik, relevan dengan tujuan survei, seperti yang dibahas di atas. g gg p dirancang untuk mendapatkan informasi yang spesifik, relevan dengan tujuan survei, seperti yang dibahas di atas. Wawancara informan kunci Informan kunci adalah orang-orang yang dianggap memiliki pengetahuan tentang isu-isu tertentu. Informan kunci akan memberikan informasi rinci kepada tim wawancara, dan terutama dalam menafsirkan sejumlah isu penting yang tidak bisa diberikan oleh anggota masyarakat lainnya. Informan kunci yang potensial harus dipilih, melalui konsultasi dengan kepala desa, pemimpin tradisional atau kepala marga, untuk diwawancarai secara mendalam oleh tim survei (Catatan Pendukung 6). Panduan praktis untuk survei mata pencaharian sosial-ekonomi dan hak dan kepemilikan lahan anduan praktis untuk survei mata pencaharian sosial-ekonomi dan hak dan kepemilikan lahan 11 Diskusi kelompok terfokus Diskusi kelompok terfokus ini bertujuan untuk memperoleh informasi mendalam tentang konsep, persepsi dan gagasan sekelompok orang berjumlah 6-12 orang. Idealnya, diskusi ini adalah suatu proses yang berulang-ulang, dimana setiap diskusi didasarkan pada diskusi sebelumnya dengan mengembangkan suatu topik atau penekanan pada aspek-aspek tertentu (Catatan Pendukung 7). Nining Liswanti, Bayuni Shantiko, Emily Fripp, Esther Mwangi dan Yves Laumonier 12 Dalam konteks perencanaan penggunaan lahan kolaboratif, diskusi kelompok terfokus mencakup diskusi tentang pengaturan kelembagaan, hak-hak masyarakat, akses masyarakat terhadap hutan, penggunaan hutan oleh masyarakat dan pengelolaan sumberdaya hutan secara berkelanjutan, Kunci keberhasilan diskusi kelompok terfokus adalah adanya fasilitator yang kuat yang akan merangsang diskusi dan memastikan bahwa proses diskusi telah sesuai dengan sasaran yang ingin dicapai tim survei. Cara ini bermanfaat jika instrumen penelitian lain yang digunakan berhubungan dengan diskusi kelompok terfokus, seperti wawancara informan kunci, wawancara mendalam atau teknik kualitatif lainnya. Formulir survei yang digunakan untuk survei desa dan rumah tangga, wawancara informan kunci dan diskusi kelompok terfokus dapat dilihat dalam Catatan Pendukung 8. Analisis dan penggunaan data yang dikoleksi harus mencerminkan tujuan survei Tingkat analisis dan penggunaan data yang dikumpulkan akan bergantung pada tujuan survei dan hasil akhir yang diharapkan. Untuk perencanaan penggunaan lahan kolaboratif, hasil survei akan menyediakan: Pertama, dasar yang kuat dari faktor sosial-ekonomi yang berhubungan dengan sumberdaya lahan dan kegunaannya, dan kedua, adanya wawasan yang mendalam dan terinci mengenai kelembagaan masyarakat, hubungan mereka dengan para perencana penggunaan lahan (misalnya, badan-badan pemerintah ) dan setiap jenis penggunaan yang berpotensi konflik. Bersama-sama, informasi ini dapat digunakan untuk mengembangkan alat pengambilan keputusan tentang penggunaan lahan kolaboratif. Nining Liswanti, Bayuni Shantiko, Emily Fripp, Esther Mwangi dan Yves Laumonier Nining Liswanti, Bayuni Shantiko, Emily Fripp, Esther Mwangi dan Yves Laumonier 14 Tahap 1 – Analisis data Pemilihan metode untuk analisis data bergantung pada volume data yang dikumpulkan dan penggunaan hasil-hasil temuan yang diharapkan. Ketika survei tingkat desa dan rumah tangga dilakukan, volume data yang dikumpulkan akan sangat banyak. Analisis data ini biasanya dilakukan dalam satu paket statistik seperti SPSS. Sebelum menggunakan paket perangkat lunak ini, data harus dimasukkan dan dibersihkan sebelum dianalisis. Selanjutnya, data dapat diekspor ke paket perangkat lunak lain seperti Microsoft Excel untuk analisis lebih lanjut dan untuk membuat tabel dan grafik. Untuk informasi kualitatif lain yang dikumpulkan, misalnya, dari diskusi kelompok terfokus dan wawancara informan kunci, analisis data didasarkan pada metode Grounded Theory yang dikembangkan oleh Glaser dan Strauss. Metode analisis ini memilah daftar kategori di dalam masing-masing pertanyaan penelitian dan berbagai bagian dari instrumen survei untuk mengidentifikasi tema-tema utama secara terpisah untuk diskusi kelompok terfokus dan wawancara informan kunci. Hasilnya diolah menggunakan nVivo, yaitu perangkat lunak untuk menganalisis data kualitatif. Sebuah skema hierarki pengelompokan tema (kode) dikembangkan atas dasar daftar awal kategori, untuk mencerminkan pertanyaan utama penelitian, dan yang dikembangkan lebih lanjut dengan tema yang muncul dari data. Data tersebut kemudian dianalisis sesuai dengan tema yang telah teridentifikasi. Untuk diskusi kelompok terfokus, data temuan dipisahkan berdasarkan jenis kelamin, usia dan lokasi, data juga diberi kode menggunakan kode ‘kasus’, dengan membuat ‘simpul’ yang terpisah untuk setiap wawancara. Berbagai ‘kueri’ untuk kode lebih lanjut digunakan untuk menganalisis pola, tren dan tanggapan terhadap berbagai pertanyaan dalam survei. Hasil dari kueri diekspor ke MS Word dan dianalisis untuk menulis bagian-bagian dari laporan yang merangkum temuan dan mengidentifikasi pola dan tren di dalam data. Analisis akhir harus mencerminkan maksud dan tujuan survei. Untuk membantu melakukan analisis secara sistematis, beberapa pertanyaan penelitian dapat dikembangkan, seperti: •• Seberapa penting akses ke sumberdaya hutan untuk mata pencaharian masyarakat? •• Apakah masyarakat miskin lebih atau kurang bergantung pada sumberdaya hutan dibandingkan dengan kelompok lain? •• Bagaimana akses ke sumberdaya hutan dan implikasi mata pencaharian yan dihasilkan berbeda-beda untuk sampel survei, misalnya, sebuah kabupaten? •• Bagaimana kepemilikan lahan, hak penggunaan dan kepemilikan lahan memengaruhi mata pencaharian masyarakat? Panduan praktis untuk survei mata pencaharian sosial-ekonomi dan hak dan kepemilikan lahan 15 Tahap 2 – Menggunakan hasil Penggunaan hasil survei akan bergantung pada tujuannya. Untuk perencanaan penggunaan lahan kolaboratif, hasilnya akan digunakan tidak hanya sebagai data dasar tetapi juga untuk menyoroti isu-isu penting – kendala dan peluang – yang dihadapi oleh masyarakat, khususnya yang berkaitan dengan penggunaan lahan dan mekanisme potensial untuk mengatasi masalah ini. Data dasar yang kuat dapat dikombinasikan dengan data biofisik untuk memberikan gambaran menyeluruh tentang situasi daerah yang disurvei (misalnya, kabupaten). Memahami masyarakat dan hubungan internal dan eksternal mereka (misalnya, dengan badan pemerintah) akan memberikan landasan yang baik dalam mengembangkan proses untuk melibatkan masyarakat dalam proses pengambilan keputusan tentang penggunaan lahan kolaboratif. Pengalaman dari CoLUPSIA (Collaborative Land Use Planning and Sustainable Institutional Arrangements - Perencanaan Penggunaan Lahan Kolaboratif dan Penataan Kelembagaan yang Berkelanjutan) dijelaskan secara lebih rinci dalam Catatan Pendukung 9. Tujuan Untuk mendukung ‘Panduan praktis untuk survei mata pencaharian sosial- ekonomi dan hak dan kepemilikan lahan untuk digunakan dalam perencanaan penggunaan lahan kolaboratif yang berbasis ekosistem’, telah disiapkan serangkaian Catatan Pendukung. Catatan Pendukung ini memberikan informasi tambahan dan panduan, yang dirancang untuk membantu para praktisi lapangan dalam melaksanakan survei sosial-ekonomi. Catatan ini didasarkan pada pengalaman yang diperoleh dari proyek CoLUPSIA. Isi catatan ini belum lengkap, tetapi bertujuan untuk menutupi hal-hal penting yang diidentifikasi dan digunakan sebagai pelajaran selama pekerjaan survei diselesaikan di dua kabupaten (Kapuas Hulu dan Maluku Tengah) di Indonesia, yang secara total telah dilakukan dan melibatkan 1.366 rumah tangga. Panduan Praktis dan Catatan Pendukung ini secara bersama-sama, termasuk contoh kuesioner yang digunakan, telah membentuk suatu alat bantu (Tool Kit) sosial-ekonomi. Alat bantu ini tersedia di situs CoLUPSIA untuk diunduh baik secara keseluruhan atau sebagian dokumen. Nining Liswanti, Bayuni Shantiko, Emily Fripp, Esther Mwangi dan Yves Laumonier Nining Liswanti, Bayuni Shantiko, Emily Fripp, Esther Mwangi dan Yves Laumonier 18 1 Berdasarkan UU No 41/1999 tentang Kehutanan (Indonesia). Akses ke lahan ‘Akses ke lahan’ diartikan sebagai cara seseorang/kelompok untuk mendapatkan akses atau menggunakan lahan. Di pedesaan, akses ke lahan berbasis adat-istiadat setempat ditentukan oleh pimpinan adat yang memberikan hak pakai kepada anggota masyarakat. Selain itu, akses ke lahan dapat diperoleh dengan cara membeli, menyewa, sistem bagi hasil, mendapatkan warisan atau menempati lahan secara tidak sah. Catatan 1 – Definisi kunci Untuk memudahkan pemahaman kuesioner dan pelaksanaan survei sosial- ekonomi, beberapa konsep kunci yang digunakan perlu dipahami. Pertanyaan tertutup Tipe pertanyaan yang membatasi kemungkinan jawaban dari responden; tipe ini dapat dijawab dengan satu kata atau kalimat singkat. Definisi ‘hutan’1 Studi tentang mata pencaharian, kepemilikan dan hak atas lahan Studi ini mengenai bagaimana mata pencarian dibentuk oleh kepemilikan dan hak lahan dan bagaimana masyarakat mengelola sumberdaya alam di suatu wilayah. Pertanyaan terbuka Struktur pertanyaan yang longgar, yang tidak memerlukan jawaban yang baku dan pasti sehingga responden dapat menyampaikan jawaban secara bebas dengan kata- katanya sendiri. Kepemilikan dan hak atas lahan Kepemilikan dan hak atas lahan ‘Kepemilikan dan hak atas lahan’ mengacu pada pengaturan kelembagaan untuk mengelola hubungan antar individu maupun kelompok orang dengan lahan secara hukum formal maupun berdasarkan adat. Definisi ‘hutan’1 •• Hutan adalah suatu kesatuan ekosistem berupa hamparan lahan berisi sumberdaya alam hayati yang didominasi pepohonan dalam persekutuan alam lingkungannya, yang satu dengan lainnya tidak dapat dipisahkan. •• Kawasan hutan adalah wilayah tertentu yang ditunjuk dan atau ditetapkan oleh pemerintah untuk dipertahankan keberadaannya sebagai hutan tetap. •• Hutan negara adalah hutan yang berada pada tanah yang tidak dibebani hak pribadi atas tanah. •• Hutan adat adalah hutan negara yang berada dalam wilayah masyarakat hukum adat. •• Hutan produksi adalah kawasan hutan yang mempunyai fungsi pokok menghasilkan hasil hutan (kayu). •• Hutan lindung adalah kawasan hutan yang mempunyai fungsi pokok sebagai perlindungan sistem penyangga kehidupan untuk mengatur tata air, mencegah banjir, mengendalikan erosi, mencegah intrusi air laut, dan memelihara kesuburan tanah. •• Hutan konservasi adalah kawasan hutan dengan ciri khas tertentu, yang mempunyai fungsi pokok pelestarian keanekaragaman tumbuhan dan satwa serta ekosistemnya. Panduan praktis untuk survei mata pencaharian sosial-ekonomi dan hak dan kepemilikan lahan anduan praktis untuk survei mata pencaharian sosial-ekonomi dan hak dan kepemilikan lahan 19 Pertanyaan semiterbuka Hampir sama dengan definisi pertanyaan terbuka, tetapi beberapa jawaban sudah dibuat kategorinya untuk memudahkan perekaman informasi dan analisisnya. Rumah tangga Rumah tangga didefinisikan sebagai 1) kelompok orang yang hidup di bawah satu atap dan mempunyai hubungan kekerabatan; 2) menggunakan dapur atau tungku dimana mereka makan bersama dan berbagi pengeluaran untuk makanan dan minuman (lebih dari 30% dari pendapatan individu); atau 3) tempat dimana kelompok individu tinggal bersama berbagi tenaga kerja, produksi dan konsumsi tanpa dibayar. Mata pencaharian Mata pencaharian mengacu kepada kemampuan seseorang untuk hidup, secara finansial, makanan maupun aset. Menentukan ukuran sampel Menentukan ukuran sampel Ukuran sampel yang tepat sangat dipengaruhi oleh tujuan suatu penelitian. Contohnya, sensus penduduk memerlukan partisipasi seluruh penduduk sehingga ukuran sampel sebanyak jumlah penduduk (100%). Faktor seperti keterbatasan sumberdaya (waktu, anggaran, manusia) akan menjadi pertimbangan dalam menentukan ukuran sampel, namun diperlukan kehati-hatian agar tidak menurunkan kualitas studi. Untuk menentukan ukuran sampel, penggunaan persentase terhadap populasi bisa digunakan, contohnya mewawancarai 5% dari total rumah tangga sebagai sampel. Cara lainnya menggunakan jumlah minimum tertentu sebagai dasar penentuan sampel. Untuk CoLUPSIA, ukuran sampel ditentukan menggunakan kombinasi pendekatan persentase dan minimum responden setiap desa (30 rumah tangga). Jumlah sampel yang disurvei sebanyak 566 rumah tangga di Maluku Tengah dan 800 rumah tangga di Kapuas Hulu. Penarikan sampel acak Untuk menarik sampel tahapan yang dilakukan adalah sebagai berikut 1. Membuat daftar dari populasi keseluruhan secara berurutan, misalnya nama kepala keluarga (KK) di desa X diurutkan dari 1-250 2. Tentukan jumlah sampel yang diinginkan, contohnya, 50 KK 3. Gunakan kocokan seperti arisan, atau pemunculan angka acak dari komputer/ kalkulator untuk menentukan 50 KK yang dipilih 4. Sampel yang terpilih disusun dalam daftar tersendiri, jika diperlukan buatlah daftar cadangan dari daftar sisa nama keluarga (200 KK) apabila ada responden yang tidak berada di tempat atau tidak bersedia diwawancarai. Kepastian hak atas lahan Adalah kepastian hak atas lahan yang diakui oleh pihak lain dan dilindungi dari ancaman tertentu. Individu dapat menghadapi risiko atas ketidakpastian hak lahan mereka berupa klaim pihak lain atau kerugian akibat pengusiran. Nining Liswanti, Bayuni Shantiko, Emily Fripp, Esther Mwangi dan Yves Laumonier 20 Membangkitkan angka acak 1. Kocokan arisan. Metode ini paling mudah dan tersedia bahan-bahannya di lapangan. Peneliti menuliskan angka sejumlah populasi kemudian dimasukkan ke dalam kocokan, kemudian diambil secara acak yang jumlahnya sebanyak jumlah sampel. anduan praktis untuk survei mata pencaharian sosial-ekonomi dan hak dan kepemilikan lahan 21 Nining Liswanti, Bayuni Shantiko, Emily Fripp, Esther Mwangi dan Yves Laumonier 22 2. Kalkulator. Jika memungkinkan membawa scientific kalkulator dilengkapi fungsi pembangkitan angka random. Tahapannya adalah sebagai berikut: 2. Kalkulator. Jika memungkinkan membawa scientific kalkulator dilengkapi fungsi pembangkitan angka random. Tahapannya adalah sebagai berikut: a. Tentukan dahulu jumlah populasi, misalnya 50 b. Untuk mendapatkan angka acak antara 0-50, tekan tombol fungsi acak di kalkulator dan dikalikan 50, yaitu ran#50 c. Angka acak yang muncul dalam bentuk desimal, untuk mendapatkan angka mutlak bisa melakukan pembulatan (round) atau membuang angka desimal (trunc) misalnya, 12,6342 (round=13; trunc=12) d. Langkah No 2c diulang kembali untuk mendapatkan sejumlah n sampel yang ingin ditarik. 3. Program komputer. Jika tersedia, pemunculan angka acak dapat dilakukan di program Microsoft excel dengan cara: 3. Program komputer. Jika tersedia, pemunculan angka acak dapat dilakukan di program Microsoft excel dengan cara: a. Ketik formula umum angka random, yaitu rand() a. Ketik formula umum angka random, yaitu rand() b. Ulangi langkah No 2a, 2b dan 2c, tulis kembali formula apabila memilih metode round maka formulanya adalah =round(50rand(),0); apabila memilih metode trunc maka formulanya menjadi =trunc(50rand()) Perlu diketahui bahwa fungsi “rand()” sangat sensitif dalam Excel, sehingga setiap ada pergerakan cursor saja angka acak akan terus bergerak. Karena itu setelah angka acak terbentuk, daftar angka acak yang dipakai adalah daftar yang pertama. Gunakan perintah copy - paste special – values – ok untuk menyalin tanpa mengubah nilai angka acak yang dibangkitkan. Perlu diketahui bahwa fungsi “rand()” sangat sensitif dalam Excel, sehingga setiap ada pergerakan cursor saja angka acak akan terus bergerak. Karena itu setelah angka acak terbentuk, daftar angka acak yang dipakai adalah daftar yang pertama. Gunakan perintah copy - paste special – values – ok untuk menyalin tanpa mengubah nilai angka acak yang dibangkitkan. Panduan praktis untuk survei mata pencaharian sosial-ekonomi dan hak dan kepemilikan lahan 23 Catatan 3 – Memilih pertanyaan yang tepat Ketika merancang survei dan melakukan wawancara, penting untuk mempertimbangkan struktur pertanyaan, apakah terbuka, tertutup atau kombinasi keduanya. Jenis pertanyaan dan cara bertanya akan memengaruhi jawaban yang diberikan. Secara umum, pertanyaan dapat diajukan dalam bentuk terbuka maupun tertutup. Perbedaan keduanya terletak pada pertanyaan terbuka memberikan kesempatan kepada responden untuk menjelaskan jawabannya, sedangkan pertanyaan tertutup tidak memungkinkan hal ini. Dengan pertanyaan tertutup, responden tidak memiliki kebebasan menjawab selain yang sudah disediakan di lembar pertanyaan. Pertanyaan semiterbuka dapat pula digunakan dalam survei sosial-ekonomi. Kuesioner survei akan menggunakan beragam jenis pertanyaan untuk memastikan agar informasi diperoleh dengan cara yang layak. Pertanyaan terbuka - contoh Apa pendapat Bapak mengenai hasil panen tahun ini? Mengapa berkurang, apa yang menyebabkannya? Pertanyaan terbuka Pertanyaan terbuka Pertanyaan terbuka - contoh Apa pendapat Bapak mengenai hasil panen tahun ini? Mengapa berkurang, apa yang menyebabkannya? Apa pendapat Bapak mengenai hasil panen tahun ini? Mengapa berkurang, apa yang menyebabkannya? Pertanyaan terbuka memungkinkan peneliti menggali lebih banyak informasi dari responden karena responden mempunyai kebebasan menjawab dan memberikan penjelasan. Pertanyaan terbuka memiliki keunggulan dan kekurangan, sebagaimana tercantum dalam Tabel 1. Nining Liswanti, Bayuni Shantiko, Emily Fripp, Esther Mwangi dan Yves Laumonier 24 Pertanyaan semiterbuka Pertanyaan semiterbuka Pertanyaan semiterbuka – contoh: Selama 5 (lima) tahun terakhir, apakah hasil hutan yang dikonsumsi rumah tangga: a. meningkat, karena ...... a. berkurang, karena ...... Tabel 1. Keunggulan dan kelemahan pertanyaan terbuka Tabel 1. Keunggulan dan kelemahan pertanyaan terbuka Keunggulan Keunggulan Kelemahan •• Dapat membantu peneliti menemukan informasi baru yang sebelumnya tidak terpikirkan •• Catatan pembicaraan dengan responden dapat menjadi ilustrasi yang menarik di dalam laporan. •• Meluaskan ruang lingkup ketika analisis sehingga memungkinkan munculnya interpretasi kesimpulan yang baru. •• Diperlukan pewawancara yang berpengalaman untuk memulai diskusi suatu topik dan mencatat temuan- temuannya •• Analisis data dan informasi membutuhkan keahlian dan waktu yang cukup. •• Dapat membantu peneliti menemukan informasi baru yang sebelumnya tidak terpikirkan •• Diperlukan pewawancara yang berpengalaman untuk memulai diskusi suatu topik dan mencatat temuan- •• Diperlukan pewawancara yang berpengalaman untuk memulai diskusi suatu topik dan mencatat temuan- temuannya •• Catatan pembicaraan dengan responden dapat menjadi ilustrasi yang menarik di dalam laporan. temuannya •• Analisis data dan informasi membutuhkan keahlian dan wakt k •• Meluaskan ruang lingkup ketika analisis sehingga memungkinkan munculnya interpretasi kesimpulan yang baru. Untuk mengatasi kelemahan pertanyaan terbuka, maka ketika melakukan survei hal-hal berikut perlu diperhatikan: Untuk mengatasi kelemahan pertanyaan terbuka, maka ketika melakukan survei hal-hal berikut perlu diperhatikan: •• Melatih pewawancara dan memberikan arahan selama kegiatan berlangsung akan membantu mendapatkan kualitas data/informasi. •• Menyiapkan daftar pertanyaan secara sistematis yang memungkinkan pewawancara menggali jawaban responden. •• Pastikan mencoba pertanyaan terbuka dan berlatih bersama anggota tim. Pertanyaan semiterbuka – contoh: Selama 5 (lima) tahun terakhir, apakah hasil hutan yang dikonsumsi rumah tangga: a. meningkat, karena ...... a. berkurang, karena ...... Selama 5 (lima) tahun terakhir, apakah hasil hutan yang dikonsumsi rumah tangga: a. meningkat, karena ...... a. berkurang, karena ...... Dalam tipe pertanyaan ini, responden diberi pilihan jawaban dalam bentuk kategori. Contoh di atas menunjukkan bahwa jawabannya hanya ‘meningkat’ atau ‘berkurang’ yang mencirikan pertanyaan tertutup, tetapi responden diberikan kesempatan menjelaskan atau alasan “karena....” (ciri pertanyaan terbuka). Tipe pertanyaan ini memiliki keunggulan dan kelemahan, diuraikan dalam Tabel 2. Panduan praktis untuk survei mata pencaharian sosial-ekonomi dan hak dan kepemilikan lahan 25 Tabel 2. Keunggulan dan kelemahan pertanyaan semiterbuka Tabel 2. Keunggulan dan kelemahan pertanyaan semiterbuka Tabel 2. Keunggulan dan kelemahan pertanyaan semiterbuka Keunggulan Kelemahan •• Jawaban mudah dicatat dan cepat, analisis mudah. •• Kemungkinan ada jawaban penting responden yang terlewat karena tidak ada kategori yang sesuai. •• Pewawancara bisa lalai karena memaksakan informasi yang masuk ke dalam kategori yang ada sementara informasi lainnya bisa hilang. •• Jika responden ragu menjawab, pewawancara kemungkinan berupaya membantu dengan kisi jawaban, hal ini berpotensi bias. Keunggulan Kelemahan Keunggulan •• Jawaban mudah dicatat dan cepat, analisis mudah. •• Kemungkinan ada jawaban penting responden yang terlewat karena tidak ada kategori yang sesuai. •• Pewawancara bisa lalai karena memaksakan informasi yang masuk ke dalam kategori yang ada sementara informasi lainnya bisa hilang. •• Jika responden ragu menjawab, pewawancara kemungkinan berupaya membantu dengan kisi jawaban, hal ini berpotensi bias. Untuk mengatasi kelemahan di atas, maka ketika melakukan survei hal-hal berikut perlu diperhatikan: Untuk mengatasi kelemahan di atas, maka ketika melakukan survei hal-hal berikut perlu diperhatikan: •• Jawaban responden agar dicatat dan menyediakan kertas yang cukup untuk mencatat informasi dari responden atau gunakan alat perekam. •• Jika responden tidak menjawab, usahakan mengulangi pertanyaan dengan cara berbeda serta jangan memberikan kisi-kisi jawaban. Pertanyaan tertutup Apa masalah atau kendala apa yang Bapak/Ibu hadapi dalam kegiatan usaha pertanian? Serangan babi Sering Kadang-kadang Jarang Serangan satwa lain Sering Kadang-kadang Jarang Kekurangan modal Sering Kadang-kadang Jarang Apa masalah atau kendala apa yang Bapak/Ibu hadapi dalam kegiatan usaha ? Apa masalah atau kendala apa yang Bapak/Ibu hadapi dalam kegiatan usaha pertanian? Dengan pertanyaan tertutup, responden harus memilih jawaban yang tersedia, tidak memungkinkan adanya penjelasan tambahan. Pertanyaan tertutup berguna ketika waktu penelitian terbatas dan hanya memerlukan informasi tertentu saja. Tipe pertanyaan ini memiliki keunggulan dan kelemahan, seperti dicantumkan dalam Tabel 3. Nining Liswanti, Bayuni Shantiko, Emily Fripp, Esther Mwangi dan Yves Laumonier 26 Untuk mengatasi kelemahan di atas, pewawancara dapat menanyakan dan menggali alasan pemilihan jawaban. Tabel 3. Keunggulan dan kelemahan pertanyaan tertutup Tabel 3. Keunggulan dan kelemahan pertanyaan tertutup Keunggulan •• Jawaban cepat dikumpulkan dan menghemat waktu. •• Membandingkan jawaban responden antar waktu dan kelompok lebih mudah. •• Analisis lanjutan dari pertanyaan tertutup dapat menyulitkan karena pertanyaan tidak dilengkapi dengan pertanyaan lain untuk menjelaskan alasan responden memilih suatu pilihan. •• Jawaban cepat dikumpulkan dan menghemat waktu. •• Membandingkan jawaban responden antar waktu dan kelompok lebih mudah. •• Analisis lanjutan dari pertanyaan tertutup dapat menyulitkan karena pertanyaan tidak dilengkapi dengan pertanyaan lain untuk menjelaskan alasan responden memilih suatu pilihan. •• Membandingkan jawaban responden antar waktu dan kelompok lebih mudah. Untuk mengatasi kelemahan di atas, pewawancara dapat menanyakan dan menggali alasan pemilihan jawaban. Panduan praktis untuk survei mata pencaharian sosial-ekonomi dan hak dan kepemilikan lahan 27 2. Meminta ijin dari kepala desa sebelum melakukan kegiatan survei. Pastikan warga desa mendapat informasi dan ijin dari kepala desa diperoleh sejak awal. Catatan 4 – Melaksanakan survei Sebelum melakukan survei, lakukan beberapa langkah berikut ini. 1. Menyiapkan secara matang dan menjadualkan kegiatan survei merupakan bagian penting kesuksesan sebuah survei. Tim survei harus menyiapkan diri, rencana kerja dan alokasi waktu penyelesaian survei harus disetujui sejak awal. Peran dan tanggung jawab tim harus jelas. Lembar data dan kuesioner harus disiapkan dan tim diberi pelatihan. 1. Menyiapkan secara matang dan menjadualkan kegiatan survei merupakan bagian penting kesuksesan sebuah survei. Tim survei harus menyiapkan diri, rencana kerja dan alokasi waktu penyelesaian survei harus disetujui sejak awal. Peran dan tanggung jawab tim harus jelas. Lembar data dan kuesioner harus disiapkan dan tim diberi pelatihan. 2. Meminta ijin dari kepala desa sebelum melakukan kegiatan survei. Pastikan warga desa mendapat informasi dan ijin dari kepala desa diperoleh sejak awal. 3. Jika memungkinkan, pertemuan dengan masyarakat atau dengan kepala desa dilaksanakan sebelum kegiatan dimulai, untuk mengenalkan anggota tim survei, menjelaskan tentang survei, tujuan dan bagaimana melakukannya, dan menyediakan waktu untuk pertanyaan dan diskusi dengan masyarakat atau kepala desa. Pelaksanaan pertemuan desa membutuhkan perencanaan dan 2. Meminta ijin dari kepala desa sebelum melakukan kegiatan survei. Pastikan warga desa mendapat informasi dan ijin dari kepala desa diperoleh sejak awal. 3. Jika memungkinkan, pertemuan dengan masyarakat atau dengan kepala desa dilaksanakan sebelum kegiatan dimulai untuk mengenalkan anggota tim 2. Meminta ijin dari kepala desa sebelum melakukan kegiatan survei. Pastikan warga desa mendapat informasi dan ijin dari kepala desa diperoleh sejak awal. 2. Meminta ijin dari kepala desa sebelum melakukan kegiatan survei. Pastikan warga desa mendapat informasi dan ijin dari kepala desa diperoleh sejak awal. 3. Jika memungkinkan, pertemuan dengan masyarakat atau dengan kepala desa dilaksanakan sebelum kegiatan dimulai, untuk mengenalkan anggota tim survei, menjelaskan tentang survei, tujuan dan bagaimana melakukannya, dan menyediakan waktu untuk pertanyaan dan diskusi dengan masyarakat atau kepala desa. Pelaksanaan pertemuan desa membutuhkan perencanaan dan 3. Jika memungkinkan, pertemuan dengan masyarakat atau dengan kepala desa dilaksanakan sebelum kegiatan dimulai, untuk mengenalkan anggota tim survei, menjelaskan tentang survei, tujuan dan bagaimana melakukannya, dan menyediakan waktu untuk pertanyaan dan diskusi dengan masyarakat atau kepala desa. Pelaksanaan pertemuan desa membutuhkan perencanaan dan 3. Jika memungkinkan, pertemuan dengan masyarakat atau dengan kepala desa dilaksanakan sebelum kegiatan dimulai, untuk mengenalkan anggota tim survei, menjelaskan tentang survei, tujuan dan bagaimana melakukannya, dan menyediakan waktu untuk pertanyaan dan diskusi dengan masyarakat atau kepala desa. Catatan 4 – Melaksanakan survei Pelaksanaan pertemuan desa membutuhkan perencanaan dan Nining Liswanti, Bayuni Shantiko, Emily Fripp, Esther Mwangi dan Yves Laumonier 28 pengorganisasian mengingat pentingnya kegiatan ini di awal survei. Tim survei dapat bekerja sama dengan pimpinan desa untuk memastikan keterlibatan masyarakat desa. 4 P ik i dib i j l d l ih P ik id k pengorganisasian mengingat pentingnya kegiatan ini di awal survei. Tim survei dapat bekerja sama dengan pimpinan desa untuk memastikan keterlibatan masyarakat desa. pengorganisasian mengingat pentingnya kegiatan ini di awal survei. Tim survei dapat bekerja sama dengan pimpinan desa untuk memastikan keterlibatan masyarakat desa. pengorganisasian mengingat pentingnya kegiatan ini di awal survei. Tim survei dapat bekerja sama dengan pimpinan desa untuk memastikan keterlibatan masyarakat desa. 4. Pastikan semua anggota tim diberi penjelasan dan pelatihan. Pastikan tidak ada perubahan tim yang telah diberi pelatihan selama survei. Ini untuk memastikan konsistensi dan kualitas data. Pelatihan harus melatih cara melakukan survei (misalnya, teknik mendengarkan, menanyakan pertanyaan terbuka) dan keterampilan mewawancarai termasuk tujuan survei dan makna setiap pertanyaan. 5. Hindari memberikan harapan kepada responden dengan menjelaskan sejelas-jelasnya mengenai kegiatan dan hasil yang mungkin dicapai. Hal ini sangat penting. Panduan praktis untuk survei mata pencaharian sosial-ekonomi dan hak dan kepemilikan lahan 29 Catatan 5 – Panduan pertemuan dengan masyarakat Pertemuan dengan masyarakat sangat penting bagi kesuksesan survei dan langkah penting untuk melibatkan masyarakat di setiap tahapan perencanaan tata guna lahan. Membangun pelibatan masyarakat memerlukan waktu dan tidak bisa dipaksa atau terburu-buru. Survei sosial-ekonomi dan diskusi kelompok terfokus, jika dilakukan dengan benar, menjadi langkah awal membangun hubungan dengan masyarakat. Informasi yang dikumpulkan selama survei sering bersifat pribadi bahkan rahasia, dan informasi ini harus dikelola secara baik. Panduan di bawah ini akan membantu pelaksanaan pertemuan dengan masyar •• Mengatur pertemuan di tempat/balai pertemuan masyarakat, dan menyepakati waktu pertemuan dengan para tokoh. •• Mulailah dengan memperkenalkan anggota tim, lalu jelaskan latar belakang penelitian ini dan peran organisasi kita, termasuk apa yang dapat dan tidak kita berikan. Pastikan kemungkinan hasil survei dapat dipahami oleh masyarakat dan tidak memberikan harapan yang berlebihan: apa yang bisa dan tidak bisa lakukan oleh tim. Hindari membuat janji (lihat Kotak 1). •• Jelaskan kegiatan-kegiatan penelitian dan tujuan yang ingin dicapai serta sumberdaya yang akan disediakan. Jelaskan bagaimana masyarakat dapat berpartisipasi, dan bagaimana kita menginginkan bantuan mereka dan memberikan saran kepada kita. •• Jelaskan aspek-aspek penting dari jadual dan kegiatan penelitian dan simak dengan baik jika ada potensi masalah atau konflik apa saja dengan kegiatan- kegiatan setempat. Berusahalah untuk menentukan jadual yang bisa diterima untuk kegiatan-kegiatan utama. •• Menyilakan masyarakat memperkenalkan diri mereka/desanya. Mendapatkan informasi latar belakang tentang desa: jumlah penduduk dan jumlah keluarga, etnis, kegiatan-kegiatan masyarakat saat ini, dsb. Apakah mereka punya waktu untuk berpartisipasi dengan cara tertentu? Jika ya, kapan waktu yang cocok bagi mereka untuk melakukannya? •• Melalui diskusi-diskusi informal, mulailah untuk mengenali informan kunci yang mempunyai pengetahuan lapangan yang terkait dengan penelitian. •• Periksa apakah anggota masyarakat senang dengan kegiatan-kegiatan yang diusulkan; berikan penjelasan jika ada aspek-aspek khusus yang mungkin tidak bisa diterima. Terimalah jika ada larangan tertentu dan ubah pendekatan dengan cara lain. Mengakhiri pertemuan. Mulailah merencanakan berbagai kegiatan berdasarkan kemungkinan yang ada. Nining Liswanti, Bayuni Shantiko, Emily Fripp, Esther Mwangi dan Yves Laumonier 30 Kotak 1. Contoh catatan penjelasan untuk mengenalkan anggota tim survei di desa Tujuan kedatangan kami ke desa ini untuk melakukan kegiatan survei yang berkaitan dengan keadaan sosial-ekonomi masyarakat. Kegiatan ini meliputi wawancara dan diskusi kelompok. Kami memahami bahwa waktu sangat berharga bagi masyarakat. Oleh karena itu kami akan mendiskusikan jadual kegiatan selama kami tinggal dengan kepala desa, berdasarkan waktu yang paling cocok bagi masyarakat. Kegiatan bisa dilakukan di pagi hari, siang maupun malam sesuai keberadaan responden. Informasi yang diberikan oleh masyarakat adalah rahasia, dan kami tidak akan memberikan informasi ini kepada pihak lain yang mempunyai kepentingan berbeda. Mohon dicatat bahwa kegiatan ini tidak ada kaitannya dengan perpajakan. Kegiatan ini tidak menyediakan manfaat tunai langsung kepada masyarakat. Lebih jauh, proyek ini tidak menjanjikan suatu kegiatan akan dilakukan di desa ini di masa depan. Namun, kami percaya bahwa hasil dari studi ini akan bermanfaat bagi masyarakat di masa depan, terutama dengan meningkatnya kemampuan mendiskusikan perencanaan tata guna lahan dengan pemerintah. Panduan praktis untuk survei mata pencaharian sosial-ekonomi dan hak dan kepemilikan lahan 31 Catatan 6 – Teknik wawancara Wawancara dapat dilakukan secara individu atau kelompok kecil sebanyak dua atau tiga orang. Sebelum wawancara, pewawancara harus menyiapkan pertanyaan dan memikirkan bagaimana wawancara akan dilakukan. •• Pakaian menunjukkan kepribadian Anda. Berpakaian yang sopan, tidak mencolok, sesuai dengan adat setempat akan meningkatkan tingkat penerimaan masyarakat desa. •• Bersikap terbuka. Proses wawancara tidak hanya sekedar ‘mendapatkan data’, tetapi lebih dari itu untuk memperoleh “pemahaman”. Karena itu hubungan yang terjalin baik dengan masyarakat dan pemikiran yang terbuka sangat diperlukan. •• Jangan terpengaruh oleh pendapat yang sudah ada sebelumnya. •• Jawaban responden kadang terdengar tidak masuk akal, jangan berhenti meminta penjelasan sampai betul-betul mendapatkan jawaban yang dapat dimengerti. •• Di kalangan masyarakat tertentu, perempuan tidak bebas bertemu dengan tamu/orang asing. Jika memerlukan wawancara khusus dengan perempuan, usahakan komposisi tim pewawancara terbagi antara laki-laki dan perempuan sehingga pewawancara perempuan dapat mengambil data yang relevan. Perhatikan isu-isu jender. Pastikan pendekatan wawancara sesuai norma yang berlaku. •• Kemampuan mendengar dan menyadari bahwa informasi yang disampaikan oleh responden itu menarik, tidak terburu-buru dan dapat membuat responden merasa nyaman tanpa merasa terancam dengan pertanyaan pewawancara. •• Apabila mendapati responden yang antusias menjelaskan sesuatu hingga keluar dari pokok bahasan, bersikaplah sopan ketika mengalihkan pembicaraan. Berikut ini kiat-kiat untuk membantu wawancara agar berjalan secara lancar, lihat Kotak 2. Berikut ini kiat-kiat untuk membantu wawancara agar berjalan secara lancar, lihat Kotak 2. Nining Liswanti, Bayuni Shantiko, Emily Fripp, Esther Mwangi dan Yves Laumonier 32 Kotak 2. Panduan wawancara •• Temui anggota masyarakat dan jalin hubungan, usahakan informan merasa nyaman dan rileks, perhatikan sikap badan dan bahasa tubuh. •• Jelaskan maksud dan tujuan survei dan jelaskan hal-hal yang menyangkut kerahasiaan. •• Tetapkan aturan-aturan dasar – jelaskan bahwa jika mereka tidak tahu jawabannya, tidak apa-apa. Usahakan waktunya singkat, perhatikan waktu dan jika mereka mulai gelisah, mengubah topik, kurang perhatian. Hentikan atau jika perlu istirahat. Jangan terburu-buru, bersabarlah dan tenang, tetapi serius. Gunakan bahasa yang sederhana, siapkan cara-cara lain untuk menanyakan hal yang sama. Jangan mengarahkan informan dengan mengusulkan jawaban atau pendapat Anda sendiri: sabar dan berilah waktu kepada responden untuk berpikir. •• Hormati berbagai pandangan, peraturan dan adat istiadat setempat (misalnya, seorang laki-laki tidak boleh datang sendirian dan mewawancarai responden perempuan). Tinggalkan isu yang sensitif atau tunda sampai wawancara ke dua. Jangan memaksa informan untuk menjawab. •• Biarkan informan berbicara dan bahkan sedikit menyimpang dari pertanyaan, tetapi jangan terlalu lama. •• Sediakan alat-alat peraga, peta atau gambar untuk membantu Anda menjelaskan suatu gagasan. •• Kegiatan-kegiatan, seperti membuat peta, merupakan kegiatan yang baik untuk menjaga minat masyarakat. Menyapa responden dengan senyum yang bersahabat. •• Terima mereka dengan ramah dan tawarkan beberapa kompensasi untuk pekerjaan yang tertunda, tetapi jangan membeli informasi. •• Jangan memberi janji-janji. •• Pastikan Anda menyampaikan terima kasih kepada para informan. Beritahu mereka bahwa Anda mungkin perlu kembali untuk mengecek. Panduan praktis untuk survei mata pencaharian sosial-ekonomi dan hak dan kepemilikan lahan 33 Catatan 7 – Diskusi kelompok terfokus Diskusi kelompok terfokus (FGD) bertujuan untuk mengumpulkan informasi yang lebih dalam tentang konsep, persepsi, dan ide-ide suatu kelompok berjumlah 6-12 orang. Idealnya suatu FGD adalah lebih dari sekedar proses interaksi bertanya dan menjawab. Pelaksanaan FGD merupakan suatu proses yang berulang- ulang, dimana setiap diskusi dikembangkan atas diskusi sebelumnya dengan membicarakan suatu topik atau menekankan pada aspek-aspek tertentu. Kegiatan ini membutuhkan seorang fasilitator untuk memandu anggota kelompok dalam mendiskusikan topik yang dibutuhkan. Hal ini bermanfaat jika sarana penelitian yang lain seperti wawancara informan kunci, wawancara mendalam, atau teknik kualitatif lainnya digunakan berkaitan dengan FGD. Dalam konteks proyek perencanaan penggunaan lahan kolaboratif yang melibatkan masyarakat lokal dalam kawasan hutan, suatu FGD dapat mencakup diskusi tentang tatanan kelembagaan, hak dan akses masyarakat ke hutan, Dalam konteks proyek perencanaan penggunaan lahan kolaboratif yang melibatkan masyarakat lokal dalam kawasan hutan, suatu FGD dapat mencakup diskusi tentang tatanan kelembagaan, hak dan akses masyarakat ke hutan, Nining Liswanti, Bayuni Shantiko, Emily Fripp, Esther Mwangi dan Yves Laumonier 34 penggunaan hutan oleh masyarakat dan pengelolaan sumberdaya hutan secara berkelanjutan. Contoh-contoh aspek yang dapat didiskusikan selama kegiatan FGD dapat dilihat di bawah ini (Tabel 4). penggunaan hutan oleh masyarakat dan pengelolaan sumberdaya hutan secara berkelanjutan. Contoh-contoh aspek yang dapat didiskusikan selama kegiatan FGD dapat dilihat di bawah ini (Tabel 4). Sebelum melakukan FGD, anggota tim harus melakukan analisis situasi, merekrut peserta, dan mengatur sesi-sesi diskusi. Sebelum melakukan FGD, anggota tim harus melakukan analisis situasi, merekrut peserta, dan mengatur sesi-sesi diskusi. 1. Analisis situasi, untuk memastikan adanya pengetahuan yang baik tentang kondisi lokal, untuk memastikan diskusi memperhitungkan isu-isu lokal dan demografi dan kondisi sosial. Ini akan membantu untuk membuat FGD yang ditargetkan. 2. Rekrutmen peserta, supaya perbedaan latar belakang yang lebar dapat terwakili di dalam kelompok. Kelompok-kelompok harus dibagi berdasarkan umur dan jender. Semua yang terlibat harus diyakinkan tentang kerahasiaan semua respon yang diberikan. Peserta harus diberitahu setidaknya satu hari sebelumnya tentang pertemuan ini untuk memastikan partisipasi yang maksimum. Jelaskan kepada peserta tentang pentingnya keragaman di dalam kelompok, yaitu untuk meyakinkan pendapat yang berbeda dapat diekspresikan. 3. Pengaturan sebelum diskusi. Komunikasi dan interaksi yang efektif menentukan kualitas hasil dari FGD. Jadi persiapan yang baik untuk FGD sangat penting. Persiapan ini termasuk pemilihan lokasi dan tempat yang Tabel 4. Contoh tema dan topik untuk bahan diskusi dalam FGD Tabel 4. Contoh tema dan topik untuk bahan diskusi dalam FGD Tema Topik •• Identifikasi pemangku kepentingan •• Situasi mengenai para pemangku kepentingan, kegiatan yang dilakukan dan potensi dampaknya •• Hak atas sumberdaya hutan •• Pemahaman tentang hak atas sumberdaya hutan •• Pola pemanfaatan sumberdaya hutan, distribusi keuntungan •• Pengelolaan hutan, penegakan aturan dan kepatuhan •• Stok sumberdaya alam •• Fungsi pengelolaan hutan •• Aturan dalam pengelolaan hutan, penegakan dan sanksi •• Pengambilan keputusan •• Persepsi •• Persepsi mengenai kepastian akses lahan dan bagaimana perubahan akses telah terjadi •• Proyek pembangunan desa •• Proyek yang pernah dilakukan dan bagaimana tingkat kesuksesannya •• Persepsi mengenai kepastian akses lahan dan bagaimana perubahan akses telah terjadi •• Proyek pembangunan desa •• Proyek yang pernah dilakukan dan bagaimana tingkat kesuksesannya •• Proyek yang pernah dilakukan dan bagaimana tingkat kesuksesannya anduan praktis untuk survei mata pencaharian sosial-ekonomi dan hak dan kepemilikan lahan 35 Nining Liswanti, Bayuni Shantiko, Emily Fripp, Esther Mwangi dan Yves Laumonier 36 dilakukan secara hati-hati, misalnya mengatur kursi secara melingkar dan tidak dalam format ruangan kelas, untuk menghindarkan gangguan dan meyakinkan lokasinya diyakini sebagai tempat yang aman oleh semua peserta. Siapkan serangkaian daftar pertanyaan terbuka secara tertulis yang akan di bahas dalam FGD. dilakukan secara hati-hati, misalnya mengatur kursi secara melingkar dan tidak dalam format ruangan kelas, untuk menghindarkan gangguan dan meyakinkan lokasinya diyakini sebagai tempat yang aman oleh semua peserta. Siapkan serangkaian daftar pertanyaan terbuka secara tertulis yang akan di bahas dalam FGD. 4. Selama sesi diskusi, pastikan pertukaran pendapat berlangsung efektif, anggota tim harus membagi tugas (misalnya, fasilitator (lihat Kotak 3), perekam/ pencatat). 4. Selama sesi diskusi, pastikan pertukaran pendapat berlangsung efektif, anggota tim harus membagi tugas (misalnya, fasilitator (lihat Kotak 3), perekam/ pencatat). Kotak 3. Peran fasilitator •• Fasilitator TIDAK harus bertindak sebagai pakar tentang topik yang didiskusikan. Peran fasilitator adalah untuk merangsang dan mengarahkan selama diskusi. •• Sebagai fasilitator, perkenalkan diri Anda dan anggota tim yang terlibat. Minta peserta mengenalkan diri dengan nama apa pun yang mereka ingin gunakan. •• Jelaskan tujuan dari FGD, jenis informasi yang dibutuhkan, dan bagaimana informasi akan digunakan. Mintalah izin untuk menggunakan alat perekam dan tekankan kembali tentang aturan kerahasiaan. •• Buat aturan diskusi yang disepakati oleh peserta, singkat dan padat. Misalnya, aturan merokok selama diskusi. •• Tunjukkan minat Anda pada ide-ide kelompok, lakukan kontak mata dengan peserta dan gunakan humor jika diperlukan. Gunakan pertanyaan yang mendorong peserta agar sebanyak mungkin mengekspresikan pandangan mereka. INGAT bahwa tidak ada jawaban ‘benar’ atau ‘salah’. Bereaksi secara netral dan wajar terhadap respon verbal dan nonverbal. •• Bangunlah hubungan baik dan berempati, dan hindari untuk berperan sebagai ahli. •• Jangan merasa Anda harus mengatakan sesuatu dalam setiap jeda dalam diskusi. Tunggu sebentar dan perhatikan apa yang terjadi. Jaga irama diskusi. Ingat peran fasilitator adalah memastikan peserta untuk berbagi pendapat. •• Dengarkan dengan seksama dan alihkan diskusi dari satu topik ke topik lainnya secara perlahan. Jika peserta spontan berpindah ke topik yang lain, biarkan diskusi berjalan sebentar hingga muncul informasi baru yang relevan, buat kesimpulan singkat dan kembalilah ke topik diskusi semula. •• Pada akhir pertemuan, jangan lupa membuat kesimpulan dan ringkasan hasil diskusi. Tanyakan peserta jika ada yang terlupakan dalam kesimpulan. Ucapkan terima kasih atas partisipasi dan waktu yang diberikan oleh peserta. Panduan praktis untuk survei mata pencaharian sosial-ekonomi dan hak dan kepemilikan lahan 37 5. Memastikan keterlibatan aktif dari semua peserta. Hal ini penting dan anggota tim perlu bekerja bersama untuk memastikan bahwa semua peserta terlibat merata dalam diskusi. Gunakan pertanyaan yang memotivasi untuk memastikan partisipasi peserta secara aktif. Bagi kelompok untuk mengakomodasi latar belakang dan pandangan yang kontras, jika hal ini akan menciptakan diskusi yang panjang. Jika diperlukan fasilitator harus bertanya kepada kelompok untuk mendapatkan respon. 6. Mencatat proses dan intisari hasil diskusi setelah FGD adalah penting. Pencatatan ini ditekankan pada topik utama yang didiskusikan; topik yang mungkin sulit untuk didiskusikan; besarnya kesepakatan di dalam kelompok; temuan yang tidak terduga; dan jika ada perubahan yang direkomendasikan untuk FGD di masa depan. 7. Perekaman semua informasi. Kotak 3. Peran fasilitator Keseluruhan diskusi dan semua pokok pembicaraan yang muncul harus direkam, karena proses ini didasarkan pada pertanyaan terbuka, untuk memastikan bahwa tidak ada yang terlupakan atau munculnya pokok-pokok yang di luar konteks. Hasil catatan harus dibagikan, jika memungkinkan, ditambahkan dengan hasil rekaman, menggunakan alat media yang memadai, seperti perekam suara atau sejenisnya. 8. Jumlah dan lamanya sesi; jangka waktu diskusi bervariasi menurut dinamika peserta, namun idealnya FGD akan memakan waktu antara 60 dan 90 menit. Sesi awal mungkin akan memakan waktu lebih lama saat peserta menerima informasi baru di kegiatan FGD. Nining Liswanti, Bayuni Shantiko, Emily Fripp, Esther Mwangi dan Yves Laumonier 38 Catatan 8 – Contoh kuesioner baku: Rumah tangga, desa, dan informan kunci Catatan 8 – Contoh kuesioner baku: Rumah tangga, desa, dan informan kunci Kuesioner 1. Profil desa – Informan kunci (Ka. desa, Ka. adat, dll.) Provinsi : .......................................................................................................................................... Kabupaten : .......................................................................................................................................... Kecamatan : .......................................................................................................................................... Desa : .......................................................................................................................................... Tanggal : .......................................................................................................................................... Pewawancara : .......................................................................................................................................... Pencatat : .......................................................................................................................................... Data informan kunci 1 Nama : .............................................................................................................................................. 2 Umur : .............................................................................................................................................. 3 Jabatan : .............................................................................................................................................. 4 Sejak kapan menjabat/kedudukan di desa ini? ...................................................................... 5 Apakah kecenderungan jabatan Kades turun temurun? ¨ Ya ¨ Tidak A. Desa A.1 Deskripsi desa A.1.1 Tipe desa: ¨ Lama ¨ Transmigrasi ¨ Baru ¨ Lainnya, sebutkan ............. A.1.2 Apakah masih ada masyarakat adat di sini, yang masih pakai hukum adat? ¨ Ya ¨ Tidak A.1.3 Siapa nama kepala adat? A.1.4 Kapan desa ini berdiri/terbentuk? ..................................................... Menurut sejarah, sekitar tahun berapa di desa ini terbentuk pemukiman? A.2 Dusun/Kampung A.2.1 Berapa banyak jumlah dusun di desa ini? .......... buah A.2.2 Nama masing-masing dusun? ........................................................... Catatan 8 – Contoh kuesioner baku: Rumah tangga, desa, dan informan kunci Kuesioner 1. Profil desa – Informan kunci (Ka. desa, Ka. adat, dll.) Provinsi : .......................................................................................................................................... Kabupaten : .......................................................................................................................................... Kecamatan : .......................................................................................................................................... Desa : .......................................................................................................................................... Tanggal : .......................................................................................................................................... Pewawancara : .......................................................................................................................................... Pencatat : .......................................................................................................................................... Data informan kunci 1 Nama : .............................................................................................................................................. 2 Umur : .............................................................................................................................................. 3 Jabatan : .............................................................................................................................................. 4 Sejak kapan menjabat/kedudukan di desa ini? ...................................................................... 5 Apakah kecenderungan jabatan Kades turun temurun? ¨ Ya ¨ Tidak A. Desa A.1 Deskripsi desa A.1.1 Tipe desa: ¨ Lama ¨ Transmigrasi ¨ Baru ¨ Lainnya, sebutkan ............. A.1.2 Apakah masih ada masyarakat adat di sini, yang masih pakai hukum adat? ¨ Ya ¨ Tidak A.1.3 Siapa nama kepala adat? A.1.4 Kapan desa ini berdiri/terbentuk? ..................................................... Menurut sejarah, sekitar tahun berapa di desa ini terbentuk pemukiman? A.2 Dusun/Kampung A.2.1 Berapa banyak jumlah dusun di desa ini? .......... buah A.2.2 Nama masing-masing dusun? ........................................................... Kuesioner 1. Profil desa – Informan kunci (Ka. desa, Ka. adat, dll.) Provinsi : .......................................................................................................................................... Kabupaten : .......................................................................................................................................... Kecamatan : .......................................................................................................................................... Desa : .......................................................................................................................................... Tanggal : .......................................................................................................................................... Pewawancara : .......................................................................................................................................... Pencatat : .......................................................................................................................................... Data informan kunci 1 Nama : .............................................................................................................................................. 2 Umur : .............................................................................................................................................. 3 Jabatan : .............................................................................................................................................. 4 Sejak kapan menjabat/kedudukan di desa ini? ...................................................................... 5 Apakah kecenderungan jabatan Kades turun temurun? ¨ Ya ¨ Tidak A. Desa A.1 Deskripsi desa A.1.1 Tipe desa: ¨ Lama ¨ Transmigrasi ¨ Baru ¨ Lainnya, sebutkan ............. Catatan 8 – Contoh kuesioner baku: Rumah tangga, desa, dan informan kunci A.1.2 Apakah masih ada masyarakat adat di sini, yang masih pakai hukum adat? ¨ Ya ¨ Tidak A.1.3 Siapa nama kepala adat? A.1.4 Kapan desa ini berdiri/terbentuk? ..................................................... Menurut sejarah, sekitar tahun berapa di desa ini terbentuk pemukiman? A.2 Dusun/Kampung A.2.1 Berapa banyak jumlah dusun di desa ini? .......... buah A.2.2 Nama masing-masing dusun? ........................................................... 39 A.2.4 Status lahan hutan yang dimanfaatkan (KOD) A. Kepemilikan lahan B. Pemanfaatan lahan Berdasarkan Hukum: 1. Hutan negara 2. Hutan hak 3. Hutan adat 4. Hak milik 5. HGU, HGB dan HPH 1. Dimanfaatkan oleh negara 2. Dimanfaatkan secara bersama 3. Dimanfaatkan oleh individu 4. Pemanfaatan dengan ijin (meminjam) 5. Dimanfaatkan tanpa ijin Berdasarkan adat/kepemilikan tradisional: 1. Hutan adat 2. Hak milik A.3 Batas desa A.3.1 Luas desa ini? (perkiraan) .......... ha A.3.2 Apakah batasan desa ini dengan desa lain, jelas/tidak jelas? ¨ Ya jelas ¨ Tidak cukup jelas A.3.3 Apa batas administratif (1), batas alam (2) di desa ini? Sekitar berapa kilo jauh dari sini (jarak dari pusat desa)? Arah Batas administratif Batas alam/fisik Keadaan Nama desa tetangga Jarak* (km) Geografi Jarak (km) Jelas Tidak Utara Selatan Barat Timur Geografi, bisa sungai, gunung, jalan, rumah besar, dan lain lain yang bisa dilihat dan dipakai sebagai referensi A.2.4 Status lahan hutan yang dimanfaatkan (KOD) A. Kepemilikan lahan B. Pemanfaatan lahan Berdasarkan Hukum: 1. Hutan negara 2. Hutan hak 3. Hutan adat 4. Hak milik 5. HGU, HGB dan HPH 1. Dimanfaatkan oleh negara 2. Dimanfaatkan secara bersama 3. Dimanfaatkan oleh individu 4. Pemanfaatan dengan ijin (meminjam) 5. Dimanfaatkan tanpa ijin Berdasarkan adat/kepemilikan tradisional: 1. Hutan adat 2. Hak milik A.3 Batas desa A.3.1 Luas desa ini? (perkiraan) .......... ha A.3.2 Apakah batasan desa ini dengan desa lain, jelas/tidak jelas? ¨ Ya jelas ¨ Tidak cukup jelas A.3.3 Apa batas administratif (1), batas alam (2) di desa ini? Sekitar berapa kilo jauh dari sini (jarak dari pusat desa)? Arah Batas administratif Batas alam/fisik Keadaan Nama desa tetangga Jarak (km) Geografi Jarak (km) Jelas Tidak Utara Selatan A.2.4 Status lahan hutan yang dimanfaatkan (KOD) 40 Nining Liswanti, Bayuni Shantiko, Emily Fripp, Esther Mwangi dan Yves Laumonier A.3.4 Apakah ada desa-desa yang melakukan kegiatan pertanian/peternakan mereka di luar batas desa? ¨ Ya ¨ Tidak A.3.5 Dimana mereka melakukan kegiatan tersebut? (lihat status lahan dan hutan, KOD) . ............................................................................................................... . ............................................................................................................... a Arah: 1 = Utara, 2 = Selatan, 3 = Barat, 4 = Timur b Jarak: 1 = 0-5 km, 2 = 5-10 km, 3 = 10-15 km, 4 = lebih dari 15 km c Lama perjalanan (jalan kaki): 1 = kurang 1 jam, 2 = 1-2 jam, 3 = 2-4 jam, 4 = lebih dari setengah hari d Hutan: 1 = hutan konservasi, 2 = hutan lindung, 3 = hutan produksi KOD diisi berdasarkan referensi status lahan (Tabel A.2.4) Catatan 8 – Contoh kuesioner baku: Rumah tangga, desa, dan informan kunci A.4 Lingkungan desa A.4.1 Hutan rakyat: Hutan rakyat (hutan HAK), dimiliki rakyat, di luar kawasan hutan negara A.4.1.1 Apakah ada hutan rakyat (hutan HAK) yang dimanfaatkan masyarakat? ¨ Ada ¨ Tidak A.4.1.2 Apa nama hutan rakyat (hutan HAK) itu? A.4.1.3 Bisa tolong jelaskan situasi hutan rakyat itu? Nama Luas Araha Jarakb Lama pergic Status hutan, KOD 40 a Arah: 1 = Utara, 2 = Selatan, 3 = Barat, 4 = Timur b Jarak: 1 = 0-5 km, 2 = 5-10 km, 3 = 10-15 km, 4 = lebih dari 15 km c Lama perjalanan (jalan kaki): 1 = kurang 1 jam, 2 = 1-2 jam, 3 = 2-4 jam, 4 = lebih dari setengah hari KOD diisi berdasarkan referensi status lahan (Tabel A.2.4) A.4.2 Hutan adat (petuanan, ulayat dll) hutan adat: Hutan milik negara, dikelola oleh adat. A.4.2.1 Apakah ada hutan adat yang dimanfaatkan masyarakat di sini? ¨ Ada ¨ Tidak ada A.4.2.2 Apa nama hutan adat itu? A.4.2.3 bisa tolong jelaskan situasi hutan adat itu? Catatan 8 – Contoh kuesioner baku: Rumah tangga, desa, dan informan kunci Nama Luas Araha Jarakb Lama pergic Di dalam hutan apad Status hutan, KOD a Arah: 1 = Utara, 2 = Selatan, 3 = Barat, 4 = Timur b Jarak: 1 = 0-5 km, 2 = 5-10 km, 3 = 10-15 km, 4 = lebih dari 15 km c Lama perjalanan (jalan kaki): 1 = kurang 1 jam, 2 = 1-2 jam, 3 = 2-4 jam, 4 = lebih dari setengah hari d Hutan: 1 = hutan konservasi, 2 = hutan lindung, 3 = hutan produksi KOD diisi berdasarkan referensi status lahan (Tabel A.2.4) a Arah: 1 = Utara, 2 = Selatan, 3 = Barat, 4 = Timur b Jarak: 1 = 0-5 km, 2 = 5-10 km, 3 = 10-15 km, 4 = lebih dari 15 km c Lama perjalanan (jalan kaki): 1 = kurang 1 jam, 2 = 1-2 jam, 3 = 2-4 jam, 4 = lebih dari setengah hari d Hutan: 1 = hutan konservasi, 2 = hutan lindung, 3 = hutan produksi KOD diisi berdasarkan referensi status lahan (Tabel A.2.4) a Arah: 1 = Utara, 2 = Selatan, 3 = Barat, 4 = Timur b Jarak: 1 = 0-5 km, 2 = 5-10 km, 3 = 10-15 km, 4 = lebih dari 15 km c Lama perjalanan (jalan kaki): 1 = kurang 1 jam, 2 = 1-2 jam, 3 = 2-4 jam, 4 = lebih dari setengah hari d Hutan: 1 = hutan konservasi, 2 = hutan lindung, 3 = hutan produksi KOD diisi berdasarkan referensi status lahan (Tabel A.2.4) Panduan praktis untuk survei mata pencaharian sosial-ekonomi dan hak dan kepemilikan lahan 41 A.4.3 Jarak ke Hutan Lindung Hutan lindung dikontrol oleh negara. Masyarakat boleh memanfaatkan hutan lindung kecuali menebang pohon. A.4.3.1 Apakah Bapak/Ibu/Saudara tahu di mana letak hutan lindung? ¨ Ya ¨ Tidak A.4.3.2 Bila ya, sebutkan namanya? A.4.3.3 Di mana arahnya dari desa ini? ¨ Utara ¨ Timur ¨ Selatan ¨ Barat A.4.3.4 Sekitar berapa kilometer jaraknya dari desa ini? .......... km A.4.3.5 Seandainya jalan kaki dari desa ini ke batas hutan lindung, berapa lama dapat ditempuh? Catatan 8 – Contoh kuesioner baku: Rumah tangga, desa, dan informan kunci ¨ Kurang satu jam ¨ 1-2 jam ¨ 2-4 jam ¨ Lebih dari setengah hari Nama hutan lindung Kategori (TN – HL – CA) Arah Jarak (km)a Lama (jam)b Jarak: 1 = 0-5 km, 2 = 5-10 km, 3 = 10-15 km, 4 = lebih dari 15 km a Jarak: 1 = 0-5 km, 2 = 5-10 km, 3 = 10-15 km, 4 = lebih dari 15 km a Jarak: 1 = 0-5 km, 2 = 5-10 km, 3 = 10-15 km, 4 = lebih dari 15 km b Lama perjalanan (jalan kaki): 1 = kurang 1 jam, 2 = 1-2 jam, 3 = 2-4 jam, 4 = lebih setengah hari A.4.4 Perusahaan A.4.4.1 Apakah desa ini berbatasan dengan perusahaan? Perusahaan ................. ¨ Ya (lanjut ke A.4.4.2) ¨ Belum (lanjut ke A.4.4.2) ¨ Tidak (lanjut ke sub B) A.4.4.2 Apakah Bapak/Ibu tahu dimana batas antara desa dan perusahaan tersebut? ¨ Ya ¨ Tidak A.4.4.3 Di mana arahnya dari desa ini? ¨ Utara ¨ Selatan ¨ Timur ¨ Barat A.4.4.4 Berapa kilometer jarak dari desa ini sampai batas perusahaan tersebut? A.4.4.5 Seandainya jalan kaki dari desa ini ke batas perusahaan tersebut, berapa lama dapat ditempuh? ¨ Kurang satu jam ¨ 1-2 jam ¨ 2-4 jam ¨ Lebih dari setengah hari Nining Liswanti, Bayuni Shantiko, Emily Fripp, Esther Mwangi dan Yves Laumonier 42 A.4.4.6 Berapa luas dan jenis kegiatan perusahaan tersebut? Nama perusahaan Luas lokasi Araha Jarak (km) Lamab Jenis kegiatanc Rencana/ Jadi Status hutan, KOD a Arah: 1 = utara, 2 = selatan, 3 = barat, 4 = timur b Lama perjalanan (jalan kaki): 1 = kurang 1 jam, 2 = 1-2 jam, 3 = 2-4 jam, 4 = lebih setengah hari c Jenis kegiatan: 1 = HPH, 2 = Perusahaan kelapa sawit, 3 = Perusahaan karet, 4 = Perusahaan coklat, 5 = Perusahaan pengalengan ikan, 6 = Perusahaan kelapa, 7 = HTI, 8 = Perusahaan kayu (sawmill), 9 = Perusahaan Peternakan besar, 10 = Perusahaan lain (sebut) KOD diisi berdasarkan referensi status lahan (Tabel A.2.4) A.4.4.6 Berapa luas dan jenis kegiatan perusahaan tersebut? Catatan 8 – Contoh kuesioner baku: Rumah tangga, desa, dan informan kunci Nama perusahaan Luas lokasi Araha Jarak (km) Lamab Jenis kegiatanc Rencana/ Jadi Status hutan, KOD a Arah: 1 = utara, 2 = selatan, 3 = barat, 4 = timur b Lama perjalanan (jalan kaki): 1 = kurang 1 jam, 2 = 1-2 jam, 3 = 2-4 jam, 4 = lebih setengah hari c Jenis kegiatan: 1 = HPH, 2 = Perusahaan kelapa sawit, 3 = Perusahaan karet, 4 = Perusahaan coklat, 5 = Perusahaan pengalengan ikan, 6 = Perusahaan kelapa, 7 = HTI, 8 = Perusahaan kayu (sawmill), 9 = Perusahaan Peternakan besar, 10 = Perusahaan lain (sebut) KOD diisi berdasarkan referensi status lahan (Tabel A 2 4) A.4.4.6 Berapa luas dan jenis kegiatan perusahaan tersebut? a Arah: 1 = utara, 2 = selatan, 3 = barat, 4 = timur b Lama perjalanan (jalan kaki): 1 = kurang 1 jam, 2 = 1-2 jam, 3 = 2-4 jam, 4 = lebih setengah hari c Jenis kegiatan: 1 = HPH, 2 = Perusahaan kelapa sawit, 3 = Perusahaan karet, 4 = Perusahaan coklat, 5 = Perusahaan pengalengan ikan, 6 = Perusahaan kelapa, 7 = HTI, 8 = Perusahaan kayu (sawmill), 9 = Perusahaan Peternakan besar, 10 = Perusahaan lain (sebut) KOD dii i b d k f i l h (T b l A 2 4) A.4.5 Masalah perbatasan A.4.5.1 Apakah batas desa ini jelas? ¨ Ya ¨ Tidak A.4.5.2 Apakah di desa ini pernah terjadi masalah mengenai batas dengan desa tetangga atau kawasan hutan negara (batas desa maupun batas hutan)? ¨ Ya ¨ Tidak A.4.5.3. Bila ya, sebutkan masalahnya? .............................................................. . ............................................................................................................... . ............................................................................................................... B. Demografi B.1 Penduduk B.1.1 Jumlah keluarga yang tinggal di desa tahun ini? .......... KK B.1.2 Jumlah keluarga yang tinggal di desa tahun yang lalu? .......... KK (Jumlah keluarga tahun ini dikurangi jumlah keluarga yang baru menikah dan keluar desa) B.1.3 Jumlah penduduk di desa ini? .......... Jiwa B.1.4 Jumlah penduduk di desa ini tahun yang lalu? .......... Jiwa 0-4 tahun 5-14 tahun 15-60 tahun >60 tahun Total tahun ini Total tahun lalu Keluarga xxxx xxxx xxxx xxxx Laki-laki Perempuan Total 43 B.2 Komposisi etnis B.2.1 Bagaimana komposisi penduduk desa B.2.1.1 Penduduk asli desa ini apa? ................................................................... B.2.1.2 Apakah ada pendatang di sini? Dari mana asal mereka? Berapa jumlah keluarga untuk pendatang? Catatan 8 – Contoh kuesioner baku: Rumah tangga, desa, dan informan kunci (tabel diisi kalau ada suku lain sebagai pendatang) Komposisi etnis (termasuk pendatang) Jumlah keluarga Jumlah jiwa 1 2 3 4 5 B.2.2 Bahasa daerah di sini bahasa apa? .......................................................... B.3 Komposisi agama B.3.1 Bagaimana komposisi penduduk desa ini menurut agama? B.3.2 Jumlah sarana ibadah yang ada di desa ini? Agama Jumlah keluarga Tempat ibadah Jumlah Kristen Protestan Gereja Katolik Katolik Gereja Protestan Islam Masjid/musholla Budha Wihara Hindu Pura Aliran kepercayaan Lain (sebut) C. Sarana dan prasarana C.1 Pendidikan C.1.1 Jenis sekolah apa saja yang ada di desa ini? C.1.2 Berapa gedung sekolah ada di desa ini? C.1.3 Berapa murid masuk sekolah tahun ini? B.2 Komposisi etnis B.2.1 Bagaimana komposisi penduduk desa B.2.1.1 Penduduk asli desa ini apa? ................................................................... B.2.1.2 Apakah ada pendatang di sini? Dari mana asal mereka? Berapa jumlah keluarga untuk pendatang? (tabel diisi kalau ada suku lain sebagai pendatang) Komposisi etnis (termasuk pendatang) Jumlah keluarga Jumlah jiwa 1 B.2 Komposisi etnis B.2.1 Bagaimana komposisi penduduk desa B.2.1.1 Penduduk asli desa ini apa? ................................................................... B.2.1.2 Apakah ada pendatang di sini? Dari mana asal mereka? Berapa jumlah keluarga untuk pendatang? (tabel diisi kalau ada suku lain sebagai pendatang) Komposisi etnis (termasuk pendatang) Jumlah keluarga Jumlah jiwa B.2.2 Bahasa daerah di sini bahasa apa? .......................................................... B.3 Komposisi agama B.3.1 Bagaimana komposisi penduduk desa ini menurut agama? B.3.2 Jumlah sarana ibadah yang ada di desa ini? Agama Jumlah keluarga Tempat ibadah Jumlah Kristen Protestan Gereja Katolik Katolik Gereja Protestan Islam Masjid/musholla Budha Wihara Hindu Pura Aliran kepercayaan Lain (sebut) C. Sarana dan prasarana C.1 Pendidikan C.1.1 Jenis sekolah apa saja yang ada di desa ini? C.1.2 Berapa gedung sekolah ada di desa ini? C.1.3 Berapa murid masuk sekolah tahun ini? C. Sarana dan prasarana C.1 Pendidikan C.1.1 Jenis sekolah apa saja yang ada di desa ini? C.1.2 Berapa gedung sekolah ada di desa ini? C.1.3 Berapa murid masuk sekolah tahun ini? 44 C.1.4 Berapa jumlah guru mengajar di sekolah yang ada di desa ini? (termasuk guru yang mengajar di sekolah tersebut, tetapi tidak tinggal di desa ini) Tingkat Jumlah murid Jumlah guru Jumlah gedung TK SD SLTP/SMP SLTA/SMA Perguruan tinggi C.1.5 Tingkat pendidikan penduduk Tingkat pendidikan Jumlah penduduk atau% Buta huruf SD atau di bawahnya Tamat SLTP/SMP Tamat SLTA/SMA Perguruan Tinggi C.2 Perumahan C.2.1 Jumlah rumah di desa ini? .......... rumah C.2.2 Apakah ada keluarga yang tidak punya rumah di desa ini dan tinggal menetap di kebun (berumah di kebun/pondok)? Catatan 8 – Contoh kuesioner baku: Rumah tangga, desa, dan informan kunci ¨ Ada ¨ Tidak ada C.2.3 Bila ya, ada berapa keluarga yang tidak mempunyai rumah di desa? .......... KK C.2.4 Berapa jumlah rumah yang dibangun dalam lima tahun terakhir? .......... buah rumah C.2.5 Berapa jumlah kayu yang diperlukan untuk membangun satu rumah? .......... m3 C.3 Kesehatan C.3.1 Jenis prasarana kesehatan yang ada di desa: ¨ Balai pengobatan ¨ Puskesmas ¨ Puskesmas pembantu ¨ Rumah bidan desa ¨ Lain ............................................................ C.3.2 Jumlah tenaga medis di desa ini: .......... orang C.3.3 Tenaga medis tertinggi yang ada di desa: ¨ Mantri ¨ Dokter ¨ Perawat ¨ Bidan desa ¨ Lain ................ C.3.4 Frekuensi kunjungan dokter? .......... kali/tahun – bulan – minggu 45 C.3.5 Penyakit paling menonjol: 1/.................. 2/.................. 3/.................. C.3.6 Jumlah bidan yang berijazah: .......... orang C.3.7 Jumlah bidan yang tidak berijazah: .......... orang C.3.8 Apakah ada orang di desa ini yang masih menggunakan pengobatan tradisional (dari tanaman dan lain-lain)? ¨ Ada ¨ Tidak ada C.3.9 Dari mana mereka ambil bahan obat itu? Dari mana? Ya Tidak Halaman rumah Sawah Kebun Ladang Hutan Belukar di sekitar desa Lain (sebutkan) C.4 Sanitasi lingkungan C.4.1 Bagaimana situasi energi dan sanitasi di desa ini? Sumber energi Jumlah rumah atau% Sejak kapan Kayu bakar Minyak tanah Arang (tempurung kelapa) Listrik PLN Listrik diesel Sumber air minum Sumur terbuka Sumur tertutup (sumur bor) Air minum dari sungai Jamban Pakai jamban C.5 Perhubungan C.5.1 Hubungan ke pasar Dimana masyarakat menjual hasil bumi? .............................................. C.5.1.1 Berapa jauh dari desa ke pasar besar terdekat? .......... km C.5.1.2 Nama pasar terdekat? C.3.5 Penyakit paling menonjol: 1/.................. 2/.................. 3/.................. C.3.6 Jumlah bidan yang berijazah: .......... orang C.3.7 Jumlah bidan yang tidak berijazah: .......... orang C.3.8 Apakah ada orang di desa ini yang masih menggunakan pengobatan tradisional (dari tanaman dan lain-lain)? ¨ Ada ¨ Tidak ada C.3.9 Dari mana mereka ambil bahan obat itu? Dari mana? Ya Tidak Halaman rumah Sawah Kebun Ladang Hutan Belukar di sekitar desa Lain (sebutkan) C.4 Sanitasi lingkungan C.4.1 Bagaimana situasi energi dan sanitasi di desa ini? Sumber energi Jumlah rumah atau% Sejak kapan Kayu bakar Minyak tanah Arang (tempurung kelapa) Listrik PLN Listrik diesel Sumber air minum Sumur terbuka Sumur tertutup (sumur bor) Air minum dari sungai Jamban Pakai jamban C.5 Perhubungan C.5.1 Hubungan ke pasar Dimana masyarakat menjual hasil bumi? .............................................. C.5.1.1 Berapa jauh dari desa ke pasar besar terdekat? .......... km C.5.1.2 Nama pasar terdekat? Catatan 8 – Contoh kuesioner baku: Rumah tangga, desa, dan informan kunci Nining Liswanti, Bayuni Shantiko, Emily Fripp, Esther Mwangi dan Yves Laumonier 46 C.5.1.3 Berapa kali seminggu kegiatan di pasar tersebut? ¨ 1 kali ¨ 2 kali ¨ 3 kali ¨ Lebih dari 3 kali ¨ Tiap hari C.5.1.4 Berapa jauh jarak dari desa ke pasar terdekat Jenis Jalan mobil Sungai Laut Lamaa Ongkos/orang Ongkos/kilogram Sejak kapan Frekuensi angkutan/minggu Aksesb a Lama perjalanan: 1 = kurang 1 jam, 2 = 1-2 jam, 3 = 2-4 jam, 4 = lebih dari 4 jam. b Mudah/sulit menghubungi desa: 1 = tetap bagus, 2 = tetap bisa tetapi sulit, 3 = bermusim tidak bisa dilalui (150-365 bisa dilalui), 4 = biasa tidak bisa dilalui (<150 hari) C.5.2 Hubungan ke kecamatan C.5.2.1 Berapa jauh dari desa ke ibu kota kecamatan? .......... km C.5.2.2 Apa nama ibu kota kecamatan? ............................................................. Jenis Jalan mobil Sungai Laut Lamaa Ongkos/orang Ongkos/kilogram Sejak kapan Frekuensi angkutan/minggu Aksesb a Lama: 1 = kurang 1 jam, 2 = 1-2 jam, 3 = 2-4 jam, 4 = lebih dari 4 jam. b Mudah/sulit menghubungi desa: 1 = tetap bagus, 2 = tetap bisa tetapi sulit, 3 = bermusim tidak bisa dilalui (150-365 bisa dilalui), 4 = biasa tidak bisa dilalui (<150 hari) D. Ekonomi desa D.1 Koperasi D.1.1 Apakah di desa ini ada KUD yang aktif? ¨ Ya ¨ Tidak (lanjut ke pertanyaan D.1.3.) D.1.2 Kegiatan KUD, antara lain (isi tabel di bawah ini) C.5.1.3 Berapa kali seminggu kegiatan di pasar tersebut? ¨ 1 kali ¨ 2 kali ¨ 3 kali ¨ Lebih dari 3 kali ¨ Tiap hari C.5.1.4 Berapa jauh jarak dari desa ke pasar terdekat Jenis Jalan mobil Sungai Laut Lamaa Ongkos/orang Ongkos/kilogram Sejak kapan Frekuensi angkutan/minggu Aksesb a Lama perjalanan: 1 = kurang 1 jam, 2 = 1-2 jam, 3 = 2-4 jam, 4 = lebih dari 4 jam. b Mudah/sulit menghubungi desa: 1 = tetap bagus, 2 = tetap bisa tetapi sulit, 3 = bermusim tidak bisa dilalui (150-365 bisa dilalui), 4 = biasa tidak bisa dilalui (<150 hari) C.5.2 Hubungan ke kecamatan C.5.2.1 Berapa jauh dari desa ke ibu kota kecamatan? .......... km C.5.2.2 Apa nama ibu kota kecamatan? ............................................................. Jenis Jalan mobil Sungai Laut Lamaa Ongkos/orang Ongkos/kilogram Sejak kapan Frekuensi angkutan/minggu Aksesb a Lama: 1 = kurang 1 jam, 2 = 1-2 jam, 3 = 2-4 jam, 4 = lebih dari 4 jam. Catatan 8 – Contoh kuesioner baku: Rumah tangga, desa, dan informan kunci b Mudah/sulit menghubungi desa: 1 = tetap bagus, 2 = tetap bisa tetapi sulit, 3 = bermusim tidak bisa dilalui (150-365 bisa dilalui), 4 = biasa tidak bisa dilalui (<150 hari) D. Ekonomi desa D.1 Koperasi D.1.1 Apakah di desa ini ada KUD yang aktif? ¨ Ya ¨ Tidak (lanjut ke pertanyaan D.1.3.) D.1.2 Kegiatan KUD, antara lain (isi tabel di bawah ini) D. Ekonomi desa D.1 Koperasi D.1.1 Apakah di desa ini ada KUD yang aktif? ¨ Ya ¨ Tidak (lanjut ke pertanyaan D.1.3.) D.1.2 Kegiatan KUD, antara lain (isi tabel di bawah ini) Kegiatan Kios bersama Simpan/pinjam Kios sarprodia Pemasaran Lain (sebut) Ya/Tidak a Sarprodi = sarana produksi Panduan praktis untuk survei mata pencaharian sosial-ekonomi dan hak dan kepemilikan lahan 47 D.1.3 Dimana masyarakat bisa memperoleh pinjaman uang/kredit? Sumber pinjaman Ya Tidak Sumber pinjaman Ya Tidak Bank IDT KUD Arisan Kas desa Lain (sebut) Tauke D.2 Prasarana dan saprodi pertanian D.2.1 Apakah di sini ada lahan irigasi? ¨ Ada ¨ Tidak ada (lanjut ke D.2.2.) Prasarana Sejak tahun Luas (ha) Jumlah keluarga Irigasi sederhana Irigasi semi-teknis Irigasi teknis D.2.2. Apa produk pertanian yang paling penting? Jenis penggunaan lahan Produk 1 Produk 2 Produk 3 Nama Ha Nama Ha Nama Ha Kebun rumah Ladang Sawah Kebun tanaman D.2.3 Berapa luas lahan yang ditanam dengan tanaman pertanian utama? D.2.3.1 Apakah di desa ini sudah menggunakan bibit unggul? ¨ Ya ¨ Tidak Komoditas D.2.3: Jenis utama tanaman pertanian D.2.3.1: Bibit unggul Luas total lahan* Status lahan, KOD Luas* Sejak Jumlah KK Jagung Padi D.1.3 Dimana masyarakat bisa memperoleh pinjaman uang/kredit? Sumber pinjaman Ya Tidak Sumber pinjaman Ya Tidak Bank IDT KUD Arisan Kas desa Lain (sebut) Tauke D.2 Prasarana dan saprodi pertanian D.2.1 Apakah di sini ada lahan irigasi? ¨ Ada ¨ Tidak ada (lanjut ke D.2.2.) Prasarana Sejak tahun Luas (ha) Jumlah keluarga Irigasi sederhana Irigasi semi-teknis Irigasi teknis D.2.2. Apa produk pertanian yang paling penting? Jenis penggunaan lahan Produk 1 Produk 2 Produk 3 Nama Ha Nama Ha Nama Ha Kebun rumah Ladang Sawah Kebun tanaman D.2.3 Berapa luas lahan yang ditanam dengan tanaman pertanian utama? D.2.3.1 Apakah di desa ini sudah menggunakan bibit unggul? ¨ Ya ¨ Tidak Komoditas D.2.3: Jenis utama tanaman pertanian D.2.3.1: Bibit unggul Luas total lahan* Status lahan, KOD Luas* Sejak Jumlah KK Jagung Padi Karet D.1.3 Dimana masyarakat bisa memperoleh pinjaman uang/kredit? Catatan 8 – Contoh kuesioner baku: Rumah tangga, desa, dan informan kunci KOD diisi berdasarkan referensi status lahan (Tabel A.2.4) Nining Liswanti, Bayuni Shantiko, Emily Fripp, Esther Mwangi dan Yves Laumonier 48 D.2.3.2 Apakah di desa ini sudah menggunakan pupuk/pestisida (obat-obatan)/ herbisida/fungisida? ¨ Ya (isi tabel di bawah) ¨ Tidak Komoditas Pupuk Pestisida Herbisida Fungisida Luas (ha) Sejak KK Luas (ha) Sejak KK Luas (ha) Sejak KK Luas (ha) Sejak KK Jagung Padi D.2.4 Aset desa D.2.4.1 Berapa jumlah gergaji rantai (chainsaw) yang beroperasi di desa ini .......... buah D.2.4.2 Berapa warga yang memiliki gergaji rantai di desa ini .......... keluarga D.2.4.3 Kepemilikan aset di desa? Jumlah total Jumlah KK yang memiliki Mobil/truk Sepeda motor TV Parabola antenna CD/VCD/DVD/player Telepon genggang/telpon Radio Perahu motor/mesin ketinting Kulkas Panduan praktis untuk survei mata pencaharian sosial-ekonomi dan hak dan kepemilikan lahan 49 D.2.5 Ongkos kegiatan pertanian D.2.5.1 Harga tanah/ha Jenis Lahan Beli Sewa/setahun Kosong Sudah ditanami Kosong Sudah ditanam Lahan kering dekat desa Lahan kering jauh dari desa Lahan buka baru D.2.5.2 Biaya produksi pertanian (upah harian kerja) Jenis lahan Membajak lahan/ menanami Merawat/menyiangi Panen Laki-laki Perempuan Laki-laki Perempuan Laki-laki Perempuan Ladang Kebun D.2.5.3 Biaya membuka tanah (1 ha)/luas membuka lahan menurut kebiasaan setempat: D.2.5.3.1 Berapa luas rata-rata orang membuka lahan pertanian? Luas ................. D.2.5.3.2 Berapa biaya pembukaan lahan kebun/belukar maupun hutan? Borongan upah harian kerja Kebun/belukar Hutan Hari kerja Upah harian Hari kerja Upah harian Menebang pohon (termasuk sewa gergaji/chainsaw) Membelah/membagi/ menggergaji pohon Mengangkut kayu Membersihkan lahan Membakar lahan D.2.5.4 Siapa di desa ini yang memutuskan untuk membuka lahan baru? ¨ Keluarga sendiri ¨ Kepala desa ¨ Kolektif tingkat masyarakat ¨ Lain (menjelaskan) .......................................................................... D.2.5.3 Biaya membuka tanah (1 ha)/luas membuka lahan menurut kebiasaan setempat: D.2.5.3.1 Berapa luas rata-rata orang membuka lahan pertanian? Luas ................. D.2.5.3.2 Berapa biaya pembukaan lahan kebun/belukar maupun hutan? Borongan upah harian kerja Kebun/belukar Hutan Hari kerja Upah harian Hari kerja Upah harian Menebang pohon (termasuk sewa gergaji/chainsaw) Membelah/membagi/ menggergaji pohon Mengangkut kayu Membersihkan lahan Membakar lahan D.2.5.4 Siapa di desa ini yang memutuskan untuk membuka lahan baru? ¨ Keluarga sendiri ¨ Kepala desa ¨ Kolektif tingkat masyarakat ¨ Lain (menjelaskan) .......................................................................... Nining Liswanti, Bayuni Shantiko, Emily Fripp, Esther Mwangi dan Yves Laumonier 50 D.2.6 Apa hasil utama di desa ini, dan bila dijual, dalam bentuk apa? Hasil pertanian Kalau dijual, apa bentuknya dan berapa harganya? Bentuk jual (kering/ basah) Pembeli ke petani/ ke pasar Kotor Harga/Kg Bersih Harga/Kg Satuan* Berapa hasil? Catatan 8 – Contoh kuesioner baku: Rumah tangga, desa, dan informan kunci Tahun lalu Tahun ini Tahun lalu Tahun ini Padi Gabah Beras D.2.6 Apa hasil utama di desa ini, dan bila dijual, dalam bentuk apa? Hasil pertanian Kalau dijual, apa bentuknya dan berapa harganya? Bentuk jual (kering/ basah) Pembeli ke petani/ ke pasar Kotor Harga/Kg Bersih Harga/Kg Satuan* Berapa hasil? Tahun lalu Tahun ini Tahun lalu Tahun ini Padi Gabah Beras Catatan: ) jika menggunakan ukuran lokal (UL), berapa UL = .......... kg D.2.7 Kelompok tani D.2.7.1 Apakah di desa ini sudah ada kelompok tani? ¨ Ya ¨ Tidak (lanjut ke D.3.) D.2.7.2 Untuk jenis tanaman apa? (sebutkan di tabel di bawah) D.2.7.3 Apakah kelompok tani itu secara ¨ Formal (resmi) ¨ Informal (isi tabel di bawah) D.2.7.4 Berapa jumlah anggota tani laki-laki? D.2.7.5 Apakah ada kelompok tani wanita? (isi tabel di bawah) Nama kelompok tani Jenis tanaman Status (formal/informal) Jumlah anggota laki-laki Jumlah anggota perempuan Catatan: ) jika menggunakan ukuran lokal (UL), berapa UL = .......... kg D.2.7 Kelompok tani D.2.7.1 Apakah di desa ini sudah ada kelompok tani? ¨ Ya ¨ Tidak (lanjut ke D.3.) D.2.7.2 Untuk jenis tanaman apa? (sebutkan di tabel di bawah) D.2.7.3 Apakah kelompok tani itu secara ¨ Formal (resmi) ¨ Informal (isi tabel di bawah) D.2.7.4 Berapa jumlah anggota tani laki-laki? D.2.7.5 Apakah ada kelompok tani wanita? (isi tabel di bawah) Nama kelompok tani Jenis tanaman Status (formal/informal) Jumlah anggota laki-laki Jumlah anggota perempuan Catatan: ) jika menggunakan ukuran lokal (UL), berapa UL = .......... kg kg D.3 Peternakan dan perikanan D.3.1 Jenis ternak di desa ini? Jenis Jumlah ekor Jumlah keluarga Sistem pemeliharaana Kerbau Sapi Kuda Babi Kambing/domba Bebek/itik Ayam Lain (sebutkan) .......... a Sistem pemeliharaan: 1 = liar, 2 = ikat, 3 = kandang malam, 4 = gembala, 5 = kandang tetap D.3 Peternakan dan perikanan D.3.1 Jenis ternak di desa ini? Jenis Jumlah ekor Jumlah keluarga Sistem pemeliharaana Kerbau Sapi Kuda Babi Kambing/domba Bebek/itik Ayam Lain (sebutkan) .......... a Status: 1 = tetap sepanjang tahun, 2 = musiman, 3 = tidak tetap dalam tahun, 4 = tidak tetap antar tahun, 5 = budidaya b Tulis jenis produksi/budidaya seperti tangkapan ikan utama, rumput laut, dsb. Catatan 8 – Contoh kuesioner baku: Rumah tangga, desa, dan informan kunci a Sistem pemeliharaan: 1 = liar, 2 = ikat, 3 = kandang malam, 4 = gembala, 5 = kandang tetap Jumlah keluarga Sistem pemeliharaana a Sistem pemeliharaan: 1 = liar, 2 = ikat, 3 = kandang malam, 4 = gembala, 5 = kandang tetap Panduan praktis untuk survei mata pencaharian sosial-ekonomi dan hak dan kepemilikan lahan 51 D.3.2 Harga ternak g Harga/ ekor Harga/ ekor Kerbau Jantan Dewasa Babi Jantan Dewasa Anak Anak Betina Dewasa Betina Dewasa Anak Anak Sapi Jantan Dewasa Anak Betina Dewasa Anak Kambing /domba Jantan Dewasa Bebek Anak Ayam Betina Dewasa .......... Anak .......... D.3.3 Perikanan (Darat/Laut) Jenis alat tangkap Status nelayana Jumlah keluarga Jenis produksib Harga/kg Keramba Jermal Jaring Bubu .......... .......... a Status: 1 = tetap sepanjang tahun, 2 = musiman, 3 = tidak tetap dalam tahun, 4 = tidak tetap antar tahun, 5 = budidaya b Tulis jenis produksi/budidaya seperti tangkapan ikan utama, rumput laut, dsb. a Status: 1 = tetap sepanjang tahun, 2 = musiman, 3 = tidak tetap dalam tahun, 4 = tidak tetap antar tahun, 5 = budidaya b Tulis jenis produksi/budidaya seperti tangkapan ikan utama, rumput laut, dsb. b Tulis jenis produksi/budidaya seperti tangkapan ikan utama, rumput laut, dsb. 52 D.4 Kegiatan ekonomi D.4.1 Kegiatan sumber penghidupan yang paling penting? (kalau bisa diurutkan dari yang paling penting) No Kegiatan Statusa Jumlah Keluarga 1 2 3 4 5 6 7 8 a Status: 1 = tetap sepanjang tahun, 2 = musiman, 3 = tidak tetap dalam tahun, 4 = tidak tetap antar tahun D.4.2 Pengambilan hasil hutan No Kegiatan Statusa Jumlah keluarga Harga/Unit Diambil dari mana (KOD) 1 2 3 4 5 6 7 8 a Status: 1 = tetap sepanjang tahun, 2 = musiman, 3 = tidak tetap dalam tahun, 4 = tidak tetap antar tahun KOD diisi berdasarkan referensi status lahan (Tabel A.2.4) D.4.2.1 Berapa jumlah kayu bakar digunakan oleh keluarga (2 dewasa, 3 anak) dalam satu bulan? .......... D.4.2.2 Berapa unit kayu dipakai untuk membangun 1 rumah biasa (dihuni oleh 2 dewasa, 2 anak)? .......... m3 D.4.2.1 Berapa jumlah kayu bakar digunakan oleh keluarga (2 dewasa, 3 anak) dalam satu bulan? .......... D.4.2.2 Berapa unit kayu dipakai untuk membangun 1 rumah biasa (dihuni oleh 2 dewasa, 2 anak)? .......... m3 Panduan praktis untuk survei mata pencaharian sosial-ekonomi dan hak dan kepemilikan lahan 53 D.5 Distribusi penggunaan lahan Luas (Hektar) Jumlah KK Ladang Kebun komoditas I.......... 5 = tidak berhasil Catatan 8 – Contoh kuesioner baku: Rumah tangga, desa, dan informan kunci Kebun komoditas II.......... Kebun komoditas III.......... Tidak memiliki lahan 0,1 < lahan < 1 hektar 1 < lahan < 1,9 hektar 2 < lahan < 4,9 hektar 5 < lahan < 10 hektar Di atas 10 hektar E. Program pembangunan E.1 Bantuan pembangunan desa (lima tahun terakhir) dari pemerintah Jenis program Tahun S Keluarga Jenis kegiatan Keberhasilana a Keberhasilan: 1 = sangat berhasil, 2 = berhasil, 3 = setengah berhasil, 4 = kurang berhasil, 5 = tidak berhasil Catatan: Inpres untuk bantuan desa tidak termasuk (karena rutin – tiap desa dapat) E.2 Kegiatan LSM Jenis program Tahun S Keluarga Jenis kegiatan Keberhasilana D.5 Distribusi penggunaan lahan Luas (Hektar) Jumlah KK Ladang Kebun komoditas I.......... Kebun komoditas II.......... Kebun komoditas III.......... Tidak memiliki lahan 0,1 < lahan < 1 hektar 1 < lahan < 1,9 hektar 2 < lahan < 4,9 hektar 5 < lahan < 10 hektar Di atas 10 hektar E. Program pembangunan E.1 Bantuan pembangunan desa (lima tahun terakhir) dari pemerintah Jenis program Tahun S Keluarga Jenis kegiatan Keberhasilana a Keberhasilan: 1 = sangat berhasil, 2 = berhasil, 3 = setengah berhasil, 4 = kurang berhasil, 5 = tidak berhasil Catatan: Inpres untuk bantuan desa tidak termasuk (karena rutin – tiap desa dapat) a Keberhasilan: 1 = sangat berhasil, 2 = berhasil, 3 = setengah berhasil, 4 = kurang berhasil, 5 id k b h il 5 = tidak berhasil Nining Liswanti, Bayuni Shantiko, Emily Fripp, Esther Mwangi dan Yves Laumonier 54 E.3 Kegiatan kehutanan Jenis program Tahun S Keluarga Jenis kegiatan Keberhasilana a Keberhasilan: 1 = sangat berhasil, 2 = berhasil, 3 = setengah berhasil, 4 = kurang berhasil, 5 = tidak berhasil F. Permasalahan hutan dan desa Bentuk-bentuk masalah Ada/ tidak ada Tahun kejadian Keterangan penting Sebagian besar warga tidak tahu status hutan Tata batas hutan belum jelas Tata batas sudah jelas tetapi belum diterima oleh masyarakat Lahan masyarakat di dalam kawasan Pengambilan kayu oleh orang luar desa Pengambilan kayu oleh orang desa Penangkapan satwa/burung Ada gangguan kegiatan tradisional di hutan Alat/barang disita pihak kehutanan Terjadi banjir/tanah longsor rutin Hama dari hutan Kebakaran hutan di sekitar desa Kebakaran kebun di sekitar desa Masa paceklik desa Persengketaan tanah antar desa Persengketaan tanah dalam desa Pencurian ternak masyarakat Wabah penyakit ternak Hama belalang, tikus, wereng Wabah penyakit manusia Masalah kekeringan/kurang air bersih Masalah lainnya, sebutkan .......... Catatan 8 – Contoh kuesioner baku: Rumah tangga, desa, dan informan kunci a Keberhasilan: 1 = sangat berhasil, 2 = berhasil, 3 = setengah berhasil, 4 = kurang berhasil, 5 = tidak berhasil F. Permasalahan hutan dan desa Bentuk-bentuk masalah Ada/ tidak ada Tahun kejadian Keterangan penting Sebagian besar warga tidak tahu status hutan Tata batas hutan belum jelas Tata batas sudah jelas tetapi belum diterima oleh masyarakat Lahan masyarakat di dalam kawasan Pengambilan kayu oleh orang luar desa Pengambilan kayu oleh orang desa Penangkapan satwa/burung Ada gangguan kegiatan tradisional di hutan Alat/barang disita pihak kehutanan Terjadi banjir/tanah longsor rutin Hama dari hutan Kebakaran hutan di sekitar desa Kebakaran kebun di sekitar desa Masa paceklik desa Persengketaan tanah antar desa Persengketaan tanah dalam desa Pencurian ternak masyarakat Wabah penyakit ternak Hama belalang, tikus, wereng Wabah penyakit manusia Masalah kekeringan/kurang air bersih Masalah lainnya, sebutkan .......... 55 Nining Liswanti, Bayuni Shantiko, Emily Fripp, Esther Mwangi dan Yves Laumonier 56 Kuesioner 2. Survei rumah tangga Provinsi : .......................................................................................................................................... Kabupaten : .......................................................................................................................................... Kecamatan : .......................................................................................................................................... Desa : .......................................................................................................................................... Responden : .......................................................................................................................................... Tanggal : .......................................................................................................................................... Pewawancara : .......................................................................................................................................... Pencatat : .......................................................................................................................................... A. Data umum A.1 Keluarga A.1.1 Nama responden: ............................................................................. A.1.2 Berapa usia Bapak/Ibu: ..................................................................... A.1.3 Jenis kelamin ¨ Laki-laki ¨ Perempuan A.1.4 Apakah Anda kepala keluarga ini? ¨ Ya (A.1.6) ¨ Tidak (A.1.5) A.1.5 Hubungan dengan kepala keluarga ¨ Suami ¨ Istri ¨ Anak ¨ Lainnya ........................................ A.1.6 Apakah Anda lahir di desa ini? ¨ Ya ¨ Tidak A.1.7 Apakah Anda asli berasal dari desa ini? ¨ Ya ¨ Tidak, ................. A.1.8 Jumlah anak ¨ .......... Laki-laki ¨ .......... Perempuan A.1.9 Jumlah tanggungan ¨ .......... Anak laki-laki ¨ .......... Anak perempuan ¨ .......... Orang tua A.1.10 Orang (selain keluarga inti) yang masih ditanggung dan tinggal di rumah (>6 bulan) ¨ .......... Laki-laki ¨ .......... Perempuan A.1.11 Komposisi rumah tangga Umur (tahun) Laki-laki Perempuan Jumlah 0 – 4 5 – 14 Kuesioner 2. Survei rumah tangga A. Data umum A.1 Keluarga A.1.1 Nama responden: ............................................................................. A.1.2 Berapa usia Bapak/Ibu: ..................................................................... A.1.3 Jenis kelamin ¨ Laki-laki ¨ Perempuan A.1.4 Apakah Anda kepala keluarga ini? ¨ Ya (A.1.6) ¨ Tidak (A.1.5) A.1.5 Hubungan dengan kepala keluarga ¨ Suami ¨ Istri ¨ Anak ¨ Lainnya ........................................ A.1.6 Apakah Anda lahir di desa ini? ¨ Ya ¨ Tidak A.1.7 Apakah Anda asli berasal dari desa ini? ¨ Ya ¨ Tidak, ................. A.1.8 Jumlah anak ¨ .......... Laki-laki ¨ .......... Perempuan A.1.9 Jumlah tanggungan ¨ .......... Anak laki-laki ¨ .......... Catatan 8 – Contoh kuesioner baku: Rumah tangga, desa, dan informan kunci Anak perempuan ¨ .......... Orang tua A.1.10 Orang (selain keluarga inti) yang masih ditanggung dan tinggal di rumah (>6 bulan) ¨ .......... Laki-laki ¨ .......... Perempuan A.1.11 Komposisi rumah tangga Umur (tahun) Laki-laki Perempuan Jumlah 0 – 4 5 – 14 15 – 60 > 60 Jumlah Panduan praktis untuk survei mata pencaharian sosial-ekonomi dan hak dan kepemilikan lahan 57 A.2 Data pendidikan A.2.1 Jumlah anak yang sedang sekolah Sekolah Laki-laki Perempuan SD SMP SMA Perguruan Tinggi (PT) Jumlah A.2.2 Ijazah tertinggi untuk anggota keluarga yang tidak bersekolah lagi SD SMP SMA PT Suami Istri Anak laki-laki Anak perempuan B. Data pertanian musim tanam yang lalu B.1 Sistem pertanian musim tanam yang lalu B.1.1 Pada musim tanam yang lalu, apakah Bapak/Ibu mengusahakan kegiatan pertanian ¨ Ya ¨ Tidak (B.1.3) B.1.2 Di lahan siapa, kegiatan pertanian dilakukan? ¨ Milik sendiri (Tabel B.1.6) ¨ Orang lain (Tabel B.1.8) B.1.3 Apakah Bapak/Ibu memiliki lahan yang dikerjakan orang lain? ¨ Ya (isi Tabel B.1.8) ¨ Tidak B.1.4 Apakah tanah yang Bapak usahakan memiliki sertifikat? ¨ Ya ¨ Tidak B.1.5 Apa jenis sertifikat tersebut Sertifikat Luas (ha) Hak milik Hak guna usaha Hak guna bangunan Girik, SKT (surat keterangan tanah) Lainnya A.2 Data pendidikan A.2.1 Jumlah anak yang sedang sekolah Sekolah Laki-laki Perempuan SD SMP SMA Perguruan Tinggi (PT) Jumlah A.2.2 Ijazah tertinggi untuk anggota keluarga yang tidak bersekolah lagi SD SMP SMA PT Suami Istri Anak laki-laki Anak perempuan B. Data pertanian musim tanam yang lalu B.1 Sistem pertanian musim tanam yang lalu B.1.1 Pada musim tanam yang lalu, apakah Bapak/Ibu mengusahakan kegiatan pertanian ¨ Ya ¨ Tidak (B.1.3) B.1.2 Di lahan siapa, kegiatan pertanian dilakukan? ¨ Milik sendiri (Tabel B.1.6) ¨ Orang lain (Tabel B.1.8) B.1.3 Apakah Bapak/Ibu memiliki lahan yang dikerjakan orang lain? ¨ Ya (isi Tabel B.1.8) ¨ Tidak B.1.4 Apakah tanah yang Bapak usahakan memiliki sertifikat? ¨ Ya ¨ Tidak B.1.5 Apa jenis sertifikat tersebut Sertifikat Luas (ha) Hak milik Hak guna usaha Hak guna bangunan Girik, SKT (surat keterangan tanah) Lainnya Ijazah tertinggi untuk anggota keluarga yang tidak bersekolah lagi B. Data pertanian musim tanam yang lalu B.1 Sistem pertanian musim tanam yang lalu B.1.1 Pada musim tanam yang lalu, apakah Bapak/Ibu mengusahakan kegiatan pertanian ¨ Ya ¨ Tidak (B.1.3) B.1.2 Di lahan siapa, kegiatan pertanian dilakukan? ¨ Milik sendiri (Tabel B.1.6) ¨ Orang lain (Tabel B.1.8) B.1.3 Apakah Bapak/Ibu memiliki lahan yang dikerjakan orang lain? Catatan 8 – Contoh kuesioner baku: Rumah tangga, desa, dan informan kunci ¨ Ya (isi Tabel B.1.8) ¨ Tidak B.1.4 Apakah tanah yang Bapak usahakan memiliki sertifikat? ¨ Ya ¨ Tidak B.1.5 Apa jenis sertifikat tersebut Sertifikat Luas (ha) Hak milik Hak guna usaha Hak guna bangunan Girik, SKT (surat keterangan tanah) Lainnya Luas (ha) Nining Liswanti, Bayuni Shantiko, Emily Fripp, Esther Mwangi dan Yves Laumonier 58 B.1.6 Kegiatan pertanian musim tanam yang lalu – menggarap lahan/ tanah sendiri Lahan milik sendiri, mengusahakan sendiri Jenis lahan Luasa Jarakb Dimanac KOD Tanaman utama Sejak Tanaman lainnya Tanaman pangan Ladang Kebun sayuran Pekarangan Sawah (irigasi, rawa ) Padang rumput penggembalaan Kebun/dusun ) Kebun karet Kebun .......... a Kalau pakai ukuran lokal,1 ukuran lokal = .......... ha atau m2 b Jarak dihitung dari lamanya jalan kaki ke lokasi (dalam menit) c Arah dan nama lokal (kalau ada) ) coret yang tidak perlu Pengisian KOD, lihat Tabel B.1.7 B.1.7 Status Lahan Hutan yang dimanfaatkan (KOD) A. Kepemilikan Lahan B. Pemanfaatan Lahan Berdasarkan Hukum 1 Hutan negara 1. Dimanfaatkan oleh negara 2 Dimanfaatkan secara bersama B.1.6 Kegiatan pertanian musim tanam yang lalu – menggarap lahan/ tanah sendiri Lahan milik sendiri, mengusahakan sendiri Jenis lahan Luasa Jarakb Dimanac KOD Tanaman utama Sejak Tanaman lainnya Tanaman pangan Ladang Kebun sayuran Pekarangan Sawah (irigasi, rawa ) Padang rumput penggembalaan Kebun/dusun ) Kebun karet Kebun .......... a Kalau pakai ukuran lokal,1 ukuran lokal = .......... ha atau m2 b Jarak dihitung dari lamanya jalan kaki ke lokasi (dalam menit) c Arah dan nama lokal (kalau ada) ) coret yang tidak perlu Pengisian KOD, lihat Tabel B.1.7 B.1.7 Status Lahan Hutan yang dimanfaatkan (KOD) A. Kepemilikan Lahan B. Pemanfaatan Lahan Berdasarkan Hukum 1. Hutan negara 2. Hutan hak 3. Hutan adat 4. Hak milik 5. HGU, HGB dan HPH 1. Dimanfaatkan oleh negara 2. Dimanfaatkan secara bersama 3. Dimanfaatkan oleh individu 4. Pemanfaatan dengan ijin (meminjam) 5. Dimanfaatkan tanpa ijin Berdasarkan adat/kepemilikan tradisional: 1 Hutan adat B.1.7 Status Lahan Hutan yang dimanfaatkan (KOD) A. Kepemilikan Lahan B. Pemanfaatan Lahan Berdasarkan Hukum 1. Hutan negara 2. Hutan hak 3. Hutan adat 4. Hak milik 5. HGU, HGB dan HPH 1. Dimanfaatkan oleh negara 2. Dimanfaatkan secara bersama 3. Dimanfaatkan oleh individu 4. Pemanfaatan dengan ijin (meminjam) 5. Dimanfaatkan tanpa ijin Berdasarkan adat/kepemilikan tradisional: 1. Hutan adat 2. Hak milik B.1.7 Status Lahan Hutan yang dimanfaatkan (KOD) A. Kepemilikan Lahan B. Pemanfaatan Lahan Berdasarkan Hukum 1. Hutan negara 2. Hutan hak 3. Catatan 8 – Contoh kuesioner baku: Rumah tangga, desa, dan informan kunci Hutan adat 4. Hak milik 5. HGU, HGB dan HPH 1. Dimanfaatkan oleh negara 2. Dimanfaatkan secara bersama 3. Dimanfaatkan oleh individu 4. Pemanfaatan dengan ijin (meminjam) 5. Dimanfaatkan tanpa ijin Berdasarkan adat/kepemilikan tradisional: 1. Hutan adat 2. Hak milik Panduan praktis untuk survei mata pencaharian sosial-ekonomi dan hak dan kepemilikan lahan 59 B.1.8 Kegiatan pertanian musim tanam yang lalu – mengusahakan lahan/tanah orang lain atau lahan/tanah dikerjakan oleh orang lain Mengerjakan lahan/tanah orang lain Jenis lahan Luasa Jarakb Dimanac KOD Tanaman utama Sejak Tanaman lainnya Sistem pembayaran Tanaman pangan Ladang Kebun sayuran Pekarangan Sawah (irigasi, rawa ) Padang rumput penggembalaan Kebun (termasuk buah-buahan) Kebun karet Kebun .......... a Kalau pakai ukuran lokal, 1 ukuran lokal = .......... ha atau m2 b Jarak dihitung dari lamanya jalan kaki ke lokasi (dalam menit) c Arah dan nama lokal (kalau ada) ) coret yang tidak perlu KOD diisi berdasarkan referensi status lahan (Tabel B.1.7) B.2 Sarana produksi yang dipakai pada musim tanam yang lalu B.2 Sarana produksi yang dipakai pada musim tanam yang lalu B.2.1 Apakah Bapak/Ibu menggunakan bibit/pupuk/insektisida/herbisida pada lahan yang ditanami tanaman pangan pada musim tanam yang lalu: ¨ Ya (isi tabel B.2.2) ¨ Tidak (B.3) B.2.2 Sarana produksi lahan tanaman pangan Komoditas Bibit Pupuk Herbisida Insektisida Fungisida Kg Rp Nama Kg Rp Kg Rp Kg Rp Kg Rp Padi ladang Jagung 60 Nining Liswanti, Bayuni Shantiko, Emily Fripp, Esther Mwangi dan Yves Laumonier B.2.3 Sarana produksi lahan perkebunan Komoditas Bibit Pupuk Herbisida Insektisida Fungisida Kg Rp Nama Kg Rp Kg Rp Kg Rp Kg Rp Karet B.3. Tenaga kerja B.3.1 Apakah Bapak/Ibu menggunakan tenaga kerja di luar keluarga? B.3.2 Biaya tenaga kerja: Lahan tanaman pangan (tahun yang lalu) Komoditas Biaya tenaga kerja di luar keluarga (dalam Rupiah) Pengolahan/ membajak tanah Menanam Merawat/ menyiangi Panen Pengolahan bahan panen Penjualan (termasuk transport) Padi Nining Liswanti, Bayuni Shantiko, Emily Fripp, Esther Mwangi dan Yves Laumonier Nining Liswanti, Bayuni Shantiko, Emily Fripp, Esther Mwangi dan Yves Laumonier 60 B.2.3 Sarana produksi lahan perkebunan Komoditas Bibit Pupuk Herbisida Insektisida Fungisida Kg Rp Nama Kg Rp Kg Rp Kg Rp Kg Rp Karet B.2.3 Sarana produksi lahan perkebunan Komoditas Bibit Pupuk Herbisida Insektisida Fungisida Kg Rp Nama Kg Rp Kg Rp Kg Rp Kg Rp Karet B.3. Tenaga kerja B.3.1 Apakah Bapak/Ibu menggunakan tenaga kerja di luar keluarga? Catatan 8 – Contoh kuesioner baku: Rumah tangga, desa, dan informan kunci B.3.2 Biaya tenaga kerja: Lahan tanaman pangan (tahun yang lalu) Komoditas Biaya tenaga kerja di luar keluarga (dalam Rupiah) Pengolahan/ membajak tanah Menanam Merawat/ menyiangi Panen Pengolahan bahan panen Penjualan (termasuk transport) Padi Jagung B.3. Tenaga kerja B.3.1 Apakah Bapak/Ibu menggunakan tenaga kerja di luar keluarga? B.3.2 Biaya tenaga kerja: Lahan tanaman pangan (tahun yang lalu) Komoditas Biaya tenaga kerja di luar keluarga (dalam Rupiah) Pengolahan/ membajak tanah Menanam Merawat/ menyiangi Panen Pengolahan bahan panen Penjualan (termasuk transport) Padi Jagung B.3. Tenaga kerja B.3.1 Apakah Bapak/Ibu menggunakan tenaga kerja di luar keluarga? B.3.2 Biaya tenaga kerja: Lahan tanaman pangan (tahun yang lalu) Komoditas Biaya tenaga kerja di luar keluarga (dalam Rupiah) Pengolahan/ membajak tanah Menanam Merawat/ menyiangi Panen Pengolahan bahan panen Penjualan (termasuk transport) Padi Jagung B.3.3 Biaya dan tenaga kerja di luar keluarga: Kebun (tahun yang lalu) Komoditas Biaya tenaga kerja di luar keluarga (dalam Rupiah) Menanam Memupuk Merawat/ menyiangi Panen Pengolahan bahan panen Penjualan (termasuk angkutan) Karet Biaya dan tenaga kerja di luar keluarga: Kebun (tahun yang lalu) Karet anduan praktis untuk survei mata pencaharian sosial-ekonomi dan hak dan kepemilikan lahan 61 B.4 Hasil Pertanian/Perkebunan B.4.1 Isilah hasil pertanian dari musim tanam yang lalu pada tabel di bawah ini: Komoditas Berapa musim/th Ukuran lokal (jika ada) Panen Makan sendiri Bibit dipisah Dijual Penjualan Nama UL 1 UL = ... kg Berapa?a Bentuk % % % Berapa kg Harga/kg Total Tanaman pangan (buah sayur hasil lainnya) B.4 Hasil Pertanian/Perkebunan B.4.1 Isilah hasil pertanian dari musim tanam yang lalu pada tabel di bawah ini: Komoditas Berapa musim/th Ukuran lokal (jika ada) Panen Makan sendiri Bibit dipisah Dijual Penjualan Nama UL 1 UL = ... kg Berapa?a Bentuk % % % Berapa kg Harga/kg Total Tanaman pangan (buah sayur hasil lainnya) B.4 Hasil Pertanian/Perkebunan B.4.1 Isilah hasil pertanian dari musim tanam yang lalu pada tabel di bawah ini: Komoditas Berapa musim/th Ukuran lokal (jika ada) Panen Makan sendiri Bibit dipisah Dijual Penjualan Nama UL 1 UL = ... kg Berapa?a Bentuk % % % Berapa kg Harga/kg Total Tanaman pangan (buah, sayur, hasil lainnya) Padi Jagung Tanaman perkebunan Karet a diisi sesuai satuan (kg atau UL) Nining Liswanti, Bayuni Shantiko, Emily Fripp, Esther Mwangi dan Yves Laumonier 62 C. Data pertanian pada musim tanam saat ini C.1.1 Jenis lahan yang digarap pada musim tanam saat ini? C.1.2 Apakah Anda telah membuka lahan baru sejak musim tanam yang lalu? C.1.3 Apakah Anda punya rencana untuk memperluas kebun Anda atau membuka lahan baru? C.1.4 Apakah Anda punya rencana untuk memperbarui (mengganti tanaman) kebun Anda? C.1.5 Apakah Anda punya rencana untuk mengurangi lahan Anda? C.1.6 Apakah Anda punya rencana untuk meninggalkan lahan Anda? C.1.7 Lahan digarap: Ladang – kebun Ladang Kebun Lainnya C.1.1 Jenis lahan digarap musim tanam sekarang Sama/tidak ) Luas Tanaman lainnya C.1.2 Pembukaan lahan baru sejak musim tanam lalu Ya/tidak ) Luas Dimana KOD C.1.3 Apakah ada rencana perluasan lahan Ya/tidak ) Luas Dimana KOD C.1.4 Apakah ada rencana pergantian tanaman Ya/tidak ) Luas Tanaman apa C.1.5 Apakah ada rencana mengurangi lahan Ya/tidak ) Luas Dimana KOD C.1.6 Apakah ada rencana meninggalkan lahan Ya/tidak ) Luas Dimana KOD Untuk mengisi KOD lihat Tabel B.1.7 Untuk mengisi KOD lihat Tabel B.1.7 Panduan praktis untuk survei mata pencaharian sosial-ekonomi dan hak dan kepemilikan lahan 63 C.2 Apakah lahan yang sekarang dimanfaatkan dapat terus dimanfaatkan sampai 5 tahun lagi? ¨ Tidak bisa (lanjutkan ke C.3) ¨ Belum tahu ¨ Ya C.3 Apa alasan lahan tidak bisa dimanfaatkan sampai 5 tahun lagi? a. Tidak punya sertifikat b. Saya hanya punya akses sementara c. Lahannya pinjaman d. Hak atas lahan bisa dicabut sewaktu-waktu e. Hak atas lahan tidak ditegakkan f. Ada pembatasan pemanfaatan lahan oleh pemerintah g. Persaingan dengan sesama penduduk desa h. Persaingan dengan penduduk desa tetangga i. Persaingan pemanfaatan lahan dengan perusahaan (dari luar desa) j. Pembangunan infrastruktur/jalan k. ................................................................................................ l. B.4 Hasil Pertanian/Perkebunan B.4.1 Isilah hasil pertanian dari musim tanam yang lalu pada tabel di bawah ini: Komoditas Berapa musim/th Ukuran lokal (jika ada) Panen Makan sendiri Bibit dipisah Dijual Penjualan Nama UL 1 UL = ... kg Berapa?a Bentuk % % % Berapa kg Harga/kg Total Tanaman pangan (buah sayur hasil lainnya) ................................................................................................ Catatan: jika jawaban lebih dari 1 (satu), minta responden memberikan peringkat alasan berdasarkan nilai pentingnya D. Peternakan dan perikanan D.1 Apakah Bapak memiliki ternak? ¨ Ya, dan dipelihara sendiri (isi tabel D.1.1) ¨ Ya, dan dipelihara oleh orang lain (isi tabel D.1.6) ¨ Tidak (D.1.7) Nining Liswanti, Bayuni Shantiko, Emily Fripp, Esther Mwangi dan Yves Laumonier 64 g p Jenis ternak Sistema Dimana KOD Luas Lahan Jumlah (ekor) Dimakan setahun (ekor) Jml dijual setahun Harga/ ekor Biaya pemeliharaanb Kerbau Jantan Dewasa Anak Betina Dewasa Anak Kambing/ domba Jantan Dewasa Anak Betina Dewasa Anak Sapi Jantan Dewasa Anak Betina Dewasa Anak Babi Jantan Dewasa Anak Betina Dewasa Anak Lain .......... Jantan Dewasa Anak Betina Dewasa Anak berlanjut ke halaman berikutnya D.1.1 Kegiatan peternakan Jenis ternak Sistema Dimana KOD Luas Lahan Jumlah (ekor) Dimakan setahun (ekor) Jml dijual setahun Harga/ ekor Biaya pemeliharaanb Kerbau Jantan Dewasa Anak Betina Dewasa Anak Kambing/ domba Jantan Dewasa Anak Betina Dewasa Anak Sapi Jantan Dewasa Anak Betina Dewasa Anak Babi Jantan Dewasa Anak Betina Dewasa Anak Lain .......... Jantan Dewasa Anak Betina Dewasa Anak berlanjut ke halaman berikutnya g p Jenis ternak Sistema Dimana KOD Luas Lahan Jumlah (ekor) Dimakan setahun (ekor) Jml dijual setahun Harga/ ekor Biaya pemeliharaanb K b J D % dimakan sendiri % dijual Berapa harga/satuan Biaya sekali melaut (pergi-pulang) a Jarak: 1 = kurang dari 1 jam, 2 = 2–4 jam, 3 = 4–8 jam, 4 = lebih dari 8 jam b Siapa: 1 = Ayah, 2 = Ibu, 3 = Ayah dan ibu, 4 = Ayah dan anak, 5 = Ibu dan anak, 6 = Anak, 7 = Seluruh keluarga c Frekuensi: 1 = tiap hari, 2 = tiap minggu, 3 = 2 kali sebulan, 4 = tiap bulan, 5 = dua bulan sekali, 6 = dua kali setahun, 7 = kurang dari 2 kali setahun d Tidak melaut karena musim: 1 = 1 minggu, 2 = 2 minggu, 3 = 1 bulan, 4 = 2 bulan, 5 = 3 bulan, 6 = lebih dari 3 bulan Untuk mengisi KOD lihat Tabel B 1 7 D.1.7 Apakah Anda memiliki kolam ikan? ¨ Ya (Tabel D.1.7.1) ¨ Tidak D.1.7.1 Kegiatan perikanan – Kolam ikan/karamba Komponen Satuan Luas Ha Jumlah kolam/karamba Buah Sistem pemeliharaana Tujuanb Dari panen ikan:% konsumsi sendiri % dijual Biaya setahun (pakan dan pemasaran) Rp/tahun Penjualan setahun Rp/tahun a 1 = tetap, 2 = musiman b 1 = konsumsi, 2 = dijual D.1.8 Apakah Anda menjadi nelayan sungai/danau/laut? ¨ Ya ¨ Tidak Hasil 1 Hasil 2 Hasil 3 Status penggunaan lahan, KOD Tangkapan ikan Jaraka Siapa yang pergib Frekuensic Tidak melautd Jumlah tangkapan 1 x melaut Satuannya apa? % dimakan sendiri % dijual Berapa harga/satuan Biaya sekali melaut (pergi-pulang) a Jarak: 1 = kurang dari 1 jam, 2 = 2–4 jam, 3 = 4–8 jam, 4 = lebih dari 8 jam b Siapa: 1 = Ayah, 2 = Ibu, 3 = Ayah dan ibu, 4 = Ayah dan anak, 5 = Ibu dan anak, 6 = Anak, 7 = Seluruh keluarga c Frekuensi: 1 = tiap hari, 2 = tiap minggu, 3 = 2 kali sebulan, 4 = tiap bulan, 5 = dua bulan sekali, 6 = dua kali setahun, 7 = kurang dari 2 kali setahun d Tidak melaut karena musim: 1 = 1 minggu, 2 = 2 minggu, 3 = 1 bulan, 4 = 2 bulan, 5 = 3 bulan, 6 = lebih dari 3 bulan Untuk mengisi KOD lihat Tabel B.1.7 D.1.8 Apakah Anda menjadi nelayan sungai/danau/laut? g p Jenis ternak Sistema Dimana KOD Luas Lahan Jumlah (ekor) Dimakan setahun (ekor) Jml dijual setahun Harga/ ekor Biaya pemeliharaanb K b J D anduan praktis untuk survei mata pencaharian sosial-ekonomi dan hak dan kepemilikan lahan 65 Jenis ternak Sistema Dimana KOD Luas Lahan Jumlah (ekor) Dimakan setahun (ekor) Jml dijual setahun Harga/ ekor Biaya pemeliharaanb Ayam xxxx` xxx Produksi telur xxxx xxxx xxx xxxx Itik/bebek xxxx xxx Produksi telur xxxx xxxx xxx xxxx a 1 = liar, 2 = ikat, 3 = kandang malam, 4 = gembala, 5 = kandang tetap b Termasuk sewa lahan, pakan, tenaga kerja, kesehatan hewan Untuk mengisi KOD lihat Tabel B.1.7 D.1.1 Lanjutan Jenis ternak Sistema Dimana KOD Luas Lahan Jumlah (ekor) Dimakan setahun (ekor) Jml dijual setahun Harga/ ekor Biaya pemeliharaanb Ayam xxxx` xxx Produksi telur xxxx xxxx xxx xxxx Itik/bebek xxxx xxx Produksi telur xxxx xxxx xxx xxxx a 1 = liar, 2 = ikat, 3 = kandang malam, 4 = gembala, 5 = kandang tetap b Termasuk sewa lahan, pakan, tenaga kerja, kesehatan hewan Untuk mengisi KOD lihat Tabel B.1.7 D.1.1 Lanjutan Nining Liswanti, Bayuni Shantiko, Emily Fripp, Esther Mwangi dan Yves Laumonier 66 D.1.2 Produk turunan dari kegiatan peternakan Jumlah panen Satuan % dipakai sendiri % dijual Harga jual/ unit Susu Mentega Kulit Pupuk D.1.2 Produk turunan dari kegiatan peternakan D.1.3 Sistem pembayaran kalau hewan dipelihara orang lain Siapa yang menanggung biaya? Berapa biayanya? Rp .......... Sistem pembayarannya ............................................ Rp ........../tahun D.1.4 Apakah Anda memelihara hewan orang lain? ¨ Ya (tabel D.1.6) ¨ Tidak (D 1.7) D.1.5 Pendapatan yang diperoleh dari memelihara hewan orang lain? Sistem pembayaran .......... Pendapatan yang diterima Rp ........../tahun D.1.6 Memelihara hewan milik orang lain Hewan Ternak Sistem pemeliharaana Luas tanah Dimana KOD Jumlah (ekor) Kerbau Panduan praktis untuk survei mata pencaharian sosial-ekonomi dan hak dan kepemilikan lahan 67 D.1.7 Apakah Anda memiliki kolam ikan? ¨ Ya (Tabel D.1.7.1) ¨ Tidak D.1.7.1 Kegiatan perikanan – Kolam ikan/karamba Komponen Satuan Luas Ha Jumlah kolam/karamba Buah Sistem pemeliharaana Tujuanb Dari panen ikan:% konsumsi sendiri % dijual Biaya setahun (pakan dan pemasaran) Rp/tahun Penjualan setahun Rp/tahun a 1 = tetap, 2 = musiman b 1 = konsumsi, 2 = dijual D.1.8 Apakah Anda menjadi nelayan sungai/danau/laut? ¨ Ya ¨ Tidak Hasil 1 Hasil 2 Hasil 3 Status penggunaan lahan, KOD Tangkapan ikan Jaraka Siapa yang pergib Frekuensic Tidak melautd Jumlah tangkapan 1 x melaut Satuannya apa? g p Jenis ternak Sistema Dimana KOD Luas Lahan Jumlah (ekor) Dimakan setahun (ekor) Jml dijual setahun Harga/ ekor Biaya pemeliharaanb K b J D ¨ Ya ¨ Tidak Hasil 1 Hasil 2 Hasil 3 Status penggunaan lahan, KOD Tangkapan ikan Jaraka Siapa yang pergib Frekuensic Tidak melautd Jumlah tangkapan 1 x melaut Satuannya apa? % dimakan sendiri % dijual Berapa harga/satuan Biaya sekali melaut (pergi-pulang) a Jarak: 1 = kurang dari 1 jam, 2 = 2–4 jam, 3 = 4–8 jam, 4 = lebih dari 8 jam b Siapa: 1 = Ayah, 2 = Ibu, 3 = Ayah dan ibu, 4 = Ayah dan anak, 5 = Ibu dan anak, 6 = Anak, 7 = Seluruh keluarga c Frekuensi: 1 = tiap hari, 2 = tiap minggu, 3 = 2 kali sebulan, 4 = tiap bulan, 5 = dua bulan sekali, 6 = dua kali setahun, 7 = kurang dari 2 kali setahun d Tidak melaut karena musim: 1 = 1 minggu, 2 = 2 minggu, 3 = 1 bulan, 4 = 2 bulan, 5 = 3 bulan, 6 = lebih dari 3 bulan Untuk mengisi KOD lihat Tabel B.1.7 akah Anda menjadi nelayan sungai/danau/laut? ¨ Ya ¨ Tidak Nining Liswanti, Bayuni Shantiko, Emily Fripp, Esther Mwangi dan Yves Laumonier 68 E. Masalah dalam kegiatan pertanian/perkebunan/peternakan/ perikanan - catat masalah/kendala yang diucapkan oleh responden secara spontan E.1 Masalah atau kendala apa yang dihadapi Bapak/Ibu dalam kegiatan usaha tani? Masalah Sering Kadang- kadang Jarang Urutan kepentingan Serangan babi Serangan satwa lain .......... Serangan hama lain Kekurangan modal Sulit memperoleh bibit unggul Harga pupuk mahal Sulit memasarkan hasil Harga jual terlalu rendah Informasi teknologi kurang Kurangnya sumber air untuk usaha tani Banjir Lahan usaha luasnya kecil Status lahan tidak jelas Sengketa lahan dengan perusahaan Lainnya .......... .................... .................... E.2 Apakah pernah mengalami situasi-situasi berikut ini selama 5 (lima) tahun terakhir? Aspek Kerentanan Ya/tidak) Apa yang dilakukan untuk mengatasinya? Mengalami penurunan/kehilangan pendapatan secara tiba-tiba Kegagalan panen Bencana alam (banjir, kekeringan, gempa, angin putting beliung, hama dan virus ) Pengusiran dari rumah oleh .......... E. Masalah dalam kegiatan pertanian/perkebunan/peternakan/ perikanan - catat masalah/kendala yang diucapkan oleh responden secara spontan E.1 Masalah atau kendala apa yang dihadapi Bapak/Ibu dalam kegiatan usaha tani? Masalah Sering Kadang- kadang Jarang Urutan kepentingan Serangan babi Serangan satwa lain .......... Serangan hama lain Kekurangan modal Sulit memperoleh bibit unggul Harga pupuk mahal Sulit memasarkan hasil Harga jual terlalu rendah Informasi teknologi kurang Kurangnya sumber air untuk usaha tani Banjir Lahan usaha luasnya kecil Status lahan tidak jelas Sengketa lahan dengan perusahaan Lainnya .......... .................... berlanjut ke halaman berikutnya berlanjut ke halaman berikutnya g p Jenis ternak Sistema Dimana KOD Luas Lahan Jumlah (ekor) Dimakan setahun (ekor) Jml dijual setahun Harga/ ekor Biaya pemeliharaanb K b J D a Jarak: 1 = kurang dari 1 jam, 2 = 2–4 jam, 3 = 4–8 jam, 4 = lebih dari 8 jam b Siapa: 1 = Ayah, 2 = Ibu, 3 = Ayah dan ibu, 4 = Ayah dan anak, 5 = Ibu dan anak, 6 = Anak, 7 = Seluruh keluarga c Frekuensi: 1 = tiap hari, 2 = tiap minggu, 3 = 2 kali sebulan, 4 = tiap bulan, 5 = dua bulan sekali, 6 = dua kali setahun, 7 = kurang dari 2 kali setahun Untuk mengisi KOD lihat Tabel B.1.7 G. Hasil hutan G.1 Apakah Anda mengambil hasil hutan bukan kayu? ¨ Ya ¨ Tidak (G.2) G.1.1 Pemanfaatan hasil hutan non-kayu (HHNK) Hasil hutan Status lahan, KOD Jarak a Siapa b Frekuensi c Jumlah panen Unit (lokal) % pakai sendiri % jual Harga satuan Persepsi status sumberdaya +/-) Laki Perempuan Madu Rotan Anggrek Daun atap Sayuran Babi hutan Getah karet Tanaman obat .................... a Jarak: 1 = kurang dari 1 jam, 2 = 2–4 jam, 3 = 4–8 jam, 4 = lebih dari 8 jam b Siapa: 1 = Ayah, 2 = Ibu, 3 = Ayah dan ibu, 4 = Ayah dan anak, 5 = Ibu dan anak, 6 = Anak, 7 = Seluruh keluarga c Frekuensi: 1 = tiap hari, 2 = tiap minggu, 3 = 2 kali sebulan, 4 = tiap bulan, 5 = dua bulan sekali, 6 = dua kali setahun, 7 = kurang dari 2 kali setahun Untuk mengisi KOD lihat Tabel B.1.7 G. Hasil hutan G.1 Apakah Anda mengambil hasil hutan bukan kayu? ¨ Ya ¨ Tidak (G.2) G.1.1 Pemanfaatan hasil hutan non-kayu (HHNK) Hasil hutan Status lahan, KOD Jarak a Siapa b Frekuensi c Jumlah panen Unit (lokal) % pakai sendiri % jual Harga satuan Persepsi status sumberdaya +/-) Laki Perempuan Madu Rotan Anggrek Daun atap Sayuran Babi hutan Getah karet Tanaman obat .................... g p Jenis ternak Sistema Dimana KOD Luas Lahan Jumlah (ekor) Dimakan setahun (ekor) Jml dijual setahun Harga/ ekor Biaya pemeliharaanb K b J D .................... E.2 Apakah pernah mengalami situasi-situasi berikut ini selama 5 (lima) tahun terakhir? Aspek Kerentanan Ya/tidak) Apa yang dilakukan untuk mengatasinya? Mengalami penurunan/kehilangan pendapatan secara tiba-tiba Kegagalan panen Apakah pernah mengalami situasi-situasi berikut ini selama 5 (lima) tahun terakhir? berlanjut ke halaman berikutnya Panduan praktis untuk survei mata pencaharian sosial-ekonomi dan hak dan kepemilikan lahan 69 Aspek Kerentanan Ya/tidak) Apa yang dilakukan untuk mengatasinya? Kebakaran rumah .................... pilih yang sesuai E.2 Lanjutan Aspek Kerentanan Ya/tidak) Apa yang dilakukan untuk mengatasinya? Kebakaran rumah .................... pilih yang sesuai F. Penyuluhan dan Organisasi Sosial F.1 Apakah Anda menjadi anggota perkumpulan/organisasi di tingkat desa? ¨ Ya ¨ Tidak F.1.1 Penyuluhan Tenaga penyuluh Topik penyuluhan Frekuensi Petugas Pertanian, PPL Petugas Keluarga Berencana Petugas Kehutanan Lainnya .......... F.2 Apakah Anda pernah menerima bantuan atau partisipasi dalam program pembangunan masyarakat yang dilakukan oleh organisasi eksternal (pemerintah, LSM, dll) ¨ Ya ¨ Tidak F.2.1 Bantuan pembangunan masyarakat selama 5 tahun terakhir Tahun Nama program Nama organisasi Deskripsi kegiatan Tingkat kesuksesana a 1 = sangat sukses, 2 = sukses, 3 = agak sukses, 4 = kurang sukses, 5 = tidak sukses F.3 Apakah Anda anggota organisasi? F.3.1 Keanggotaan organisasi Nama organisasi Status organisasia Anggota sejak tahun? Deskripsi kegiatan E.2 Lanjutan F. Penyuluhan dan Organisasi Sosial F.1 Apakah Anda menjadi anggota perkumpulan/organisasi di tingkat desa? ¨ Ya ¨ Tidak F.1.1 Penyuluhan Tenaga penyuluh Topik penyuluhan Frekuensi Petugas Pertanian, PPL Petugas Keluarga Berencana Petugas Kehutanan Lainnya F. Penyuluhan dan Organisasi Sosial a 1 = formal, 2 = informal Nining Liswanti, Bayuni Shantiko, Emily Fripp, Esther Mwangi dan Yves Laumonier 70 G. Hasil hutan G.1 Apakah Anda mengambil hasil hutan bukan kayu? ¨ Ya ¨ Tidak (G.2) G.1.1 Pemanfaatan hasil hutan non-kayu (HHNK) Hasil hutan Status lahan, KOD Jarak a Siapa b Frekuensi c Jumlah panen Unit (lokal) % pakai sendiri % jual Harga satuan Persepsi status sumberdaya +/-*) Laki Perempuan Madu Rotan Anggrek Daun atap Sayuran Babi hutan Getah karet Tanaman obat .................... g p Jenis ternak Sistema Dimana KOD Luas Lahan Jumlah (ekor) Dimakan setahun (ekor) Jml dijual setahun Harga/ ekor Biaya pemeliharaanb K b J D a Jarak: 1 = kurang dari 1 jam, 2 = 2–4 jam, 3 = 4–8 jam, 4 = lebih dari 8 jam b Siapa: 1 = Ayah, 2 = Ibu, 3 = Ayah dan ibu, 4 = Ayah dan anak, 5 = Ibu dan anak, 6 = Anak, 7 = Seluruh keluarga c Frekuensi: 1 = tiap hari, 2 = tiap minggu, 3 = 2 kali sebulan, 4 = tiap bulan, 5 = dua bulan sekali, 6 = dua kali setahun, 7 = kurang dari 2 kali setahun Untuk mengisi KOD lihat Tabel B.1.7 G.2 Apakah hutan yang sekarang dimanfaatkan dapat terus dimanfaatkan sampai 5 tahun lagi? ¨ Tidak bisa (lanjutkan ke G.3) ¨ Belum tahu ¨ Ya G 3 Ap l h t tid k bi di f tk p i 5 t h l i? a Jarak: 1 = kurang dari 1 jam, 2 = 2–4 jam, 3 = 4–8 jam, 4 = lebih dari 8 jam b Siapa: 1 = Ayah, 2 = Ibu, 3 = Ayah dan ibu, 4 = Ayah dan anak, 5 = Ibu dan anak, 6 = Anak, 7 = Seluruh keluarga c Frekuensi: 1 = tiap hari, 2 = tiap minggu, 3 = 2 kali sebulan, 4 = tiap bulan, 5 = dua bulan sekali, 6 = dua kali setahun, 7 = kurang dari 2 kali setahun Untuk mengisi KOD lihat Tabel B.1.7 G.2 Apakah hutan yang sekarang dimanfaatkan dapat terus dimanfaatkan sampai 5 tahun lagi? ¨ Tidak bisa (lanjutkan ke G.3) ¨ Belum tahu ¨ Ya G.3 Apa alasan hutan tidak bisa dimanfaatkan sampai 5 tahun lagi? a. Status hutan tidak jelas b. Batas hutan tidak jelas G.2 Apakah hutan yang sekarang dimanfaatkan dapat terus dimanfaatkan sampai 5 tahun lagi? ¨ Tidak bisa (lanjutkan ke G.3) ¨ Belum tahu ¨ Ya G.3 Apa alasan hutan tidak bisa dimanfaatkan sampai 5 tahun lagi? a. Status hutan tidak jelas b. Batas hutan tidak jelas Panduan praktis untuk survei mata pencaharian sosial-ekonomi dan hak dan kepemilikan lahan 71 c. Hak atas lahan bisa dicabut sewaktu-waktu d. Hak atas lahan tidak ditegakkan e. Ada pembatasan pemanfaatan oleh pemerintah f. Persaingan dengan sesama penduduk desa g. Persaingan dengan penduduk desa tetangga h. Persaingan pemanfaatan lahan dengan perusahaan (dari luar desa) i. Pembangunan infrastruktur/jalan j. ......................................................................................................... k. ......................................................................................................... g p Jenis ternak Sistema Dimana KOD Luas Lahan Jumlah (ekor) Dimakan setahun (ekor) Jml dijual setahun Harga/ ekor Biaya pemeliharaanb K b J D Catatan: jika jawaban lebih dari 1 (satu), minta responden memberi peringkat alasan berdasarkan nilai pentingnya G.4 Hasil kayu G.4.1 Pemanfaatan hasil kayu (kayu bakar dan kayu gergajian/untuk bahan bangunan) Kayu bakar Kayu bangunan 1 Kayu bangunan 2 Nama jenis kayu Lokasia Status lahan, KOD Jarak (lamanya)b Siapa yang pergic Frekuensi Jumlah diambil Satuannya apa? (ikat, m3, .........) % dipakai sendiri % dijual Berapa harganya/satuan a Lokasi: 1 = Hutan, 2 = Ladang, 3 = Belukar b Jarak: 1 = kurang dari 1 jam, 2 = 2–4 jam, 3 = 4–8 jam, 4 = lebih dari 8 jam c Siapa: 1 = Ayah, 2 = Ibu, 3 = Ayah dan ibu, 4 = Ayah dan anak, 5 = Ibu dan anak, 6 = Anak, 7 = Seluruh keluarga Untuk mengisi KOD lihat Tabel B.1.7 c. Hak atas lahan bisa dicabut sewaktu-waktu d. Hak atas lahan tidak ditegakkan e. Ada pembatasan pemanfaatan oleh pemerintah f. Persaingan dengan sesama penduduk desa g. Persaingan dengan penduduk desa tetangga h. Persaingan pemanfaatan lahan dengan perusahaan (dari luar desa) i. Pembangunan infrastruktur/jalan j. ......................................................................................................... k. ......................................................................................................... Catatan: jika jawaban lebih dari 1 (satu), minta responden memberi peringkat alasan berdasarkan nilai pentingnya G.4 Hasil kayu G.4.1 Pemanfaatan hasil kayu (kayu bakar dan kayu gergajian/untuk bahan bangunan) Kayu bakar Kayu bangunan 1 Kayu bangunan 2 Nama jenis kayu Lokasia Status lahan, KOD Jarak (lamanya)b Siapa yang pergic Frekuensi Jumlah diambil Satuannya apa? (ikat, m3, .........) % dipakai sendiri % dijual Berapa harganya/satuan a Lokasi: 1 = Hutan, 2 = Ladang, 3 = Belukar b Jarak: 1 = kurang dari 1 jam, 2 = 2–4 jam, 3 = 4–8 jam, 4 = lebih dari 8 jam c Siapa: 1 = Ayah, 2 = Ibu, 3 = Ayah dan ibu, 4 = Ayah dan anak, 5 = Ibu dan anak, 6 = Anak, 7 = Seluruh keluarga Untuk mengisi KOD lihat Tabel B.1.7 Berapa harganya/satuan a Lokasi: 1 = Hutan, 2 = Ladang, 3 = Belukar b Jarak: 1 = kurang dari 1 jam, 2 = 2–4 jam, 3 = 4–8 jam, 4 = lebih dari 8 jam c Siapa: 1 = Ayah, 2 = Ibu, 3 = Ayah dan ibu, 4 = Ayah dan anak, 5 = Ibu dan anak, 6 = Anak, 7 = Seluruh keluarga Untuk mengisi KOD lihat Tabel B.1.7 a Lokasi: 1 = Hutan, 2 = Ladang, 3 = Belukar b Jarak: 1 = kurang dari 1 jam, 2 = 2–4 jam, 3 = 4–8 jam, 4 = lebih dari 8 jam c Siapa: 1 = Ayah, 2 = Ibu, 3 = Ayah dan ibu, 4 = Ayah dan anak, 5 = Ibu dan anak, 6 = Anak, 7 = Seluruh keluarga Untuk mengisi KOD lihat Tabel B.1.7 a Lokasi: 1 = Hutan, 2 = Ladang, 3 = Belukar Nining Liswanti, Bayuni Shantiko, Emily Fripp, Esther Mwangi dan Yves Laumonier 72 H. Persepsi tentang perubahan H.1 Dalam 5 (lima) tahun terakhir, apakah ketersediaan lahan hutan untuk dibuka: a. meningkat (ke H.2) b. tidak berubah c. berkurang (ke H.3) d. tidak tahu H.2 Jika ketersediaan lahan hutan untuk dibuka MENINGKAT, apa alasannya? a. kurangnya persaingan antara penduduk desa/kampung b. kurangnya persaingan dengan penduduk desa tetangga c. berkurangnya pembatasan dari pemerintah .......... H.3 Jika ketersediaan lahan hutan untuk dibuka BERKURANG, apa alasannya? a. Persaingan antara penduduk desa/kampung b. Persaingan dengan penduduk desa tetangga c. Adanya pembatasan dari pemerintah .................... H.4 Selama 5 (lima) tahun terakhir, apakah hasil hutan yang dikonsumsi rumah tangga: a. meningkat, karena .......... b. tidak berubah c. berkurang, karena .......... d. tidak tahu H.5 Selama 5 (lima) tahun terakhir, apakah pendapatan rumah tangga dari hasil hutan (yang dijual): a. meningkat, karena .......... b. tidak berubah c. berkurang, karena .......... d. tidak tahu H.6 Bagaimana Bapak menggambarkan kehidupan keluarga dibandingkan 5 (lima) tahun yang lalu? a. lebih baik, karena .......... b. tidak berubah c. lebih susah, karena .......... Panduan praktis untuk survei mata pencaharian sosial-ekonomi dan hak dan kepemilikan lahan 73 I. Persepsi tentang ketersediaan hutan antargenerasi Nama Jenis Kelamin Umur Generasi Kakek/nenek Diri sendiri Anak/Cucu J. Pendapatan di luar pertanian J.1 Kegiatan apa yang merupakan kegiatan pokok? ¨ Pertanian ¨ Perkebunan ¨ Peternakan ¨ Melaut/Nelayan ¨ Kehutanan ¨ Lain ................................ J.2 Apakah masih ada pendapatan dari luar pertanian, perkebunan, peternakan, perikanan dan hutan? berlanjut ke halaman berikutnya Berapa harganya/satuan ¨ Ya ¨ Tidak (AKHIR KUESIONER) J.3 Apa jenis pekerjaan tersebut? J.4 Apakah pekerjaan tersebut bersifat ¨ tetap ¨ musiman J.5 Kalau hasil dari kerja tersebut di atas dibandingkan dengan hasil kegiatan pokok, ¨ lebih besar ¨ sama ¨ lebih kecil J.6 Berapa besar pendapatan dari kegiatan tersebut di atas dalam setahun? Rp .................... K Kepemilikan aset K.1 Gunakan pengamatan untuk mengetahui aset yang dimiliki Nama Barang Jumlah Tahun beli Harga (Rp) Keterangan Generator listrik Lampu petromak Televisi parabola Radio tape Chainsaw Kulkas Persepsi tentang ketersediaan hutan antargenerasi J. Pendapatan di luar pertanian J.1 Kegiatan apa yang merupakan kegiatan pokok? ¨ Pertanian ¨ Perkebunan ¨ Peternakan ¨ Melaut/Nelayan ¨ Kehutanan ¨ Lain ................................ J.2 Apakah masih ada pendapatan dari luar pertanian, perkebunan, peternakan, perikanan dan hutan? ¨ Ya ¨ Tidak (AKHIR KUESIONER) J.3 Apa jenis pekerjaan tersebut? J.4 Apakah pekerjaan tersebut bersifat ¨ tetap ¨ musiman J.5 Kalau hasil dari kerja tersebut di atas dibandingkan dengan hasil kegiatan pokok, ¨ lebih besar ¨ sama ¨ lebih kecil J.6 Berapa besar pendapatan dari kegiatan tersebut di atas dalam setahun? Rp .................... K Kepemilikan aset K.1 Gunakan pengamatan untuk mengetahui aset yang dimiliki Nama Barang Jumlah Tahun beli Harga (Rp) Keterangan Generator listrik Lampu petromak Televisi parabola Radio tape Chainsaw Kulkas Sepeda Sepeda motor Motor ketinting/ tempel b l j t k h l b ik t J. Pendapatan di luar pertanian J.1 Kegiatan apa yang merupakan kegiatan pokok? ¨ Pertanian ¨ Perkebunan ¨ Peternakan ¨ Melaut/Nelayan ¨ Kehutanan ¨ Lain ................................ J.2 Apakah masih ada pendapatan dari luar pertanian, perkebunan, peternakan, perikanan dan hutan? ¨ Ya ¨ Tidak (AKHIR KUESIONER) J.3 Apa jenis pekerjaan tersebut? J.4 Apakah pekerjaan tersebut bersifat ¨ tetap ¨ musiman J.5 Kalau hasil dari kerja tersebut di atas dibandingkan dengan hasil kegiatan pokok, ¨ lebih besar ¨ sama ¨ lebih kecil J.6 Berapa besar pendapatan dari kegiatan tersebut di atas dalam setahun? Rp .................... K Kepemilikan aset K.1 Gunakan pengamatan untuk mengetahui aset yang dimiliki Nama Barang Jumlah Tahun beli Harga (Rp) Keterangan Generator listrik Lampu petromak Televisi parabola Radio tape Chainsaw Kulkas Sepeda Sepeda motor Motor ketinting/ tempel berlanjut ke halaman berikutnya Nining Liswanti, Bayuni Shantiko, Emily Fripp, Esther Mwangi dan Yves Laumonier 74 Nama Barang Jumlah Tahun beli Harga (Rp) Keterangan Perahu dayung Mesin jahit Tempayan, guci Kamera Senapan Mesin ketam padi K.2 Halaman kontrol Pengeluaran Rumah Tangga Sebulan Setahun Jumlah Biasa (Sehari-hari) Makanan/minuman Energi (listrik, kayu, gas, minyak) Transportasi (termasuk bensin) Kebersihan (sabun, sabun cuci, ..........) Baju/sepatu Sekolah Kesehatan Pengembalian pinjaman Rokok Sumbangan (rutin) Lainnya .......... berlanjut ke halaman berikutnya Persepsi tentang ketersediaan hutan antargenerasi Total Pengeluaran tak terduga (investasi atau lain-lain) Rumah xxxxxxx Transportasi (motor, mobil ..........) xxxxxxx Tanah xxxxxxx Ternak xxxxxxx Chainsaw xxxxxxx Alat-alat lain xxxxxxx Emas xxxxxxx K.1 Lanjutan Nama Barang Jumlah Tahun beli Harga (Rp) Keterangan Perahu dayung Mesin jahit Tempayan, guci Kamera Senapan Mesin ketam padi K.1 Lanjutan g g p g Perahu dayung Mesin jahit Tempayan, guci Kamera Senapan Mesin ketam padi K.2 Halaman kontrol Pengeluaran Rumah Tangga Sebulan Setahun Jumlah Biasa (Sehari-hari) Makanan/minuman Energi (listrik, kayu, gas, minyak) Transportasi (termasuk bensin) Kebersihan (sabun, sabun cuci, ..........) Baju/sepatu Sekolah Kesehatan Pengembalian pinjaman Rokok Sumbangan (rutin) Lainnya .......... Total Pengeluaran tak terduga (investasi atau lain-lain) Rumah xxxxxxx Transportasi (motor, mobil ..........) xxxxxxx Tanah xxxxxxx Ternak xxxxxxx Chainsaw xxxxxxx Alat-alat lain xxxxxxx Emas xxxxxxx Bantuan non rutin (keluarga, gereja dsb) xxxxxxx Kesehatan Lainnya .......... xxxxxxx Total xxxxxxx b l j k h l b ik Kamera Senapan Mesin ketam padi K.2 Halaman kontrol Pengeluaran Rumah Tangga Sebulan Setahun Jumlah Biasa (Sehari-hari) Makanan/minuman Energi (listrik, kayu, gas, minyak) Transportasi (termasuk bensin) Kebersihan (sabun, sabun cuci, ..........) Baju/sepatu Sekolah Kesehatan Pengembalian pinjaman Rokok Sumbangan (rutin) Lainnya .......... Total Pengeluaran tak terduga (investasi atau lain-lain) Rumah xxxxxxx Transportasi (motor, mobil ..........) xxxxxxx Tanah xxxxxxx Ternak xxxxxxx Chainsaw xxxxxxx Alat-alat lain xxxxxxx Emas xxxxxxx Bantuan non rutin (keluarga, gereja dsb) xxxxxxx Kesehatan Lainnya .......... xxxxxxx Total xxxxxxx berlanjut ke halaman berikutnya Pengeluaran tak terduga (investasi atau lain-lain) Rumah xxxxxxx Transportasi (motor, mobil ..........) xxxxxxx Tanah xxxxxxx Ternak xxxxxxx Chainsaw xxxxxxx Alat-alat lain xxxxxxx Emas xxxxxxx Bantuan non rutin (keluarga, gereja dsb) xxxxxxx Kesehatan Lainnya .......... xxxxxxx Total xxxxxxx berlanjut ke halaman berikutnya Pengeluaran tak terduga (investasi atau lain-lain) Panduan praktis untuk survei mata pencaharian sosial-ekonomi dan hak dan kepemilikan lahan 75 K.2 Lanjutan Pengeluaran Rumah Tangga Sebulan Setahun Jumlah Pendapatan dari penjualan tak terduga atau dari pinjaman tahun lalu Kendaraan bermotor xxxxxxx Tanah xxxxxxx Ternak xxxxxxx Chainsaw xxxxxxx Alat-alat lain xxxxxxx Pinjaman xxxxxxx Bantuan dari keluarga xxxxxxx Jual emas xxxxxxx Lainnya .......... xxxxxxx Total xxxxxxx Pendapatan dari menyewakan Kerbau/kuda Sepeda motor/mobil Chainsaw Rumah/kamar Lain .......... Total Total Ucapkan terimakasih kepada responden atas partisipasi dan untuk berbagi pengalaman, pikiran dan waktu Lanjutan Lanjutan K.2 K.2 Lanjutan Pengeluaran Rumah Tangga Sebulan Setahun Jumlah Pendapatan dari penjualan tak terduga atau dari pinjaman tahun lalu Kendaraan bermotor xxxxxxx Tanah xxxxxxx Ternak xxxxxxx Chainsaw xxxxxxx Alat-alat lain xxxxxxx Pinjaman xxxxxxx Bantuan dari keluarga xxxxxxx Jual emas xxxxxxx Lainnya .......... Persepsi tentang ketersediaan hutan antargenerasi xxxxxxx Total xxxxxxx Pendapatan dari menyewakan Pengeluaran Rumah Tangga Ucapkan terimakasih kepada responden atas partisipasi dan untuk berbagi pengalaman, pikiran dan waktu Nining Liswanti, Bayuni Shantiko, Emily Fripp, Esther Mwangi dan Yves Laumonier 76 Kuesioner 3. Diskusi Kelompok Terfokus (FGD) (Kelompok: jender, kelompok umur, suku, dan mata pencaharian) Propinsi : ........................................................................................................................................ Kabupaten : ........................................................................................................................................ Kecamatan : ........................................................................................................................................ Desa : ........................................................................................................................................ Kelompok : ........................................................................................................................................ Peserta FGD : ........................................................................................................................................ Tanggal/Waktu : ........................................................................................................................................ Fasilitator : ........................................................................................................................................ Pencatat : ........................................................................................................................................ A. Hak atas sumberdaya hutan, penegakan dan kepatuhan 1. Apa yang Anda ketahui tentang istilah hak atas tanah atau hak-hak atas sumberdaya hutan? a. Apakah Anda mengetahui aturan (adat dan negara) yang memengaruhi hak Anda atas lahan atau sumberdaya hutan? Sebutkan dan jelaskan! Bagaimana Anda memahami peraturan tersebut? b. Siapa pemilik hutan berdasarkan aturan hukum? Apakah masyarakat memiliki hak atau sertifikat? 2. Apa pandangan Anda tentang hutan berikut ini. Tolong jelaskan alasan pemilihan pandangan Anda dan bagaimana pandangan Anda memengaruhi penggunaan hutan?: a. Hutan sebagai tempat keramat/suci? b. Hutan sebagai sumberdaya ekonomi? c. Hutan sebagai tempat suci dan sumberdaya ekonomi? d. Hutan dengan fungsi lainnya? 3 Bagaimana Anda memanfaatkan lahan dan sumberdaya hutan? Kuesioner 3. Diskusi Kelompok Terfokus (FGD) (Kelompok: jender, kelompok umur, suku, dan mata pencaharian) Propinsi : ........................................................................................................................................ Kabupaten : ........................................................................................................................................ Kecamatan : ........................................................................................................................................ Desa : ........................................................................................................................................ Kelompok : ........................................................................................................................................ Peserta FGD : ........................................................................................................................................ Tanggal/Waktu : ........................................................................................................................................ Fasilitator : ........................................................................................................................................ Pencatat : ........................................................................................................................................ Kuesioner 3. Diskusi Kelompok Terfokus (FGD) A. Hak atas sumberdaya hutan, penegakan dan kepatuhan Apakah manfaat lain dari hutan yang berhubungan dengan kegiatan pengelolaan hutan atau kehutanan masyarakat? Bagaimana manfaat hutan yang berhubungan dengan lapangan pekerjaan atau pendapatan dibagikan di antara masyarakat? Apakah Anda puas dengan pembagian tersebut? Jika tidak, mengapa? Tolong beri contoh. Bagaimana pembagian keuntungan antara pengguna hutan dapat diperbaiki? Harap berikan peringkat dari semua saran Anda. 5. Atribut/ciri apa yang harus dimiliki penduduk setempat agar bisa masuk ke hutan untuk keperluan rekreasi, agama atau kegiatan nonkonsumtif lainnya? (Contoh atribut/ciri: marga/suku tertentu, agama tertentu, laki-laki, perempuan, tua, muda) A. Hak atas sumberdaya hutan, penegakan dan kepatuhan p y g g sumberdaya hutan? a. Apakah Anda mengetahui aturan (adat dan negara) yang memengaruhi hak Anda atas lahan atau sumberdaya hutan? Sebutkan dan jelaskan! Bagaimana Anda memahami peraturan tersebut? a. Apakah Anda mengetahui aturan (adat dan negara) yang memengaruhi hak Anda atas lahan atau sumberdaya hutan? Sebutkan dan jelaskan! Bagaimana Anda memahami peraturan tersebut? b. Siapa pemilik hutan berdasarkan aturan hukum? Apakah masyarakat memiliki hak atau sertifikat? 2. Apa pandangan Anda tentang hutan berikut ini. Tolong jelaskan alasan pemilihan pandangan Anda dan bagaimana pandangan Anda memengaruhi penggunaan hutan?: a. Hutan sebagai tempat keramat/suci? b. Hutan sebagai sumberdaya ekonomi? c. Hutan sebagai tempat suci dan sumberdaya ekonomi? d. Hutan dengan fungsi lainnya? 2. Apa pandangan Anda tentang hutan berikut ini. Tolong jelaskan alasan pemilihan pandangan Anda dan bagaimana pandangan Anda memengaruhi penggunaan hutan?: a. Hutan sebagai tempat keramat/suci? b. Hutan sebagai sumberdaya ekonomi? c. Hutan sebagai tempat suci dan sumberdaya ekonomi? d. Hutan dengan fungsi lainnya? 3. Bagaimana Anda memanfaatkan lahan dan sumberdaya hutan? Apa produk utama yang Anda peroleh dari hutan? Coba urutkan berdasarkan urutan kepentingannya. (Sebutkan 3 jenis untuk subsisten dan 3 jenis untuk dijual) Panduan praktis untuk survei mata pencaharian sosial-ekonomi dan hak dan kepemilikan lahan 77 Apakah mereka mendapatkan hasil tersebut berdasarkan musim atau diperoleh sepanjang tahun? 4. Apakah manfaat lain dari hutan yang berhubungan dengan kegiatan pengelolaan hutan atau kehutanan masyarakat? Bagaimana manfaat hutan yang berhubungan dengan lapangan pekerjaan atau pendapatan dibagikan di antara masyarakat? Apakah Anda puas dengan pembagian tersebut? Jika tidak, mengapa? Tolong beri contoh. Bagaimana pembagian keuntungan antara pengguna hutan dapat diperbaiki? Harap berikan peringkat dari semua saran Anda. 5. Atribut/ciri apa yang harus dimiliki penduduk setempat agar bisa masuk ke hutan untuk keperluan rekreasi, agama atau kegiatan nonkonsumtif lainnya? (Contoh atribut/ciri: marga/suku tertentu, agama tertentu, laki-laki, perempuan, tua, muda) B. Pengelolaan hutan 6. Menurut Anda, berapa banyak (ukuran lokal) kebutuhan Anda diambil dari hutan? Misalnya, untuk makanan ternak, kayu bakar, kayu perumahan, makanan, atau untuk kebutuhan lain dll. (Untuk 3 produk subsisten dan 3 produk dijual) Apakah kebutuhan ini meningkat atau menurun dalam 5-10 tahun terakhir? Jelaskan alasan penurunan atau peningkatan dan berikan peringkat berdasarkan urutan kepentingan (Gunakan Tabel berikut untuk membuat daftar hasil hutan yang digunakan, prioritaskan 3 jenis subsisten dan 3 jenis untuk dijual, tumbuhan dan binatang) Hasil Hutan 2000 2005 2010 Apakah mereka mendapatkan hasil tersebut berdasarkan musim atau diperoleh sepanjang tahun? 4. B. Pengelolaan hutan anduan praktis untuk survei mata pencaharian sosial-ekonomi dan hak dan kepemilikan lahan 79 e. Menurut Anda, apakah orang di desa mengikuti aturan untuk pemanenan, pengolahan, atau penjualan produk dari hutan atau menggunakan hutan untuk tujuan nonkonsumtif. Tolong jelaskan jawaban Anda. •• Tidak pernah atau jarang •• Kadang-kadang •• Sering •• Kadang-kadang •• Hampir selalu mengikuti •• Hampir selalu mengikuti Jelaskan jawaban Anda. Jelaskan jawaban Anda. f. Menurut Anda, apa saja masalah paling serius dalam mengelola hutan untuk lima tahun ke depan. Bagaimana cara memecahkannya? g. Menurut Anda, apa kesempatan dan peluang yang mungkin terjadi dalam 5 (lima) tahun ke depan yang terkait dengan pengelolaan hutan? Bagaimana merealisasikan peluang tersebut, apa yang perlu dilakukan? Siapa yang perlu dilibatkan? B. Pengelolaan hutan g 6. Menurut Anda, berapa banyak (ukuran lokal) kebutuhan Anda diambil dari hutan? Misalnya, untuk makanan ternak, kayu bakar, kayu perumahan, makanan, atau untuk kebutuhan lain dll. (Untuk 3 produk subsisten dan 3 produk dijual) Apakah kebutuhan ini meningkat atau menurun dalam 5-10 tahun terakhir? Jelaskan alasan penurunan atau peningkatan dan berikan peringkat berdasarkan urutan kepentingan (Gunakan Tabel berikut untuk membuat daftar hasil hutan yang digunakan, prioritaskan 3 jenis subsisten dan 3 jenis untuk dijual, tumbuhan dan binatang) Hasil Hutan 2000 2005 2010 (Gunakan Histoecological matrix, BAG Nomor 5 PDM) 6. Menurut Anda, berapa banyak (ukuran lokal) kebutuhan Anda diambil dari hutan? Misalnya, untuk makanan ternak, kayu bakar, kayu perumahan, makanan, atau untuk kebutuhan lain dll. (Untuk 3 produk subsisten dan 3 produk dijual) Apakah kebutuhan ini meningkat atau menurun dalam 5-10 tahun terakhir? Jelaskan alasan penurunan atau peningkatan dan berikan peringkat berdasarkan urutan kepentingan (Gunakan Tabel berikut untuk membuat daftar hasil hutan yang digunakan, prioritaskan 3 jenis subsisten dan 3 jenis untuk dijual, tumbuhan dan binatang) (Gunakan Histoecological matrix, BAG Nomor 5 PDM) (Gunakan Histoecological matrix, BAG Nomor 5 PDM) Nining Liswanti, Bayuni Shantiko, Emily Fripp, Esther Mwangi dan Yves Laumonier 78 7. Apa bentuk tanggung jawab Anda terhadap hutan (sehubungan dengan fungsi pengelolaan dan konservasi hutan)? Gunakan Tabel berikut untuk menjelaskan: Fungsi Manajemen Aktor yang seharusnya terlibat (internal dan eksternal) Menetapkan/melindungi batas- batas (SDA) Mengembangkan/menerapkan aturan/peraturan Memantau kepatuhan Menyelesaikan konflik Memberikan kepemimpinan/ organisasi Menilai denda/sanksi Contoh: Jika pengguna hutan diminta untuk memperbaiki kondisi hutan dengan melakukan kegiatan regenerasi atau kegiatan lain (misalnya, membasmi perambahan oleh jenis invasif, membuat pembibitan untuk mendistribusikan bibit, mengurangi tingkat panen, atau menanam modal dalam teknologi untuk perbaikan hutan, misalnya peternakan lebah madu, menanam pohon, atau metode lain untuk melindungi, menjaga atau meningkatkan sumberdaya hutan. a. Jika pernah, sebutkan. Seberapa sering Anda melakukan kegiatan yang a. Jika pernah, sebutkan. Seberapa sering Anda melakukan kegiatan yang disebutkan di atas selama tahun lalu? b. Apakah ada anggota masyarakat tertentu yang mencoba membatasi pemanfaatan hutan dengan pemanenan hasil hutan dari lahan komunal lain atau hutan milik pemerintah? c. Jika ya, di hutan mana yang telah digunakan secara intensif sehingga membatasi penggunaan hutan di desa ini? (misalnya, hutan milik swasta, hutan pemerintah, hutan komunal kelompok lain yang memiliki hak atas hutan). d. Apakah ada pembatasan pemanenan hasil hutan selama 5 tahun terakhir? Jika ya, bagaimana perubahannya? Apa yang menjadi dasar pembatasan tersebut. C. Persepsi mengenai kepastian lahan 8. Apakah menurut Anda, Anda akan terus memiliki akses ke lahan dan sumberdaya hutan selama 5 tahun ke depan? 8. Apakah menurut Anda, Anda akan terus memiliki akses ke lahan dan sumberdaya hutan selama 5 tahun ke depan? a. Jelaskan bagaimana perasaan Anda dan apa yang menyebabkan munculnya perasaan tersebut? b. Jika persepsinya negatif: Apa yang harus dilakukan agar ketakutan/ keraguan tersebut dipulihkan? Apa tindakan yang dapat diambil untuk membuat hak Anda atas tanah dan hutan lebih aman? Dengan siapa? Apa yang menjadi tanggung jawab (desa Anda)? 9. Apakah kerapatan pepohonan di hutan berubah dalam lima tahun terakhir? Jika ya, sebutkan 3 alasan yang terpenting bagi perubahan kepadatan pohon. 9. Apakah kerapatan pepohonan di hutan berubah dalam lima tahun terakhir? Jika ya, sebutkan 3 alasan yang terpenting bagi perubahan kepadatan pohon. 10. Selama 5 (lima) tahun terakhir, adakah perubahan di daerah di mana terdapat vegetasi di dalam hutan? Jika meningkat, sebutkan 3 alasan kenapa meningkat. Jika menurun, sebutkan 3 alasan kenapa menurun. Nining Liswanti, Bayuni Shantiko, Emily Fripp, Esther Mwangi dan Yves Laumonier 80 D. Proyek pembangunan di desa Wawancara mendalam dengan informan kunci (Kepala desa, kepala adat, wakil masyarakat, kepala marga) Propinsi : ............................................................................................................................ Kabupaten : ............................................................................................................................ Kecamatan : ............................................................................................................................ Desa : ............................................................................................................................ Nama Informan/ : ............................................................................................................................ Umur/Suku/Marga : ............................................................................................................................ Tanggal/Waktu : ............................................................................................................................ Fasilitator : ............................................................................................................................ Pencatat : ............................................................................................................................ D. Proyek pembangunan di desa 11. Sebutkan proyek pembangunan yang telah dilakukan di desa ini dalam 10 (sepuluh) tahun terakhir untuk meningkatkan partisipasi masyarakat? a. Pernahkah ada proyek tentang akses/hak atas hutan dan lahan dan pengelolaannya? (termasuk perubahan iklim, tanaman obat, dll) b. Apakah ada proyek-proyek khusus yang mendorong hak-hak dan kepemilikan masyarakat? c. Apakah ada proyek-proyek tertentu yang turut mendorong partisipasi perempuan dan laki-laki dalam pengelolaan hutan? Jelaskan proyek- proyek tersebut: •• Nama; •• Sponsor (LSM/Pemerintah/Donor); •• Tujuan dan kegiatan kunci; •• Periode kegiatan Lihat catatan untuk menentukan tingkat partisipasi masyarakat dalam masing-masing proyek. •• Tujuan dan kegiatan kunci; •• Periode kegiatan Lihat catatan untuk menentukan tingkat partisipasi masyarakat dalam masing-masing proyek. 12. Bagaimana tingkat kesuksesan proyek tersebut? Tolong jelaskan mengenai peringkat pilihan Anda. Jika gagal, sebutkan 3 cara yang bisa dilakukan untuk memperbaiki kegagalan tersebut. a. Sukses, b. Sedikit sukses, c. Biasa d. Agak tidak berhasil, e. Gagal Jelaskan siapa yang mengatakan hal itu. Jika proyek gagal, jelaskan 3 cara bagaimana kegagalan tersebut bisa dihindari. 12. Bagaimana tingkat kesuksesan proyek tersebut? Tolong jelaskan mengenai peringkat pilihan Anda. Jika gagal, sebutkan 3 cara yang bisa dilakukan untuk memperbaiki kegagalan tersebut. a. Sukses, b. Sedikit sukses, c. Biasa d. Agak tidak berhasil, e. Gagal 12. Bagaimana tingkat kesuksesan proyek tersebut? Tolong jelaskan mengena peringkat pilihan Anda. Jika gagal, sebutkan 3 cara yang bisa dilakukan untuk memperbaiki kegagalan tersebut. Jelaskan siapa yang mengatakan hal itu. Jika proyek gagal, jelaskan 3 cara bagaimana kegagalan tersebut bisa dihindari. Jelaskan siapa yang mengatakan hal itu. Jika proyek gagal, jelaskan 3 cara bagaimana kegagalan tersebut bisa dihindari. Panduan praktis untuk survei mata pencaharian sosial-ekonomi dan hak dan kepemilikan lahan 81 Laporan hasil FGD Tanggal: Fasilitator: Waktu: Pencatat: Desa: Peserta diskusi: Catatan hasil diskusi: Topik yang menguras energi Bagaimana konsensus dicapai (sulit/ mudah)? Jelaskan Tuliskan petikan pendapat yang menarik Apakah ada temuan yang khusus dan tak terduga Diskusi yang spontan dan relevan saat istirahat atau setelah diskusi telah ditutup Laporan hasil FGD Tanggal: Waktu: Desa: Peserta diskusi: Catatan hasil diskusi: Topik yang menguras energi Bagaimana konsensus dicapai (sulit/ mudah)? Jelaskan Tuliskan petikan pendapat yang menarik Apakah ada temuan yang khusus dan tak terduga Diskusi yang spontan dan relevan saat istirahat atau setelah diskusi telah ditutup Laporan hasil FGD Tanggal: Fasilitator: Waktu: Pencatat: Desa: Peserta diskusi: Nining Liswanti, Bayuni Shantiko, Emily Fripp, Esther Mwangi dan Yves Laumonier 82 Kuesioner 4. A. Kelembagaan yang terkait pengaturan hutan 1. Apakah di desa ini ada aturan resmi dan atau aturan tidak resmi I (termasuk norma adat, mitos, tradisi) – yang terkait dengan penggunaan atau masuk ke hutan (pertimbangkan juga dunia roh, yang mendasari tradisi yang membolehkan (atau melarang) akses ke hutan). Apakah aturan ini diterapkan secara berbeda untuk laki-laki dan perempuan? Tolong jelaskan. 2. Apakah ada aturan khusus yang membatasi kegiatan yang dilakukan di hutan seperti tersebut di bawah ini? 2. a. Pemeliharaan/rehabilitasi (misalnya, membasmi jenis invasif; meningkatkan regenerasi, membuat pembatas untuk mencegah ternak berkeliaran ke hutan dll). Jika ada, jelaskan. Siapakah yang membuat aturan ini(desa atau pemerintah daerah)? b. Pemanenan hasil hutan (misalnya, produk apa yang harus dipanen, waktu panen, kuantitas/tahun?). Jika ada, jelaskan. Siapakah yang membuat aturan ini(desa atau pemerintah daerah)? b. Pemanenan hasil hutan (misalnya, produk apa yang harus dipanen, waktu panen, kuantitas/tahun?). Jika ada, jelaskan. Siapakah yang membuat aturan ini(desa atau pemerintah daerah)? Panduan praktis untuk survei mata pencaharian sosial-ekonomi dan hak dan kepemilikan lahan 83 c. Apakah ada aturan yang membatasi kegiatan lainnya? (misalnya, pengolahan hasil hutan, penjualan hasil hutan? Jika ada, harap jelaskan. Siapakah yang membuat aturan ini (desa atau pemerintah daerah)? Apakah produk yang diolah itu untuk konsumsi sendiri atau dijual? 3. Sanksi hukuman apa yang diberikan kepada anggota masyarakat desa, jika mereka melanggar aturan-aturan yang terkait dengan hutan. a. Melanggar satu kali? b. Melanggar dua kali? c. Melanggar berkali-kali/sering? Siapa yang memutuskan untuk memberikan hukuman tersebut? 4. a. Siapa yang bertanggung jawab di desa ini untuk membuat aturan tentang hutan? Siapakah mereka? Bagaimana Anda menggambarkan mereka? b. Bagaimana Anda menggambarkan individu-individu yang tidak membuat aturan? Apakah individu-individu tersebut berasal dari satu kelompok agama, satu kelompok suku, kelompok laki-laki atau perempuan? 5. Apabila aturan adat/desa ditegakkan, sejauh mana sanksinya ditaati oleh anggota masyarakat? a. Tidak satupun anggota masyarakat mematuhi sanksi b. Beberapa anggota masyarakat mematuhi sanksi c. Hampir setengah anggota masyarakat (50%) mematuhi sanksi d. Lebih dari sebagian besar anggota masyarakat mematuhi sanksi e. Seluruh anggota masyarakat mematuhi sanksi 6. Jika anggota masyarakat yang menggunakan hutan kehilangan hak untuk memanen/mengambil hasil hutan tertentu, bagaimana hak mereka bisa dik b lik ? a. Siapa yang bertanggung jawab di desa ini untuk membuat aturan tentang hutan? Siapakah mereka? Bagaimana Anda menggambarkan mereka? b. Bagaimana Anda menggambarkan individu-individu yang tidak membuat aturan? Apakah individu-individu tersebut berasal dari satu kelompok agama, satu kelompok suku, kelompok laki-laki atau perempuan? 5. A. Kelembagaan yang terkait pengaturan hutan Apabila aturan adat/desa ditegakkan, sejauh mana sanksinya ditaati oleh anggota masyarakat? a. Tidak satupun anggota masyarakat mematuhi sanksi b. Beberapa anggota masyarakat mematuhi sanksi c. Hampir setengah anggota masyarakat (50%) mematuhi sanksi d. Lebih dari sebagian besar anggota masyarakat mematuhi sanksi e. Seluruh anggota masyarakat mematuhi sanksi 6. Jika anggota masyarakat yang menggunakan hutan kehilangan hak untuk memanen/mengambil hasil hutan tertentu, bagaimana hak mereka bisa dikembalikan? Nining Liswanti, Bayuni Shantiko, Emily Fripp, Esther Mwangi dan Yves Laumonier 84 7. Selama lima tahun terakhir, apakah ada pihak luar (aparat pemerintah) yang pernah diminta untuk memberikan sanksi hukuman terhadap anggota masyarakat yang melanggar? Jika pernah, jelaskan peristiwa tersebut? B. Ancaman dan konflik 8. Jelaskan siapa saja yang memanfaatkan hutan di wilayah desa ini? a. Masyarakat desa, marga tertentu, suku tertentu, masyarakat desa lain? b. Siapa mereka dan darimana mereka datang? c. Seberapa sering mereka menggunakan hutan di wilayah ini? d. Bagian hutan mana yang mereka gunakan dan untuk apa? e. Apakah mereka perlu izin untuk menggunakan hutan? Siapa yang memberikan ijin kepada mereka untuk menggunakan hutan? (Kepala desa? Kepala Adat? Camat?) 9. Jika masyarakat yang menggunakan hutan di wilayah ini berasal dari marga atau suku yang berbeda, apakah mereka memahami dengan baik semua aturan yang berlaku? Apakah mereka mengikuti aturan tersebut? 10. Apakah ada konflik sehubungan dengan penggunaan lahan dan hutan antar amarga atau suku di desa ini selama lima tahun terakhir? a. Jika ada, jelaskan asal usul konflik dan bagaimana konflik tersebut dapat diselesaikan? 10. Apakah ada konflik sehubungan dengan penggunaan lahan dan hutan antar amarga atau suku di desa ini selama lima tahun terakhir? a. Jika ada, jelaskan asal usul konflik dan bagaimana konflik tersebut dapat diselesaikan? b. Berapa lama waktu yang dibutuhkan untuk menyelesaikan konflik? c. Apa tantangan yang Anda hadapi ketika mencoba untuk menyelesaikan konflik?l a. Jika ada, jelaskan asal usul konflik dan bagaimana konflik tersebut dapat diselesaikan? b. Berapa lama waktu yang dibutuhkan untuk menyelesaikan konflik? b. Berapa lama waktu yang dibutuhkan untuk menyelesaikan konflik? c. Apa tantangan yang Anda hadapi ketika mencoba untuk menyelesaikan konflik? c. Apa tantangan yang Anda hadapi ketika mencoba untuk menyelesaikan konflik? 11. Apakah ada perbedaan pendapat atau konflik atas akses hutan atau alokasi lahan dengan pihak lain di luar desa ini selama lima tahun terakhir? 11. Apakah ada perbedaan pendapat atau konflik atas akses hutan atau alokasi lahan dengan pihak lain di luar desa ini selama lima tahun terakhir? Panduan praktis untuk survei mata pencaharian sosial-ekonomi dan hak dan kepemilikan lahan 85 a. Jika ya, siapakah mereka? (misalnya, LSM, dinas, departemen, kementerian, atau pihak lain?) b. Kapan konflik tersebut mulai dan kapan berakhir? c. Apa alasan yang mendasari terjadinya konflik? d. Bagaimana Anda menyelesaikan konflik tersebut? e. Siapakah pihak yang terlibat dalam menyelesaikan konflik? f. Apa yang mereka lakukan untuk menyelesaikan konflik? g. Apakah ada orang yang dominan dalam menyelesaikan konflik? Siapa mereka dan bagaimana mereka melakukannya? h. Apakah Anda puas dengan penyelesaian konflik tersebut? Jika tidak, apa yang Anda lakukan untuk mengatasi ketidakpuasan Anda? Apakah Anda berhasil? Apa saja kendala utama yang Anda hadapi saat menyelesaikan konflik? 12. B. Ancaman dan konflik Selama lima tahun terakhir, apakah pernah timbul masalah yang menyebabkan konflik atas akses ke hutan atau alokasi lahan di antara masyarakat sendiri? a. Jika ya, jelaskan konflik tersebut? b. Bagaimana konflik internal di desa ini dapat diselesaikan? c. Apakah ada cara atau mekanisme untuk menyelesaikan konflik ini? d. Apa hambatan utama untuk menyelesaikan konflik di desa ini? a. Jika ya, siapakah mereka? (misalnya, LSM, dinas, departemen, kementerian, atau pihak lain?) b. Kapan konflik tersebut mulai dan kapan berakhir? c. Apa alasan yang mendasari terjadinya konflik? d. Bagaimana Anda menyelesaikan konflik tersebut? e. Siapakah pihak yang terlibat dalam menyelesaikan konflik? f. Apa yang mereka lakukan untuk menyelesaikan konflik? g. Apakah ada orang yang dominan dalam menyelesaikan konflik? Siapa mereka dan bagaimana mereka melakukannya? h. Apakah Anda puas dengan penyelesaian konflik tersebut? Jika tidak, apa yang Anda lakukan untuk mengatasi ketidakpuasan Anda? Apakah Anda berhasil? Apa saja kendala utama yang Anda hadapi saat menyelesaikan konflik? 12. Selama lima tahun terakhir, apakah pernah timbul masalah yang menyebabkan konflik atas akses ke hutan atau alokasi lahan di antara k di i? h. Apakah Anda puas dengan penyelesaian konflik tersebut? Jika tidak, apa yang Anda lakukan untuk mengatasi ketidakpuasan Anda? Apakah Anda berhasil? Apa saja kendala utama yang Anda hadapi saat menyelesaikan konflik? h. Apakah Anda puas dengan penyelesaian konflik tersebut? Jika tidak, apa yang Anda lakukan untuk mengatasi ketidakpuasan Anda? Apakah Anda berhasil? Apa saja kendala utama yang Anda hadapi saat menyelesaikan konflik? 12. Selama lima tahun terakhir, apakah pernah timbul masalah yang menyebabkan konflik atas akses ke hutan atau alokasi lahan di antara masyarakat sendiri? a. Jika ya, jelaskan konflik tersebut? b. Bagaimana konflik internal di desa ini dapat diselesaikan? c. Apakah ada cara atau mekanisme untuk menyelesaikan konflik ini? d. Apa hambatan utama untuk menyelesaikan konflik di desa ini? Nining Liswanti, Bayuni Shantiko, Emily Fripp, Esther Mwangi dan Yves Laumonier 86 e. Apakah ada hal-hal lain yang dapat membantu untuk mengelola konflik dan bagaimana menyelesaikan konflik secara internal? e. Apakah ada hal-hal lain yang dapat membantu untuk mengelola konflik dan bagaimana menyelesaikan konflik secara internal? e. Apakah ada hal-hal lain yang dapat membantu untuk mengelola konflik dan bagaimana menyelesaikan konflik secara internal? 13. Selama lima tahun terakhir, bagaimana pendapat Anda mengenai tingkat konflik di dalam kelompok telah (mohon jelaskan): a. Meningkat b. Sama saja c. Menurun d. Mengganggu kegiatan sehari-hari e. Mengganggu sewaktu-waktu f. B. Ancaman dan konflik Disalurkan dengan cara-cara yang tidak mengganggu kegiatan sehari‑hari 14. Bagaimana masyarakat menyatakan keinginan dan keprihatinan mereka tentang lahan dan hutan kepada pihak berwenang? (misalnya, pejabat lokal, pejabat pemerintah, dll)? jawaban boleh lebih dari satu: a. Petisi resmi b. Dengar pendapat resmi c. Demonstrasi d. Pertemuan Umum e. Penyanderaan pejabat untuk ditukarkan (cara ilegal) f. Lainnya (jelaskan) 15. Apabila Anda menggunakan salah satu cara di atas, jelaskan cara tersebut diterapkan untuk kasus apa? Apa keinginan/permintaan masyarakat? Apakah pemerintah bisa mengatasi masalah tersebut? Jika tidak, mengapa tidak? Jika ya, apakah Anda puas dengan solusi mereka? Panduan praktis untuk survei mata pencaharian sosial-ekonomi dan hak dan kepemilikan lahan 87 Catatan 9 – Pengalaman dari proyek CoLUPSIA Proyek CoLUPSIA adalah proyek penelitian yang didanai oleh Uni Eropa. Proyek ini dilaksanakan oleh CIRAD (Centre de coopération internationale en recherche agronomique pour le développement) melalui kolaborasi dengan mitra lembaga penelitian yaitu CIFOR (Center for International Forestry Research), Universitas Pattimura di Ambon, Universitas Gadjah Mada di Yogyakarta dan LSM (Telapak, HuMA, TOMA dan Riak Bumi). Salah satu komponen utama CoLUPSIA adalah melengkapi survei sosial-ekonomi (survei rumah tangga dan profil desa), bersamaan dengan diskusi kelompok Salah satu komponen utama CoLUPSIA adalah melengkapi survei sosial-ekonomi (survei rumah tangga dan profil desa), bersamaan dengan diskusi kelompok terfokus dan wawancara informan kunci, di desa-desa di Kapuas Hulu di Provinsi i terfokus dan wawancara informan kunci, di desa-desa di Kapuas Hulu di Provinsi Kalimantan Barat dan Maluku Tengah (Pulau Seram) di Provinsi Maluku. Berikut ini adalah gambaran hasil dan fakta yang penting. terfokus dan wawancara informan kunci, di desa-desa di Kapuas Hulu di Provinsi Kalimantan Barat dan Maluku Tengah (Pulau Seram) di Provinsi Maluku. Berikut ini adalah gambaran hasil dan fakta yang penting. •• Sampel desa dipilih dari wilayah percontohan: empat di Kapuas Hulu dan lima di Maluku Tengah. Wawancara dilakukan di wilayah sampel di masing-masing kabupaten, sebagaimana ditunjukkan pada Gambar 2 dan 3. •• Sampel desa dipilih dari wilayah percontohan: empat di Kapuas Hulu dan lima di Maluku Tengah. Wawancara dilakukan di wilayah sampel di masing-masing kabupaten, sebagaimana ditunjukkan pada Gambar 2 dan 3. Nining Liswanti, Bayuni Shantiko, Emily Fripp, Esther Mwangi dan Yves Laumonier 88 •• Selama 2010 dan 2011, 566 rumah tangga diwawancarai di Maluku Tengah dan 800 di Kapuas Hulu. •• Survei telah dilakukan di tingkat desa dengan jumlah total 39 desa (20 di Kapuas Hulu dan 19 di Maluku Tengah). •• Survei seratus informan kunci telah diselesaikan (40 di Kapuas Hulu dan 60 di Maluku Tengah). •• Data telah dikoleksi, dibersihkan dan dianalisis menggunakan SPSS, Excel dan nVivo (perangkat lunak untuk analisis data kualitatif). Informasi yang dikumpulkan digunakan untuk menghasilkan dua laporan yang merangkum penemuan di masing-masing kabupaten. Kedua laporan tersebut tersedia dalam dokumen terpisah. Informasi yang dikumpulkan digunakan untuk menghasilkan dua laporan yang merangkum penemuan di masing-masing kabupaten. Kedua laporan tersebut tersedia dalam dokumen terpisah. Hasil survei akan digunakan sebagai data dasar untuk perencanaan penggunaan lahan, dan juga untuk menekankan pada isu-isu yang relevan – kendala dan peluang – dalam menghadapi masyarakat terkait dengan penggunaan lahan dan mekanisme yang potensial untuk mengatasi masalah yang ada. Catatan 9 – Pengalaman dari proyek CoLUPSIA Data dasar sosial-ekonomi yang kuat ini dapat dikombinasikan dengan data biofisik untuk memberikan suatu pandangan langsung tentang situasi dalam wilayah studi. anduan praktis untuk survei mata pencaharian sosial-ekonomi dan hak dan kepemilikan lahan 89 Memahami masyarakat dan hubungan internal dan eksternal mereka (misalnya, badan pemerintah) memberikan landasan yang baik untuk proses pengembangan yang melibatkan masyarakat dalam pelaksanaan pembuatan keputusan tentang perencanaan penggunaan lahan secara kolaboratif. Pengalaman dari CoLUPSIA menawarkan beberapa pelajaran umum yang dapat digunakan untuk mereka yang melakukan survei sosial-ekonomi serupa di masa depan. •• Waktu survei adalah penting. Melaksanakan survei selama musim tanam mungkin akan bermasalah karena penduduk desa sangat sibuk. Untuk mengatasi hal seperti ini, tim CoLUPSIA secara sopan bisa mengundang penduduk untuk mengambil bagian dalam wawancara singkat di malam hari dan, dimana diperlukan, dilanjutkan wawancaranya di pagi hari. Sebagai alternatif, wawancara dapat dijadualkan setelah penduduk tiba di rumah dari kebun mereka. Juga sangat sulit untuk mengumpulkan orang dan melakukan diskusi kelompok selama musim tanam. Masalah serupa juga muncul jika mengunjungi desa selama musim panen, saat desa sedang melakukan selamatan/perayaan. •• Insentif sebagai kompensasi. Dalam kondisi tertentu, bagi mereka yang berpartisipasi dalam pekerjaan survei mungkin berharap untuk menerima suatu insentif (misalnya, uang) dari proyek. Karena itu, sejak awal survei sangat penting untuk dijelaskan kepada masyarakat mengenai ada atau tidak adanya insentif. •• Dalam merencanakan proses wawancara, penting sekali mempertimbangkan waktu yang tersedia dan teknik yang paling tepat saat mengajukan pertanyaan. Menjaga minat mereka yang diwawancarai merupakan tantangan, terutama jika survei sangat panjang. Dua jam adalah waktu maksimum untuk wawancara, tetapi jika responden yang diwawancarai kehilangan minat, maka kegiatan survei akan selesai lebih cepat. Gunakan istilah yang sederhana dan jelas, sebaiknya dalam dialek atau bahasa lokal, ketika mengajukan pertanyaan. •• Memeriksa informasi yang dikumpulkan. Pemimpin tim bersama anggotanya harus memeriksa semua data dengan lengkap sebelum meninggalkan desa. Ini akan menghindari masalah yang sering timbul, karena informasi yang dicatat tidak lengkap hanya setelah tim meninggalkan desa dan dalam beberapa kasus ketika telah kembali ke kantor mereka. Metodologi yang digunakan oleh tim CoLUPSIA Tujuan umum penelitian ini adalah untuk memahami faktor sosial-ekonomi yang memengaruhi pola penggunaan sumberdaya alam di tingkat kabupaten. Proyek ini bekerja sama dengan universitas lokal dan LSM. Survei telah selesai dilakukan 90 Nining Liswanti, Bayuni Shantiko, Emily Fripp, Esther Mwangi dan Yves Laumonier Gambar 2. Desa-desa penelitian di empat lokasi pilot di Kapuas Hulu, Kalimantan Barat Gambar 3. Desa-desa penelitian di lima lokasi pilot di Maluku Tengah (Pulau Seram), Maluku Nining Liswanti, Bayuni Shantiko, Emily Fripp, Esther Mwangi dan Yves Laumonier 90 Gambar 2. Desa-desa penelitian di empat lokasi pilot di Kapuas Hulu, Kalimantan Barat Gambar 2. Desa-desa penelitian di empat lokasi pilot di Kapuas Hulu, Kalimantan Barat Gambar 3. Desa-desa penelitian di lima lokasi pilot di Maluku Tengah (Pulau Seram), Maluku Gambar 3. Desa-desa penelitian di lima lokasi pilot di Maluku Tengah (Pulau Seram), Maluku Gambar 3. Desa-desa penelitian di lima lokasi pilot di Maluku Tengah (Pulau Seram), Maluku anduan praktis untuk survei mata pencaharian sosial-ekonomi dan hak dan kepemilikan lahan 91 di 19 desa dan 30 rumah tangga per desa di Maluku Tengah, dan 20 desa dan 40 rumah tangga per desa di Kabupaten Kapuas Hulu. Rumah tangga dipilih secara acak menggunakan metode pengambilan sampel acak sistematis. Proyek ini mengumpulkan data melalui survei rumah tangga, diskusi kelompok dan wawancara informan kunci. Survei ini menggunakan dua kuesioner: kuesioner desa dan rumah tangga. Kuesioner desa digunakan untuk informan kunci, seperti kepala desa dan tokoh adat untuk mendapatkan informasi menyeluruh tentang desa mereka dan isu-isu yang terkait dengan pengelolaan hutan, sejarah konflik dan sudut pandang lokal mengenai pemanfaatan sumberdaya alam. Kuesioner rumah tangga digunakan untuk wawancara dalam rangka mengumpulkan informasi tentang demografi, kegiatan ekonomi dan persepsi tentang jaminan kepemilikan dan penggunaan sumberdaya. Diskusi kelompok terfokus dilakukan untuk membahas isu-isu tentang kepemilikan lahan. Pemilihan kelompok dilakukan berdasarkan jenis kelamin, usia dan keragaman komposisi etnis/suku. Panduan untuk melakukan diskusi kelompok disusun, yaitu meliputi hak milik, jaminan kepemilikan, pengelolaan hutan, konflik/ancaman dan persepsi masyarakat tentang sumberdaya mereka. Kebutuhan informasi Sebagaimana diuraikan dalam panduan sosial-ekonomi, suatu survei harus didesain sedemikian rupa untuk memastikan bahwa informasi yang dikumpulkan dapat memenuhi tujuan umum survei. Misalnya, jika surveinya tentang penggunaan lahan, maka pertanyaan-pertanyaan yang dibuat harus mencerminkan tujuan ini. Survei tidak harus bertujuan untuk mengumpulkan semua informasi sosial- ekonomi yang memungkinkan, melainkan harus ditargetkan untuk tujuan survei yang lebih spesifik. Ini akan menjamin bahwa hasil survei sesuai target dan surveinya dapat dilakukan dalam jangka waktu yang wajar. Tabel 5 memberikan beberapa contoh jenis data yang dapat dikumpulkan dan sarana yang paling tepat untuk mengumpulkan informasi yang digunakan dalam perencanaan penggunaan lahan kolaboratif. Nining Liswanti, Bayuni Shantiko, Emily Fripp, Esther Mwangi dan Yves Laumonier 92 Tabel 5. Data sosial-ekonomi yang dibutuhkan untuk kolaboratif perencanaan penggunaan lahan Informasi Bahan dan metoda •• Demografi (umur, jender, komposisi rumah tangga, pendidikan) •• Pertanian (sekarang dan masa lalu); hewan ternak dan perikanan; dan masalah terkait pertanian •• Layanan penyuluhan •• Hasil hutan •• Pola mata pencaharian (sumber pendapatan, aset, biaya produksi, biaya rumah tangga) •• Persepsi ‘perubahan’ dan ketersediaan hutan •• Sumberdaya alam dan penggunaan lahan (total wilayah setiap jenis pola penggunaan lahan, status lahan, produk pertanian, produk kebun, ternak, ikan, koleksi hasil hutan (kayu dan non-kayu), persepsi tentang hutan, persepsi tentang status lahan, dan kendala) •• Sumber pendapatan dari non- pertanian •• Aset rumah tangga •• Aspek kelembagaan (keanggotaan organisasi, layanan penyuluhan) •• Kuesioner •• Survei rumah tangga (30 rumah tangga) •• Profil desa •• Infrastruktur •• Situasi perekonomian desa •• Program pembangunan •• Masalah khusus degan desa dan/ atau hutan •• Hutan yang berhubungan dengan pengaturan kelembagaan (jenis lembaga, peraturan, denda) •• Ancaman dan konflik (termasuk cara- cara untuk menyelesaikan konflik) •• Kuesioner •• Wawancara dengan kepala desa, kepala adat, dan kepala suku atau marga •• Hak atas sumberdaya hutan, penegakan dan kepatuhan •• Diskusi kelompok terfokus (kelompok dibagi berdasarkan umur dan jender) Tabel 5. Data sosial-ekonomi yang dibutuhkan untuk kolaboratif perencanaan penggunaan lahan Tabel 5. Data sosial-ekonomi yang dibutuhkan untuk kolaboratif perencanaan penggunaan lahan •• Pertanian (sekarang dan masa lalu); hewan ternak dan perikanan; dan masalah terkait pertanian •• Pola mata pencaharian (sumber pendapatan, aset, biaya produksi, biaya rumah tangga) •• Persepsi ‘perubahan’ dan ketersediaan hutan •• Sumberdaya alam dan penggunaan lahan (total wilayah setiap jenis pola penggunaan lahan, status lahan, produk pertanian, produk kebun, ternak, ikan, koleksi hasil hutan (kayu dan non-kayu), persepsi tentang hutan, persepsi tentang status lahan, dan kendala) •• Sumber pendapatan dari non- pertanian •• Kuesioner •• Wawancara dengan kepala desa, kepala adat, dan kepala suku atau marga •• Hutan yang berhubungan dengan pengaturan kelembagaan (jenis lembaga, peraturan, denda) •• Ancaman dan konflik (termasuk cara- cara untuk menyelesaikan konflik) •• Persepsi tentang kepemilikan lahan •• Proyek pembangunan desa Rujukan Abdrabo, M.A. dan M.A. Hassaan 2003. From river catchments areas to the sea: comparative and integrated approach to the ecology of Mediterranean coastal zones for sustainable management (MEDCORE). A manual for socioeconomic study. Centre for Environment and Development for the Arab Region and Europe (CEDARE). Colfer, C.J.P., M.A. Brocklesby, C. Diaw, P. Etuge, M. Günter, E. Harwell, C. McDougall, N.M. Porro, R. Porro dan R. Prabhu 1999. The BAG: basic assessment guide for human well-being. The criteria & indicator toolbox series No. 5. CIFOR, Bogor, Indonesia. Dawson, S., dan L. Manderson 1993. A manual for the use of focus groups. Methods for social research in disease. International Nutrition Foundation for Developing Countries (INFDC), Boston, MA, USA. De Brune dan R. Santoso 1998. Socio-economic for land use planning. FIMP- BPKP Technical Series No. 11. Food and Agriculture Organization of the United Nations (FAO) 2002. FAO land tenure studies 3: land tenure and rural development. FAO, Rome. Glaser, B.G. dan A.L. Strauss 1967. The Discovery of Grounded Theory: Strategies for Qualitative Research, Chicago, Aldine Publishing Company Leroy-Iskandar, C. dan R. Santoso 2002. Socio-economic for land use planning 1: general issues and methodologies. FIMP-BPKP Technical Series No. 20. Liswanti, N. dan I. Basuki 2009. Guidelines for adapted Multidisciplinary Landscape Assessment methods for fire management project in India. CIFOR, Bogor, Indonesia. Markelova, H. 2012. Report on the findings from the focus group discussion surveys and key informant interviews for the ‘Collaborative land use planning and sustainable institutional arrangements for strengthening land tenure, forest, and community right in Indonesia. CoLUPSIA project. Bogor, Indonesia. Mendenhall, W., R.J. Beaver dan B.M. Beaver 2003. Introduction to Probability and Statistics. 11th Edisi, Brooks/Cole, USA. NVivo 9 2010. NVivo qualitative data analysis software Version 9, 2010. QSR International Pty Ltd. Roche, R. 2007. Livelihoods approaches as a conservation tool. IGERT Program. University of Rhode Island. Kingston, RI, USA. Nining Liswanti, Bayuni Shantiko, Emily Fripp, Esther Mwangi dan Yves Laumonier 94 Shantiko, B., Y. Andries, B. Morialkosu dan D. Amarduan 2004. Socio economic analysis of rural Tanimbar. Tanimbar Land Use Planning Project. Technical Series No. 3. Jakarta. Sheil, D. dan N. Liswanti 2006. Scoring the importance of tropical forest landscapes with local people: Patterns and insights. Environmental Management 38:126–136. Sheil, D., R.K. Puri, I. Basuki, M. van Heist, M. Wan, N. Liswanti, Rukmiyati, M.A. Agung Sardjono, I. Samsoedin dan K. Sidiyasa 2003. Rujukan Exploring biological diversity, environment and local people’s perspectives in forest landscapes. Methods for a multidisciplinary landscape assessment. 2nd edition. CIFOR, Bogor, Indonesia. Turell, G., C. Patterson, B. Oldenburg, T. Gould dan M.A. Roy 2003. The socio-economic patterning of survey participation and non-response error in multilevel study of food purchasing behavior: area and individual level characteristics. Public Health Nutrition 6:181–89. Varkevisser, C., I. Pathmanathan dan A. Brownlee 2003. Designing and conducting health system research projects. Volume 1: proposal development and fieldwork. World Health Organization/International Development Research Centre. KIT Publishers, Amsterdam. Penelitian ini dilakukan sebagai bagian dari proyek perencanaan penggunaan lahan kolaboratif dan penataan kelembagaan yang berkelanjutan, didanai oleh Uni Eropa, dan dijalankan oleh CIRAD yang bermitra dengan CIFOR, TELAPAK dan beberapa LSM lokal dan universitas. Tujuan penelitian ini adalah memberikan kontribusi untuk menghindari degradasi lingkungan dan memperkuat hak kepemilikan lahan dan hak masyarakat dengan mengintegrasikan pandangan semua pemangku kepentingan dalam proses-proses perencanaan penggunaan lahan. Hasil yang diharapkan adalah adanya hubungan antara perencanaan tata guna lahan, alokasi lahan, dan penyediaan dan potensi pembayaran jasa ekosistem. Proyek ini fokus pada dua kabupaten yaitu Kapuas Hulu dan Maluku Tengah di Indonesia. Situs web Badan Pusat Statistik (BPS) (Indonesia’s Central Statistics Agency): http://www.bps.go.id CoLUPSIA: http://www.colupsia.org/. Badan Pusat Statistik (BPS) (Indonesia’s Central Statistics Agency): http://www.bps.go.id CoLUPSIA: http://www.colupsia.org/. Publikasi ini memuat hasil penelitian pendahuluan, dan signifikan untuk isu-isu yang berkaitan dengan hutan tropis, yang perlu dipublikasikan secara tepat waktu. Publikasi ini diproduksi sebagai informasi dan promosi ajang diskusi. Isi publikasi ini telah diulas secara internal namun belum mengalami proses lebih lebih lanjut untuk diulas oleh pihak eksternal. Publikasi ini memuat hasil penelitian pendahuluan, dan signifikan untuk isu-isu yang berkaitan dengan hutan tropis, yang perlu dipublikasikan secara tepat waktu. Publikasi ini diproduksi sebagai informasi dan promosi ajang diskusi. Isi publikasi ini telah diulas secara internal namun belum mengalami proses lebih lebih lanjut untuk diulas oleh pihak eksternal. Alat survei sosial-ekonomi dirancang untuk mengumpulkan informasi sebagai sarana meningkatkan pemahaman tentang sistem pengelolaan sumberdaya lokal, penggunaan sumberdaya dan kepentingan relatif masing-masing untuk rumah tangga dan desa. Survei juga memberikan informasi tentang interaksi antara sistem pengambilan keputusan oleh pemerintah, tren persepsi masyarakat dan isu-isu prioritas, dan pengetahuan tentang lembaga berbasis masyarakat dan peran mereka dalam pemanfaatan berkelanjutan dan konservasi sumberdaya alam. Penggunaan survei merupakan langkah awal yang penting dalam melibatkan masyarakat lokal dan memastikan bahwa suara-suara lokal didengar dan bahwa hubungan masyarakat dengan sumber daya, akses dan hak atas lahan dimasukkan dalam proses perencanaan tata guna lahan kolaboratif. Perencanaan penggunaan lahan kolaboratif melibatkan kerja sama dengan semua pemangku kepentingan – pemerintah, masyarakat lokal, sektor swasta dan individu lain yang relevan – untuk memastikan bahwa lahan digunakan secara berkelanjutan, menghindari dampak negatif atau ancaman dari degradasi lingkungan dan hilangnya hutan sambil memastikan bahwa pertimbangan sosial dan ekonomi dari semua pengguna dipertimbangkan. Dalam kaitan khususnya dengan masyarakat, tujuan perencanaan penggunaan lahan kolaboratif adalah sebagai berikut: Untuk memastikan bahwa keputusan perencanaan penggunaan lahan diambil dengan mempertimbangkan pendapat masyarakat lokal, kebutuhan penggunaan lahan dan kondisi sosial-ekonomi (peluang dan kendala), termasuk hak akses ke lahan dan untuk menggunakannya Tujuan dari panduan ini (dan Catatan Pendukung) adalah untuk memberikan langkah-langkah praktis (desain survei, pengambilan sampel dan kebutuhan data; perencanaan dan pelatihan; melakukan survei) untuk para praktisi di lapangan dalam melakukan survei sosial ekonomi rumah tangga dan desa, termasuk diskusi kelompok terfokus dan wawancara informan kunci. Informasi yang diperoleh dapat digunakan secara langsung dalam proses perencanaan penggunaan lahan kolaboratif yang membantu untuk melancarkan atau memperkuat konsensus tentang kepemilikan lahan dan hak-hak di daerah, sekarang dan di masa depan. Situs web blog.cifor.org cifor.org Center for International Forestry Research CIFOR memajukan kesejahteraan manusia, konservasi lingkungan dan kesetaraan melalui penelitian yang berorientasi pada kebijakan dan praktik kehutanan di negara berkembang. CIFOR merupakan salah satu Pusat Penelitian Konsorsium CGIAR. CIFOR berkantor pusat di Bogor, Indonesia dengan kantor wilayah di Asia, Afrika dan Amerika Selatan. Center for International Forestry Research y CIFOR memajukan kesejahteraan manusia, konservasi lingkungan dan kesetaraan melalui penelitian yang berorientasi pada kebijakan dan praktik kehutanan di negara berkembang. CIFOR merupakan salah satu Pusat Penelitian Konsorsium CGIAR. CIFOR berkantor pusat di Bogor, Indonesia dengan kantor wilayah di Asia, Afrika dan Amerika Selatan. CIFOR memajukan kesejahteraan manusia, konservasi lingkungan dan kesetaraan melalui penelitian yang berorientasi pada kebijakan dan praktik kehutanan di negara berkembang. CIFOR merupakan salah satu Pusat Penelitian Konsorsium CGIAR. CIFOR berkantor pusat di Bogor, Indonesia dengan kantor wilayah di Asia, Afrika dan Amerika Selatan.
https://openalex.org/W4386705491	https://jmir.org/api/download?alt_name=resprot_v12i1e43654_app2.pdf&filename=ad7befc75541b66234f835a323410a39.pdf	English	null	A Tailored mHealth Intervention for Improving Antenatal Care Seeking and Health Behavioral Determinants During Pregnancy Among Adolescent Girls and Young Women in South Africa: Development and Protocol for a Pilot Randomized Controlled Trial	JMIR research protocols	2,023	cc-by	8,563	Questions) Page 1 of 31 Survey Flow y Standard: Introduction message (1 Question) Standard: Introduction and welcome (0 Questions) Standard: Basic information (4 Questions) Standard: Section A: Sociodemographic information (21 Questions) Standard: History of pregnancy (10 Questions) Standard: Current pregnancy (9 Questions) Standard: Access to cell phone (6 Questions) Standard: Tobacco, alcohol and drug use (4 Questions) Standard: Experiences of violence (2 Questions) Standard: Healthy lifestyle and includes nutrition, physical activity, HIV, TB and stress (2 Questions) Standard: Symptoms of Depression (8 Questions) Standard: HIV and Tuberculosis (TB) (15 Questions) Standard: Knowledge (1 Question) Standard: Risk perceptions (2 Questions) Standard: Peer norms (2 Questions) Standard: Family norms (2 Questions) Standard: Partner/boyfriend norms (2 Questions) Standard: School norms (2 Questions) Standard: Self-efficacy (2 Questions) Standard: Intention (2 Questions) Standard: Action planning (2 Questions) Block: (0 Questions) Page Break Page 1 of 31 Page 1 of 31 Start of Block: Introduction message A Pilot Study of Improving Outcomes in Teenage Pregnancy Using a Combined Tailored M- Health Program and Motivational Interviewing Intervention Thank you for participating in this study on improving the outcomes of teenage pregnancy. We greatly value your participation in this research study and your willingness to share your knowledge and experiences. Some of the information we will ask you may be sensitive which may make you a bit uncomfortable. We would like you to take your time and think about the answers and respond as honestly as possible to assist with this important research. End of Block: Introduction message Basic information Q2 Cellphone/telephone number (Kindly use the following format XXXXXXXXXX) Q2 Cellphone/telephone number (Kindly use the following format XXXXXXXXXX) Q2 Cellphone/telephone number (Kindly use the following format XXXXXXXXXX) o Main number (1) ________________________________________________ o Alternative number (2) ________________________________________________ o Alternative number (3) ________________________________________________ Q3 Was consent obtained? Q3 Was consent obtained? Q3 Was consent obtained? Sociodemographic information Q4 Date of birth (dd/mm/yyyy) Page 2 of 31 Page 2 of 31 Q5 ID number (Kindly use the following format 0201400635086) Q6 Passport number Q7 Race o Black African (1) o White (2) o Coloured (3) o Indian/Asian (4) o Q8 Nationality Q9 Are you in school? Q9 Are you in school? Skip To: Q12 If Do you attend a FET college ? = Yes Skip To: Q14 If Do you attend a FET college ? = No Skip To: Q12 If Do you attend a FET college ? = Yes Skip To: Q14 If Do you attend a FET college ? = No Q12 What are you studying? Q13 In which community is your school located? Q14 Which community do you live in? Q9 Are you in school? o Yes (1) o No (2) Q10 What grade are you in? (6,7,8,9,10,11,12) Skip To: Q13 If What grade are you in? (6,7,8,9,10,11,12) Is Not Empty Skip To: Q11 If What grade are you in? (6,7,8,9,10,11,12) Is Empty Page 3 of 31 Page 3 of 31 Q11 Do you attend a FET college ? o Yes (1) o No (2) o Yes (1) o No (2) o Yes (1) o No (2) Q15 Is your pregnancy registered at a clinic? Q15 Is your pregnancy registered at a clinic? o Yes (1) o No (2) Skip To: Q16 If Is your pregnancy registered at a clinic? = Yes Skip To: Q17 If Is your pregnancy registered at a clinic? = No Q16 What is the name of the clinic that your pregnancy is registered at? Q17 What is the name of the clinic where you want to register your pregnancy at? Q18 What is the due date of your pregnancy, if known? (dd/mm/yyyy) o Q18 What is the due date of your pregnancy, if known? (dd/mm/yyyy) o Due date (1) ________________________________________ Page 4 of 31 Page 4 of 31 Q19 What was the date of your last menstrual period? (dd/mm/yyyy) Q19 What was the date of your last menstrual period? (dd/mm/yyyy) o Date of last menstrual period (1) ________________________________________ Q20 What type of dwelling do you live in? Q20 What type of dwelling do you live in? Q20 What type of dwelling do you live in? o Brick house (1) o Flat / apartment (2) o A shack (3) o A house made of mud and stick (4) o A house made of mud, brick and stick (5) o Other (6) ________________________________________________ o A house made of mud and stick (4) o A house made of mud, brick and stick (5) Q21 Does your father (male guardian) have a paid job? (Paid job also refers to those who are self- employed e.g. your father has a shop at home) Q21 Does your father (male guardian) have a paid job? (Paid job also refers to those who are self- employed e.g. your father has a shop at home) o Yes, works 5 or more days a week (1) o Yes, works less than 5 days a week (2) o No, gets a social grant (3) o No, unemployed (4) o My father is deceased (7) Page 5 of 31 Page 5 of 31 Q22 Does your mother (female guardian) have a paid job? (Paid job also refers to those who are self- employed e.g. your mother has a shop at home) Q22 Does your mother (female guardian) have a paid job? (Paid job also refers to those who are self- employed e.g. your mother has a shop at home) o Yes, works 5 or more days a week (1) o Yes, works less than 5 days a week (2) o No, gets a social grant (3) o No, gets a social grant (3) o No, unemployed (4) o No, is ill/disabled (5) o No, is retired (6) o My mother is deceased (7) o I don’t know (8) Q23 The cost of living is a concern for many families. Can you tell me which option best describes your household’s situation? o Not enough money for basic things like food and clothes (1) o Money for food and clothes, but short on many other things (2) o We have most of the important things, but few luxury goods (3) o Money for extra things such as holidays and luxury goods (4) o Money for extra things such as holidays and luxury goods (4) End of Block: Section A: Sociodemographic information Start of Block: History of pregnancy History of pregnancy History of pregnancy Q24 Have you been pregnant before? o Yes (1) o No (2) Skip To: Q25 If Have you been pregnant before? = Yes Skip To: Q26 If Have you been pregnant before? = Yes Skip To: End of Block If Have you been pregnant before? = No Q25 If yes, how many times? Page 6 of 31 Page 6 of 31 Q26 Did you experience bleeding during pregnancy or after delivery? Q26 Did you experience bleeding during pregnancy or after delivery? Q26 Did you experience bleeding during pregnancy or after delivery? o Yes (1) o No (2) o Yes (1) o No (2) o Yes (1) o No (2) Q27 What kind of delivery did you have? o Vaginal delivery (1) o Caesarean Section (C-Section) (2) o Other, please specify (3) ________________________________________________ Q27 What kind of delivery did you have? o Vaginal delivery (1) o Caesarean Section (C-Section) (2) o Other, please specify (3) ________________________________________________ Q27 What kind of delivery did you have? o Vaginal delivery (1) o Caesarean Section (C-Section) Q28 Was it an assisted delivery? (Vaginal delivery of a baby performed with the help of forceps or a vacuum device) o Yes (1) o No (4) Q28 Was it an assisted delivery? (Vaginal delivery of a baby performed with the help of forceps or a vacuum device) o Yes (1) o No (4) it an assisted delivery? (Vaginal delivery of a baby performed with the help of forceps or a i ) o Yes (1) o No (4) Q29 Have you had a stillbirth before? o Yes (1) o No (3) o Yes (1) o No (3) Q30 Have you had a miscarriage before? o Yes (1) o No (3) Q31 Is your child still alive? o Yes (1) o No (3) Q31 Is your child still alive? Page 7 of 31 Page 7 of 31 Q32 Have you been registered on Mom Connect for previous pregnancies? o Yes (1) o No (2) o Yes (1) o No (2) o Yes (1) o No (2) End of Block: History of pregnancy Q33 Current pregnancy Q33 Current pregnancy Q34 Did you agree to have sex when you became pregnant? Q34 Did you agree to have sex when you became pregnant? o Yes (1) o No (3) o Yes (1) o No (3) Q35 What is your relationship to the person that made you pregnant? Q35 What is your relationship to the person that made you pregnant? o o o o o o o o o o Boyfriend (1) o Neighbour (17) o Relative (18) o Teacher (19) o Friend (20) o Classmate (21) o Stranger (22) o Casual boyfriend (23) o Someone who paid you for sex (24) Someone who paid you for sex (24) Q36 Was the person who made you pregnant __________________ older than you? _ older than you? Q37 How do you feel about this pregnancy? Q37 How do you feel about this pregnancy? Q37 How do you feel about this pregnancy? o Happy (1) o Sad (8) o Angry (9) o Afraid (10) o Embarrassed/Ashamed (11) o Confused (12) Q38 Did you consider having an abortion? Q38 Did you consider having an abortion? o Yes (1) o No (3) o Yes (1) o No (3) o Yes (1) o No (3) Skip To: Q39 If Did you consider having an abortion? = Yes Skip To: Q40 If Did you consider having an abortion? = No Q39 Where did you have an abortion? Q39 Where did you have an abortion? o A Private facility (1) o A government hospital (5) o A traditional healer (6) o A backstreet abortion (7) o A Private facility (1) Q40 Did anyone ask you to have an abortion? o Yes (1) o No (3) o Yes (1) o No (3) Skip To: Q41 If Did anyone ask you to have an abortion? = Yes Skip To: End of Block If Did anyone ask you to have an abortion? = No Skip To: Q41 If Did anyone ask you to have an abortion? = Yes Skip To: End of Block If Did anyone ask you to have an abortion? = No Page 9 of 31 Q26 Did you experience bleeding during pregnancy or after delivery? o Younger (1) o Same age (6) o 1- 5 years older (7) o 6 -10 years older (8) o 11 or more years older (9) Page 8 of 31 Page 8 of 31 Start of Block: Access to cell phone Access to cell phone Q42 Is your cellphone: o Your own (1) o I share a cellphone with someone (2) Q43 Is your cellphone a basic phone (can only send SMS’s and receive calls), or is your cellphone a smart phone (can download WhatsApp, BBM, Facebook, and other cell phone applications)? Please select. o Basic phone (1) o Smart phone (2) Page 10 of 31 Page 10 of 31 No one asked me to have an abortion (13) End of Block: Current pregnancy Start of Block: Access to cell phone Page 9 of 31 Page 9 of 31 Q41 Please select all the people who asked you? (Multiple responses possible) ▢ Boyfriend (1) ▢ Relative/s (such as parent, sister, brother, grandparent, aunts, other relatives ▢ Friend (9) ▢ Teacher (10) ▢ Neighbour (11) ▢ Health care worker (12) ▢ No one asked me to have an abortion (13) ▢ Boyfriend (1) ▢ Relative/s (such as parent, sister, brother, grandparent, aunts, other relatives) (8) ▢ Friend (9) ▢ Teacher (10) ▢ Neighbour (11) ▢ Health care worker (12) ▢ No one asked me to have an abortion (13) ▢ ▢ ▢ ▢ ▢ ▢ ▢ ▢ ▢ ▢ ▢ ▢ ▢ ▢ Relative/s (such as parent, sister, brother, grandparent, aunts, other relatives) (8) Q45 Have you been registered on Mom Connect for your current pregnancy? o Yes (1) o No (2) o Yes (1) o No (2) o Yes (1) o No (2) Q46 What language would you prefer to receive your messages? o English (1) o Afrikaans (2) o IsiXhosa (3) o English (1) o Afrikaans (2) o IsiXhosa (3) o English (1) End of Block: Access to cell phone Start of Block: Tobacco, alcohol and drug use Tobacco, alcohol and drug use Q47 During the past month, on how many days did you have at least one drink of alcohol? o 0 (1) o 1 to 5 (7) o 6 to 9 (8) o 10 to 19 (9) o e. 20 to 30 (10 o 0 (1) o 1 to 5 (7) o 6 to 9 (8) o 10 to 19 (9) o e. 20 to 30 (10) Page 11 of 31 Page 11 of 31 Page 10 of 31 Q44 What do you use your cellphone for? (you may choose more than one option) Q44 What do you use your cellphone for? (you may choose more than one option) ▢ I use my cellphone for making and receiving calls (1) ▢ I use my cellphone for social networks (e.g. Facebook, WhatsApp, Twitter, BBM, or chat zone services) (7) ▢ I use my cellphone for SMSs (8) ▢ I use my cellphone for surfing the internet (9) Q45 Have you been registered on Mom Connect for your current pregnancy? Page 11 of 31 Page 11 of 31 Q48 During the past month (30 days), on how many days did you smoke cigarettes, cigars, pipes, or e- cigarettes, or use smokeless tobacco such as snuff or chewing tobacco? Q48 During the past month (30 days), on how many days did you smoke cigarettes, cigars, pipes, or e- cigarettes, or use smokeless tobacco such as snuff or chewing tobacco? o 0 (1) o 1 or 2 (13) o 3 to 5 (14) o 6 to 9 (15) o 10 to 19 (16) o 20 to 29 (17) o All 30 (18) o 0 (1) o 1 or 2 (13) o 3 to 5 (14) o 6 to 9 (15) o 10 to 19 (16) o 20 to 29 (17) o All 30 (18) Q49 In the past 30 days, how many times have you used drugs such as weed/dagga(?), tik, ecstasy, or mandrax? Q49 In the past 30 days, how many times have you used drugs such as weed/dagga(?), tik, ecstasy, or mandrax? Q49 In the past 30 days, how many times have you used drugs such as weed/dagga(?), tik, ecstasy, or mandrax? o 0 (1) o 1 or 2 (8) o 3 to 9 (9) o 10 to 19 (10) o 20 or more (11) o 0 (1) o 1 or 2 (8) o 3 to 9 (9) o 10 to 19 (10) o 20 or more (11) o 0 (1) o 1 or 2 (8) o 3 to 9 (9) o 10 to 19 (10) o 20 or more (11) o 20 or more (11) End of Block: Tobacco, alcohol and drug use Start of Block: Experiences of violence Page 12 of 31 Page 12 of 31 Experiences of violence Q50 In your whole lifetime, how many times have you experienced the following? Page 11 of 31 (54) o o o o o 51.8 Foods high in fats and sugar are good for my pregnancy (55) o o o o o 51.9 I can drink as much tea/coffee as I want during my pregnancy (56) o o o o o 51.10 Multivitamins are important to take during my pregnancy as they contain the important nutrients I need for my body (2) o o o o o 51.11 If I eat a lot of green leafy vegetables, I don’t need to take an iron supplement. (57) o o o o o 51.12 Pregnant women should take folic acid before and during pregnancy (58) o o o o o 51.13 Foods rich in iron such as meat, poultry, pork and fish are important during pregnancy (59) o o o o o 51.14 Milk, maas and yoghurt are good for me during pregnancy (60) o o o o o 51.15 Calcium is important for healthy teeth and bones (61) o o o o o 51.16 I can lose weight during my pregnancy (62) o o o o o 51.17 It is okay to gain up to 16kgs weight during pregnancy (63) o o o o o 51.1 Pregnancy is a good time for me to focus on eating correctly (1) 51.2 I want to eat a healthy balanced diet during pregnancy (49) 51.3 It is okay to miss meals in a day (50) 51.4 Healthy snacks between meals are good (51) 51.5 Healthy eating keeps my energy levels up (52) 51.6 If I miss meals my baby will still be fine (53) 51.7 I can eat as much potatoes or bread, as I want in my pregnancy. (54) 51.8 Foods high in fats and sugar are good for my pregnancy (55) 51.9 I can drink as much tea/coffee as I want during my pregnancy (56) 51.10 Multivitamins are important to take during my pregnancy as they contain the important nutrients I need for my body (2) 51.11 If I eat a lot of green leafy vegetables, I don’t need to take an iron supplement. Page 11 of 31 1= Never (1) 2=Once or twice (2) 3=A few times (3) 4=Many times (4) 50.1Threat by a relative (1) o o o o 50.2 Physical violence by a relative (30) o o o o 50.3 Been touched by a relative in a way that makes you uncomfortable (31) o o o o 50.4 Threat by a teacher (32) o o o o 50.5 Physical violence by a teacher (33) o o o o 50.6 Been touched by a teacher in a way that makes you uncomfortable (34) o o o o 50.7 Threat by a boyfriend/partner (35) o o o o 50.8 Physical violence by a boyfriend/partner (36) o o o o 50.9 Been touched by a boyfriend/partner in a way that makes you uncomfortable (37) o o o o 50.10 Threat by a health care worker (10) o o o o 50.11 Physical violence by a health care worker (38) o o o o 50.12 Been touched by a health care worker in a way that makes you uncomfortable (39) o o o o 50.13 Been physically forced to have sex (when the penis enters the vagina or anus) when you did not want to (40) o o o o Page 13 of 31 Page 13 of 31 Healthy lifestyle and includes nutrition, physical activity, HIV, TB and stress Q51 This section is about a healthy lifestyle and includes nutrition, physical activity, HIV, TB and stress. You will not be judged based on the way you answer. Please answer to the best of your ability. Please select your response. Page 14 of 31 Page 14 of 31 Page 14 of 31 Strongly Disagree (1) Disagree (2) Agree (3) Strongly Agree (4) I don’t know (5) 51.1 Pregnancy is a good time for me to focus on eating correctly (1) o o o o o 51.2 I want to eat a healthy balanced diet during pregnancy (49) o o o o o 51.3 It is okay to miss meals in a day (50) o o o o o 51.4 Healthy snacks between meals are good (51) o o o o o 51.5 Healthy eating keeps my energy levels up (52) o o o o o 51.6 If I miss meals my baby will still be fine (53) o o o o o 51.7 I can eat as much potatoes or bread, as I want in my pregnancy. Page 11 of 31 (57) 51.12 Pregnant women should take folic acid before and during pregnancy (58) 51.13 Foods rich in iron such as meat, poultry, pork and fish are important during pregnancy (59) 51.14 Milk, maas and yoghurt are good for me during pregnancy (60) 51.15 Calcium is important for healthy teeth and bones (61) 51.16 I can lose weight during my pregnancy (62) 51.17 It is okay to gain up to 16kgs weight during pregnancy (63) Page 15 of 31 51.18 Constipation can be minimised by high fibre intake, water and exercise during pregnancy (64) o o o o o 51.19 It is important to walk for a few minutes every day during my pregnancy (3) o o o o o 51.20 Activities such as dancing, running, and walking fast are safe to do during pregnancy (65) o o o o o 51.21 Activities with rapid movements and contact is safe for me at the end of my pregnancy (66) o o o o o Symptoms of Depression Symptoms of Depression Q52 In the past 6 months, have you ever felt sad or hopeless? Q52 In the past 6 months, have you ever felt sad or hopeless? o Yes (1) o No (2) Q53 In the past 6 months, have you had problems concentrating, recalling details, and making decisions? o Yes (1) o No (2) o o Yes (1) o No (2) Q54 In the past 6 months, have you been crying more often than usual? o Yes (1) o No (2) Page 16 of 31 Page 16 of 31 Q55 In the past 6 months, did you ever seriously consider attempting suicide (that is, take some action to end your life)? Q55 In the past 6 months, did you ever seriously consider attempting suicide (that is, take some action to end your life)? o Yes (1) o No (2) o Yes (1) o No (2) o I don’t know (3) Q56 In the past 6 months, did you make a plan about how you would attempt suicide (that is take some action to end your life)? o Yes (1) o No (2) Q57 In the past 6 months, how many times did you actually attempt suicide (that is take some action to end your life)? o 0 times (1) o 1 times (2) o 2 or 3 times (3) o 4 or 5 times (4) o 0 times (1) o 1 times (2) o 2 or 3 times (3) o 4 or 5 times (4) o 6 or more times (5) o 6 or more times (5) Q58 In the past 6 months, if you attempted suicide, did any attempt result in an injury, poisoning, or overdose that had to be treated by a doctor or nurse? Q58 In the past 6 months, if you attempted suicide, did any attempt result in an injury, poisoning, or overdose that had to be treated by a doctor or nurse? o I did not attempt suicide (that is take some action to end my life) during the past 6 months (1) o I did not attempt suicide (that is take some action to end my life) during the past 6 months (1) o Yes (4) o No (5) o Yes (4) o No (5) o Yes (4) o No (5) End of Block: Symptoms of Depression Q60 When did you find out you were HIV+? Q52 In the past 6 months, have you ever felt sad or hopeless? o Before the pregnancy (1) o During the pregnancy (5) o Do not know (6) o Do not want to answer (7) Q60 When did you find out you were HIV+? Q64 Are you currently on treatment? Q64 Are you currently on treatment? o Yes (1) o No (2) Skip To: Q66 If Are you currently on treatment? = Yes Skip To: Q66 If Are you currently on treatment? = No Skip To: Q66 If Are you currently on treatment? = Yes Skip To: Q66 If Are you currently on treatment? = No Q60 When did you find out you were HIV+? Q60 When did you find out you were HIV+? o Before the pregnancy (1) o During the pregnancy (5) o Do not know (6) o Do not want to answer (7) Q61 Are you currently on anti-retroviral treatment (ART)? Skip To: Q63 If Are you currently on anti-retroviral treatment (ART)? = Yes Skip To: Q63 If Are you currently on anti-retroviral treatment (ART)? = No Skip To: Q63 If Are you currently on anti-retroviral treatment (ART)? = Do not want to answer Q62 How many times have you been tested during your pregnancy for HIV? Q62 How many times have you been tested during your pregnancy for HIV? o Zero (1) o Once (5) o Twice (6) o Zero (1) o Once (5) o Twice (6) o Do not want to tell you (8) o Do not want to tell you (8) Page 18 of 31 Q63 What is your TB status? o Positive (1) o Negative (5) o Do not know (6) o Do not want to answer (7) Skip To: Q64 If What is your TB status? = Positive Skip To: Q65 If What is your TB status? = Negative Skip To: Q65 If What is your TB status? = Do not know Skip To: Q65 If What is your TB status? = Do not want to answer Q63 What is your TB status? Q63 What is your TB status? Q63 What is your TB status? o Negative (5) o Do not know (6) Q72 I will be able to breastfeed my baby even if I have TB or HIV o Yes (1) o No (5) Q65 Have you been tested for TB during your pregnancy? Q65 Have you been tested for TB during your pregnancy? Q65 Have you been tested for TB during your pregnancy? o Yes (1) o No (2) o Don’t want to answer (4) o Don’t want to answer (4) Q66 If you are infected with HIV or with TB, do you think you can infect your new-born baby with TB/HIV if you are not on treatment? o Yes (1) o No (5) o Yes (1) o No (5) o Do not know (6) o Do not know (6) o Do not want to answer (7) Page 19 of 31 Page 19 of 31 Q67 If I am diagnosed with HIV early, the outcome for the baby is good Q67 If I am diagnosed with HIV early, the outcome for the baby is good o Yes (1) o No (6) o Do not know (7) o Do not know (7) o Do not want to answer (8) o I do not have HIV (9) Q68 If I am diagnosed with TB early, the outcome for the baby is good o Yes (1) o No (6) o Do not know (7) o Do not want to answer (8) o I do not have TB (9) o Do not want to answer (8) Q69 If the diagnosis and treatment for HIV are delayed it can increase the risk of early labour o Yes (1) o No (6) o Yes (1) o No (6) o Do not know (7) o Do not want to answer (8) o Do not want to answer (8) Q70 If the diagnosis and treatment for TB are delayed it can increase the risk of early labou o Yes (1) o No (6) o Yes (1) o No (6) o Do not know (7) o Do not know (7) o Do not want to answer (8) o Do not want to answer (8) Page 20 of 31 Page 20 of 31 Q71 TB can be cured with special antibiotics, which can be taken safely during pregnancy. o Yes (1) o No (6) o Yes (1) o No (6) o Yes (1) o No (6) o Do not know (7) o Do not know (7) o Do not want to answer (8) Q72 I will be able to breastfeed my baby even if I have TB or HIV Start of Block: Knowledge o Do not want to answer (7) End of Block: HIV and Tuberculosis (TB) Start of Block: Knowledge Page 21 of 31 Page 21 of 31 Q73 The section is about your current knowledge on antenatal care ability. Please select your response. 73.1 Antenatal care appointment benefits the baby, not the pregnant woman. (1) 73.2 There is no need to have the first antenatal appointment booked as early as possible (before 12 weeks of pregnancy), because it can be done at any time during pregnancy. (17) 73.3 If a pregnant woman is underweight, she needs extra antenatal care. (18) 73.4 If a pregnant woman is over-weight, she needs extra antenatal care. (19) 73.5 Pregnant women who do not receive antenatal care are more likely to give birth to a low-weight baby. (20) 73.6 Pregnant women who do not receive antenatal care are more likely to have miscarriages and give still birth (baby not alive). (21) 73.7 Pregnant women who receive late antenatal care (after 28 weeks of pregnancy) and their babies, are more likely to have health problems. (22) 73.8 Preeclampsia is a serious pregnancy problem characterised by raised blood pressure. (23) 73.9 Antenatal care can prevent maternal mortality (death of a pregnant woman). (24) 73.10 Antenatal care can prevent child mortality (death of the baby). (25) 73.11 Attending clinic appointments prepare a pregnant woman to be physically and mentally fit for child birth. (26) 73.12 Smoking while pregnant is harmful for the development of the unborn baby. (27) 73.13 Drinking alcohol will affect the health of a pregnant woman’s baby (28) 73.14 Physical exercise is not recommended for pregnant women. (29) 73.15 If a pregnant woman contracts a sexually transmitted infection and is not treated, it can be transmitted to the baby and cause serious harm. (30) 73.16 Infants, born to women with untreated TB, may be of lower birth weight. (31) Q73 The section is about your current knowledge on antenatal care. Please answer to the best of your ability. Please select your response. 73.1 Antenatal care appointment benefits the baby, not the pregnant woman. (1) ▼ Agree (1) ... I don’t know (3) 73.2 There is no need to have the first antenatal appointment booked as early as possible (before 12 weeks of pregnancy), because it can be done at any time during pregnancy. (17) ▼ Agree (1) ... o Do not want to answer (7) I don’t know (3) 73.3 If a pregnant woman is underweight, she needs extra antenatal care. (18) ▼ Agree (1) ... I don’t know (3) 73.4 If a pregnant woman is over-weight, she needs extra antenatal care. (19) ▼ Agree (1) ... I don’t know (3) 73.5 Pregnant women who do not receive antenatal care are more likely to give birth to a low-weight baby. (20) ▼ Agree (1) ... I don’t know (3) 73.6 Pregnant women who do not receive antenatal care are more likely to have miscarriages and give still birth (baby not alive). (21) ▼ Agree (1) ... I don’t know (3) 73.7 Pregnant women who receive late antenatal care (after 28 weeks of pregnancy) and their babies, are more likely to have health problems. (22) ▼ Agree (1) ... I don’t know (3) 73.8 Preeclampsia is a serious pregnancy problem characterised by raised blood pressure. (23) ▼ Agree (1) ... I don’t know (3) 73.9 Antenatal care can prevent maternal mortality (death of a pregnant woman). (24) ▼ Agree (1) ... I don’t know (3) 73.10 Antenatal care can prevent child mortality (death of the baby). (25) ▼ Agree (1) ... I don’t know (3) 73.11 Attending clinic appointments prepare a pregnant woman to be physically and mentally fit for child birth. (26) ▼ Agree (1) ... I don’t know (3) 73.12 Smoking while pregnant is harmful for the development of the unborn baby. (27) ▼ Agree (1) ... I don’t know (3) 73.13 Drinking alcohol will affect the health of a pregnant woman’s baby (28) ▼ Agree (1) ... I don’t know (3) 73.14 Physical exercise is not recommended for pregnant women. (29) ▼ Agree (1) ... I don’t know (3) 73.15 If a pregnant woman contracts a sexually transmitted infection and is not treated, it can be transmitted to the baby and cause serious harm. (30) ▼ Agree (1) ... I don’t know (3) 73.16 Infants, born to women with untreated TB, may be of lower birth weight. (31) ▼ Agree (1) ... I don’t know (3) Please answer to the best of your ▼ Agree (1) ... I don’t know (3) ▼ Agree (1) ... I don’t know (3) ▼ Agree (1) ... I don’t know (3) ▼ Agree (1) ... I don’t know (3) ▼ Agree (1) ... I don’t know (3) ▼ Agree (1) ... o Do not want to answer (7) I don’t know (3) ▼ Agree (1) ... I don’t know (3) ▼ Agree (1) ... I don’t know (3) ▼ Agree (1) ... I don’t know (3) ▼ Agree (1) ... I don’t know (3) ▼ Agree (1) ... I don’t know (3) ▼ Agree (1) ... I don’t know (3) ▼ Agree (1) ... I don’t know (3) ▼ Agree (1) ... I don’t know (3) ▼ Agree (1) ... I don’t know (3) ▼ Agree (1) ... I don’t know (3) ▼ Agree (1) ... I don’t know (3) ▼ Agree (1) ... I don’t know (3) ▼ Agree (1) ... I don’t know (3) ▼ Agree (1) ... I don’t know (3) ▼ Agree (1) ... I don’t know (3) ▼ Agree (1) ... I don’t know (3) ▼ Agree (1) ... I don’t know (3) ▼ Agree (1) ... I don’t know (3) ▼ Agree (1) ... I don’t know (3) ▼ Agree (1) ... I don’t know (3) ▼ Agree (1) ... I don’t know (3) ▼ Agree (1) ... I don’t know (3) ▼ Agree (1) ... I don’t know (3) ▼ Agree (1) ... I don’t know (3) ▼ Agree (1) ... I don’t know (3) ▼ Agree (1) ... I don’t know (3) 73.7 Pregnant women who receive late antenatal care (after 28 weeks of pregnancy) and their babies, are more likely to have health problems. (22) 73.8 Preeclampsia is a serious pregnancy problem characterised by raised blood pressure. (23) 73.9 Antenatal care can prevent maternal mortality (death of a pregnant woman). (24) 73.10 Antenatal care can prevent child mortality (death of the baby). (25) 73.11 Attending clinic appointments prepare a pregnant woman to be physically and mentally fit for child birth. (26) 73.12 Smoking while pregnant is harmful for the development of the unborn baby. (27) 73.13 Drinking alcohol will affect the health of a pregnant woman’s baby (28) End of Block: Knowledge Start of Block: Risk perceptions Page 22 of 31 Page 22 of 31 Risk perceptions Q74 This section is about your perceptions on the clinic visits. Please answer to the best of your ability. Please select your response. Strongly Disagree (1) Disagree (2) Agree (3) Strongly Agree (4) I don’t know (5) 74.1 My risk of having pregnancy problems is low. (1) o o o o o 74.2 The risk of experiencing preeclampsia (e.g. o Do not want to answer (7) high blood pressure) is higher, if I don’t attend my clinic appointments. (11) o o o o o 74.3 The risk of experiencing heavy bleeding during pregnancy or childbirth is higher, if I don’t attend my clinic appointments. (12) o o o o o 74.4 I think pregnancy problems can develop into something serious and life threatening. (13) o o o o o 74.5 Compared to other pregnant teenagers, I am less likely to suffer from complications of pregnancy (e.g. hypertension, preeclampsia, or heavy bleeding during childbirth). (14) o o o o o 74.6 Attending clinic visits is important for me (15) o o o o o 74.7 Attending clinic visits is hard for me (16) o o o o o 74.8 Missing my clinic appointment ONCE will not affect my pregnancy. (17) o o o o o 74.9 Attending clinic visits is an inconvenience for me (18) o o o o o 74.10 Missing my clinic appointment more than TWICE will affect my pregnancy. (19) o o o o o End of Block: Risk perceptions Risk perceptions Q74 This section is about your perceptions on the clinic visits. Please answer to the best of your ability. Please select your response. s n is about your perceptions on the clinic visits. Please answer to the best of your ability. our response. End of Block: Risk perceptions Start of Block: Attitudes Page 23 of 31 Page 23 of 31 Attitudes Q75 This section is to understand your attitudes about going to clinic appointments. Please answer to the best of your ability. Please select your response. Strongly Disagree (1) Disagree (2) Agree (3) Strongly Agree (4) I don’t know (5) 75.1 I think it is important for me to attend ALL the clinic appointments that are arranged for me. (1) o o o o o 75.2 I think it is okay/fine for me to miss/skip SOME of the clinic appointments that are arranged for me. (16) o o o o o 75.3 Going to clinic appointments will help me understand if my pregnancy is progressing well. (17) o o o o o 75.4 Going to clinic appointments will help me detect any potential health problems of my pregnancy. (18) o o o o o 75.5 Going to clinic appointment helps me keep track of my baby’s health and development. o Do not want to answer (7) (19) o o o o o 75.6 Going to clinic appointments helps me keep track of my own health. (20) o o o o o 75.7 Going to clinic appointment will help me detect any health problems with me and my unborn baby early. (21) o o o o o 75.8 Going to clinic appointment is helpful because I can find out useful information about my pregnancy. (22) o o o o o 75.9 Going to clinic appointment is a waste of time, because it usually takes a long time and it is costly. (23) o o o o o 75. 10 I do NOT want to attend my clinic appointments because the health care workers make me afraid by shouting at me. (24) o o o o o 75.11 I do NOT want to attend my clinic appointments because the health care workers make it clear that I am not welcomed at the clinic by being hostile. (25) o o o o o 75.12 I do NOT want to attend my clinic appointments because I feel that I am being judged and discriminated against by the health care workers. (26) o o o o o 75.13 I do NOT want attend my clinic appointments because I am afraid that other people might find out about my pregnancy. (27) o o o o o Attitudes Q75 This section is to understand your attitudes about going to clinic appointments. Please answer to the best of your ability. Please select your response. Attitudes Q75 This section is to understand your attitudes about going to clinic appointments. Please answer to the best of your ability. Please select your response. Attitudes Q75 This section is to understand your attitudes about going to clinic appointments. Please answer to the best of your ability. Please select your response. Page 24 of 31 Page 24 of 31 End of Block: Attitudes Start of Block: Social influences Social influences Q76 This section is about the social support for your pregnancy. Please answer to the best of your ability. Please select your response Strongly disagree (1) Disagree (2) Agree (3) Strongly agree (4) Not applicable (5) 76.1 My family encourages me to go to clinic appointments. (1) o o o o o 76.2 My school teachers encourage me to go to clinic appointments. (6) o o o o o 76.3 My friends encourage me to go to clinic appointments. o Do not want to answer (7) (7) o o o o o 76.4 My boyfriend/partner/father of child encourages me to go to clinic appointments. (8) o o o o o End of Block: Social influences Start of Block: Peer norms End of Block: Attitudes n is about the social support for your pregnancy. Please answer to the best of your ability. our response Page 25 of 31 Page 25 of 31 Peer norms Q77 This section is about your friends’ behaviours and attitudes of pregnancy. Please answer to the best of your ability. If you do NOT have a friend who has been pregnant, please select 'not applicable' Strongly disagree (1) Disagree (2) Agree (3) Strongly Agree (4) Not applicable (5) 77.1 My friends who are/have been pregnant go to the clinic on the day of their appointment. (1) o o o o o 77.2 My friends, who are/ have been pregnant think that it is only necessary to go to the clinic at the end of the pregnancy. (27) o o o o o 77.3 My friends, who are /have been pregnant think that if I go to my clinic appointment, I will get helpful advice for maintaining a healthy pregnancy. (28) o o o o o 77.4 My friends, who are/ have been pregnant think that the health care workers at the clinic are unfriendly, threatening and rude. (29) o o o o o 77.5 My friends, who are/ have been pregnant think that the health care workers at the clinic give them information that is confusing. (30) o o o o o 77.6 My frineds, who are /have been pregnant think that the health care workers will prepare me for a safe delivery (31) o o o o o Strongly disagree (1) Disagree (2) Agree (3) Strongly Agree (4) Not applicable (5) End of Block: Peer norms Start of Block: Family norms Page 26 of 31 Page 26 of 31 Family norms Q78 This section is about your family members’ attitudes of your pregnancy. Please answer to the best of your ability. Please select your response. Family norms Q78 This section is about your family members’ attitudes of your pregnancy. Please answer to the best of your ability. Please select your response. Family norms Q78 This section is about your family members’ attitudes of your pregnancy. Please answer to the best of your ability. Please select your response. y y y p Strongly Disagree (1) Disagree (2) Agree (3) Strongly Agree (4) I don’t know (5) 78.1 My family members feels that if I go to my clinic appointment, It is only necessary for me to go at the end of the pregnancy. (1) o o o o o 78.2 My family members feel that I do not need to go to the clinic but take traditional pregnancy medicine (13) o o o o o 78. 3 My family members feels that it is helpful for me to get correct information about my pregnancy. (14) o o o o o 78.4 My family members feels that it is helpful for me to learn about my baby’s health and development. (15) o o o o o 78.5 My family members feels that I will receive good advice and health care from the health care workers. (16) o o o o o 78.6 My family members feels that It will prepare me for a safe delivery (17) o o o o o 78.7 My family members feels that I will embarrass them and bring shame to the family. (18) o o o o o End of Block: Family norms End of Block: Family norms Start of Block: Partner/boyfriend norms Page 27 of 31 Partner/boyfriend norms Q79 This section is about your partner/boyfriend’s attitudes of pregnancy. Please answer as honestly as you can, and select the number that is appropriate for you. If you do NOT have a partner/boyfriend, please circle “not applicable”. Partner/boyfriend norms Q79 This section is about your partner/boyfriend’s attitudes of pregnancy. Please answer as honestly as you can, and select the number that is appropriate for you. If you do NOT have a partner/boyfriend, please circle “not applicable”. Page 26 of 31 Strongly disagree (1) Disagree (2) Agree (3) Strongly Agree (4) Not applicable (5) 79.1 My boyfriend/partner feels that it is only necessary for me to go to the clinic at the end of the pregnancy. (1) o o o o o 79.2 My boyfriend/partner feels that if I go to my clinic appointment, it is helpful for me to get correct information about my pregnancy. (11) o o o o o 79.3 My boyfriend/partner feels that if I go to my clinic appointment, it is helpful for me to learn about my baby’s health and development. (12) o o o o o 79.4 My boyfriend/partner feels that if I go to my clinic appointment, I will receive good advice and health care from the health care workers. (13) o o o o o 79.5 My boyfriend/partner feels that if I go for an HIV test that I don’t trust and love him (14) o o o o o 79.6 My boyfriend/partner feels that if I have had an HIV test he does not have to have one (15) o o o o o 79.7 My boyfriend/partner feels that since I am already pregnant, we don’t have to use a condom when we have sex. (16) o o o o o 79.8 Now that I am pregnant, I am worried that my partner/boyfriend will sleep with other girls. (17) o o o o o End of Block: Partner/boyfriend norms Start of Block: School norms Page 28 of 31 Page 28 of 31 School norms Q80 This section is about your school teachers’ attitudes of your pregnancy. Please answer to the best of your ability and select the number that is appropriate for you. If you do NOT attend school, or if you have not talked about your pregnancy with your teacher/s, then please tick "not applicable". Strongly Disagree (1) Disagree (2) Agree (3) Strongly Agree (4) Not applicable (5) Strongly Disagree (1) Disagree (2) Agree (3) Strongly Agree (4) Not applicable (5) 80.1 My school teachers think that it is only necessary for me to go to the clinic at the end of the pregnancy. (1) o o o o o 80.2 My school teachers think that if I go to my clinic appointment, it is helpful for me to get correct information about my pregnancy. Page 26 of 31 (10) o o o o o 80.3 My school teachers think that if I go to my clinic appointment, it is helpful for me to learn about my baby’s health and development. (11) o o o o o 80.4 My school teachers think that If I go to my clinic appointment, I will receive good advice and health care from the health care workers. (12) o o o o o 80.5 My school teachers feel very uncomfortable that I am pregnant and at school. (13) o o o o o 80.6 My school teachers feel that I am an embarrassment to the school and a bad influence on the other learners. (14) o o o o o End of Block: School norms End of Block: School norms Start of Block: Self-efficacy Page 29 of 31 Page 29 of 31 Self-efficacy Q81 This section is about your feelings of confidence in attending the clinic appointments. Please answer to the best of your ability. Please select your response. Self-efficacy Q81 This section is about your feelings of confidence in attending the clinic appointments. Please answer to the best of your ability. Please select your response. Strongly Disagree (1) Disagree (2) Agree (3) Strongly Agree (4) Not applicable (5) 81.1 I am confident in my ability to attend clinic appointments, even if the clinic is far from my home. (1) o o o o o 81.2 I am confident in my ability to attend clinic appointments, even if I am feeling ill/sick. (10) o o o o o 81.3 I am confident in my ability to attend my clinic appointments, when I am feeling lazy and tired. (11) o o o o o 81.4 I am confident in my ability to attend my clinic appointments, even if I am embarrassed or ashamed to go to the clinic. (12) o o o o o 81.5 I am confident in my ability to attend my clinic appointments, even if I am afraid of the health care workers. (13) o o o o o 81.6 I am confident in my ability to attend my clinic appointments, even if the health care workers will gossip about my pregnancy. (14) o o o o o 81.7 I am confident in my ability to attend my clinic appointments, even if my family, teachers, boyfriend/partner will not allow me to go (15) o o o o o 81.8 I am confident in my ability to attend my clinic appointments, even if other patients will gossip, make judgemental remarks about my being pregnant. (16) o o o o o End of Block: Self-efficacy End of Block: Self-efficacy Start of Block: Intention Page 30 of 31 Page 30 of 31 Intention Q82 This section is about your intention related to clinic appointments. Please answer to the best of your ability. Please select your response. Intention Q82 This section is about your intention related to clinic appointments. Please answer to the best of your ability. Please select your response. y y p Strongly Disagree (1) Disagree (2) Agree (3) Strongly Agree (4) 82.1 I intend to attend my NEXT clinic appointment. (1) o o o o 82.2 I intend to attend ALL the clinic appointments. Page 29 of 31 (7) o o o o 82.3 I intend to take the health advice given by the maternal health care workers (e.g. nurses, doctors, counsellors, pharmacists, health promoters) seriously, and use it. (8) o o o o 82.4 I intend to be honest about everything that the maternal health care workers ask me. (9) o o o o 82.5 I intend to ask questions if I don’t understand something at my clinic appointment. (10) o o o o End of Block: Intention Start of Block: Action planning Action planning Q83 This section is your plans related to clinic appointments. Please answer to the best of your ability. Please select your response. Strongly Disagree (1) Disagree (2) Agree (3) Strongly Agree (4) Not Applicable (5) 83.1 I have made/going to make a plan to remind myself to attend my next clinic appointment so that I don’t forget. (1) o o o o o 83.2 I have made/going to make a plan to travel (walk/take a taxi/ask someone for a lift) to the facility where my clinic appointment is arranged. (5) o o o o o 83.3 If I miss my clinic appointment, I will make a plan to reschedule so I do not miss any appointment. (6) o o o o o 83.4 If I miss an appointment, I will wait until the next one. (7) o o o o o End of Block: Action planning our plans related to clinic appointments. Please answer to the best of your ability. ing ction is your plans related to clinic appointments. Please answer to the best of your ability. ct your response. Page 31 of 31 Page 31 of 31 Page 31 of 31
https://openalex.org/W2118962150	https://actavetscand.biomedcentral.com/counter/pdf/10.1186/1751-0147-52-25	English	null	Variant -and individual dependent nature of persistent Anaplasma phagocytophilum infection	Acta veterinaria Scandinavica	2,010	cc-by	7,104	RESEARCH Open Access BioMed Central © 2010 Granquist et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. * Correspondence: ErikGeorg.Granquist@nvh.no 1 Department of Production Animal Clinical Sciences, Section of Small Ruminant Research, Norwegian School of Veterinary Science, Sandnes, N-4325, Norway Full list of author information is available at the end of the article Abstract Background: Anaplasma phagocytophilum is the causative agent of tick-borne fever in ruminants and human granulocytotropic anaplasmosis (HGA). The bacterium is able to survive for several months in immune-competent sheep by modifying important cellular and humoral defence mechanisms. Little is known about how different strains of A. phagocytophilum propagate in their natural hosts during persistent infection. Methods: Two groups of five lambs were infected with each of two 16S rRNA gene variants of A. phagocytophilum, i.e. 16S variant 1 which is identical to GenBank no M73220 and 16S variant 2 which is identical to GenBank no AF336220, respectively. The lambs were infected intravenously and followed by blood sampling for six months. A. phagocytophilum infection in the peripheral blood was detected by absolute quantitative real-time PCR. Results: Both 16S rRNA gene variants of A. phagocytophilum established persistent infection for at least six months and showed cyclic bacteraemias, but variant 1 introduced more frequent periods of bacteraemia and higher number of organisms than 16S rRNA gene variant 2 in the peripheral blood. Conclusion: Organisms were available from blood more or less constantly during the persistent infection and there were individual differences in cyclic activity of A. phagocytophilum in the infected animals. Two 16S rRNA gene variants of A. phagocytophilum show differences in cyclic activity during persistent infection in lambs. Granquist et al. Acta Veterinaria Scandinavica 2010, 52:25 http://www.actavetscand.com/content/52/1/25 Granquist et al. Acta Veterinaria Scandinavica 2010, 52:25 http://www.actavetscand.com/content/52/1/25 Erik G Granquist1, Kjetil Bårdsen1, Karin Bergström2 and Snorre Stuen1 Erik G Granquist1, Kjetil Bårdsen1, Karin Bergström2 and Snorre Stuen1 * Correspondence: ErikGeorg.Granquist@nvh.no Experimental infection of lambs and blood sampling Experimental infection of lambs and blood sampling Eleven 5-months-old lambs of the Norwegian Dala breed, were housed indoors before and during the experimental period lasting for 184 days. The experiment was approved by the National Animal Research Authority in Norway. Five lambs (L1-L5) were intravenously inocu- lated with 1 ml of a whole blood dimethyl sulphoxide (DMSO) stabilate of the 16S rRNA gene variant 1 (Identi- cal to GenBank no M73220), containing 9.03 × 107 A. phagocytophilum organisms ml-1. Another five lambs (L6-L10) were inoculated with 1 ml whole blood DMSO stabilate of the 16S rRNA gene variant 2 (Identical to GenBank no AF336220), containing 1.6 × 108 A. phagocy- tophilum organisms ml-1. The inocula had been stored as heparinised blood with 10% DMSO at -75°C, and had earlier been used in several inoculation trials [15]. The infection doses were quantified according to the protocol described under materials and methods section "DNA isolation and qPCR analysis of A. phagocytophilum infec- tion". One lamb (L11) was kept as an uninfected control. All lambs were examined and found negative for Myco- plasma ovis (formerly Eperythrozoon ovis) infection by blood smear analysis prior to inoculation. EDTA blood samples were collected from the jugular vein on day 0 (before infection), day 3 (post infection) and then every second or third day for a six month period. Blood samples were stored at -75°C for later PCR analysis. In addition, total and differential neutrophil counts were determined electronically (ADVIA, Bayer) for the first 24 days of the infection. Serum samples were collected in week 0, 2, 4, 8, 12, 16, 20 and 26 of the experimental infection. The rectal Materials and methods temperature was recorded before each blood sampling and the incubation period was defined as the number of days between inoculation and the first day of fever (> 40°C). The duration of fever was recorded as the number of days with fever [16]. Background The inocula had heparinised blood with 10% DMSO at - earlier been used in several inoculation t infection doses were quantified according described under materials and methods isolation and qPCR analysis of A. phagocyt tion". One lamb (L11) was kept as an unin All lambs were examined and found nega plasma ovis (formerly Eperythrozoon ovi blood smear analysis prior to inoculation samples were collected from the jugular (before infection), day 3 (post infection) a second or third day for a six month period. were stored at -75°C for later PCR analys total and differential neutrophil counts we electronically (ADVIA, Bayer) for the first infection. Serum samples were collected in 12, 16, 20 and 26 of the experimental infect Table 1: Primer -and plasmid sequences for Primer Sequen ApMSP2252 5'ACAGTCCAGCGTT ApMSP2459 5'GCACCACCAATAC 5'GAATTCGCC TAAGGGAGAG TGGAAAGAGT ATCCTCGGATT GTTGGTTATG A Primer binding sites are indicated (underscored) i the final reaction mix. Background known to cause severe clinical disease in domestic sheep (Variant 1) [5,9]. Different genetic variants have been found within the same flock of sheep and even within sin- gle animals [5]. Anaplasma phagocytophilum is an obligate intracellular bacterium, transmitted by Ixodes ticks, and is recognized as the causative agent of TBF (tick-borne fever) in sheep and HGA (human granulocytotropic anaplasmosis) in humans [1-3]. Estimates suggest that approximately 300.000 lambs are infected by A. phagocytophilum on pastures in Norway each year, inflicting considerable eco- nomic and animal welfare consequences [4,5]. It is sug- gested that tick-borne diseases and particularly anaplasmosis are underreported in veterinary and human medicine in Norway [6]. One reason for this underesti- mation might be attributed to the diversity in virulence and thus clinical manifestation among genetic variants of A. phagocytophilum [7,8]. Five 16S rRNA gene variants of A. phagocytophilum have previously been identified to infect sheep in Norway, and one of these variants is By modifying important cellular and humoral defence mechanisms, A. phagocytophilum is able to survive and propagate for several months in immune-competent sheep [2,10,11], which may be crucial for the survival of the organisms due to the lack of transovarial transmission between generations of ticks [12,13]. Because of the brev- ity of acute A. phagocytophilum infection, transmission may rely on the tick's ability to acquire the organism from persistently infected sheep [14]. The ability for some 16S rRNA gene variants to establish sustained and persistent bacteraemias may contribute to enhanced transmission from host and tick and favour the spread and propagation of certain genetic strains in nature [14,15]. The present study aims to investigate in vivo propaga- tion of two naturally occurring sheep variants of A. phagocytophilum during persistent infection in lambs by qPCR (absolute quantitative real-time PCR). Page 2 of 7 Granquist et al. Acta Veterinaria Scandinavica 2010, 52:25 http://www.actavetscand.com/content/52/1/25 Materials and methods Experimental infection of lambs and blood s Eleven 5-months-old lambs of the Norweg were housed indoors before and during th period lasting for 184 days. The ex approved by the National Animal Researc Norway. Five lambs (L1-L5) were intrave lated with 1 ml of a whole blood dimet (DMSO) stabilate of the 16S rRNA gene va cal to GenBank no M73220), containing phagocytophilum organisms ml-1. Anoth (L6-L10) were inoculated with 1 ml whole stabilate of the 16S rRNA gene variant GenBank no AF336220), containing 1.6 × 1 tophilum organisms ml-1. DNA isolation and qPCR analysis of Anaplasma phagocytophilum infection An automated isolation procedure based on magnetic bead technology was performed by the application of the MagNA Pure LC instrument (Roche) and the MagNA Pure LC DNA Isolation Kit I Blood Cells High Perfor- mance (Roche). Briefly, a total number of 81 EDTA blood samples from each of the infected animals were thawed at room temperature and 200 μl blood was transferred to the DNA isolation procedure according to the instruction manual (Roche). The isolated DNA was eluted with 100 μl low salt buffer and stored at -20°C awaiting PCR analy- sis. The concentration of DNA in each sample was deter- mined by OD260 spectrophotometry (GeneQuant II, Pharmacia Biotech, Cambridge, UK). The samples were diluted 1:100 before PCR analysis. y A 275 bp plasmid was designed from the N-terminal conserved part of the msp2(p44) expression site for abso- lute quantification against a standard curve. The plasmid represents a region between 364 and 112 bps upstream of the highly conserved 5' sequence that encodes the LAKT amino acid residue which flanks the N-terminal end of the hyper variable region in the msp2(p44) expression site (Table 1) [17]. The primers were ApMSP2252 5' ACAGTCCAGCGTTTAGCAAGA and ApMSP2 459 5' CACCACCAATACCATAACCA amplifying a product of Table 1: Primer -and plasmid sequences for qPCR of the A. phagocytophilum msp2 (p44) expression site (partial sequence). Primer Sequence Conc. Tm °C ApMSP2252 5'ACAGTCCAGCGTTTAGCAAGA-3' 0.5 μM 57.0 ApMSP2459 5'GCACCACCAATACCATAACCA-3' 0.5 μM 56.5 Plasmid construct 5'GAATTCGCCCTTACAGTCCAGCGTTTAGCAAGATAAGAGATTTTAGTA TAAGGGAGAGTAACGGAGAGACTAAGGCAGTATATCCATACTTAAAGGA TGGAAAGAGTGTAAAGCTTGAGTCTAACAAGTTTGACTGGAACACTCCTG ATCCTCGGATTGGGTTTAAGGACAACATGCTTGTAGCTATGGAAGGCAGT GTTGGTTATGGTATTGGTGGTGCCAGGGTTGAGCTTGAGATTGGTTACG AGCGCTTCAAGACCAAAAGGGCGAATTCT-3' Primer binding sites are indicated (underscored) in the sequence of the plasmid construct. The concentrations of primers are calculated for the final reaction mix. mid sequences for qPCR of the A. phagocytophilum msp2 (p44) expression site (partial sequence). Table 1: Primer -and plasmid sequences for qPCR of the A. phagocytophilum msp2 (p44) expression s Granquist et al. Acta Veterinaria Scandinavica 2010, 52:25 http://www.actavetscand.com/content/52/1/25 Granquist et al. Acta Veterinaria Scandinavica 2010, 52:25 http://www.actavetscand.com/content/52/1/25 Page 3 of 7 208 bp covering both the plasmid and the conserved N- terminal region of the expression site for msp2(p44) (Table 1). Plasmid and primers were manufactured by TIB Molbiol (Germany). The plasmid was included as duplicates in every PCR run at 10 fold dilution series ranging from 101 to 106 copies. A Light Cycler 480 instru- ment (Roche) was used for the real-time absolute quanti- fication PCR analysis. Serology The standard curve was made from the mean of dupli- cate readings of the plasmid amplification. The quantifi- cation against the standard curve was automatically done using the LightCycler 480 Software (release 1.5.0) (Roche) (Fig. 1 and 2). The Cq values (quantification cycle) were determined by the 2nd derivative maximum method, and quantitative data (X) were estimated as the mean of tripli- cate Cq values divided by the concentration (μg/ml) of DNA in each sample to correct for the differences in the efficacy of DNA preparations. Triplicate readings with mean values below 1 were regarded as zero and the ampl- icons were verified by melting point analysis (Tm) (Fig. 3). The quantitative data are presented as Log10 (1+X) on a linear graph for the longitudinal development of bacter- aemia over time. A threshold was set at the lowest plas- mid dilution (101 copies) and calculated as follows (Log10 (1+101) = 1.04) to better distinguish the periods of bacte- raemias (Fig. 4, 5 and 6). Briefly, an indirect immunofluorescence antibody assay (IFA) was used to determine the developing polyvalent antibody titres to A. phagocytophilum in serum from the infected lamb. Two-fold dilutions of sera were added to slides pre-coated with antigen obtained from horses (for- merly Ehrlichia equi) (Protatek, St. Paul. Minn.). Bound antibodies were visualized by fluorescein-isothiocyanate (FITC)-conjugated rabbit-anti-sheep immunoglobulin (Cappel, Organon Teknika, West Chester, PA). Sera were screened for antibodies at dilution 1:40. If positive, the serum was further diluted and retested. A titre of 40 or more was regarded as positive [18]. The titres were pre- sented as Log10(1+titre) (Fig. 7). Results Figure 1 qPCR amplification of the plasmid dilution series. A plas- mid dilution series was produced containing 101 to 106 copies of the msp2 (p44) gene of A. phagocytophilum. The figure shows the amplifi- cation cycles of the dilution series used as standard curve for quantifi- cation of the infection. In addition the figure shows the lack of amplification of the non template controls. Statistics For statistical analyses, two-sample t-tests were per- formed in the Statistix, version 4.0 (Analytical software). P values < 0.05 (two-tailed) were regarded as significant. DNA isolation and qPCR analysis of Anaplasma phagocytophilum infection 96 well white plates were loaded with a reaction mix consisting of 1 μl (10 μM) ApMSP2252 primer, 1 μl (10 μM) ApMSP2459 primer, 3 μl RNAse free H2O, 10 μl LightCycler 480 DNA SYBR Green I Master and 5 μl sample. Plates were sealed by sealing foil and centrifuged at 1200 rpm for two minutes. Samples and non-template controls were run in tripli- cates on each plate. Figure 2 Standard curve of the plasmid dilution series. The stan- dard curve was used for the quantification of A. phagocytophilum or- ganisms in the blood from infected lambs. Figure 2 Standard curve of the plasmid dilution series. The stan- dard curve was used for the quantification of A. phagocytophilum or- ganisms in the blood from infected lambs. Clinical manifestation and haematology All lambs reacted with fever (> 40.0°C) within five to six days after inoculation. Table 2 summarizes the main clin- Figure 3 Melting point analysis of the amplicons. The figure shows the melting point analyses (Tm) of the plasmid, A. phagocytophilum or- ganisms isolated from blood and non template controls. No primer di- mers were formed. Figure 1 qPCR amplification of the plasmid dilution series. A plas- mid dilution series was produced containing 101 to 106 copies of the msp2 (p44) gene of A. phagocytophilum. The figure shows the amplifi- cation cycles of the dilution series used as standard curve for quantifi- cation of the infection. In addition the figure shows the lack of amplification of the non template controls. Figure 3 Melting point analysis of the amplicons. The figure shows the melting point analyses (Tm) of the plasmid, A. phagocytophilum or- ganisms isolated from blood and non template controls. No primer di- mers were formed. Granquist et al. Acta Veterinaria Scandinavica 2010, 52:25 http://www.actavetscand.com/content/52/1/25 Granquist et al. Acta Veterinaria Scandinavica 2010, 52:25 http://www.actavetscand.com/content/52/1/25 Page 4 of 7 Figure 4 qPCR of infection with 16S rRNA gene variant 1 of A. phagocytophilum. Cyclic bacteraemia of persistent A. phagocytophi- lum infection in five lambs (L1-L5) inoculated with the Norwegian 16S rRNA gene variant 1, monitored by qPCR for six months. The horizontal line shows the threshold for bacteraemia and represents the lowest (10 copies) plasmid dilution for the standard curve calibration. The re- sults are presented as logarithm transformed means of triplicate Cq readings (X) for each sample, calculated as Log10 (1+X). Clinical manifestation and haematology L1 0 1 2 3 4 5 6 7 0 8 15 24 34 43 52 62 71 80 90 99 108 118 127 136 146 155 164 174 183 Days of infection Absolute quantity L2 0 1 2 3 4 5 6 7 0 8 15 24 34 43 52 62 71 80 90 99 108 118 127 136 146 155 164 174 183 Days of infection Absolute quantity L5 0 1 2 3 4 5 6 7 0 8 15 24 34 43 52 62 71 80 90 99 108 118 127 136 146 155 164 174 183 Days of infection Absolute quantity L3 0 1 2 3 4 5 6 7 0 8 15 24 34 43 52 62 71 80 90 99 108 118 127 136 146 155 164 174 183 Days of infection Absolute quantity L4 0 1 2 3 4 5 6 7 0 8 15 24 34 43 52 62 71 80 90 99 108 118 127 136 146 155 164 174 183 Days of infection Absolute quantity Figure 5 qPCR of infection with 16S rRNA gene variant 2 of A. phagocytophilum. Cyclic bacteraemia of persistent A. phagocytophi- lum infection in five lambs (L6-L10) inoculated with the Norwegian 16S rRNA gene variant (GenBank no) AF336220 monitored by qPCR for six months. The horizontal line shows the threshold for bacteraemia and represents the lowest (10 copies) plasmid dilution for the standard curve calibration. The results are presented as logarithm transformed means of triplicate Cq readings (X) for each sample, calculated as Log10 (1+X). Clinical manifestation and haematology L9 0 1 2 3 4 5 6 7 0 8 15 24 34 43 52 62 71 80 90 99 108 118 127 136 146 155 164 174 183 Days of infection Absolute quantity L7 0 1 2 3 4 5 6 7 0 8 15 24 34 43 52 62 71 80 90 99 108 118 127 136 146 155 164 174 183 Days of infection Absolute quantity L6 0 1 2 3 4 5 6 7 0 8 15 24 34 43 52 62 71 80 90 99 108 118 127 136 146 155 164 174 183 Days of infection Absolute quantity L8 0 1 2 3 4 5 6 7 0 8 15 24 34 43 52 62 71 80 90 99 108 118 127 136 146 155 164 174 183 Days of infection Absolute quantity L10 0 1 2 3 4 5 6 7 0 8 15 24 34 43 52 62 71 80 90 99 108 118 127 136 146 155 164 174 183 Days of infection Absolute quantity L6 0 1 2 3 4 5 6 7 0 8 15 24 34 43 52 62 71 80 90 99 108 118 127 136 146 155 164 174 183 Days of infection Absolute quantity L7 0 1 2 3 4 5 6 7 0 8 15 24 34 43 52 62 71 80 90 99 108 118 127 136 146 155 164 174 183 Days of infection Absolute quantity L3 0 1 2 3 4 5 6 7 0 8 15 24 34 43 52 62 71 80 90 99 108 118 127 136 146 155 164 174 183 Days of infection Absolute quantity L8 0 1 2 3 4 5 6 7 0 8 15 24 34 43 52 62 71 80 90 99 108 118 127 136 146 155 164 174 183 Days of infection Absolute quantity L4 0 1 2 3 4 5 6 7 0 8 15 24 34 43 52 62 71 80 90 99 108 118 127 136 146 155 164 174 183 Days of infection Absolute quantity L9 0 1 2 3 4 5 6 7 0 8 15 24 34 43 52 62 71 80 90 99 108 118 127 136 146 155 164 174 183 Days of infection Absolute quantity L5 0 1 2 3 4 5 6 7 0 8 15 24 34 43 52 62 71 80 90 99 108 118 127 136 146 155 164 174 183 Days of infection Absolute quantity L10 0 1 2 3 4 5 6 7 0 8 15 24 34 43 52 62 71 80 90 99 108 118 127 136 146 155 164 174 183 Days of infection Absolute quantity Figure 4 qPCR of infection with 16S rRNA gene variant 1 of A. Clinical manifestation and haematology L9 0 1 2 3 4 5 6 7 0 8 15 24 34 43 52 62 71 80 90 99 108 118 127 136 146 155 164 174 183 Days of infection Absolute quantity L7 0 1 2 3 4 5 6 7 0 8 15 24 34 43 52 62 71 80 90 99 108 118 127 136 146 155 164 174 183 Days of infection Absolute quantity L6 0 1 2 3 4 5 6 7 0 8 15 24 34 43 52 62 71 80 90 99 108 118 127 136 146 155 164 174 183 Days of infection Absolute quantity L8 0 1 2 3 4 5 6 7 0 8 15 24 34 43 52 62 71 80 90 99 108 118 127 136 146 155 164 174 183 Days of infection Absolute quantity L10 0 1 2 3 4 5 6 7 0 8 15 24 34 43 52 62 71 80 90 99 108 118 127 136 146 155 164 174 183 Days of infection Absolute quantity Figure 4 qPCR of infection with 16S rRNA gene variant 1 of A. phagocytophilum. Cyclic bacteraemia of persistent A. phagocytophi- lum infection in five lambs (L1-L5) inoculated with the Norwegian 16S rRNA gene variant 1, monitored by qPCR for six months. The horizontal line shows the threshold for bacteraemia and represents the lowest (10 copies) plasmid dilution for the standard curve calibration. The re- sults are presented as logarithm transformed means of triplicate Cq readings (X) for each sample, calculated as Log10 (1+X). Clinical manifestation and haematology L1 0 1 2 3 4 5 6 7 0 8 15 24 34 43 52 62 71 80 90 99 108 118 127 136 146 155 164 174 183 Days of infection Absolute quantity L2 0 1 2 3 4 5 6 7 0 8 15 24 34 43 52 62 71 80 90 99 108 118 127 136 146 155 164 174 183 Days of infection Absolute quantity L5 0 1 2 3 4 5 6 7 0 8 15 24 34 43 52 62 71 80 90 99 108 118 127 136 146 155 164 174 183 Days of infection Absolute quantity L3 0 1 2 3 4 5 6 7 0 8 15 24 34 43 52 62 71 80 90 99 108 118 127 136 146 155 164 174 183 Days of infection Absolute quantity L4 0 1 2 3 4 5 6 7 0 8 15 24 34 43 52 62 71 80 90 99 108 118 127 136 146 155 164 174 183 Days of infection Absolute quantity Figure 5 qPCR of infection with 16S rRNA gene variant 2 of A. phagocytophilum. Cyclic bacteraemia of persistent A. phagocytophi- lum infection in five lambs (L6-L10) inoculated with the Norwegian 16S rRNA gene variant (GenBank no) AF336220 monitored by qPCR for six months. The horizontal line shows the threshold for bacteraemia and represents the lowest (10 copies) plasmid dilution for the standard curve calibration. The results are presented as logarithm transformed means of triplicate Cq readings (X) for each sample, calculated as Log10 (1+X). qPCR amplification of the msp2/p44 expression site The lambs in both groups developed acute infections with detectable levels of bacteraemia by PCR within three days after inoculation with 16S rRNA gene variant 1 and within three to five days with variant 2. The initial peri- ods of bacteraemia had a mean duration of 25.6 (± 6.96) days with 16S rRNA gene variant 1 and 11.4 (± 1.28) days with 16S rRNA gene variant 2, respectively (P = 0.02) (Fig. 4 and 5). The initial bacteraemias were followed by lower cyclic levels of bacteraemia in all animals at indi- vidual, -and variant dependent frequencies and ampli- tudes. 16S rRNA gene variant 1 cycled more frequently compared with 16S rRNA gene variant 2 and had more periods of bacteraemia above threshold (mean of 10 peri- ods compared to 6.2 periods respectively) (Fig. 4 and 5). The number of organisms detected in the blood was higher in periods of bacteraemia with variant 1 during the persistent phase of infection (Fig. 4). One lamb (L1) had exceptionally high numbers of organisms during the peri- It has previously been confirmed that A. phagocytophi- lum can establish long term infections in immune-com- petent sheep [2,20-22]. During this experiment, both 16S rRNA gene variants persisted for at least six months, although the level of bacteraemia gradually diminished towards the end of the experiment (Fig. 4 and 5). Both variants showed a cyclic pattern with numbers of organ- isms waxing and waning at different frequencies, indicat- ing that both individual and variant dependent differences exist. Periods of bacteraemia terminated by sharp reductions in number of organisms as previously described for the closely related organism, Anaplasma marginale [23,24]. This was hypothesized to reflect an antigen variant-specific immune response to A. phagocy- tophilum [25]. A. marginale seems to cycle regularly at approximate intervals of 5-8 weeks during persistent bacteraemia [14,23]. However, the present study indicates a much more frequent and inconsistent cyclic activity of A. phagocytophilum. Figure 7 Serology of A. phagocytophilum infection. Mean antibody titre ± SD in lambs inoculated with 16S rRNA gene variant 1 (continu- ous line) and 16S rRNA gene variant 2 (dotted line) of A. phagocytophi- lum during a six months infection period. * P < 0.05. 0 0,5 1 1,5 2 2,5 3 3,5 4 0 2 4 8 12 16 20 26 Weeks of infection Log10(1+titre) * * * Variant 1 Variant 2 Transovarial transmission of A. Discussion In the present study, differences in clinical manifestation between two 16S rRNA gene variants of A. phagocytophi- lum were observed during the acute phase of the infec- tion. Lambs infected with gene variant 1 had a longer period of fever, significantly longer periods of initial bacteraemia, and a more severe neutropenia compared to lambs infected with gene variant 2. These results are in accordance with earlier observations [8]. Earlier studies have also reported that gene variant 1 of A. phagocytophi- lum is involved in the majority of fatal TBF cases in sheep [19]. ical and haematological parameters during the acute phase of the infection. Serology Figure 6 qPCR of uninfected control animal (L11). The uninfected control animal was monitored by qPCR as described for the infected lambs throughout the experimental period. The horizontal line shows the threshold for bacteraemia and represents the lowest (10 copies) plasmid dilution for the standard curve calibration. The results are pre- sented as logarithm transformed means of triplicate Cq readings (X) for each sample, calculated as Log10 (1+X). Figure 6 qPCR of uninfected control animal (L11). The uninfected control animal was monitored by qPCR as described for the infected lambs throughout the experimental period. The horizontal line shows the threshold for bacteraemia and represents the lowest (10 copies) plasmid dilution for the standard curve calibration. The results are pre- sented as logarithm transformed means of triplicate Cq readings (X) for each sample, calculated as Log10 (1+X). All lambs, except one (L7), seroconverted before day 17 after inoculation. This lamb did not have positive titre until day 59. Mean titres were higher for the lambs infected with 16S rRNA gene variant 1 than lambs infected with variant 2 (Fig. 7). Clinical manifestation and haematology The results are pre- sented as logarithm transformed means of triplicate Cq readings (X) for each sample, calculated as Log10 (1+X). L11 0 1 2 3 4 5 6 7 0 8 15 22 29 36 50 57 64 71 78 85 92 99 106 113 120 127 134 141 148 155 162 169 176 183 Days of infection Absolute quantity Granquist et al. Acta Veterinaria Scandinavica 2010, 52:25 http://www.actavetscand.com/content/52/1/25 Page 5 of 7 Granquist et al. Acta Veterinaria Scandinavica 2010, 52:25 http://www.actavetscand.com/content/52/1/25 L11 0 1 2 3 4 5 6 7 0 8 15 22 29 36 50 57 64 71 78 85 92 99 106 113 120 127 134 141 148 155 162 169 176 183 Days of infection Absolute quantity ods of bacteraemia (Fig. 4). In contrast, another lamb (L10) had low numbers of organisms and showed very lit- tle cyclic activity during the persistent phase with only one period of bacteraemia (Fig. 5). Lamb 8 (L8) had even levels of cyclic activity with very few days below threshold (10 organisms), during the persistent infection (Fig. 5). The uninfected control lamb (L11) was negative during the whole experimental period (Fig. 6). Clinical manifestation and haematology phagocytophilum. Cyclic bacteraemia of persistent A. phagocytophi- lum infection in five lambs (L1-L5) inoculated with the Norwegian 16S rRNA gene variant 1, monitored by qPCR for six months. The horizontal line shows the threshold for bacteraemia and represents the lowest (10 copies) plasmid dilution for the standard curve calibration. The re- sults are presented as logarithm transformed means of triplicate Cq readings (X) for each sample, calculated as Log10 (1+X). Figure 4 qPCR of infection with 16S rRNA gene variant 1 of A. Figure 5 qPCR of infection with 16S rRNA gene variant 2 of A. Figure 5 qPCR of infection with 16S rRNA gene variant 2 of A. phagocytophilum. Cyclic bacteraemia of persistent A. phagocytophi- lum infection in five lambs (L6-L10) inoculated with the Norwegian 16S rRNA gene variant (GenBank no) AF336220 monitored by qPCR for six months. The horizontal line shows the threshold for bacteraemia and represents the lowest (10 copies) plasmid dilution for the standard curve calibration. The results are presented as logarithm transformed means of triplicate Cq readings (X) for each sample, calculated as Log10 (1+X). Figure 5 qPCR of infection with 16S rRNA gene variant 2 of A. phagocytophilum. Cyclic bacteraemia of persistent A. phagocytophi- lum infection in five lambs (L6-L10) inoculated with the Norwegian 16S rRNA gene variant (GenBank no) AF336220 monitored by qPCR for six months. The horizontal line shows the threshold for bacteraemia and represents the lowest (10 copies) plasmid dilution for the standard curve calibration. The results are presented as logarithm transformed means of triplicate Cq readings (X) for each sample, calculated as Log10 (1+X). g q g phagocytophilum. Cyclic bacteraemia of persistent A. phagocytophi- lum infection in five lambs (L1-L5) inoculated with the Norwegian 16S rRNA gene variant 1, monitored by qPCR for six months. The horizontal line shows the threshold for bacteraemia and represents the lowest (10 copies) plasmid dilution for the standard curve calibration. The re- sults are presented as logarithm transformed means of triplicate Cq readings (X) for each sample, calculated as Log10 (1+X). Granquist et al. Acta Veterinaria Scandinavica 2010, 52:25 http://www.actavetscand.com/content/52/1/25 Figure 6 qPCR of uninfected control animal (L11). The uninfected control animal was monitored by qPCR as described for the infected lambs throughout the experimental period. The horizontal line shows the threshold for bacteraemia and represents the lowest (10 copies) plasmid dilution for the standard curve calibration. qPCR amplification of the msp2/p44 expression site phagocytophilum has been reported in certain tick species [26]. However, the maintenance of A. phagocytophilum within I. ricinus tick populations is unlikely, due to lack of evidence that trans- ovarial transmission of A. phagocytophilum in female ixo- did ticks occurs [13,27]. For the pathogens to survive in Figure 7 Serology of A. phagocytophilum infection. Mean antibody titre ± SD in lambs inoculated with 16S rRNA gene variant 1 (continu- ous line) and 16S rRNA gene variant 2 (dotted line) of A. phagocytophi- lum during a six months infection period. * P < 0.05. Granquist et al. Acta Veterinaria Scandinavica 2010, 52:25 http://www.actavetscand.com/content/52/1/25 Granquist et al. Acta Veterinaria Scandinavica 2010, 52:25 http://www.actavetscand.com/content/52/1/25 Page 6 of 7 Table 2: Mean ± SD values of clinicalvariables in the two groups of lambs inoculated with each of the two 16S rRNA gene variants of A. phagocytophilum, variant 1 and 2 respectively. 16S gene variants of A. phagocytoph ilum N Incubation period (days) Maximum fever (°C) Duration of fever (days) Nadir of neutropenia Duration of neutropenia (days) 1 5 5.6 ± 0.89 41.8 ± 0.23 9.4 ± 3.21 0.29 ± 0.108* 6.3 ± 3.20* 2 5 6.6 ± 0.55 41.0 ± 0.34 2.2 ± 0.45 0.73 ± 0.236 0.3 ± 0.50 The incubation period is the number of days from inoculation until the first day of fever. Duration of fever is the number of days with fever during the infection. Nadir of neutropenia (< 0.7 × 109 Litre-1) is the lowest neutrophil count. * P < 0.05, ** P < 0.01. ues of clinicalvariables in the two groups of lambs inoculated with each of the two 16S rRNA gene tophilum, variant 1 and 2 respectively. Table 2: Mean ± SD values of clinicalvariables in the two groups of lambs inoculated with each of the variants of A. phagocytophilum, variant 1 and 2 respectively. The incubation period is the number of days from inoculation until the first day of fever. Duration of fever is the num during the infection. Nadir of neutropenia (< 0.7 × 109 Litre-1) is the lowest neutrophil count. * P < 0.05, ** P < 0.01. an Ixodes-host cycle, the duration of host infectivity must span the gap between seasons of tick activity [28]. Acknowledgements h k l d l We thank Eli Brundtland and Kristin Sæbø Pettersen for technical support. This study was partly funded by Haalands legat. Authors' contributions GG f d h The cyclic behaviour of A. phagocytophilum during persistent infection may be influenced by the immuno- logical response towards changing surface proteins as previously suggested, although different intrinsic immu- nogenic properties may be carried by the various 16S rRNA genotypes [10,25,33]. The stronger immune response with variant 1 than variant 2 in the present study may indicate differences in immune stimulatory potential between the variants [5], differences in individ- ual humoral responses or higher antigenic relatedness of variant 1 with the used test antigen. Authors contributions EGG performed the quantification PCR and drafted the manuscript. KBa assisted in interpretation of quantification data. KBe performed the serology. SS designed -and supervised the study and performed the statistical calculations. All authors have read and approved the final manuscript. Conclusion ll Two naturally occurring sheep strains of A. phagocytophi- lum established persistent infections for at least six months. Both variants performed cyclic bacteraemia, but variant 1 showed more frequent bacteraemias and higher number of organisms during the infection. In the present study, organisms were available from blood more or less constantly during the entire study period. Future studies should use ticks to investigate whether the transmission efficacy is different among gene variants of A. phagocyto- philum and determine if ticks are able to take up organ- isms continuously during the persistent phase. The ability of A. phagocytophilum to establish long term persistence combined with a large repertoire of genetic variants make the movement of sheep between flocks and geographical locations likely to introduce novel genetic variants to new areas. qPCR amplification of the msp2/p44 expression site In the present study, there were marked differences between individual animals with respect to the amplitude of bacteraemia, number of bacteraemia periods, time of serological conversion -and response regardless of genetic variant. The reason for this strong individual vari- ation is however unknown, and has to be further eluci- dated. Ixodid ticks normally remain attached for days to weeks depending on for instance tick species and their develop- mental stage [29]. The majority of the blood meal and thus host to tick transmission mainly occurs during the rapid feeding phase at the end of the attachment period [29], while tick to host transmission takes place during the first or second day after attachment in the prepara- tory feeding phase [30]. As lambs infected with variant 1 had more frequent cycles and higher number of circulat- ing organisms than variant 2 for prolonged periods dur- ing the persistent infection, ticks are more likely to be exposed to this variant during feeding in the persistent period [14]. This may favour the spread of this variant in nature and explain why gene variant 1 is more frequently encountered than gene variant 2 by diagnostic sampling in randomly selected sheep flocks [5]. However, this statement has to be further elucidated in experimental transmission trials using ticks. For instance, the transmis- sion efficacy of gene variant 1 and 2 in Ixodes ticks, respectively, are unknown and requires further investiga- tion. Previous studies have shown that the efficacy of transmission is influenced by the number of circulating neutrophils and possibly the number of feeding ticks [31]. The ticks also seem to promote leukocyte trafficking to the tick bite site, thus directly influencing the efficacy of transmission [32]. Competing interests Th h d l h Competing interests The authors declare that they have no competing interests. Granquist et al. Acta Veterinaria Scandinavica 2010, 52:25 http://www.actavetscand.com/content/52/1/25 25. Granquist EG, Stuen S, Crosby L, Lundgren AM, Alleman AR, Barbet AF: Variant-specific and diminishing immune responses towards the highly variable MSP2(P44) outer membrane protein of Anaplasma phagocytophilum during persistent infection in lambs. Vet Immunol Immunopathol 2010, 133:117-124. References 26. 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Foggie A: Studies on the infectious agent of tick-borne fever in sheep. J Pathol Bacteriol 1951, 63:1-15. 27. Ogden NH, Casey AN, French NP, Woldehiwet Z: A review of studies on the transmission of Anaplasma phagocytophilum from sheep: implications for the force of infection in endemic cycles. Exp Appl Acarol 2002, 28:195-202. Pathol Bacteriol 1951, 63:1-15. 3. Bakken JS, Dumler JS, Chen SM, Eckman MR, Van Etta LL, Walker DH: Human granulocytic ehrlichiosis in the upper Midwest United States. A new species emerging? JAMA 1994, 272:212-218. 4. Brodie TA, Holmes PH, Urquhart GM: Some aspects of tick-borne diseases of British sheep. Vet Rec 1986, 118:415-418. 5. Stuen S, Pol I Van De, Bergstrom K, Schouls LM: Identification of Anaplasma phagocytophila (formerly Ehrlichia phagocytophila) variants in blood from sheep in Norway. J Clin Microbiol 2002, 40:3192-3197. 6. Stuen S, Bergstrom K: [Human anaplasmosis--a hidden disease in Norway?] Tidsskr Nor Laegeforen 2008 128:2579 2581 Pathol Bacteriol 1951, 63:1 15. 3. Bakken JS, Dumler JS, Chen SM, Eckman MR, Van Etta LL, Walker DH: Human granulocytic ehrlichiosis in the upper Midwest United States. A new species emerging? JAMA 1994, 272:212-218. 4. Brodie TA, Holmes PH, Urquhart GM: Some aspects of tick-borne diseases of British sheep. Vet Rec 1986, 118:415-418. 5. Stuen S, Pol I Van De, Bergstrom K, Schouls LM: Identification of Anaplasma phagocytophila (formerly Ehrlichia phagocytophila) variants in blood from sheep in Norway. J Clin Microbiol 2002, 40:3192-3197. 6. Stuen S, Bergstrom K: [Human anaplasmosis--a hidden disease in 28. Randolph SE: The shifting landscape of tick-borne zoonoses: tick-borne encephalitis and Lyme borreliosis in Europe. References Philos Trans R Soc Lond B Biol Sci 2001, 356:1045-1056. 4. Brodie TA, Holmes PH, Urquhart GM: Some aspects of tick-borne diseases of British sheep. Vet Rec 1986, 118:415-418. 5. Stuen S, Pol I Van De, Bergstrom K, Schouls LM: Identification of Anaplasma phagocytophila (formerly Ehrlichia phagocytophila) variants in blood from sheep in Norway. J Clin Microbiol 2002, 40:3192-3197. 29. Bowman A, Nuttall PA: Ticks: biology, disease and control. New York, N.Y.; Cambridge: Cambridge University Press; 2008. 30. Katavolos P, Armstrong PM, Dawson JE, Telford SR: Duration of tick attachment required for transmission of granulocytic ehrlichiosis. J Infect Dis 1998, 177:1422-1425. y 6. Stuen S, Bergstrom K: [Human anaplasmosis--a hidden disease in Norway?]. Tidsskr Nor Laegeforen 2008, 128:2579-2581. 7. Tuomi J: Experimental studies on bovine tick-borne fever. 2. Differences in virulence of strains in cattle and sheep. Acta Pathol Microbiol Scand 1967, 70:577-589. 31. Ogden NH, Casey AN, Woldehiwet Z, French NP: Transmission of Anaplasma phagocytophilum to Ixodes ricinus ticks from sheep in the acute and post-acute phases of infection. Infect Immun 2003, 71:2071-2078. 8. Stuen S, Bergstrom K, Petrovec M, Pol I Van de, Schouls LM: Differences in clinical manifestations and hematological and serological responses after experimental infection with genetic variants of Anaplasma phagocytophilum in sheep. Clin Diagn Lab Immunol 2003, 10:692-695. 32. Borjesson DL, Simon SI, Hodzic E, DeCock HE, Ballantyne CM, Barthold SW: Roles of neutrophil beta 2 integrins in kinetics of bacteremia, extravasation, and tick acquisition of Anaplasma phagocytophila in mice. Blood 2003, 101:3257-3264. 9. Ladbury GA, Stuen S, Thomas R, Bown KJ, Woldehiwet Z, Granquist EG, Bergstrom K, Birtles RJ: Dynamic transmission of numerous Anaplasma phagocytophilum genotypes among lambs in an infected sheep flock in an area of anaplasmosis endemicity. 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Author Details 1Department of Production Animal Clinical Sciences, Section of Small Ruminant Research, Norwegian School of Veterinary Science, Sandnes, N-4325, Norway and 2National Veterinary Institute, Uppsala 75189, Sweden Page 7 of 7 Granquist et al. Acta Veterinaria Scandinavica 2010, 52:25 http://www.actavetscand.com/content/52/1/25 References Woldehiwet Z: Anaplasma phagocytophilum in ruminants in Europe. Ann N Y Acad Sci 2006, 1078:446-460. 13. Woldehiwet Z, Scott GR: Tick-borne (pasture) fever. 1st edition. Oxford: Pergamon Press; 1993. 14. Eriks IS, Stiller D, Palmer GH: Impact of persistent Anaplasma marginale rickettsemia on tick infection and transmission. J Clin Microbiol 1993, 31:2091-2096. 15. Stuen S, Braten M, Bergstrom K, Bardsen K: Cyclic variation in lambs infected with Anaplasma phagocytophilum. Vet Rec 2008, 163:338-340. 16. Woldehiwet Z, Scott GR: Immunological studies on tick-borne fever in sheep. J Comp Pathol 1982, 92:457-467. 17. Barbet AF, Lundgren AM, Alleman AR, Stuen S, Bjoersdorff A, Brown RN, Drazenovich NL, Foley JE: Structure of the expression site reveals global diversity in MSP2 (P44) variants in Anaplasma phagocytophilum. Infect Immun 2006, 74:6429-6437. 18. Stuen S, Bergstrom K: Serological investigation of granulocytic Ehrlichia infection in sheep in Norway. Acta Vet Scand 2001, 42:331-338. 19. Stuen S, Nevland S, Moum T: Fatal cases of Tick-borne fever (TBF) in sheep caused by several 16S rRNA gene variants of Anaplasma phagocytophilum. Ann N Y Acad Sci 2003, 990:433-434. 20. Stuen S, Djuve R, Bergstrom K: Persistence of granulocytic Ehrlichia infection during wintertime in two sheep flocks in Norway. Acta Vet Scand 2001, 42:347-353. 21. Stuen S, Engvall EO, Artursson K: Persistence of Ehrlichia phagocytophila infection in lambs in relation to clinical parameters and antibody responses. Vet Rec 1998, 143:553-555. 22. 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https://openalex.org/W4200190730	https://figshare.com/articles/thesis/Language_Policy_Language_Practices_and_Language_Shift_in_Tabriz/17142848/1/files/31698563.pdf	English	null	Language Policy, Language Practices and Language Shift in Tabriz	null	2,021	cc-by	83,488	Language Policy, Language Practices and Language Shift in Tabriz By Abstract This thesis examines how Azeri, a minority language with the largest number of speakers in Iran, is marginalized by de facto monolingual language policies of the state favoring Farsi, the only official language, over Azeri in the three selected domains. The research provides insights into how family language policies, i.e. attitudes, ideologies and practices in the home, are influenced by macro policies of multilingual nation-states, leading to language maintenance/shift among minority groups. The investigation adopted and integrated a number of complementary theoretical frameworks and paradigms. An ecology of language paradigm (Haugen, 1972; Hornberger & Hult, 2008; Mühlhäusler, 1996) was used to situate the research within a broader sociopolitical, historical and economic context. The ethnolinguistic vitality model (Giles, Bourhis, & Taylor, 1977), and language policy and planning (LPP) frameworks proposed by Shohamy (2006) and Lo Bianco (2005, 2008c, 2012a; 2013) were utilized to explore the complex interaction between macro level LPP activities and micro level attitudes and practices. The integrated model demonstrates how language policies implemented within state-run domains and institutions produce particular Discourses. The proposed framework further illustrates how such Discourses may influence people at the grass roots level which in turn could lead to language maintenance/shift in different communities and groups. The data base for the study comprised two phases: the first phase involved ethnographic observations of the public sphere (linguistic landscape data), language use in the home (three case studies), and the local channel for Azeris (media data), interviews with fifty children, and authorities of ten kindergartens and preschools. A focus-group interview was also conducted in this phase to assist with designing an attitude questionnaire which was administered in the second phase to 150 parents of young children. The empirical data suggests that family language policies among Azeris in Tabriz are constantly and increasingly influenced by monolingual policies of the state. The institutionalization and legitimization of Farsi through de facto LPP activities has resulted in formation of uncommitted, if not negative, attitudes among Azeri parents regarding their ethnic language. The analysis shows how a Farsi-only education system cajoles kindergarten principals into favoring Farsi over Azeri, leading them to suggest that parents and children speak Farsi in the home to ease their integration into the education system. Abstract ii The linguistic landscape data demonstrates the absence of Azeri both in top-down governmental and private individual signage indicating its low status compared to Farsi and English, the two prevalent languages in public signage in Tabriz. Exploring the broadcasting media suggests Azeris' inclination towards Farsi, and then in a second place, Turkish channels. As a result, having attracted only one percent of Azeri audience, the only available channel provided by the government for Azeris, Sahand TV, provides arguably no institutional support for Azeri. The findings suggest that although family members may be viewed as free agents to choose a particular language to speak in the home, in reality such choices are highly constrained by the ecology surrounding the home which is shaped by LPP decisions and activities. Overall, this thesis sheds light on the complex nature of language policy and planning in multilingual nation-states, and how they impact on language maintenance/shift processes among minority groups, whilst also illuminating how language ecologies are manipulated by nation-states to achieve particular non-linguistic goals. iii iii Acknowledgements I find writing these acknowledgments very joyful as I look back and remember those wonderful people who helped and supported me along this long but beautiful and fulfilling experience. My grateful thanks firstly go to my parents and two brothers for their unending love, encouragement, and support without which I could not have undertaken PhD, let alone completed it. I shall never forget my father's interest to know about my research findings concerning language change in Tabriz! I'm also grateful for the help and encouragement I have received from my past colleagues at Azerbaijan Shahid Madani University and Iran Language Institute, as well as my present colleagues and friends at Victoria University. I wish to thank both academic and administrative staff in the School of Linguistics and Applied Language Studies as well whose continuous support made this PhD possible. In the realm of statistics, I received invaluable advice from Dalice Sim, Peter Gu, and Mohsen Joshanloo. Without their advice and guidance, the quantitative strand of this PhD would not have been possible. My thanks are also due to Victoria Doctoral Scholarship and Faculty Research Grants which enabled me to conduct my research and present my findings at different conferences without financial concerns. I should also like to thank those who assisted me with collecting data for this PhD research. I'd like to thank Mahya Alaei who helped with piloting my interview questions in Tabriz while I was in New Zealand. I would also like to express my thanks to all those parents and children, as well as preschools and kindergartens authorities, especially Hossein Razavi and Rahim Farzaee, whose tremendous help and participation in the research enabled me to collect the data for my research. Lastly but surely not least, I would like to extend my greatest heartfelt gratitude to my dearest supervisors, Professor Janet Holmes and Dr Meredith Marra, for their invaluable support, insights into critical thinking, flexibility and accessibility. Their continuous encouragement and inspirations stimulated my ever-increasing enthusiasm in sociolinguistics. I learned tremendously from not only their academic knowledge and expertise but also their attitudes and manners in everyday life. Without Janet and Meredith, I could not have completed my PhD thesis. iv Table of Contents Table of Contents v Table of Contents Abbreviations .................................................................................................................... viii Tables ................................................................................................................................... ix Figures ................................................................................................................................... x Chapter one: Introduction .................................................................................................. 1 1.0 Aim .................................................................................................................................. 2 1.1 Research setting ............................................................................................................... 3 1.1.1 Iran: A historical account .......................................................................................... 3 1.1.2 Tabriz: An important city for Azeris in Iran ............................................................. 8 1.2 Theoretical frameworks ................................................................................................... 9 1.3 Overview of chapters ..................................................................................................... 15 Chapter Two: Literature Review ..................................................................................... 17 2.0 Introduction ................................................................................................................... 17 2.1 Linguistic diversity and language shift .......................................................................... 17 2.2 Ecology of language ...................................................................................................... 21 2.3 Ecology of language and LPP ....................................................................................... 24 2.4 Policy as text, discourse, and performance.................................................................... 29 2.5 A proposed model of the interplay between language policy and language maintenance/shift ................................................................................................................. 35 2.6 Conclusion ..................................................................................................................... 39 Chapter Three: Methodology ........................................................................................... 40 3.0 Introduction ................................................................................................................... 40 3.1 Mixed methods .............................................................................................................. 41 3.2 Why a mixed methods approach? .................................................................................. 41 3.3 Ethical issues ................................................................................................................. 44 3.4 Data collection instruments ........................................................................................... 45 3.4.1 Interviews with kindergarten authorities ................................................................ 46 3.4.2 Linguistic landscape data ........................................................................................ 47 3.4.3 Data from Sahand TV ............................................................................................. 48 3.4.4 Interviews with Children......................................................................................... 48 v v 3.4.5 Case studies ............................................................................................................ 50 3.4.6 An attitude questionnaire: A quantitative measure ................................................ 51 3.4.6.1 Focus group interview ..................................................................................... 52 3.4.6.2 Questionnaire ................................................................................................... 53 3.4.6.2.1 Designing questions .................................................................................. 53 3.4.6.2.2 Reliability .................................................................................................. 54 3.4.6.2.3 Piloting the questionnaire ......................................................................... 54 3.4.6.2.4 Questionnaire administration .................................................................... 55 3.5 Conclusion .................................................................................................................... 56 Chapter Four: The Education System: A Discourse Planning Mechanism ................ 57 4.0 Introduction ................................................................................................................... 57 4.1 Language laws: Policy as text ....................................................................................... 58 4.2 Interviews with kindergarten authorities: Policy as discourse ...................................... 62 4.3 The education system, a vehicle for Discourse planning: Policy as performance ........ 70 4.4 Conclusion .................................................................................................................... 73 Chapter Five: Linguistic landscapes: Discourses in Public .......................................... 75 5.0 Introduction ................................................................................................................... 75 5.1 Linguistic landscapes .................................................................................................... 76 5.2 Language use in the linguistic landscape ...................................................................... 76 5.3 Top-down and bottom-up linguistic landscapes ........................................................... 78 5.4 Top-down signs in Tabriz: An indicator of ideologies and a Discourse planning mechanism .......................................................................................................................... 79 5.5 Bottom-up signs in Tabriz: An indicator of language change ...................................... 86 5.6 Discussion ..................................................................................................................... 91 5.7 Conclusion .................................................................................................................... 94 Chapter Six: The Media: A Discursive Space ............................................................... 96 6.0 Introduction ................................................................................................................... 96 6.1 Media and language policy and planning ..................................................................... 96 6.2 Representation of languages on Sahand TV ............................................................... 102 6.3 Representations of communities on Sahand TV ......................................................... 105 6.4 Conclusion .................................................................................................................. 109 vi Chapter Seven: Home: A pivotal domain ..................................................................... Table of Contents 111 7.0 Introduction ................................................................................................................. 111 7.1 Family language policy ............................................................................................... 111 7.2 Azeri parents' attitudes towards language use in the home ......................................... 113 7.3 Azeri children's linguistic choices in the home ........................................................... 115 7.4 Case studies ................................................................................................................. 118 7.5 Azeris' attitudes towards educational policies ............................................................. 125 7.6 Azeri children's attitudes and behavior towards television channels .......................... 129 7.7 Azeri parents' attitudes and behavior towards television channels ............................. 134 7.8 Discussion .................................................................................................................... 136 7.9 Conclusion ................................................................................................................... 139 Chapter Eight: Conclusion ............................................................................................. 140 8.0 Introduction ................................................................................................................. 140 8.1 Theoretical frameworks ............................................................................................... 140 8.2 Synthesis of findings ................................................................................................... 143 8.3 Areas for further research ............................................................................................ 146 8.4 Conclusion ................................................................................................................... 148 References ......................................................................................................................... 150 Appendix One: English and Farsi Questionnaires ............................................................. 182 Appendix Two: Interview Schedules ................................................................................ 195 vii Abbreviations IRIB Islamic Republic of Iran's Broadcasting LL Linguistic landscapes LPP Language policy and planning viii Tables Table 5.1: The number of signs in different languages in top-down signage in Tabriz......79 Table 5.2: The number of signs in different languages in bottom-up signage in Tabriz.....87 Table 5.3: The mean for Azeris' abilities in four skills in Azeri and Farsi......................... 88 Table 5.4: Paired samples t-test results .............................................................................. 89 Table 6.1: Airtime for Arabic, Azeri, and Farsi programs ................................................103 Table 6.2: The mean for airtime for Arabic, Azeri, and Farsi programs in seven days.... 104 Table 6.3: Types of programs broadcast in Azeri and Farsi on Sahand TV..................... 106 Table 6.4: Mainstream media (media-about) versus minority media, taken from Caspi and Elias (2011) ...................................................................................................................... 107 Table 7.1: The mean for attitudes towards Farsi in the home........................................... 114 Table 7.2: The mean for "attitudes towards Azeri in the home.........................................114 Table 7.3: Attitudes towards Azeri education....................................................................126 Table 7.4: Results for question number 2..........................................................................127 Table 7.5: Results for question number 23........................................................................127 Table 7.6: Results for question number 18........................................................................127 Table 7.7: Results for question number 33........................................................................128 Table 7.8: Results for question number 29....................................................................... 128 ix 1 The language is also known as Torki inside Iran. However, Azeri is used in this thesis to refer to this language because Azeri (and sometimes Azerbaijani) is commonly used in the research literature to refer to the language. Figures Figures Figure 1.1: Linguistic map of Iran ........................................................................................ 4 Figure 2.1: A proposed model of the interplay between language policy and language maintenance/shift ................................................................................................................ 35 Figure 5.1: Farsi-English bilingual sign...............................................................................81 Figure 5.2: Azeri/Farsi bilingual sign (Type C)...................................................................83 Figure 5.3: Azeri only sign (Type D)...................................................................................84 Figure 5.4: Azeri/English sign (Type E)..............................................................................86 Figure 5.5: Farsi-only bottom-up sign.................................................................................87 Figure 7.1: Language used with the father at home...........................................................115 Figure 7.2: Language used with the mother at home.........................................................117 Figure 7.3: Language used with siblings at home..............................................................117 Figure 7.4: Azeri children’s preference to watch cartoons in different languages............131 Figure 7.5: The number of children watching Sahand TV (in number)............................132 Figure 7.6: Channels children watch most often (in percentage)......................................132 Figure 7.7: Channels parents watch most often (in percentage)........................................135 x x A personal anecdote: Before I commenced my PhD in 2010, I used to teach English in my hometown, Tabriz in Iran, in which people speak Azeri1 (a Turkic language), a language quite different from Farsi, the official language of the country which is an Iranian language of Indo-European origin. In the English institute in which I worked, a pre-final test was given to children to prepare them for the final test. Pre-final tests were to be corrected in that session, and papers were to be returned to children so that they could improve their weaknesses at home. In one of the classes in 2008, I told the students their grades in their mother tongue, i.e. Azeri. To my astonishment, I was asked to repeat the scores in Farsi by some students because they did not understand their grades in Azeri, i.e. numbers between 0-100. I probed into the issue by asking those students about their hometown and their first language (mother tongue). I found out that those students were from Tabriz and their first language was Azeri. This made me wonder why they had difficulty understanding simple lexicon in their own language. As an MA student majoring in applied linguistics, I decided to undertake a pilot study for my sociolinguistics course to investigate on a larger scale the language attrition that I had observed. I devised a questionnaire with fifty words selected from the children's immediate environment including some numbers, colors, animals, body parts, etc. Two hundred children, aged 10-14, and two hundred adults, aged 15 and over, were asked to write the Azeri equivalent for each Farsi word on the questionnaire. The findings of the study, i.e. adults answering about 95%, and children 65% of correct answers, sounded a warning bell to me that language attrition had already begun. The attrition and shifting to Farsi provoked several questions in my mind; why are Azeri children not learning their own language? What are the possible influencing factors? Why is no one noticing or caring about it? What will happen to the language if this trend goes on? To find answers to my questions, I decided to begin my sociolinguistic journey with a focus on the interplay between language policy and planning and language maintenance and shift. 1 1.0 Aim Language shift and its final stage, i.e. language endangerment and death, is an unsettling issue in the world. Defining "moribund languages" as those which are not learned as mother tongues by children, Krauss (1992) estimated that up to fifty percent of the world's languages might already fit this category. He further predicted that only ten percent of languages seemed safe in the long term, while the remainder were in danger of becoming moribund or even extinct by the end of this century. Although Krauss's (1992) estimations and predictions are critiqued as too dire and pessimistic after two decades (Simons & Lewis, 2011), research findings demonstrate that linguistic diversity has declined twenty percent globally over the period 1970-2005 (Harmon & Loh, 2010). Iran as a multilingual and multi-ethnic country is no exception. Referring to the history of language death in Iran, in particular as the focus of my research, Moseley (2007) writes that "older stages of Iranian languages, such as Avestan, Old and Middle Persian, Pehlevi, Parthian, Sogdian, Chorasmian, Bactrian, Sarmatian and Khotanese, have gone extinct or have effectively been superseded by later stages of Iranian" (p. 315). It is commonly believed that the decline in linguistic diversity, or in other words language loss, reflects larger-scale socioeconomic, cultural, historical, and political processes influencing communities and their languages within multilingual nation-states (Nettle & Romaine, 2000). This thesis explores the importance and complexity of language maintenance/shift processes, as well as how those processes are affected by language policy and planning (LPP) of multilingual nation-states. Using the case of Azeri in Iran, this study examines the three aspects of language policy proposed by Spolsky, i.e. language management, language practices and language beliefs (Spolsky, 2004), in relation to language maintenance/shift within a multilingual nation-state. The research analyzes how the ecology within which Azeri is being transmitted to the next generation has been affected by LPP activities, and how Azeris are responding to those policies. This chapter begins with a historical description of the research setting, illuminating how Farsi has become the official language of the country. Information about the positions of languages in Iran is then provided. Taking social, political, economic, religious, demographic, educational and cultural factors into account in any sociolinguistic research is significant since these factors make up "the full ecology of human life" (Spolsky, 2004, p. 1). 1.1 Research setting This section explores the research setting. The first part presents a historical and sociopolitical account of how Farsi has become the official language of Iran, illuminating the impact of such sociopolitical and historical processes on minority languages. The second section provides a brief description of the city of Tabriz where this research was undertaken, rationalizing why Tabriz, rather than other Azeri-speaking cities, was chosen to conduct this study. 1.0 Aim The second part of the chapter outlines the theoretical frameworks 2 used in this thesis to examine the interplay between language policy of a nation-state and language maintenance among minorities. The concept of language policy used in this research as well as the broad research question of the thesis is introduced. Part three provides a broad overview of this thesis by briefly summarizing each chapter. 2 Please note that there is a slight discrepancy between statistics stated by Tohidi (2009) and statistics shown on the map. 1.1.1 Iran: A historical account Iran, officially the Islamic Republic of Iran, formerly known as Persia, is a Iran, officially the Islamic Republic of Iran, formerly known as Persia, is a multilingual and multi-ethnic country situated in central Eurasia and Western Asia with an area of 628000 square miles (1,648,000 sq. km), making it the sixteenth largest country in the world (Daniel, 2001; Nercissians, 2001). Its population is approximately eighty million with a literacy rate of 77 percent who can read and write the official language of the country, i.e. Farsi (also known as Persian) (CIA, estimate 2013). The country is comprised of minorities like Azeris, Kurds, Gilakis, Baluchis, and Turkmen who speak a language different from the official language, and who mak up nearly half of the population of Iran (Axworthy, 2008). The ethnic groups residing in Iran are Persian (51 percent), Azeri (24 percent), Gilaki and Mazandarani (8 percent), Kurd (7 percent), Arab (3 percent), Lur (2 percent), Baluch (2 percent), Turkmen (2 percent), and other groups (Tohidi, 2009) (see the linguistic map below2). All of Iran's sixty eight regional and minority languages (c.f. Ethnologue), with a special attention to Farsi, the official language (see Sadeghi, 2001), and Arabic as a liturgical language are now officially recognized (Spolsky, 2004, pp. 144, 174). 3 Figure 1.1: Linguistic map of Iran Figure 1.1: Linguistic map of Iran Figure 1.1: Linguistic map of Iran Special and systematic attention to Farsi began in Iran when fear of European colonization, Special and systematic attention to Farsi began in Iran when fear of European colonization, Special and systematic attention to Farsi began in Iran when fear of European colonization, experienced in India where Persian served as the official language until the 1830s, was felt Special and systematic attention to Farsi began in Iran when fear of European colonization, experienced in India where Persian served as the official language until the 1830s, was felt inside Iran (Tavakoli-Targhi, 2009). The British government's abolition of Persian as the official language of India in 1834 led to a desire for neologism, lexicography, the writing of grammar texts, and other Persian purist movements inside Iran (Tavakoli-Targhi, 2009). The rise of a Persian print culture in the late eighteenth century and the nineteenth century governmentalization of everyday life resulted in publications of dictionaries, as well as restyling the language, i.e. simplifying and de-Arabizing the Persian language (Kia, 1998; Tavakoli-Targhi, 2009). 1.1.1 Iran: A historical account Finally, the first Constitution of Iran in 1906 declared Persian as the official language of the country, prescribing that all members of the parliament "had to possess the ability to speak Persian, read and write Persian, and be Iranian subjects of Iranian extraction" (see Kia, 1998). A supplementary law was added in 1907 mandating that compulsory instruction in Persian should be regulated by Ministry of Science and Arts 4 (Kia, 1998, p. 32). However, as Sheyholislami (2012) writes, the policy was not implemented until a more centralized government in Tehran was established. The nationalist and purist movements succeeded to a large extent by transforming language and history into ideological tools to present Iran as one state with one language (Tavakoli-Targhi, 2009). However, what was overlooked in these movements was the multi-ethnic identities and multilingual nature of the country; Iran was not only Persia or Persian. It was "only by denying the existence of non-Persian identities [that] this nationalist discourse [could] present Iran as an ancient and unified nation with one history, one culture, and one literary language" (Kia, 1998, p. 9). The denial and neglect turned into intolerance during Reza Shah and his son's monarchy (1925-1979) (Sheyholislami, 2012). Reza Shah (originally Reza Khan, 1878-1944, the founder of Pahlavi dynasty) was an officer who seized power in 1921 in Tehran through a coup, and made himself Shah of Iran with the support of the British government in 1925. Adopting Pahlavi, the name of the middle Persian language, and renaming the country from Persia to Iran in 1934, presumably meaning "the birthplace of Aryan race" (Asgharzadeh, 2007), Reza Shah showed his nationalistic longing by using the Persian language as a tool. His regime was characterized by modernization, provision of infrastructures, secularization, and determination to build a modern nation-state (Sheyholislami, 2012). What was evident was his awareness of the role of language as a nation-building tool (see Hassanpour, Sheyholislami, & Skutnabb-Kangas, 2012; Sheyholislami, 2012). In 1924 a year before he seized the throne of Iran, Reza Khan ordered the ministry of war to form a committee to create new Persian equivalents for European and Turkish words used in the army. He formed a second committee in the army after a year when he became the Shah of Iran, asking the members to translate the widely-used European and Turkish words into Persian. 1.1.1 Iran: A historical account Between 1921 and 1925, the army in fact became the first institution to modernize and purify Persian (Tavakoli-Targhi, 2009). In line with the Shah's measures, the Teachers' Training College of Tehran established a society in1932 to create new words and terminologies resulting in close to 3000 new words, 400 of which were applied in text books. The linguistic endeavors finally led to the formation of Iranian Academy in 1935 whose principles were closely related to Académie Française (Axworthy, 2008; Daniel, 2001; Kia, 1998, pp. 20-22; Sheyholislami, 2012). Alongside his language purification and modernization activities, and soon after Reza Shah centralized his authority in Tehran, he terminated the semi-autonomous status 5 of regions such as Azerbaijan, Arabistan (Khuzistan), Luristan, and Kurdistan. Using non- Persian languages in any form of writing was prohibited and Farsi was legitimized as the only Iranian language. Other ethnic languages were repressed either by "dialectalising" languages, i.e. labeling languages as an 'imperfect dialect' of Farsi such as Kurdish and Luri, or "minoritising" non-Indo European languages such as Turkic languages, i.e. Azeri, and Arabic (see May, 2008b, for dialectalising and minoritising). All the ethnic languages were required "to be assimilated to "the superior Aryan/Persian race and culture," and if they did not acknowledge the "superiority of Aryan/Persian race," they would then become subjected to humiliation, marginalization, and exclusion" (Asgharzadeh, 2007, p. 87). Farsi was elevated as the 'national language' to unify all Iranians and present Iran as one nation with one language. Reza Shah's son, Mohammad Reza Pahlavi (1919-1980) came to power during World War II after an Anglo-Soviet invasion which forced Raza Shah's abdication in 1941. Like his father, Mohammad Reza Shah followed nationalistic and purist goals denying non-Persian ethnic minority groups' rights. It was during his reign that severe linguistic genocide took place. As Asgharzadeh reports, after approximately being an autonomous, but not a separatist, region for a year, Azerbaijan, and then Kurdistan, was invaded by Pahlavi's army, killing many people in Azerbaijan and Kurdistan. Finally, Azerbaijan collapsed after a period of resistance. Shortly after the fall of the autonomous regions, "book-burning ceremonies became a source of celebration and entertainment for the members of the dominant group and their invading army." Ultimately, the young Mohammad Reza Shah was admired as the hero of "Azerbaijan Crisis" and "the Bringer of Azerbaijan to the Bosom of the Mother Land" (Asgharzadeh, 2007, pp. 101, 102)3. 3 It is worth mentioning here that there are ongoing debates and doubts about the true nature and intentions of the autonomous regional governments as well as the truth of historical accounts of events in the 1950s in Iran. While some believe the governments were not separatists others argue that their ultimate intention was the disintegration from Iran. In this thesis, I focus on the historical impact of events on the fate of languages. The summary of event in the 1950s presented here is necessarily simplistic but aims to provide the historical context for the research. 1.1.1 Iran: A historical account The Pahlavi dynasty ended when Mohammad Reza Shah was dethroned in the Islamic revolution in 1979. May (2008a, p. 7) argues that nation-states today are under pressures from above and below to respect language rights of their minority groups. From above, nation-states under pressures of "multinational corporations and supranational political organizations along with the rise of globalization, nation-states are required to "reevaluate their political 6 and economic sovereignty." From below, minority languages within nation-states are pressuring nation-states to exert their right to either build their own nation-states or for greater representation within the existing nation-state (also see Williams, 2012). Following this global trend perhaps, the linguistic situation for minorities in Iran can be said to have been mitigated after the revolution in 1979. Language rights of ethnic minority groups are 'tolerated' if not 'promoted' (see May, 2011). Currently, according to the Article 15 of the Iranian Constitution, all minority languages in Iran are officially recognized. Minorities and their languages are "allowed" to enjoy institutional support such as teaching minority languages in the education system, having them in the mass media, etc. Nonetheless, owing to the lack of proactive policies obliging protection of minority languages, rather than merely relying on granting permission, minority languages in Iran can be arguably claimed to be threatened. Unlike the period during the Pahlavi dynasty where minorities were forced to assimilate into the mainstream Persian culture, it seems that they are now covertly 'persuaded' to join the mainstream Persian culture through a variety of mechanisms. The highly centralized education system where all teaching materials are in Farsi appears to be one strong assimilatory tool. Minority and regional languages are neither taught nor tested in the educational institutions. Given the role of education systems in shaping particular de facto policies (Shohamy, 2008), such policies towards minority languages in the education system in Iran needs to be taken into account. In the same way, the highly centralized media funded and controlled by the central government is yet another assimilatory and unifying tool (although, in the last fifteen years, Islamic Republic of Iran Broadcasting (IRIB) has established provincial channels which broadcast programs in regional and minority languages). The airtime for programs in the regional language, and Farsi if any, varies from channel to channel. These channels can be said to be some institutional support that minority languages currently receive from the government. 1.1.1 Iran: A historical account Yet, their impact on the ecology of minority languages should be carefully examined. Another domain where minority languages in Iran seem to be overwhelmed by Farsi is linguistic landscapes, i.e. language in public signage, which is closely related to educational policies and literacy in minority languages (Shiohata, 2012). Linguistic landscapes offer space for minority languages to appear in public which can have major implications for the status of languages. Obviously, this primarily requires literacy in those minority languages. The interrelation between literacy in Azeri and its presence/absence in the linguistic landscapes of Tabriz as well as the symbolic presence of other languages in this domain is also 7 7 examined in this thesis. The investigation sheds light on the language policies associated with language use in public, and explores to what extent Azeri people use their language in a written domain like linguistic landscape. This thesis explores de facto language policies within these three domains, the education system, linguistic landscapes, and broadcast media. Grass roots language attitudes and practices are then examined to illuminate the extent to which they are affected by state's language policies implemented in those domains. 1.1.2 Tabriz: An important city for Azeris in Iran Tabriz is the center of one of the thirty one provinces of Iran, i.e. the province of East Azerbaijan - contiguous with, but not to be confused with, the new republic of Azerbaijan to the north. The city has been always considered a politically, economically and historically important city in Iran. As Daniel (2001, p. 8) puts it, "Tabriz, a former capital and center of the province of Azerbaijan, has often been second only to Tehran, the present capital, in importance." Tabriz is considered to be one of the oldest cities in Iran or even in the world (Fisher, 1968), with a population of approximately 2 million people. According to Fisher (1986), the city may date from early Sasanid times (c. third or fourth century A.D.) or, more likely, from the seventh century A.D. Azeri in Tabriz has been chosen to investigate the interplay between language maintenance/shift and language policy processes for demographic and sociopolitical reasons. Azeri is the largest minority group in Iran. There are at least thirteen million Azeri speakers living in Iran, that is, almost one fifth of the whole population, making them the largest ethnic minority group after Farsis (Persians). Azeris live mostly in the northwest cities and towns of the country with Tabriz as the main city (Boeschoten, 1998). The fate of Azeri can have implications for, and potentially an impact on, other smaller minority languages in Iran. Tabriz can be considered the most important city where Azeri can be maintained because of its sociopolitical position. Historically, the city has functioned as the capital of Iran (Fisher, 1968), and an autonomous region for a short period when Azeris adopted a plethora of modern developments earlier than the rest of the country. (Asgharzadeh, 2007; Atabaki, 2000). The city is consequently renowned as the City of Firsts (Hawes & Mirvahedi, 2013). Azeris in Tabriz have been sensitive to their regional identity which can be of great significance in language contact situations. Therefore, whatever occurs in 8 Tabriz is likely to impact on other Azeri-speaking communities in other parts of the country. The dynamics of language policy with respect to Azeri in this city is thus of paramount importance (Daniel, 2001, p. 7). Against this backdrop, this research both describes and explains the linguistic situation in Tabriz by examining language policies in the domains of education, linguistic landscape, broadcast media, and home. 1.1.2 Tabriz: An important city for Azeris in Iran To do so, a number of theoretical frameworks have been utilized. The following section briefly discusses the theoretical frameworks and paradigms used in the thesis. 1.2 Theoretical frameworks This thesis explores three complex issues, namely language maintenance/shift processes, language policies and planning in multilingual states, and the interplay between the two. Given the presence of around 7000 languages in 200 nation-states, and the official recognition and institutionalization of only 200-300 languages by nation-states, language shift is predicted to take place at an alarming speed in near future resulting in vast loss of world languages. This has led some to predict that only 300-600 languages will survive in the long term (see May, 2008a, pp. 2-3). Examination of states' language policies with a focus on their impact on language maintenance/shift processes among minority groups can illuminate how some languages are maintained while others are lost. Language shift is a process whereby a speech community begins learning one or more languages at the cost of its own language, which is often an ethnic and heritage language. The main reason for language shift and/or language loss is commonly believed to be the intergenerational discontinuity of languages in the domain of home (Fishman, 1991; 2001; Manley, 2008; Suslak, 2009), i.e. when the next (younger) generation does not acquire the language. The domain of home is seen as the most important site in which a language in maintained because it is the first site where children encounter their first linguistic experience. Positive attitudes towards a language in the home can be arguably a major contributing factor to, if not guarantee, the vitality of a language. By contrast, negative attitudes may be a prime cause of intergenerational discontinuity of a language (Baker, 2006). Fishman is perhaps the most prominent figure who has argued that language maintenance or reversing language shift cannot take place unless face-to-face micro-level interactions in a language within the domain of home and neighborhood are realized. He concludes that language policies in the macro domains of education, media and other 9 institutions should not be taken as a sign and a contributing factor to the vitality of a language per se (Fishman, 1991). Despite Fishman's persuasive argument regarding the importance of the home domain, a glance at nation-states across the globe implies a strong link between macro domains and home. That is, languages which are dominantly used in macro domains of education, media, public sphere, etc. 1.2 Theoretical frameworks and officially supported by the state tend to be safe with healthy intergenerational transmission in the domain of home, perhaps with a few exceptions such as Irish (e.g. see Lo Bianco, 2012a; O'Connell, 2007; Ó hIfearnáin, 2010; Watson, 1996). In fact, it is the sociopolitical and economic dominance of some groups over others that is often cited for marginalized and dominated communities' inclination to shift to dominant languages (Grenoble, 2011; Sallabank, 2012). In the modern era, language shift is said to happen, often through transitional bilingualism, when speakers of a language decide to stop speaking their own tongue in favor of a politically and/or economically dominant neighboring language (Grenoble, 2011). In other words, family language policy dynamics which can lead to language maintenance/shift do not occur in a vacuum. Rather, such dynamics are under constant pressure from policies implemented within macro domains. Given the sociopolitical and economic dominance of certain groups in the wider society, family language policy is often influenced by the ecology surrounding home. It is therefore argued that family language policy in the home is not always "consciously planned," but rather has essentially been "predetermined by history and circumstances beyond the family's control" (Caldas, 2012, p. 351). This ecology surrounding the home clearly influences family language policies. In Spolsky's words, "each domain has its own policy, with some features managed internally and others under the influence of forces external to the domain," and "language management in the family is partly under the control of family members, but its goals are regularly influenced by the outside community" (Spolsky, 2009, p. 4). In this sense, as Pakir (2003) puts it, family language policy is "invisible." It is perhaps why some have concluded that "all meaningful language policy is ultimately played out in the home," and policies can be said to have succeeded if favorable attitudes and perceptions about the use of the dominant language in the home are formed (e.g. Caldas, 2012, p. 351). One of the major factors influencing ecologies of languages today is believed to be ti t t ' l li d l i ti iti U lik th t diti l d fi iti f One of the major factors influencing ecologies of languages today is believed to be nation-states' language policy and planning activities. 1.2 Theoretical frameworks Unlike the traditional definition of language policy and planning (LPP) predominantly engaged with seemingly non-political activities of "preparing a normative orthography, grammar, and dictionary for the guidance 10 of writers and speakers in a non-homogeneous speech community" (Haugen, 1959, p. 8), modern LPP activities are defined as those political processes which involve "intervening in the linguistic ecologies of a society with the aim of influencing its future linguistic practices" (Liddicoat, 2013, p. 2). Hence, it is argued that ecolinguistic situations can be modified by "the actions of the state power such as the choice of linguistic polices, schooling, literacy, the media, etc." (Calvet, 2006, p. 46). This necessitates that language maintenance/shift processes be explored in relation to the impact of language policies on language ecologies. Despite recent theoretical developments within the field of language policy and planning (e.g. Lo Bianco, 2005; Mühlhäusler, 2000; Shohamy, 2006; Spolsky, 2004), a comprehensive model has not been developed to investigate language maintenance/shift processes, language policy and planning activities, and the interplay between the two. The research accordingly integrates a number of theoretical frameworks as, firstly, a more thorough and rich way to understand how policy works within multilingual nation-states which may result in language shift among some groups, and secondly, as a response to a widely recognized criticism of the LPP field, i.e. a lack of an overarching theory for LPP (Cooper, 1989; Kaplan & Baldauf, 1997; Ricento, 2006). The model contributes to the understanding of how policies can shape certain attitudes, ideologies and behavior among people at the grass roots level leading to their preference for some languages over others. The ecology of language (e.g. Haugen, 1972; Hornberger & Hult, 2008; Mühlhäusler, 1992) is the overarching paradigm used here to emphasize the fact that (a) languages and language-related issues do not take place in a vacuum; (b) languages need a supportive ecology to survive, and (c) languages and their speakers can be manipulated through the manipulation of their ecology by LPP decisions and processes. Frameworks proposed by Giles, Bourhis, and Taylor (1977) and Shohamy (2006) are used to explore how and through what mechanisms LPP works to manipulate the ecology of a language. Integrating the models proposed by Giles et al. and Shohamy illuminates the political nature of domains and institutions and helps us explore who controls LPP devices, for what purposes, and why. 1.2 Theoretical frameworks That is, depending on who uses macro institutions and domains, for what purposes, and why, the same institutions and domains can function, and therefore be considered, as either institutional support promoting minority languages, or LPP mechanisms serving as assimilative tools. The inclusion of these questions in the analysis stresses the political nature of language planning and policy. 11 In an attempt to build an overarching framework, Lo Bianco's "policy as text, discourse, performance" model (2005, 2008c, 2012a) is adopted to include a discursive dimension of policy in the analysis. The extended version of this model allows the analysis of policy documents and texts, discourse preceding and/or following policies, and policy implementation. The benefit of this framework is that it enables researchers to consider the discrepancy between policies outlined in documents, and their implementation and outcome. To make the framework more useful and relevant for the purpose of this thesis, it is extended and integrated with other models mentioned above. The resultant integrated model (presented in chapter two) links the language management dimension of policy to language practices (Spolsky, 2004), and helps us recognize people's agency while taking macro pressures into account. Policy as text, in this model, is defined as written formal policies about language use in a particular society, i.e. language laws. "Language laws and officiality" are seen as the strongest policy devices (Shohamy, 2006, pp. 59-63) because they bestow different functions, statuses, and values on languages although there is no guarantee that they achieve their desired goals, or if they are implemented at all. Policies can be viewed as interventions into language ecologies and they are considered to be "part of the ideological state apparatus" (Liddicoat, 2013, p. 4). A distinction is made between (big 'D') Discourse and (small 'd') discourse in this thesis. The Discursive function of LPP mechanisms, as I argue in chapter two, plays a significant role in the formation of particular attitudes and ideologies resulting in legitimization and normalization of policies. This thesis therefore assumes that policy exists both as (small 'd') discourse and (big 'D') Discourse. Firstly, policy exists as discourses which are defined as "instances of language in-use, being communicative acts composed of words, phrases, sentences and utterances" (Liddicoat, 2013, p. 11). In Lo Bianco's terms, "statements, discussions, and public attitudes that accompany, or respond to, or precede public texts" (Lo Bianco, 2005, 2008c; 2010a, p. 49). 1.2 Theoretical frameworks An analysis of policy as (small 'd') discourse "represent the space for public debate and understanding, appreciation and adoption, dissemination or contest and resistance against promulgated texts of policy and the formulation of alternative courses of action" (Lo Bianco, 2010a, p. 49). Secondly, policy can be also said to exist as (big 'D') Discourse. Gee (1999, 2011) defines (big 'D') Discourse as "ways of combining and integrating language, actions, interactions, ways of thinking, believing, valuing, and using various symbols, tools, and 12 objects to enact a particular sort of socially recognizable identity." He further argues that Discourses are always "language plus other stuff" (p. 34), and they are "embedded in a medley of social institutions, and often involve various "props" like books and magazines of various sorts, laboratories, classrooms, buildings of various sorts, various technologies" (p. 35). This means that policies implemented in institutions and domains such as the education system, media, linguistic landscape, etc enact and disseminate particular Discourses (world views) about the value and position of languages. Because these institutions and domains are often state-run or under the auspices of dominant groups, Discourses promulgated through those domains can be claimed to be political and ideological. This suggests that policy enactment and implementation of state-dominated institutions and domains enable the dominant group to plan particular Discourses. In such institutional activities of the state language is used in an attempt to "block out alternative objects to enact a particular sort of socially recognizable identity." He further argues that Discourses are always "language plus other stuff" (p. 34), and they are "embedded in a medley of social institutions, and often involve various "props" like books and magazines of various sorts, laboratories, classrooms, buildings of various sorts, various technologies" (p. 35). This means that policies implemented in institutions and domains such as the education system, media, linguistic landscape, etc enact and disseminate particular Discourses (world views) about the value and position of languages. Because these institutions and domains are often state-run or under the auspices of dominant groups, Discourses promulgated through those domains can be claimed to be political and ideological. This suggests that policy enactment and implementation of state-dominated institutions and domains enable the dominant group to plan particular Discourses. 1.2 Theoretical frameworks In such institutional activities of the state, language is used in an attempt to "block out alternative understandings or meanings so that the thinking process of an individual reflects what a powerful outsider desires" (Lo Bianco, 2010a, p. 53). Discourse planning can play a significant role in the fate of languages as it can influence people's attitudes and perceptions about languages and their speakers. Language policies, especially in these domains and institutions, can be therefore studied as "ideological construction of the world" (Liddicoat, 2013, p. 12). objects to enact a particular sort of socially recognizable identity." He further argues that Discourses are always "language plus other stuff" (p. 34), and they are "embedded in a medley of social institutions, and often involve various "props" like books and magazines of various sorts, laboratories, classrooms, buildings of various sorts, various technologies" (p. 35). This means that policies implemented in institutions and domains such as the education system, media, linguistic landscape, etc enact and disseminate particular Discourses (world views) about the value and position of languages. Because these institutions and domains are often state-run or under the auspices of dominant groups, The policy as performance component of this model is defined as the implementation of policies. It is through the implementation of policies that particular role model behavior is realized in those domains and institutions providing linguistic and cultural models for emulation (Lo Bianco, 2010a, p. 49). In a sense, policy as performance is closely related to Discourse planning. That is, when policies are performed at the institutional level, particular Discourses are created and promulgated (c.f. Lo Bianco, 2005). As noted above, these Discourses are highly likely to influence people's attitudes and perception, and most-often their behavior, at the grass roots level. The interplay between the different levels of policy-making, policy implementation, and policy consuming can have useful implications for language maintenance/shift processes. In sum, this thesis follows Fishman's (1991) argument that languages are, and should be, primarily maintained in the domain of home. Without parents' decision to pass on a language to the next generation, language maintenance or reversing language shift becomes highly unlikely. 1.2 Theoretical frameworks However, using the ecology of language paradigm, this study acknowledges the fact that language-related issues, language shift in this case, do not happen in a sociopolitical and historical vacuum, and therefore, takes sociopolitical and 13 economic context, and the role of LPP processes in shaping language ecologies into account. That is, family members' agency and choice within the family unit are influenced by the ecology surrounding home. Moreover, the research assumes a strong link between a change in ecology and language policy and planning processes. Using Shohamy's (2006) model and an extended version of Lo Bianco's framework, the role of LPP mechanisms along with their Discursive impact, such as language rules and regulations, education system, the media, and public sphere are examined here. This thesis attempts to explore how family language policies are affected by macro state policies with respect to minorities in Tabriz, Iran, addressing the primary research question of the thesis:  How, if at all, is Azeri being transmitted to Azeri children in Tabriz? In the light of arguments made above, addressing this question requires close scrutiny of the dynamics of family language policies of Azeris as well as the policies within state-run domains and institutions. Therefore, the following research questions are investigated in this research to explore how, if at all, policies in the domain of education system, linguistic landscape, and broadcasting media affect family language policies within the domain of home. The primary reason for choosing the three domains of the education system, media, and linguistic landscape was vulnerability of Azeri in these domains (see Holmes, 2013, p. 55).  What are the language policies in the kindergartens in Tabriz?  What are the language policies in the kindergartens in Tabriz? To what extent are they being implemented?  What are the de facto language policies in the linguistic landscape in Tabriz?  What is the children’s behavior with respect to watching TV and listening to the radio? (a) Which TV channels do they watch and for how long? (b) Why do they watch certain channels?  What are Azeri parents’ attitudes towards using Azeri at home?  What is the current language behavior of Azeri children in the home? Examining policies in these domains in relation to the dynamics of family language policies will demonstrate to what extent, if at all, and in favor of which language the ecology inside the home is affected by the ecology outside the home. Analyses of data are 14 presented in four chapters. The following section provides an overview of the chapters of the thesis. 1.3 Overview of chapters This thesis is divided into four parts with each representing a different focus. Part one, including chapters two and three, situates this research in the existing literature, justifying the research questions and outlining the methodology. Part two, including chapters four, five, and six, is devoted to analyzing language policies in the state-run domains and institutions, namely, the education system, linguistic landscape, and the media, which arguably play a significant role in formation of ecology outside home. Part three, comprising chapter seven, explores family language policies in Tabriz in light of the macro policies of state. Finally, part four synthesizes the findings of the thesis and presents the conclusions drawn from this thesis. Chapter two begins with reviewing the literature associated with language maintenance/shift processes. A link between language maintenance/shift processes and language policy and planning activities is established. Drawing on a number of paradigms and frameworks, chapter two situates this research in the field of LPP and highlights gaps in the existing research. The research questions of this thesis are outlined in chapter two. Finally, an integrated comprehensive model is presented. Chapter three discusses the methodology used to examine language policies in four different domains. Reviewing the merits and weaknesses of quantitative and qualitative approaches in research, chapter three argues for the application of mixed-methods approaches to better explore policy at different levels, i.e. macro, meso, and micro level. The rationale, data collection process, and challenges and difficulties are discussed. Chapter four explores language policy in kindergartens and preschools in Tabriz using Lo Bianco's notion of policy as text, and policy as discourse. The chapter demonstrates how using Farsi in the education system has enhanced its socioeconomic value resulting in its promotion in kindergarten and authorities in Tabriz even though there is seemingly no explicit policy for kindergartens and preschools to promote Farsi. Analysing discourse produced in interviews with preschool authorities, chapter four argues that the policy implementers' attitudes and ideologies have become uncommitted and sometimes negative towards Azeri and positive and supportive towards Farsi. Chapter five examines de facto language policies in the linguistic landscape, i.e. public signage, in Tabriz, linking the findings of chapter four to language use in a written 15 domain. The study of language use in official governmental signs illustrates inattention to and lack of support for Azeri use in public sphere. 2.0 Introduction Recognizing the influential role of language policy and planning (LPP) in the fate of languages, this chapter reviews literature which explores the complex interplay between language maintenance/shift processes and language policy and planning. To examine the interaction between micro-level beliefs, attitudes and actual behavior of people, and macro level policy-making and meso-level policy implementation, a number of theoretical frameworks are critically reviewed. The ecology of language paradigm is used as an overarching framework where linguistic phenomena, e.g. language maintenance/shift, take place. An examination of the ecology of language paradigm presented in the chapter provides support for a broader ecological approach to LPP, as well as considering LPP "a kind of social practice, specifically, a practice of power" (Levinson, Sutton, & Winstead, 2009, p. 767). Taking an interdisciplinary approach, a number of theoretical frameworks from discourse analysis and critical social studies are integrated to illuminate the complexity of language maintenance/shift processes in multilingual nation-states. The discussion of those frameworks provides a basis for identifying different dimensions of language policy as well as the mechanisms through which the policies may be translated into beliefs, attitudes or actual behavior, which in turn may have an impact on vitality of languages in language-contact situations. An integrated model is finally presented. 1.3 Overview of chapters Examination of language use in bottom- up private signage also demonstrates the absence of Azeri, suggesting Azeris' low literacy as well as uncommitted if not negative attitudes towards Azeri. The stark absence of Azeri in a written domain like the linguistic landscape, both in top-down and bottom-up, suggests Azeri is not officially supported, stressing its status as an oral language. ests Azeri is not officially supported, stressing its status as an oral language. Chapter six examines policies in another macro domain, namely the broadcasting media. The examination of programs aired on Sahand TV, a local Azeri channel, shows how bilingualism is encouraged for Azeris. Closer scrutiny, however, suggests the channel's role in formation of particular attitudes and ideologies about languages. Chapter seven explores family language policies in relation to language policies in macro domains which shape the ecology outside home. The de facto family language policies, i.e. Azeri parents' attitudes and behavior, clearly show how their attitudes and behavior have been influenced by macro policies of state. Chapter eight concludes the thesis by presenting some final reflections on the link between state policies and language maintenance/shift among minorities. 16 2.1 Linguistic diversity and language shift The decline of linguistic and cultural diversity is a widespread and unsettling phenomenon around the globe. Of an estimated number of 6000-7000 languages of the world, it is argued that 3000 can be classified as threatened (Wurm, 2001). Concern about the loss of diversity and vitality of the world's languages has been earnestly building for over two decades since Krauss (1992) predicted the loss or endangerment of the majority of the world's languages in the long run. Although Krauss' (1992) estimation that only ten percent of languages are safe has been critiqued as too pessimistic (Simons & Lewis, 2011), empirical studies now show that linguistic and cultural diversity has globally declined twenty percent in just thirty five years, between 1970 and 2005 (Harmon & Loh, 2010). Nettle and Romaine (2000), for example, report that of an estimated 300 languages spoken in the United States of America when Columbus arrived in 1492, nearly half of the languages are extinct today. Australia also loses one or two of its Aboriginal languages per 17 year. Although more than 250 languages may have existed in Australia before European contact, it is predicted that all the Aboriginal languages will die if nothing is done to protect them (McKay, 2007). A brief look at other parts of the world confirms the same dismal picture. In China, a process of intergenerational language shift toward Mandarin (Chinese) is firmly underway in many regions. Minority nationalities consequently lack confidence in their own mother tongues, believing that mastery of Chinese will help them secure more opportunities (Bradley, 2005). In Pakistan, Rahman (2003) notes that there are six major languages and over fifty nine minor languages. Since the policy of the state favors two languages, Urdu and English, languages like Aer (200 speakers reported in 1998), Gowro (200 speakers reported in 1990), Kundal Shahi (500 speakers reported in 2003), and others appear to be on the verge of extinction. In Africa where approximately 2000 languages (nearly one third of the world's languages) are spoken, a ten percent loss of linguistic diversity is considered likely by the end of this century (Batibo, 2005). As a part of a more general pattern of declining linguistic diversity around the globe, language loss in Iran has been also reported. 2.1 Linguistic diversity and language shift "Older stages of Iranian languages, such as Avestan, Old and Middle Persian, Pehlevi, Parthian, Sogdian, Chorasmian, Bactrian, Sarmatian and Khotanese, have gone extinct or have effectively been superseded by later stages of Iranian" (Moseley, 2007, p. 315). Research findings about the current linguistic situation in Iran suggest that Iran's minority languages such as Azeri, Kurdish, and other smaller languages are overwhelmed by Farsi, the official language of the country, in different domains and institutions (e.g. Hassanpour, et al., 2012; Hawes & Mirvahedi, 2013; Holmes, 2013; Mirvahedi, 2012). Of all the many different reasons cited for language loss, such as natural catastrophes, famine, disease, war and genocide, a high emigration rate, a high in- migration rate, and/or a differential birth or death resulting in a statistically marked change in the ratio of speakers for two languages in a community, today language shift is regarded as the most prevalent reason (Dorian, 1980; Grenoble, 2011; Sallabank, 2012). That is, a process whereby a community begins to gradually add a language to its linguistic repertoire over generations at the cost of its ethnic heritage language, resulting in "a reduction in the number of speakers of a language, a decreasing saturation of language speakers in the population, a loss in language proficiency, or a decreasing use of that language in different domains." It is consequently considered a "downward language movement" and a "negative language development" which can lead to language endangerment and finally language death (Baker, 2001, p. 59). By contrast, the opposite 18 process, i.e. language maintenance, can be said to be an additive and/or positive language development which leads to "relative language stability in number and distribution of its speakers, its proficient usage by children and adults, and its retention in specific domains (e.g. home, school, religion)" (Baker, 2001, p. 59). The processes are seen as "two sides of the same coin," and thus, a good understanding of language shift process can contribute to language maintenance endeavors, illuminating how linguistic diversity can be maintained, and how language shift may possibly be reversed (Gafaranga, 2010). Family members, i.e. parents and children (and grandparents in extended families) within the domain of home are recognized as playing a key role in language maintenance/shift processes (Fishman, 1991; 2001; Pfaff, 1990; Queen, 2003; Spolsky, 2011). Exploring language ideologies, attitudes, and practices of family members in the home, i.e. 2.1 Linguistic diversity and language shift family language policy, can arguably provide not only the earliest evidence for the path of contact-induced language change, but it can also shed light on the nature of such changes (Letsholo, 2009; Pfaff, 1990; Spolsky, 2011). The current state of the language can thus be inferred by referring to the age of youngest speaker, and the absence of children speaking the language. Evidence that intergenerational transmission of the language has ceased predicts that a variety may soon disappear (Spolsky, 2011). It is, therefore, maintained that favorable family language policy towards language(s) can determine, and potentially guarantee, their vitality within a community to a great extent. Positive attitudes towards a language can serve as a contributing factor in the vitality of a language. By contrast, negative attitudes may be a prime cause of intergenerational discontinuity of a language (Baker, 2006). Family language policy towards languages is considered so important that some believe that "all meaningful language policy is ultimately played out in the home," and policies can be said to have succeeded if favorable attitudes and perceptions about the use of the dominant language in the home are formed (Caldas, 2012, p. 351). Intergenerational transmission of a language is, therefore, viewed as the "gold standard of language vitality" (Sallabank, 2012, p. 106), and the main factor in language survival (Fishman,1991, 2001). Against this background, this thesis addresses the following question as its principal and overarching question in an under-researched language-contact setting, i.e. Tabriz, Iran: Primary research question: Primary research question: Primary research question:  How, if at all, is Azeri being transmitted to Azeri children in Tabriz? 19 Although intergenerational transmission and parents and children's role and decisions in this process are regarded as the most important factor in language maintenance/shift, family members' choices and decisions are constrained by factors beyond the family's control (Caldas, 2012; Lane, 2010; Sicoli, 2011; Spolsky, 2009; Tollefson, 1991). Lane, for example, argues that despite the fact that the loss of intergenerational language transmission can be seen, in part, as a choice parents make, in reality "they do not always have a choice" (Lane, 2010, p. 63). Their actions are influenced by large-scale social factors such as language policies and attitudes toward minority languages. This reinforces an argument by Spolsky (2009) that each domain has its own policy which is influenced both internally and externally. Similarly, Sicoli (2011) writes that it is commonly held that language shift takes place because speakers "choose" to do so, achieving some gain (such as socioeconomic mobility). However, "agency, rather than the free will of an individual acting from a rational position, is emergent in social practice." Actions are "socioculturally constrained both in the possibility of their deployments and in their effects." Sicoli further argues that because acts changing and/or sustaining a society are situated in sociocultural matrices, it is problematic to assume that agency only lies in individual action (Sicoli, 2011, p. 162). This, as Blommaert (2005) argues, does not mean eliminating creativity, choice, or freedom from our analysis. Rather, situating individual agency within a wider frame of constraints brings the issue of agency, creativity and choice analytically into sharper focus. This argument suggests two main points about language maintenance/shift processes. Firstly, it seems safe to say that it is not only parents and/or children who influence the choice of language(s) of the home. In other words, language maintenance/shift processes are situated in an ecology of language where the home is one of the key domains, and not the only key domain. Secondly, intergenerational discontinuity of language, often leading to language shift on a community scale, can be taken as a sign of sociopolitical, economic, and cultural subordination on a larger scale. As Sallabank (2012) argues, of all the reasons cited for language loss, it is sociocultural, economic and political dominance of some groups over others that brings about motivations among dominated and marginalized groups to shift to dominant languages. 2.2 Ecology of language "Ecology of language," or ecolinguistics (cf. Fill, 1997), can be used as a comprehensive explanatory model to study language-related issues (Calvet, 2006). Taken originally from research in the natural sciences to protect endangered species and preserve diversity, the ecology of language paradigm was first introduced into linguistic studies to examine the interaction of a language with its environment, defined as the interaction of a language with "other languages in the minds of bi- and multilingual speakers . . ." as well as "with the society in which it functions as a medium of communication" (Haugen, 1972, p. 325). The key concept behind the term "ecology of language," as Creese and Martin (2003) argue, is that language is not viewed as a separate entity from a society which uses it (c.f. Steffensen & Nash, 2007). An ecological approach to linguistic phenomena, "Ecology of language," or ecolinguistics (cf. Fill, 1997), can be used as a comprehensive explanatory model to study language-related issues (Calvet, 2006). Taken originally from research in the natural sciences to protect endangered species and preserve diversity, the ecology of language paradigm was first introduced into linguistic studies to examine the interaction of a language with its environment, defined as the interaction of a language with "other languages in the minds of bi- and multilingual speakers . . ." as well as "with the society in which it functions as a medium of communication" (Haugen, 1972, p. 325). The key concept behind the term "ecology of language," as Creese and Martin (2003) argue, is that language is not viewed as a separate entity from a society which uses it (c.f. Steffensen & Nash, 2007). An ecological approach to linguistic phenomena, consequently, involves an exploration of "the relationship of languages to each other and to the society in which these languages exist" which includes "the geographical, socio- economic and cultural conditions in which the speakers of a given language exist, as well as the wider linguistic environment" (Creese & Martin, 2003, p. 1). In other words, the ecology of language paradigm investigates the interrelations between linguistic ecologies and social, historical, sociolinguistic, and political forces at different levels of individual, community, and society (Mühlhäusler, 1996). Within this perspective, language is thus seen as part of "larger meaning-making resources," including " all the affordances that the physical, social, and symbolic worlds have to offer" (van Lier, 2008, p. Primary research question: As a result of this process, minority languages come to be stigmatized, leading to negative attitudes and ideologies towards such languages (c.f. Dye & Dye, 2012). In other words, language shift and language attrition, are the primary causes for language loss, and it happens, often through transitional bilingualism, when speakers of a language decide to stop speaking 20 their own tongue in favor of a politically and/or economically dominant neighboring language (Grenoble, 2011; Wang & Chong, 2011). It is, thus, simplistic to hold only family and family language policy responsible for language maintenance/shift phenomena and ignore the influencing pressures surrounding home. This perspective opens possibilities of investigating why such phenomena take place in a society as it allows the examination of the ecology of language and factors influencing such ecologies. 2.2 Ecology of language 599), enabling the researcher to "map all aspects of the language environment, from the sociological to the psychological" (Hornberger & Hult, 2008, p. 281; Kramsch & Vork Steffensen, 2008). consequently, involves an exploration of "the relationship of languages to each other and to the society in which these languages exist" which includes "the geographical, socio- economic and cultural conditions in which the speakers of a given language exist, as well as the wider linguistic environment" (Creese & Martin, 2003, p. 1). In other words, the ecology of language paradigm investigates the interrelations between linguistic ecologies and social, historical, sociolinguistic, and political forces at different levels of individual, community, and society (Mühlhäusler, 1996). Within this perspective, language is thus seen as part of "larger meaning-making resources," including " all the affordances that the physical, social, and symbolic worlds have to offer" (van Lier, 2008, p. 599), enabling the researcher to "map all aspects of the language environment, from the sociological to the psychological" (Hornberger & Hult, 2008, p. 281; Kramsch & Vork Steffensen, 2008). Because language is not separated from social, cultural, political, economic and linguistic factors, but rather, seen to be in constant interaction with those ecological factors (Adamou, 2010; Grenoble, 2011), the ecology of language paradigm allows the examination of a wide range of relevant possible explanations for linguistic phenomena in 21 a given area. "Interrelated sequences of causes and effects" are examined to explain "changes in the traditional language behavior of one group under the influence of another" which might result in a switch in the language of one of the groups (Mackey, 2001, p. 68). Within this paradigm, language loss is thus seen as a result of a language losing its oikos, the Greek root meaning "home." An oikos, defined as "a complex ecological support system," is considered vital for languages' sustained well-being, and loss of such a supportive system rather than speakers and planners' intentions is regarded as the major reason for language loss (Mühlhäusler, 1992, 1996). In this view, "human communities need to be sustainable in order to maintain their languages, and in order to support a language it is necessary to support the group that speaks it" (Sallabank, 2012, p. 122). 2.2 Ecology of language Language shift becomes likely when one or some of the ecological factors, such as, "the number of speakers, relationship with other languages, patterns of transmission, speakers' attitudes, domains of use, institutional support" (Mühlhäusler, 1992, pp. 173-177), undermine a language in its ecology. As a consequence, the language loses its "ecological niche" assigned in a linguistic ecosystem which is defined on the basis of its relation with other languages and with its milieu, the place it occupies in the ecosystem, and its functions (Calvet, 2006, p. 24). Clearly, language loss or death becomes imminent when a language loses its functions and place in a language-contact context. The traditional language maintenance and preservation measures which were concerned with preserving the structure of languages by providing dictionaries, grammar, and "high literature," are, therefore, considered unlikely to succeed unless the "question of language ecology" is asked (Mühlhäusler, 1992, p. 164). a given area. "Interrelated sequences of causes and effects" are examined to explain "changes in the traditional language behavior of one group under the influence of another" which might result in a switch in the language of one of the groups (Mackey, 2001, p. 68). Within this paradigm, language loss is thus seen as a result of a language losing its oikos, the Greek root meaning "home." An oikos, defined as "a complex ecological support system," is considered vital for languages' sustained well-being, and loss of such a supportive system rather than speakers and planners' intentions is regarded as the major reason for language loss (Mühlhäusler, 1992, 1996). In this view, "human communities need to be sustainable in order to maintain their languages, and in order to support a language it is necessary to support the group that speaks it" (Sallabank, 2012, p. 122). Language shift becomes likely when one or some of the ecological factors, such as, "the number of speakers, relationship with other languages, patterns of transmission, speakers' attitudes, domains of use, institutional support" (Mühlhäusler, 1992, pp. 173-177), undermine a language in its ecology. As a consequence, the language loses its "ecological niche" assigned in a linguistic ecosystem which is defined on the basis of its relation with other languages and with its milieu, the place it occupies in the ecosystem, and its functions (Calvet, 2006, p. 24). Clearly, language loss or death becomes imminent when a language loses its functions and place in a language-contact context. 2.2 Ecology of language The traditional language maintenance and preservation measures which were concerned with preserving the structure of languages by providing dictionaries, grammar, and "high literature," are, therefore, considered unlikely to succeed unless the "question of language ecology" is asked (Mühlhäusler, 1992, p. 164). Language shift becomes likely when one or some of the ecological factors, such as, "the number of speakers, relationship with other languages, patterns of transmission, speakers' attitudes, domains of use, institutional support" (Mühlhäusler, 1992, pp. 173-177), Although the ecology of language paradigm has opened a window into a more contextualized examination of linguistic issues, and it promotes diversity, and multilingualism, offering the possibility of discussion and inclusion of linguistic rights of speakers of all languages in research (May, 2003; Phillipson & Skutnabb-Kangas, 1996; Skutnabb-Kangas & Phillipson, 2008), the paradigm has not been without its critiques and criticisms. The main criticism leveled against the paradigm is a terminological one with important political implications. It is argued that "biologisation of languages" (Pennycook, 2004) and associating biodiversity with linguistic diversity can be misleading, leading us into the fallacy of dealing with languages as an organism (Edwards, 2009; Mackey, 2001). The wholesale adoption of ecological and biomorphic metaphors such as ecology, survival, death, and adaptation to the environment implies the view that "language loss is an inevitable part of the cycle of social and linguistic evolution." One thus can view language 22 loss as "simply a failure on its part, or its speakers, to compete adequately in the modern world, where of course, only the fittest languages can (and should) survive" (May, 2008a, p. 3). It is believed that the reinforcement of such social Darwinism leads to "depoliticization of language diversity" neglecting and obscuring the wider historical, social and political factors at work in language loss (May, 2008a, p. 3; Pennycook, 2004, p. 216). Accordingly, Pennycook (2004, p. 223) argues that "language ecology downplays human agency and linguistic creativity; and by locating a notion of diversity only in the enumeration of languages, it draws attention away from other forms of linguistic diversity and political action" (see also May, 2005, 2012a). loss as "simply a failure on its part, or its speakers, to compete adequately in the modern world, where of course, only the fittest languages can (and should) survive" (May, 2008a, p. 3). 2.2 Ecology of language It is believed that the reinforcement of such social Darwinism leads to Liddicoat (2013) argues that language ecologies incorporating speech communities are subject to "hierarchies of prestige." These hierarchical differences are inseparably connected with "ideological and cultural constructions." Greater value is thus attributed to large dominant languages whereas lesser value is given to smaller, minority languages. These differences of value ultimately influence "what gets planned in the language ecology, what needs to be planned, and reactions accorded to particular interventions in language" (Liddicoat, 2013, p. 5). This necessitates that ecology of language be viewed as ideologically and discursively constructed, rather than a peaceful, kind and gentle environment/space for and among languages and their speakers. Commenting on Mühlhäusler's (2000, p. 308) wishful and utopian thinking that "metaphors of 'struggle of life and survival' taken from the first Darwinian insights entailing 'adaptations of all kinds with the web of life,' should be replaced by the "appreciation of all kinds and their abilities to cooperate and co-exist, rather than kill, exploit and suppress," Edwards (2004, 2010), for instance, brings the issue of ideology to the fore. He writes that the key word here is "should," and the key question is whether there is a real desire to achieve that goal (Edwards, 2004, 2008, 2010), indicating the political nature of language ecologies. It is thus argued that language ecologies can be better investigated if complemented with examination of ideologies of language (Blackledge, 2008). Liddicoat (2013) argues that language ecologies incorporating speech communities are subject to "hierarchies of prestige." These hierarchical differences are inseparably connected with "ideological and cultural constructions." Greater value is thus attributed to large dominant languages whereas lesser value is given to smaller, minority languages. In sum, languages need a complex social, cultural, economical, and political support system to survive and thrive. Because the continuity of languages is bound up with the life of its users, language decline and death may indicate changes in the circumstances of their speakers (Edwards, 2010, p. 38). This truth should not, however, ignore the fact that the life of some can be altered by others. In fact, "languages become minorized because speaker communities are marginalized" (Sallabank, 2012, p. 122), and that is because planning in human ecologies is "ultimately contingent upon the ideological 23 positions of those in power" (Edwards, 2009, p. 227). 2.2 Ecology of language As noted above, the ecology of language paradigm has not paid adequate attention to the most common cause of language endangerment, i.e. sociocultural, economic and political dominance of some over others. Neglecting the role of human agency in language maintenance/shift processes hinders investigations of why language shift takes place on the one hand, and effective policies to reverse language shift processes, on the other. Ricento (2000) points out that we need a conceptual framework to bridge the gap between micro-sociolinguistics work on language choice, dealing with language behavior, identity and agency, and macro-sociolinguistic work on language policy. As a result, as Ricento (2000) argues, LPP should be responsive to, and potentially apply, developments in other fields such as discourse analysis, ethnography and critical social theory. An interdisciplinary approach to LPP studies and the integration of micro-level research (the sociolinguistics of language) and macro-level investigations (the sociolinguistics of society) can provide an answer to this important, yet unanswered, question, "why do individuals opt to use (or cease to use) particular languages and varieties for specified functions in different domains, and how do these choices influence - and how are they influenced by - institutional language policy decision-making (local, national or supranational)?" (Ricento, 2000, p. 208). The development of a conceptual framework (ecology of language or perhaps some other) will lead us to "the next - as yet unnamed - phase of language policy and planning research and scholarship" (Ricento, 2000, p. 209). Proposing the title "integrationist" for the new unnamed era of LPP, Pennycook (2004) argues that integrationist research could maintain elements of the language ecology paradigm, mainly the embedded and complex relationship between languages and their environments, while not reducing languages to biological entities, insisting on the importance of seeing language as a "cultural artefact and part of human endeavours to create new worlds" (Pennycook, 2004, p. 213). In response to criticisms and suggestions reviewed above, the following sections explore a number of theoretical frameworks within the field of language policy to integrate the ecology of language paradigm with language policy and planning activities. 2.3 Ecology of language and LPP Language policy and planning, initially described as ‘language engineering’ (see Cooper, 1989; Karam, 1974) was traditionally proposed as a rational solution to linguistic problems at nation-state level, generally known as "treatment of language problems" 24 (Neustupný, 1974). Early language policy and planning approaches tended to avoid the larger social, economic, and political setting where language change, use, and development took place (Cooper, 1989; Tollefson, 2008). Defined as "the activity of preparing a normative orthography, grammar, and dictionary for the guidance of writers and speakers in a non-homogeneous speech community" (Haugen, 1959, p. 8), LPP, in a period labeled as "decolonization, structuralism, and pragmatism" (Ricento, 2000), was primarily associated with seemingly ideologically-neutral activities (Luke, Mchoul, & Mey, 1990, p. 26). During this stage, LPP was regarded solely as a non-political, non-ideological, nation- unifying and pragmatic paradigm which focused on establishing a stable diglossic society where majority languages were promoted as public languages of wider communication (cf. Tauli, 1974, p. 64). Influenced by the positivist views prevailing in social sciences and linguistics, LPP theories and models of the time promulgated the naive belief that major problems of societies could be solved through scientific methods and planning (Baldauf, 2004; Luke, et al., 1990). As a result of such beliefs, a series of technical distinctions, such as "selection, codification, elaboration, and implementation" (Haugen, 1959), and "status planning" and "corpus planning" (Kloss, 1969), were developed aiming "to provide linguists with the theoretical vocabulary to systematically approach and diagnose LPP- related issues" (Wee, 2011, p. 12). As a consequence, little attention was given to questions of how LPP processes might help "sustain dominance and dependency relations between groups" (Wee, 2011, p. 13). In those early approaches to LPP, labeled by Mühlhäusler as "non-ecological approaches to LPP," the aim was to achieve the dominance of a (national) language over other languages through "reserving a number of important public domains for a single language (e.g. law, army, broadcasting), compulsory education in a national language, making other languages invisible by not naming, renaming, banning publications and similar methods, and by resettlement of minorities or redrawing of administrative boundaries" (Mühlhäusler, 2000, p. 329). Such LPP approaches did not take into account the effects of those LPP decisions and processes leading to the destruction of language ecologies resulting in language loss, reducing linguistic diversity all over the world (e.g. Nettle & Romaine, 2000; Crystal, 2000; Dixon, 1980; Rahman, 2003). 2.3 Ecology of language and LPP Language policy and planning in the modern era has been noted for its impact on Language policy and planning in the modern era has been noted for its impact on the ecology of languages (Kaplan & Baldauf, 2008). In fact, language policy and planning can be defined as a process that involves "intervening in the linguistic ecologies of a society with the aim of influencing its future linguistic practices" (Liddicoat, 2013, p. 2). 25 In the same vein, Calvet (2006, p. 46) argues that an ecolinguistic situation can be modified mainly by two factors, namely "the habits of the speakers" such as immigration, and disruption in transmission of the dominated languages from one generation to another, and "the actions of the state power such as the choice of linguistic polices, schooling, literacy, the media, etc.," indicating human agency in shaping and manipulation of language ecologies. These domains and institutions, such as the education system, media, language regulations, linguistic landscape, etc., become a tool to bring about changes, for good or ill, in language ecologies. The domains and institutions whose functions can be governed by states, or dominant groups were first recognized and labeled as institutional support in Giles and his colleagues' model, "ethnolinguistic vitality model" (Giles, et al., 1977). They proposed that three main factors, i.e. the number of speakers, status, and the institutional support a group receives, were most likely to influence the linguistic vitality of groups. These factors were considered particularly significant to minority groups. According to this model, the more status a linguistic group has and the more speakers use the language, the more linguistic vitality the group can be said to possess. Moreover, because the vitality of language is related to its use in various institutions and domains, the institutional support a linguistic group receives may influence the vitality of language to a great extent (Giles, et al., 1977, p. 309). They argue that a group's language is vital to the extent that its language is well represented in domains and institutions such as mass media, education system, parliament, work, etc. (Giles, et al., 1977, pp. 315-316). Providing a group with more institutional support which makes its language use possible in different domains and institutions may raise its status both in the eyes of the minority and majority group members. 2.3 Ecology of language and LPP These factors are highly likely to go hand in hand and work as contributing factors for maintaining languages. What was not explicitly taken into account in Giles and his colleagues' predominantly descriptive model was the point that those critical domains and institutions are controlled and thus can be manipulated by dominant groups in a given context leading to language shift or loss. The framework did not address how institutional supports can act as mechanisms to control and manipulate groups. Including a strong link between LPP decisions and presence (or lack thereof) of institutional support for a group in analyses enables researchers to connect language policy and planning processes with their outcome and impact on groups and their languages. 26 Shohamy (2006), for example, argues that the provision or lack of such institutional support for groups is ideological: language policy and planning operate through mechanisms, including rules and regulations, language education, language tests, language in public space, ideology-myth-propaganda-coercion, and so on, which are usually opaque to the public. As she argues, it is these overt and/or covert mechanisms that create the "real policies" (Shohamy, 2006). Capable of manipulating state ideologies, these mechanisms are said to be covertly and implicitly perpetuating language practices. Rules and regulations, for example, are used by states along with other "symbolic markers" to "determine who is 'in' and who is 'out'" (Shohamy, 2006, p. 26). National languages are usually assigned certain prestigious functions through rules and regulations which are used as "devices of power and control," and they are regarded as "symbols of inclusion and exclusion." People who know those languages are thus associated with prestige and power, and those who can/do not are seen as having low class and prestige (Shohamy, 2006, p. 29). Acknowledging the role of states in accelerating language shift processes, Spolsky (2011) similarly argues that educational systems and other forms of management, e.g. language laws, the media, etc., are used by governments to encourage people to switch from vernaculars to official standard languages. Because policies behind language management are not necessarily written, Spolsky's (2004) suggests that policy may be implicit, or the explicit one may not be implemented. Accordingly, "there is no obvious answer to the question of what the language policy of a nation is" (Spolsky, 2004, p. 39). The answer to the "real policy" question lies in the language practices, and language beliefs and ideologies of a community. 2.3 Ecology of language and LPP Within this school of thought, "language policy falls in the midst of these manipulations and battles, between language ideology and practice" (Shohamy, 2006, p. xv). Both Spolsky’s (2004) and Shohamy’s (2006) frameworks address criticisms of early LPP work. These frameworks extend language policy research to include not just explicit aspects of language policy (language policy statements), but also implicit aspects of language policy (the practices and beliefs of a community, or the de facto policy). Both Spolsky’s (2004) and Shohamy’s (2006) frameworks address criticisms of early LPP work. These frameworks extend language policy research to include not just explicit aspects of language policy (language policy statements), but also implicit aspects of language policy (the practices and beliefs of a community, or the de facto policy). Shohamy extends Spolsky’s framework to investigate the complex relationship between language management, practices and beliefs/ideology through the means of mechanisms. This expanded view of language policy has been developed specifically because of the Both Spolsky’s (2004) and Shohamy’s (2006) frameworks address criticisms of early LPP work. These frameworks extend language policy research to include not just explicit aspects of language policy (language policy statements), but also implicit aspects of language policy (the practices and beliefs of a community, or the de facto policy). Shohamy extends Spolsky’s framework to investigate the complex relationship between language management, practices and beliefs/ideology through the means of mechanisms. This expanded view of language policy has been developed specifically because of the discrepancy between declared policies and de facto policies (Shohamy 2006: 52–53). Nonetheless, despite understandings Shohamy's (2006) model offers, it has been critiqued for its major weakness, i.e. assuming "much more focused and successful effort on the part 27 of bureaucrats and politicians than the evidence warrants" (Spolsky, 2008a, p. 141). of bureaucrats and politicians than the evidence warrants" (Spolsky, 2008a, p. 141). Because "language planning does not solely depend upon the imprimatur of the powerful; it also requires acceptance from those whose linguistic habits are to be affected" (Edwards, 2009, p. 228; Liddicoat & Baldauf, 2008), an integrationist approach, as noted above, should also account for not only written policies and the mechanisms through which they are implemented but also policy implementers' and policy consumers' attitudes and perceptions at different level of analysis, i.e. the macro, meso and micro levels. 2.3 Ecology of language and LPP Addressing the following question, chapter six investigates Azeri parents and children's attitudes and behavior (in other words, family language policy) towards broadcast media available to them, including Sahand TV, a local channel for Azeris.  What are the parents and children’s attitudes and behavior with respect to watching TV? (a) Which TV channels do they watch and for how long? (c) Why do they watch certain channels? (c) Why do they watch certain channels? To be able to investigate these questions comprehensively, another model is explored here. What follows is the development of Lo Bianco's (2005, 2008c) model. The expansion and integration of this model with the models presented above provides us with a zoom lens. At its widest angle, macro sociopolitical and economic structures can be analyzed. It also enables us to examine policies at meso and institutional level. Focusing on the micro level, the model allows us to explore grass roots attitudes and behavior as well. 2.3 Ecology of language and LPP Therefore, in order to examine the main research question of this thesis regarding the intergenerational transmission of Azeri in Tabriz, more specific research questions of this thesis are outlined below. These questions concern four domains, home, the education system, linguistic landscapes, and media.  What are Azeri parents’ attitudes towards using Azeri at home?  What are Azeri parents’ attitudes towards using Azeri at home?  What is the current language behavior of Azeri children in the home?  What is the current language behavior of Azeri children in the home? As argued above, family language policy, including parents and children's attitudes and behavior towards languages, does not happen in a vacuum. Rather, family language policy is constantly influenced by the ecology surrounding the home. Reviewing the critiques and criticisms of the ecology of language paradigm, I contended that language ecologies are ideological constructs, and thus, the role of language policy and planning and LPP mechanisms (Shohamy, 2006) needs to be considered. Consequently, three domains and institutions most relevant to the case of Azeri in Tabriz are chosen for investigating the role of LPP in shaping the ecology within which Azeri exists. Kindergartens within the education system, as the first domain where children encounter "social policy of the broadest kind (cultural pluralism or assimilation for example)" (Edwards & Giles, 1984, p. 120), are selected as the first site to investigate the role of LPP in the Azeri language ecology (see chapter four), addressing the following question:  What are the language policies in the kindergartens in Tabriz? And to what extent are they being implemented? Linguistic landscape (public signage) research is a recent development within the field of LPP and a domain which can illuminate the dominant and marginalized discourses on language (Puzey, 2012; Reershemius, 2011). The linguistic landscape in Tabriz is investigated here to shed light on "how authorities wish to portray a local linguistic 28 situation," and whether, if at all, that particular portrayal is accepted by the general population (Puzey, 2012, p. 141). The following research question is consequently examined in chapter five:  What are the (de facto) language policies in the linguistic landscape in Tabriz? The last domain investigated as a mechanism of LPP is the broadcast media. 2.4 Policy as text, discourse, and performance To better understand the interplay between macro policies, the institutions through which policies are enacted, and the people's attitudes and behavior with respect to those policies, this thesis uses Lo Bianco's (2005, 2008c) "policy as text, discourse, and performance" model, or what he recently labeled as "intention, interpretation, and implementation" model (Lo Bianco & Aliani, 2013). By examining policy at three levels (namely macro, meso, and micro levels), the analyses help us investigate the complex relation between formal written policies at the macro level, how those policies are actually interpreted and implemented by policy implementers at the meso level, and how such implementations of policies are received by policy consumers at the micro level. Policy as text, in this model, is defined as written formal policies about "intended language futures" (Lo Bianco & Aliani, 2013, p. 3) in a particular society, i.e. language laws, or in Spolsky's words, "the formulation and proclamation of an explicit plan about 29 language use" (Spolsky, 2004, p. 11). "Language laws and officiality" are seen as the strongest of policy devices (Shohamy, 2006, pp. 59-63) because they bestow different functions, statuses, and values on different languages (although there is no guarantee that they are implemented the way they are stated, if they are at all implemented, i.e. the relation between policy and practice is complex and a cause-and-effect relation cannot be assumed (e.g. see Cincotta-Segi, 2011)). Yet, language policy documents are seen as "interventions into the language ecology that seek to shape that ecology to particular ends by mobilizing the resources of the state for language objectives." As such, they are considered to be "part of the ideological state apparatus" (Liddicoat, 2013, p. 4). Formal policies consequently define a set of possibilities for languages and their speakers which play a significant part in empowering certain groups and disempowering others. However, given that there is no direct cause-and-effect relation between policy and practice and the relation between the two is complex, the examination of "intermediate organizational entities" (Fairclough, 2011, p. 120) such as education systems, media, etc. becomes very important. The investigation of practices of such institutions can shed light on the relation between social structures (such as political, economic, structure, etc) which define a set of possibilities and what actually happens (Fairclough, 2011; van Dijk, 2008). 2.4 Policy as text, discourse, and performance It is through such intermediary practices that certain structural possibilities in particular areas of life are selected and retained while others are excluded (Fairclough, 2011, p. 120). This model can advance our understanding of how written formal policies which define a set of possibilities are mediated and finally performed by policy implementers and how such To include the role of the intermediate organizational entities in policy analyses, the discursive nature of such institutions should be considered in the study. It is argued that the political nature of policies may be obscured if policies are analyzed only as text, but not discourse (Ball, 1993; Lo Bianco, 2005). The definition of discourse in this model is extended here to refer to two types of discourses. Firstly, policy exists as discourses which are defined as "instances of language in-use, being communicative acts composed of words, phrases, sentences and utterances" (Liddicoat, 2013, p. 11), what Gee (1999, 2011) terms (little 'd') discourse. In language policy research, this is what Lo Bianco defines as "statements, discussions, and public attitudes that accompany, or respond to, or precede public texts" (Lo Bianco, 2005, 2008c; 2010a, p. 49). An analysis of policy as (small 'd') discourse "represent the space for public debate and understanding, appreciation and 30 adoption, dissemination or contest and resistance against promulgated texts of policy and the formulation of alternative courses of action" (Lo Bianco, 2010a, p. 49). In other words, this type of discourse arises because "official texts require legitimacy and confirmation to succeed" (Lo Bianco & Aliani, 2013, p. 3). Secondly, policy can be also said to exist as (big 'D') Discourse. Gee (2011) defines (big 'D') Discourse as "ways of combining and integrating language, actions, interactions, ways of thinking, believing, valuing, and using various symbols, tools, and objects to enact a particular sort of socially recognizable identity" (29). He further argues that Discourses are always "language plus other stuff" (p. 34), and they are "embedded in a medley of social institutions, and often involve various "props" like books and magazines of various sorts, laboratories, classrooms, buildings of various sorts, various technologies" (p. 35). In a sense, it can be arguably said that Discourses are enacted and disseminated through, by, and in the macro domains and institutions such as the education system, media, linguistic landscape, etc. (e.g. Mumby & Mease, 2011; van Dijk, 2008). 2.4 Policy as text, discourse, and performance These domains construct and project implicit, often-ideological, messages, and images onto their audience. These Discursively constructed messages and images are often about the value and position of different languages and their speakers (Blackledge, 2008). This is closely related to Lo Bianco's policy as performance. He defines performance as the role-model as well as subversive, transgressive and undermining behavior that powerful and significant institutions and individuals perform to provide linguistic and cultural models for emulation (Lo Bianco, 2010a, p. 49). Because such domains and institutions, or LPP mechanisms, are usually state-run or work under the auspices of dominant groups, Discourses promulgated through those domains can be claimed to be political and ideological. I consequently suggest that this type of (big 'D') Discourse can be accordingly planned by dominant groups to construct and impose a particular way of seeing the world (c.f. Gee, 2011). Policies, especially in these domains and institutions, can be therefore studied as "ideological construction of the world" (Liddicoat, 2013, p. 12). In such ideological constructions, language is used in an attempt to "block out alternative understandings or meanings so that the thinking process of an individual reflects what a powerful outsider desires" (Lo Bianco, 2010a, p. 53). Discourse planning can play a significant role in the fate of languages as it can influence people's attitudes and perceptions about languages and their speakers. Exploring discourse in policy analyses and the distinction between policy as (small Exploring discourse in policy analyses and the distinction between policy as (small 'd') discourse and (big 'D') Discourse has two major implications for language policy 31 research. Firstly, viewing functions of macro domains and institutions as essentially Discursive can be a response to criticisms of Shohmay's (2006) model. Rather than assuming a cause and effect relation between LPP mechanisms and behavior at grass roots level, examining them as Discourse planning devices leaves space for grass roots agency and activism. Within this framework, although certain types of Discourses are constructed and promulgated through those domains, people have a choice to either endorse or contest such Discourses. This is not, however, to suggest that those domains and institutions function only Discursively and do not provide any tangible benefits, e.g. literacy in a language. In fact, when those Discourses and socioeconomic gain work in tandem within a domain, they make the strongest Discourse planning device, e.g. in education systems (see chapter four). 2.4 Policy as text, discourse, and performance Secondly, Lo Bianco (2005) often uses policy as discourse and discourse planning interchangeably and without distinguishing between discourse and Discourse, implying that discourse, i.e. interactions in their social context, can be planned through policy processes. The distinction made above helps understand the two concepts as two distinct concepts at two different levels, discourse at the micro and meso level, and Discourse at the macro level. (Small 'd') discourse , i.e. instances of interaction, can be examined at the meso level (those who are involved in policy implementations), as well as the micro level (the policy consumers), which can be distinguished from the (big 'D') Discourse. Secondly, Lo Bianco (2005) often uses policy as discourse and discourse planning interchangeably and without distinguishing between discourse and Discourse, implying that discourse, i.e. interactions in their social context, can be planned through policy processes. The distinction made above helps understand the two concepts as two distinct concepts at two different levels, discourse at the micro and meso level, and Discourse at the macro level. (Small 'd') discourse , i.e. instances of interaction, can be examined at the meso level (those who are involved in policy implementations), as well as the micro level (the policy consumers), which can be distinguished from the (big 'D') Discourse. Moreover, in contrast to Lo Bianco (2005, p. 262) who asserts that "discourse planning is rarely conscious," defining Discourse planning as above makes it clear that Discourse planning like other types of planning is a political, ideological, and conscious process to bring about a desired effect. As I argue throughout this thesis, it is through Discourse planning processes that social structures are (re)produced (see also van Dijk, 2008, p. 23). Accepting the dominant Discourse(s) in favor of the dominant language(s) is likely to result in negative attitudes and perceptions about the minority and regional languages, which in turn may lead to language shift. Moreover, in contrast to Lo Bianco (2005, p. 262) who asserts that "discourse planning is rarely conscious," defining Discourse planning as above makes it clear that Discourse planning like other types of planning is a political, ideological, and conscious process to bring about a desired effect. As I argue throughout this thesis, it is through Discourse planning processes that social structures are (re)produced (see also van Dijk, 2008, p. 23). 2.4 Policy as text, discourse, and performance Accepting the dominant Discourse(s) in favor of the dominant language(s) is likely to result in negative attitudes and perceptions about the minority and regional languages, which in turn may lead to language shift. The inclusion of micro and meso level discourse, and macro level Discourse in policy analysis can provide useful information. The examination of policy implementers and policy consumers' discourse can demonstrate how policies are interpreted and enacted at the micro and meso level, and to what extent, if at all, those discourses derive their meaning from macro level Discourses (Liddicoat, 2013, p. 11). It is through the examination of both types of discourses and Discourses, rather than only discourses as Lo Bianco's work suggests, that "the precise interpretation of the intended language policy, its 32 level of seriousness or otherwise, covert messages it carries or conceals" can be revealed (Lo Bianco, 2008c, p. 168). Consequently, investigations of this type may serve researchers to shed light on latent attitudes of policy implementers as well as policy consumers. The issue of attitudes are indeed regarded very important in LPP studies and some have argued that the success/failure of LPP decisions depend on the extent to which they have been successful to change attitudes (Baker, 1992, 2006). The success of policies, whatever their goals might be, through intermediary practices of major state-run domains depend on Discourse practices which may take different forms in different contexts. Discourse practices as a "political practice" are defined as those (Discursive) practices which aim to naturalize particular power relations and ideologies and eliminate as much resistance as possible (Lo Bianco, 2012b, p. 225; Woodside-Jiron, 2011, pp. 167, 169). In van Dijk's (2008) words, the crucial factor A to exert mental control over B is that B must know about "A's wishes, wants, preferences, or intentions." Apart from direct communication, such messages are delivered through (public) Discourse which is often advertised in domains such as mass media, education systems, etc. In other words, the practices of politics as a social domain are "virtually exclusively discursive" which brings about the reproduction of political ideologies through Discourse. It is through (public) Discourse that public's minds, i.e. their knowledge, opinions, attitudes and ideologies, and consequently the behavior and actions are influenced (and often controlled). In such a scenario, van Dijk (2008, p. 2.4 Policy as text, discourse, and performance 15) argues that since people are "persuaded, seduced, indoctrinated, or manipulated," there is no need for coercion (see also Lo Bianco, 2008a; Nye, 2004). The view of policy as Discourse dictated by a powerful authority through state-run institutions as an attempt to influence another group's attitudes, perceptions, and thoughts is of great relevance and significance to the language contact situations where a dominated minority group is under pressure by a dominant group, as is the case for Azeri in Tabriz. As Ball (1993, p. 15) notes, discourse constructs and allows certain power relations, "redistributing voice," so that "it does not matter what some people say or think, only certain voices can be heard as meaningful or authoritative" (see also Heller, 2008). Referring to the discursive effect of policies, Ball (1993, p.12) further argues that policies may not directly tell someone what to do, but they "create circumstances in which the range of options available in deciding what to do are narrowed or changed." As I argue in this thesis, particular language ecologies are often Discursively created by/through state- run domains and institutions. The view of policy as Discourse dictated by a powerful authority through state-run institutions as an attempt to influence another group's attitudes, perceptions, and thoughts is of great relevance and significance to the language contact situations where a dominated minority group is under pressure by a dominant group, as is the case for Azeri in Tabriz. As Ball (1993 p 15) notes discourse constructs and allows certain power relations 33 The policy as performance component of my adapted version of Lo Bianco's model is defined in this thesis as the implementation of policies within macro domains and institutions. It is through the implementation of policies that particular role model behavior is realized in those domains and institutions. Unlike Lo Bianco who does not explicitly refer to the outcome of the performance of policies, I argue that when policies are performed, in other words implemented, at the institutional level, particular Discourses are created and promulgated. In a sense, policy as performance can be said to be closely related to Discourse planning. As noted above, these Discourses are highly likely to influence people's attitudes and perception, and most-often their behavior, at the grass roots level. The interplay between the different levels of policy-making, policy implementation, and policy consuming has useful implications for language maintenance/shift processes. Drawing on Ball's (1993, p. 2.4 Policy as text, discourse, and performance 10) definition of policy as "text and action, words and deeds, it is what is enacted as well as what is intended," Lo Bianco argues that policy is "an ensemble of activities, some of which are textual (laws, reports, authorisations), others of which are discursive (speeches, radio debates), while still others involve the public performance of behaviors that powerful individuals or institutions hold up as models to be followed" (Lo Bianco, 2008c, p. 157). Although this definition of policy is useful, I suggest here that the distinction between (big 'D') Discourse and (small 'd') discourse and including the notion of Discourse planning as defined above could provide a more comprehensive definition, enabling us to explore policy simultaneously at macro, meso, and micro levels. Supplementing the analysis of formal textual announcements of policy with additional analyses of discourses around them as well as investigating the functions of macro domains and institutions provides access to discursive aspects of language planning. Within this framework, these domains and institutions can function as vehicles for Discourse planning, such as when particular programs are broadcast in certain language(s) on a TV channel, or when a particular language dominates schools and the linguistic landscapes of a bi/multilingual region. The Discourses generated through these mechanisms becomes a model for the target audience to follow. Discourse planning should not, however, be viewed as a sinister act in nature. Rather, we should investigate 'what type of discourse' is produced 'by whom,' 'for what purposes,' 'through what mechanisms,' and 'with what effects' (c.f. Cooper, 1989). It is contended that the discursive context indicates how policy as text is to be "interpreted, evaluated and enacted" (Lo Bianco, 2008c, p. 168). Drawing on Ball's (1993, p. 10) definition of policy as "text and action, words and deeds, it is what is enacted as well as what is intended," Lo Bianco argues that policy is "an ensemble of activities, some of which are textual (laws, reports, authorisations), others of which are discursive (speeches, radio debates), while still others involve the public performance of behaviors that powerful individuals or institutions hold up as models to be followed" (Lo Bianco, 2008c, p. 157). 2.4 Policy as text, discourse, and performance Although this definition of policy is useful, I suggest here that the distinction between (big 'D') Discourse and (small 'd') discourse and including the notion of Discourse planning as defined above could provide a more comprehensive definition, enabling us to explore policy simultaneously at macro, meso, and micro levels. Supplementing the analysis of formal textual announcements of policy with additional analyses of discourses around them as well as investigating the functions of macro domains and institutions provides access to discursive aspects of language planning. 34 Ball (1993) argues that in the analysis of complex social issues such as policy and language maintenance/shift, we need a "a toolbox of diverse concepts and theories" (p. 10). The following section presents a comprehensive model as an integrationist approach to exploring the interplay between language policy and planning and language maintenance/shift processes, particularly in the domain of home. The model proposes a way in understanding how policies work in favor of particular language(s), disempowering other language(s) and group(s), especially within multilingual nation-states. Figure 2.1: A proposed model of the interplay between language policy and language maintenance/shift 2.5 A proposed model of the interplay between language policy and Language maintenance/shift is a complex issue because it involves human agency on the one hand, and macro sociopolitical, historical, cultural, and economic structures, on the other. The issue becomes even more complicated when the interplay between human agency and macro structures are considered, realizing that human agency, freedom, and choice is constrained by "normatives, determined by the general patterns of inequality" (Blommaert, 2005, p. 99). An investigation of the connection between agency and structure in relation to language maintenance/shift processes can shed light on questions like "why do individuals opt to use (or cease to use) particular languages and varieties for specified functions in different domains, and how do these choices influence - and how are they influenced by - institutional language policy decision-making (local, national or supranational)?" (Ricento, 2000, p. 208). Figure 2.1: A proposed model of the interplay between language policy and language maintenance/shift Figure 2.1: A proposed model of the interplay between language policy and language maintenance/shift 35 35 The model proposed here is an attempt to demonstrate why a minority group may stop using its ethnic language in different domains (in favor of dominant language(s)), and how that choice is influenced by language policy and planning activities as well as the macro sociopolitical, historical and economic structures within a nation-state. As mentioned above, the model is built on a number of theoretical frameworks, including the ecology of language paradigm and recent developments in the field of language policy and planning. The model proposed here is an attempt to demonstrate why a minority group may stop using its ethnic language in different domains (in favor of dominant language(s)), and how that choice is influenced by language policy and planning activities as well as the macro sociopolitical, historical and economic structures within a nation-state. As ti d b th d l i b ilt b f th ti l f k i l di th mentioned above, the model is built on a number of theoretical frameworks, including the ecology of language paradigm and recent developments in the field of language policy and planning. 2.5 A proposed model of the interplay between language policy and The ecology of language is taken here as an overarching paradigm to include every factor which might have an impact, directly or indirectly, on language use, including a wide range of factors at different scales and levels, such as socieconomic, political, and historical structures at the macro level, as well as human agency at the meso (e.g. policy implementers) or micro (e.g. policy consumers) levels. Language is thus seen as embedded within a larger ecology (Nettle & Romaine, 2000) within my approach. Language policy documents here are viewed as interventions into language ecologies (Liddicoat, 2013), which are themselves influenced by sociopolitical, historical and economic structures. The framework enables the researcher to examine both formal, declared and written language policies and informal, unwritten and undeclared ones in the analysis. As McCarthy (2011, p. 2) puts it, formal and written policies should not be dismissed, rather they need to be placed in "contexts as part of the larger sociocultural system." Referring to some kind of official text in defining policy alone, therefore, will cause the dehumanization, decontextualization, and dehistoricization of official state policies (McCarty, 2011, p. xii; Schiffman, 1996; Spolsky, 2004). Given that policies, whatever their goals might be, are not directly transferred to people, but rather, they are mediated, interpreted, and enacted through and within intermediary domains and institutions such as education systems, the media, language in public space, etc. (Cincotta-Segi, 2011; Fairclough, 2011), the role of those intermediary institutions as LPP mechanisms (Shohamy, 2006) is taken into account here. As I argued above, the implementation of policies within these domains and institutions create and disseminate certain Discourses, i.e. ways of thinking and seeing the world, influence people's attitudes, and potentially their behavior, at the micro level (illustrated by ). Thus the use of the (upside down) wifi sign implies that the Discourses (re)produced at the institutional level, just like wifi signals which are invisible, are subtle and implicit making them hidden from the public eye. The model assumes space for local and micro level reactions, and agency and language activism (shown by different facial expressions and 36 arrows respectively) (e.g. see Sicoli, 2011). The use of arrows to represent agency means that agency at the grass roots level is often in the form of actions, e.g. forming NGOs, mother tongue schooling, establishing a television and radio channel, etc. 2.5 A proposed model of the interplay between language policy and This is not, however, to suggest the insignificance of those macro domains and their impact on language maintenance. If the mere reliance on the macro domains to maintain a language is like blowing air into a flat tire, we could liken the mere reliance on the domain of home to save or maintain a language to nails on the road which may cause a puncture in the tire. That is, it is wishful thinking to think languages can be maintained or saved only in the home without being recognized and used in macro domains. In other words, not only do languages need home as an important domain of language use, but they also need an oikos for survival, i.e. "a complex ecological support system" (Mühlhäusler, 1992, 1996). In other words, for a language to have its natural intergenerational transmission there needs to be a complex ecological support system. A language should be actively used in the ecology outside home. Otherwise, as Wang and Chong (2011) argue, the maintenance of languages which are used only in restricted settings, including home, is not optimistic. The lack of any of the ecological requirements, e.g. institutional support (Giles, et al., 1977) of a language can consequently have a detrimental effect on its survival. What is of paramount significance here is the fact that decisions as to what extent and to which group(s) and their language(s) institutional support should be provided is a political and ideological act (Shohamy, 2006; Spolsky, 2009). This means that language ecologies can be governed by dominant groups which may result in better language maintenance among some communities, and faster language shift among others. Although nation-states' language policies do not access family units directly, the apparatuses they possess, e.g. the education system, media, language rules and regulations, etc., can Discursively shape particular language ecologies In more abstract terms, language policies can be viewed as a three-dimensional process in this framework which traverses and influences the dimension of time, space, and place simultaneously. Policies affect and are affected by time because, while being products of history, they are future-oriented (see Blommaert, 2005, about layered simultaneity). Policy documents tend to define a status in future for a particular group and its language(s) within a territory (dimension of place). Policies further influence space in the form of Discourses they create in the society. 2.5 A proposed model of the interplay between language policy and The mere reliance on the macro domains to make the intergenerational transmission take place while 37 ignoring the role of family has been criticized and likened to blowing air into a tire which has a puncture (Fishman, 1991, p. xii). This is not, however, to suggest the insignificance of those macro domains and their impact on language maintenance. If the mere reliance on the macro domains to maintain a language is like blowing air into a flat tire, we could liken the mere reliance on the domain of home to save or maintain a language to nails on the road which may cause a puncture in the tire. That is, it is wishful thinking to think languages can be maintained or saved only in the home without being recognized and used in macro domains. In other words, not only do languages need home as an important domain of language use, but they also need an oikos for survival, i.e. "a complex ecological support system" (Mühlhäusler, 1992, 1996). In other words, for a language to have its natural intergenerational transmission there needs to be a complex ecological support system. A language should be actively used in the ecology outside home. Otherwise, as Wang and Chong (2011) argue, the maintenance of languages which are used only in restricted settings, including home, is not optimistic. The lack of any of the ecological requirements, e.g. institutional support (Giles, et al., 1977) of a language can consequently have a detrimental effect on its survival. What is of paramount significance here is the fact that decisions as to what extent and to which group(s) and their language(s) institutional support should be provided is a political and ideological act (Shohamy, 2006; Spolsky, 2009). This means that language ecologies can be governed by dominant groups which may result in better language maintenance among some communities, and faster language shift among others. Although nation-states' language policies do not access family units directly, the apparatuses they possess, e.g. the education system, media, language rules and regulations, etc., can Discursively shape particular language ecologies favoring dominant languages. Such ecologies, as I argue in this thesis, can ultimately lead to language shift among minority communities. ignoring the role of family has been criticized and likened to blowing air into a tire which has a puncture (Fishman, 1991, p. xii). 2.5 A proposed model of the interplay between language policy and However, as noted above, human agency and freedom is taken to be constrained by macro structures (Blommaert, 2005; Tollefson, 1991). The model allows the researcher to investigate LPP processes at the macro level, while exploring and conducting "ethnography of language policy" (Canagarajah, 2006). "While LPP operates from the macro-level of state and international institutions, ethnography focuses on the micro level of interpersonal relationships, conversation, and everyday life" (Canagarajah, 2006, p. 153). It has been argued that the integration of micro-level research ("the sociolinguistics of language") with macro-level investigations ("the sociolinguistics of society") is likely to offer a more complete and satisfactory explanation for language behavior than is available at present (Kelly-Holmes, 2010; Ricento, 2000). Ethnography of language policy was, thus, proposed (Hornberger & Johnson, 2007) to, metaphorically speaking, "unpeel the layers of the LPP onion" (Ricento & Hornberger, 1996). Ethnography of language policy makes it possible for the researcher to consider and examine "agents," "goals," "processes," "discourses which engender and perpetuate the policy," and "the dynamic social and historical contexts in which the policy exists" (Johnson, 2009, p. 144). The model also considers "local actors’ sense making in the implementation of policy" at the meso level (Levinson, et al., 2009, p. 780). Introducing "institutional ethnography," Levinson et al. believe that institutional structures and practices shape and organize everyday experience and the examination of such experience can shed light on the social dynamics of those institutions which are central to the formation of policy. Seeing policy as a practice of power, they argue that the ethnographic study of policy enables us "to see the practice that goes into creating and sustaining the sedimented common sense of policy and….to see the practice of policy appropriation, for which local interests and meanings (often in COP) provide the basis" (Levinson, et al., 2009, p. 789). The model shows the complex interplay between the layers of language policy and planning, and how and through what mechanisms those LPP decisions are turned into practices, which might result in particular family language policies in the domain of home. In other words, it demonstrates that the domain of home and the interactions between family members in the home are under the constant influence of outside ecology. 2.5 A proposed model of the interplay between language policy and Such Discourses seem to be the strongest impact of policies because without successful Discourses planned policies tend to fail to achieve their goals. 38 In sum, the review of existing theoretical frameworks and paradigms in this chapter stresses that an integrationist approach to exploring language maintenance/shift processes in relation to language policy and planning of nation-states needs to be applied. This can better demonstrate how and through what mechanisms macro language policies dictated/advertised in domains outside the home may work their ways into the domain of home, resulting in a change in attitudes and perceptions in favor of the dominant language. Such a comprehensive analysis includes a study of grass-roots level practices and attitudes, macro state policies with respect to languages, and the mechanism and their functionality regarding those languages. This research in Tabriz, Iran attempts to address such issues by exploring grass-roots level behavior and attitudes in the domain of home with respect to Azeri and Farsi, the language policies in pre-schools and the media, a television channel, which is available to Azeri speakers as an institutional support. 2.6 Conclusion The first part of the chapter reviewed the definition and causes of language maintenance/shift. It was argued the most common cause for language shift in this era is sociopolitical and cultural dominance of some groups over others. This part consequently situated the research as one viewing LPP as political and ideological which influences languages and their functions and vitality through impacting on their ecology. Distinguishing between (big 'D') Discourse and (small 'd') discourse within language policy research, the notion of Discourse planning was defined. It was suggested that the macro policies, intermediary domains and institutions, and micro grass roots level practices should be critically examined. It was argued that the domain of home, as the location for intergenerational transmission cannot and should not be considered in a vacuum. Rather, the external domains and institutions and macro sociopolitical and economic factors should be included in the analysis. This can provide a comprehensive picture of a given language contact situation within a state. Based on the gaps identified and suggestions made, research questions for the investigation of language shift in Tabriz have been developed. In the next chapter, the methodology to explore these research questions is discussed. The first part of the chapter reviewed the definition and causes of language maintenance/shift. It was argued the most common cause for language shift in this era is sociopolitical and cultural dominance of some groups over others. This part consequently situated the research as one viewing LPP as political and ideological which influences languages and their functions and vitality through impacting on their ecology. Distinguishing between (big 'D') Discourse and (small 'd') discourse within language policy research, the notion of Discourse planning was defined. It was suggested that the macro policies, intermediary domains and institutions, and micro grass roots level practices should be critically examined. It was argued that the domain of home, as the location for intergenerational transmission cannot and should not be considered in a vacuum. Rather, the external domains and institutions and macro sociopolitical and economic factors should be included in the analysis. This can provide a comprehensive picture of a given language contact situation within a state. Based on the gaps identified and suggestions made, research questions for the investigation of language shift in Tabriz have been developed. In the next chapter, the methodology to explore these research questions is discussed. 39 3.0 Introduction Chapter two discussed the necessity of and need for understanding and systematic analysis of the relationship between macro and micro dimensions of language policy and planning as a perennial issue (Fishman, 1972a; Hornberger & Johnson, 2011; Hult, 2010a). An integrated model derived from a number of theoretical frameworks was then proposed to address this issue. In this chapter, I argue that operationalization of an integrated model requires that a variety of methods and techniques be used in research. That is, a mixed- methods approach needs to be taken. While mirroring the complexity and multidisciplinary of the field (Baldauf, 2002, p. 394), the "diversity of methods" (Kirk & Miller, 1986) used in language policy and planning (LPP) research enhances the validity of research findings or "inference quality" (Teddlie & Tashakkori, 2003). The validity of the findings are often augmented through triangulation which can include "data triangulation, theory triangulation, methodological triangulation, and investigator triangulation" (Brewer & Hunter, 2006; Deniz, 1970, 1989). The underlying premise of triangulation, or "multiple operationalism" (Campbell & Fiske, 1959), as a design strategy is that all methods have their own limitations and biases, and, thus, using only one method to investigate a given phenomenon may yield biased results. Therefore, through triangulation, that is, "the use of multiple methods, with offsetting or counteracting biases" Greene et al. (1989, p. 256), the validity of inquiry results is enhanced. In other words, mixed methods research and the possibility of triangulation enable us to "attack a research problem with an arsenal of methods that have nonoverlapping weaknesses in addition to their complementary strengths" (Brewer & Hunter, 2006, p. 4). This chapter describes the mixed methods approach used to address the research questions outlined in chapter two, concentrating on the qualitative and quantitative components, associated with phase one and phase two of data collection respectively. Different qualitative data collection instruments, namely, structured, semi-structured, and focus group interviews, case studies, and photographs (of linguistic landscapes), alongside a quantitative data collection instrument, a questionnaire, used to explore the macro-level policies alongside the micro-level practices and attitudes are discussed. Further, the 40 rationale and the design of the present mixed methods study and the data collection issues related to each approach, i.e. qualitative and quantitative approaches, are detailed in this chapter. 3.1 Mixed methods The research questions of this thesis are addressed by applying two types of approaches, i.e. qualitative and quantitative, sequentially in two phases. Phase one, i.e. the qualitative strand of the research (January 2011- June 2011), takes a broader perspective in describing the research context by collecting exploratory qualitative data in a range of contexts, public signage, education, broadcast media, and home. Photos were taken of language use pattern on public signage in Tabriz. In-depth and detailed data were also collected by interviewing fifty children as well as ten kindergarten authorities. Three case studies were undertaken as well to explore language practices in the home. The first phase of the research also involved a focus-group interview with six mothers of young children. The data from this stage of the research provided useful direction to the second phase of the data collection, i.e. designing and using a questionnaire to administer to 150 families later on in 2012. The second phase explored parents’ attitudes towards the languages of Azeri, Farsi, and Turkish, the local channel of Tabriz, and their linguistic behavior in different domains. Below, the rationale for using the mixed methods approach and the advantages and challenges of such an approach are discussed. 4 Mixed methods has been given different labels in the last three decades; e.g. ‘‘a methodological approach’’ (Tashakkori & Teddlie, 1998), ‘‘third methodological movement’’ (Teddlie & Tashakkori, 2003), ‘‘a research paradigm’’ (Johnson & Onwuegbuzie, 2004), "the multiple ways of seeing and hearing" Greene (2007). 3.2 Why a mixed methods approach? The mixed methods approach4 has grown out of the idea that the historical schism within the social sciences between qualitative and quantitative research (Castro, Kellison, Boyd, & Kopak, 2010; Johnson & Gray, 2010) should be bridged and a truce should be established between the "paradigm warriors" (Johnson, Onwuegbuzie, & Turner, 2007). Ending the "paradigm war" (Gage, 1989), mixed methods research designs were proposed in the hope that they offer strengths that counterbalance the weaknesses of both quantitative and qualitative research, and act as an incentive for the researcher to use multiple paradigms (Creswell & Plano Clark, 2011). 4 Mixed methods has been given different labels in the last three decades; e.g. ‘‘a methodological approach’’ (Tashakkori & Teddlie, 1998), ‘‘third methodological movement’’ (Teddlie & Tashakkori, 2003), ‘‘a research paradigm’’ (Johnson & Onwuegbuzie, 2004), "the multiple ways of seeing and hearing" Greene (2007). 41 A paradigm is defined as "a conceptual model of a person's worldview, complete with the assumptions that are associated with that view" (Mertens, 2003, p. 139), or in Plowright’s (2011) words, "a system of ideas or theoretical principles that determine, maintain and reinforce our way of thinking about an issue or a topic" (p. 177). It has been proposed that three major paradigms guide current research practices: positivist/postpositivist, interpretive/constructivist, and pragmatism and/or transformative/emancipatory (DeCuir-Gunby, 2008; Mertens, 2003; Tashakkori & Teddlie, 2003). These paradigms underlie quantitative research, qualitative research, and mixed methods research respectively. To use different methods and to advocate for a change for better language maintenance, the present study adopts a pragmatic and transformative/emancipatory paradigm as its underlying paradigm. Mixed methods research with its underlying paradigm(s), i.e. pragmatism and/or transformative/emancipatory, does not, however, dismiss positivism/postpositivism or constructivism. In fact, it attempts to make the most of these paradigms. In DeCuir- Gunby’s terms, mixed methods is "the best of both worlds" (DeCuir-Gunby, 2008, p. 126). It is worth mentioning that pragmatism and transformative/emancipatory can be regarded as two separate paradigms in their own right as they allow and promote different functions in mixed methods research (see below). A paradigm is defined as "a conceptual model of a person's worldview, complete with the assumptions that are associated with that view" (Mertens, 2003, p. 139), or in Plowright’s (2011) words, "a system of ideas or theoretical principles that determine, maintain and reinforce our way of thinking about an issue or a topic" (p. 177). 3.2 Why a mixed methods approach? It has been positivist/postpositivist, interpretive/constructivist, and pragmatism and/or transformative/emancipatory (DeCuir-Gunby, 2008; Mertens, 2003; Tashakkori & Teddlie, 2003). These paradigms underlie quantitative research, qualitative research, and mixed methods research respectively. To use different methods and to advocate for a change for better language maintenance, the present study adopts a pragmatic and transformative/emancipatory paradigm as its underlying paradigm. Mixed methods research with its underlying paradigm(s), i.e. pragmatism and/or Pragmatism as a "well-developed and attractive philosophy for integrating perspectives and approaches" (Johnson, et al., 2007) offers "historical strands and warrants for the new discourses of social science research, which embraces plurality of method and multiple methods philosophies" (Maxcy, 2003, p. 52). Further, as Tashakkori and Teddlie (1998) assert, ‘truth’ for pragmatists is "what works" rather than "metaphysical truths." In other words, in pragmatism as the underlying paradigm for mixed methods research "only results count" (Maxcy, 2003, p. 85). That is, the researcher adopts the most appropriate methods and measures whichever paradigm or research traditions they are associated with. In this sense, pragmatism rejects "the incompatibility thesis" (Howe, 1988), i.e. that quantitative and qualitative research paradigms alongside their associated methods cannot and should not be mixed. Within pragmatism, the antagonism between paradigms is considered to be "unproductive" (Johnson, et al., 2007, p. 127), and the separation of the two approaches considered unhelpful (Tashakkori & Creswell, 2008). Basing his argument on the fact that the paradigms are not competing ones, Bergman (2010, p. 173) similarly argues that "if we were indeed faced with two competing paradigms, then it would not be possible to combine qualitative and quantitative elements within one research question." 42 Mixed methods research, therefore, is "an approach to knowledge (theory and practice) that attempts to consider multiple viewpoints, perspectives, positions, and standpoints (always including the standpoints of qualitative and quantitative research)" (Johnson, et al., 2007, p. 113). The second paradigm assumed in this research in tandem with pragmatism is The second paradigm assumed in this research in tandem with pragmatism is transformative/emancipatory because the research aims to promote a change towards better language maintenance among a minority people and also promote greater social equity and justice (Creswell, Clark, Gutmann, & Hanson, 2003; Teddlie & Tashakkori, 2003). This is achieved by focusing on the lives of and experiences of those who suffer oppression and discrimination (Mertens, 2003). It is worth noting that these two paradigms, i.e. 3.2 Why a mixed methods approach? pragmatism and/or transformative/emancipatory, are not mutually exclusive and they can be applied alongside each other in a single study, although some advocate one over the other (Teddlie & Tashakkori, 2003). The second paradigm assumed in this research in tandem with pragmatism is transformative/emancipatory because the research aims to promote a change towards better language maintenance among a minority people and also promote greater social equity and justice (Creswell, Clark, Gutmann, & Hanson, 2003; Teddlie & Tashakkori, 2003). This is achieved by focusing on the lives of and experiences of those who suffer oppression and discrimination (Mertens, 2003). It is worth noting that these two paradigms, i.e. pragmatism and/or transformative/emancipatory, are not mutually exclusive and they can be applied alongside each other in a single study, although some advocate one over the other (Teddlie & Tashakkori, 2003). Given the preceding discussion, this thesis adopts the definition proposed by Johnson et al. (2007). According to these authors, mixed method research is ….the type of research in which a researcher or team of researchers combines elements of qualitative and quantitative research approaches (e.g., use of qualitative and quantitative viewpoints, data collection, analysis, inference techniques) for the broad purposes of breadth and depth of understanding and corroboration (Johnson et al., 2007, p. 123). This definition affords the use of mixed methods for two main purposes. Firstly, the definition emphasizes the shared fundamental tenet of the mixed methods researchers’ community, that is, "a strict qualitative–quantitative dichotomy is not necessary or productive for answering research questions" (Tashakkori, 2009, p. 288), and "methods should be mixed in a way that has complementary strengths and nonoverlapping weaknesses" (Johnson & Turner, 2003, p. 299). This leads to the second affordance that the definition offers, i.e. "triangulation, complementarity, development, initiation, and expansion" (Greene, Caracelli, & Graham, 1989, p. 255). Accordingly, this research applies four types of triangulation outlined by Deniz (1989), that is, (a) data triangulation, (b) theory triangulation, (c) methodological triangulation, and (d) investigator triangulation. It is worth noting, however, that due to the nature of PhD research, i.e. independent but supervised research, the fourth type of triangulation, namely, investigator triangulation, is relatively weak compared to other types of triangulation. Therefore, a 43 variety of sources and multiple methods will be used to triangulate the data, and multiple perspectives and theories will be used to interpret those data. 3.2 Why a mixed methods approach? Although triangulation is used in research to cross-check the findings and achieve convergence, it can also yield inconsistent or contradictory outcomes. Whichever of the three outcomes prevails, the researcher can construct superior explanations of the observed social phenomena (Deniz, 1989), and produce a more complete picture (Morse, 2003). In a similar vein, Bergman (2011) argues that although mixed methods should be used in a way that convergence occurs between the qualitative components of the research and quantitative results, divergences in such a context can be also used to qualify the research findings. This enables the researcher applying mixed methods designs to "verify and generate theory in the same study" (Teddlie & Tashakkori, 2003, p. 15), and "access knowledge or insights unavailable to a qualitative study and a quantitative study undertaken independently" (O’Cathain, Murphy, & Nicholl, 2007, p. 147). Although the advantages of mixed methods outweigh the challenges, conducting this kind of research is not easy (Creswell & Plano Clark, 2011). These challenges can act as a hindrance to the research. Because both qualitative and quantitative research are undertaken, the main challenge for the researcher in practice is to develop skills in both approaches (Molina-Azorı´n, 2011, p. 8). Furthermore, dealing with a variety of instruments to collect data from participants of different ages and positions requires careful consideration of ethical issues. The following section explores how ethical issues were addressed in this research. 3.3 Ethical issues The consideration of ethics as an essential part of the research plan in linguistics has gains a considerable amount of attention in the last 20 years. Questions such as "who undertakes research and in whose interest, who the research belongs to, who writes and gets credit for authorship, how public the findings are, and what effect and status they have" are considered important (Trechter, 2013, p. 35). As is appropriate for all research, the Human Ethics Committee (HEC) of Victoria University of Wellington requires that ethical approval be sought for research involving human participants. This is to ensure and protect the participants’ right to privacy and autonomy. Ethical approval for this research, accordingly, was sought twice, before phase one and phase two of data collection in December 2010 and December 2011, respectively. 44 Phase one of the data collection (including the interviews with kindergarten authorities, interviews with children, a focus group interview, case studies, and collecting linguistic landscape data) was undertaken from January 2011 to June 2011. As a part of the HEC application for this phase, the interview questions and information sheets for both kindergarten authorities and children, were submitted for HEC approval in order to obtain "fully informed voluntary consent" (Gregory, 2003) from participants. In addition, consent forms to be signed by children’s parents were designed so that the interviews could be undertaken with parents’ permission (see Appendix Two). Information sheets clearly stated in Farsi (see chapter four and five for Azeris' literacy in Azeri and Farsi) what the goals, methods and processes of data collection of the research were. Participants were also reassured about their anonymity and the confidentiality of the information they provided. Phase two of data collection began in February 2012. In this phase, an anonymous questionnaire designed on the basis of the focus group was administered. Before distributing the questionnaire, questions were submitted for HEC approval. Making sure of the anonymity of the questionnaire, questions were approved by the HEC committee. The anonymity of the questionnaire helped ensure the confidentiality, which in return encouraged the participants to take part in the research (Gregory, 2003). Questions focused on the participants’ attitudes toward languages, and their language behavior in different domains (For full description, see below). 3.4.1 Interviews with kindergarten authorities Authorities and principals of ten kindergartens in Tabriz were interviewed in order to explore language policies within the domain of education. To access kindergarten authorities, the "friend of a friend" approach (Milroy, 1980) was used. Hence, I was introduced to the first kindergarten authority by a friend who arranged my first meeting with one of the kindergarten authorities. I was then introduced by the first interviewee to her colleagues. This chain of introduction continued until I had interviewed authorities in ten kindergartens. The process went smoothly because the first interviewee was a member of a decision-making committee (see chapter four), which made accessing the other nine kindergartens relatively easy. Appointments for the interviews with the other nine kindergartens were made by phone. All the interviews were undertaken in Azeri because Azeri is considered and spoken as an oral language on a daily basis. The interviews were conducted within the kindergartens enabling me to undertake brief observations as well. For example, while I was interviewing in one of the kindergartens, a mother with her child came into the office. The dynamics of language contact and change could be observed in the interactions between the child, the mother, and the manager of the kindergarten. While the mother and the manager spoke in Azeri, the child was addressed in Farsi by both the mother and manager. Such observations, though brief and spontaneous, provided additional insights concerning the role that these kindergartens play in language change. Semi-structured interviews were conducted with kindergarten authorities because this type of interview makes it possible for the participants to speak about what they find salient rather than the researcher dictating the direction of the interview, as would be in the structured approaches (Barbour, 2008). They also give the researcher just enough latitude to probe into issue using follow-up questions while staying on the track. The interview schedule was devised to include questions mainly regarding the presence or absence of national or regional policies (see Appendix Two). In the case of the absence of such policies, the authorities were asked about the internal de facto policies within the kindergarten regarding language choice as the medium of instruction. Before each interview, I briefly outlined the general purposes of the research, and reassured the interviewees of the confidentiality of the material collected attempting to encourage them to talk freely about the issues raised in the interviews. 3.4 Data collection instruments The following sections explore the qualitative and quantitative strands of the data collection. The sections are ordered in a way that corresponds to following chapters (chapters 4-7). The data collection in kindergartens is presented first; this set of data is analyzed in chapter four. Next, linguistic landscape data collection process is discussed; the analysis of this set of data is presented in chapter five. The section is later followed by the data collected through interviews with children and the data collected on the local channel in Tabriz (Sahand TV); the analysis of this set of data is presented in chapter six. The final section discusses how case studies were carried out, and how the questionnaire was designed. This set of data is analyzed in chapter seven. 45 3.4.1 Interviews with kindergarten authorities This was very important in order to earn their trust and elicit valid information. Nonetheless, some participants were still concerned about the information they revealed due to the sensitivity of language policies in 46 general. This posed challenges in obtaining the information in some instances. In such cases, follow-up questions were raised. The most common area in which those follow-up questions were used concerned the presence of clear language policy as to which language should be used as the medium of instruction. In the case of not receiving a clear answer, the question was broken down into questions such as "are there any language policies online you are aware of?" or "have you received any recommendations from a governmental organization as to which language you should use in the classroom?" The recorded data was later transferred into the computer for transcription and analysis. The data analysis of this set of data is presented in chapter five. 3.4.2 Linguistic landscape data Language in public signage (linguistic landscapes) has gained considerable attention in sociolinguistic studies. Serving two basic functions of "informational" and "symbolic," linguistic landscape acts as an indicator of the relative power and status of the linguistic communities in a particular territory, and is considered important in language policy and planning literature as it has the capacity to reveal valuable information about the vitality of ethnolinguistic groups residing in multilingual settings (Landry & Bourhis, 1997). It is argued that language in the environment is not arbitrary and it delivers messages about societies, people, the economy, policy, class, identities, multilingualism, etc. (Shohamy & Gorter, 2009). In this research, linguistic landscape data was collected in the hope that it may shed more light on the language contact situation in Tabriz, Iran. The main challenge with collecting linguistic landscape data, as Gorter and Cenoz note, was "sampling and representativity" (Gorter & Cenoz, 2008, p. 352), that is, which street(s) or neighborhood(s) I should choose in order to present a representative picture of the linguistic landscape of the city (see also Androutsopoulos, 2014). The challenge was a serious one considering the size of the city of around two million people living in Tabriz. Hence, I first mapped out the streets taking notes of which streets or areas might best offer a typical picture of the linguistic landscape situation. My notes consisted of information about the different types of signs, e.g. traffic signs, shop windows, graffiti, etc., and language use patterns relating to each type. Nearly all public signage in different regions in the city had the same linguistic pattern, Farsi and English being the two dominant languages. Three streets in the downtown area were finally selected as the research setting; there were several schools in that area. The premise underlying such a choice was the potential impact of linguistic landscape on children's attitudes and ideologies (see chapter 47 five). Having identified the streets, I started taking photos of any written signs on those streets. I undertook the photo-taking task on public holidays when streets were not busy. Three hundred and seventy photos, including photos of shop windows, traffic signs, graffiti, personal notes, and anything written aimed at a public audience, were captured and counted. The data was later transferred into the computer for analysis. 3.4.2 Linguistic landscape data The analysis of this data set is presented in chapter five illuminating language policies and linguistic behavior of Azeris regarding language use within a written domain. 3.4.3 Data from Sahand TV Islamic Republic of Iran Broadcasting (IRIB) is a state-run corporation. Studying language use patterns in the programs broadcast through such a corporation can consequently reveal attitudes and ideologies of the government towards languages. Similar to the domain of linguistic landscape, the main methodological issue here was also sampling reliable data. Because there was a great deal of available material for collection, i.e. programs broadcast every day, decisions needed to be made about what and when to record. To collect data on Sahand TV, a local channel for Azeris, a "constructed week sampling for content analysis" was selected as an efficient sampling method (Hester & Dougall, 2007). The data was collected for a period of seven weeks. One day from each week was selected for recording programs on Sahand TV. Such a sampling method is believed to yield an objective representative of the content for analysis (see Hester & Dougall, 2007, for a comparison of sampling methods for content analysis). This method of data collection was important for this research because the type of programs vary on days associated with certain religious and cultural events and ceremonies. Recording programs over a single week could have resulted in a biased data set. To ensure that recording took place without interruptions, I stayed home and regularly checked on the television and computer which were doing the recording. The analysis of Sahand TV programs are presented in chapter six. 3.4.4 Interviews with Children It has been argued that because many children develop ideologies and certain attitudes about languages at a young age, collecting data from children can provide evidence for incipient change (Pfaff, 1990). Examining children's ideologies about languages may arguably provide useful information about the nature of this change (Queen, 2003). To investigate children's attitudes, structured interviews were used. 48 Interviews are often considered to be a very effective means of eliciting "relevant, valuable, and analytically rich data" (Barbour, 2008, p. 114). Face-to-face, structured interviews were, thus, selected as the most appropriate method to elicit in-depth information from fifty children in this research. These young schoolchildren were studying English at different English institutes. To avoid any potential biased data, they were chosen from different areas of the city from different socioeconomic backgrounds. Other information-eliciting techniques such as questionnaires were not used in this case because of the difficulty of administering questionnaires to young children. I undertook pilot-tests for my questions in the interview protocol (see Nunan, 1992; Oppenheim, 1992, for piloting). This allowed me to learn whether the questions elicit the type of information I expected to obtain or not before undertaking the research with the target participants. To achieve such a goal, having devised the interview questions, I e- mailed them to a colleague in Tabriz where he was able to pilot the questions in the context of the research with four children with the same characteristics as the target participants. Having received positive feedback on the questions regarding their capacity to yield valuable information, I was satisfied that the questions elicited the information I hoped to collect. Fifty (seven to twelve year-old) children were interviewed in Azeri. As mentioned above, these children were students at a language school in Tabriz, Iran. Before the interviews started, consent forms were sent to each child’s parents asking for permission to have an interview with the child (see Appendix Two). Having received permission from the parents, each child was allocated a specific date and time. To avoid any stress during the interviews and establish good "verbal and non- verbal rapport" (Fiksdal, 1988; Spencer-Oatey, 2008) with the children, two measures were taken; one before the interviews and the other during the interviews. Before the interviews, I attended the children’s classes for a few sessions. The teacher introduced me as a friend and a researcher. 3.4.5 Case studies Case studies offer interesting and real-world insightful information allowing researchers to find a balance between theory and practice, and enabling researchers to see "what is going on" as an ethnographer in the research setting (Wolcott, 2008). These ethnographic observations were conducted to study "how the members of [Azeri] community behave and why they behave in that way" (Levon, 2013, p. 69). Three case studies were carried out to demonstrate the dynamics of family language policy in the domain of the home, and illuminate how and to what degree parents' and children's choices and decisions are influenced by the ecology outside the home. Undertaking case studies and complementing the findings with data from an attitude questionnaire (see below) in this research was based on the premise that there could be a discrepancy between parents' reported attitudes and beliefs and their actual behavior in the home (Yu, 2010). The cases in this research were selected on the basis of their availability in a "convenience sample" (Nunan, 1992). To reduce the effects of the "observer’s paradox," (Labov, 1972), those cases were selected through my network of close friends and relatives. Helping reducing the effects of observer’s paradox, the slight familiarity with the family and child in advance typically put the child at ease soon after the first meeting. This encouraged the child to act in my presence as he/she normally did, thus allowing the collection of valuable data. Ethnographic research is often conducted through "prolonged observation" (Levon, 2013). In this research, however, case studies served as a tool to triangulate the main data sets collected through interviews and the questionnaire, providing a richer account of the language contact situation in Tabriz (see Duff, 2008). Each family was thus observed in their home during three days for only about 2 hours each day. The observation focused on the linguistic behavior of the child in relation to the people around him/her as well as the broadcasting media, especially television. Because the families did not consent to audio or video-recording, I had to take notes of what was going on. Note-taking was a challenge around these children. Because I was sometimes involved in a game with the child and could not take notes of what was happening on the spot, I needed to write my detailed description afterwards. Taking notes was more difficult with one of the children in particular. 3.4.4 Interviews with Children This enabled me to establish good rapport with children even before the interviews started. The other measure which was a great aid in putting children at ease was their teacher’s presence during the interviews. Each interview lasted about five minutes, and all the interviews were recorded for further analysis. The only challenge in this stage of data collection was having enough willing participants. Some parents displayed skepticism. One family called the institute asking about the identity of the interviewer and the confidentiality of the information despite reading the information 49 sheet. She was reassured by the manager of the institute about the issues she was concerned about. Finally, 50 participants were willing to be interviewed. 3.4.5 Case studies Because she was six years old, and attended a pre-school, she was quite aware 50 of me taking notes of what she was doing. To avoid unnatural and biased data, I adopted the same strategy of writing a description after the observation ended. Because of the older age of this child compared to the other two, who were both four, I was also careful not to talk about my research in front of the child. While case studies provided in-depth insights into attitudes and behavior of parents and children in the home, they could not be generalized to a larger population (Woodside, 2010). To address this gap, the research utilized an attitude questionnaire which was administered to 150 parents of young children. 5 The focus group interview was carried out in the first phase of data collection. However, the focus group interview is discussed in this section since it was used in this thesis as an instrument which assisted with designing the questionnaire. 3.4.6 An attitude questionnaire: A quantitative measure Quantitative approaches, unlike qualitative approaches, are used in research so that the researcher can generalize the findings and examine the interplay between the variables (Castro, et al., 2010). This allows the researcher using a mixed methods design to compensate for the shortcomings associated with the qualitative approach. This is achieved by quantitative measures producing "numerical" (Plowright 2011), "objective and generalizable" data (Nunan, 1992). In metaphorical terms, by mixing "hard" and "soft" data (Nunan, 1992, p.3), the researcher enjoys greater latitude in the analysis and interpretation of the data and findings. An attitude questionnaire was used in this research as a quantitative measure (see Appendix One). It was designed to gather three types of data, namely "factual, behavioral, and attitudinal" (Dornyei & Taguchi, 2010). Attitudinal questions addressed the three basic aspects of attitudes, namely cognitive, affective and behavioral (Albarracin, Johnson, & Zanna, 2005; Garrett, 2010). The three aspects respectively concerned Azeri parents' beliefs about languages and language policies, the extent to which they approve or disapprove of language(s) and/or policies, and how they report they act with respect to particular language(s) and/or policies. Questions focused on eliciting the parents' attitudes towards the official language of Farsi, the mother tongue of Azeri and other languages such as Turkish in Tabriz, Iran. It was also designed to find out what linguistic practices the respondents reported for different domains. The questionnaire was administered in Farsi rather than Azeri to insure a higher response rate. Administering questionnaires in Azeri could have decreased the number of questionnaires completed and returned by parents because of Azeris' low reading and writing proficiency in Azeri (see chapter four and five). A focus group interview preceded the questionnaire which assisted with designing relevant questions. 51 3.4.6.1 Focus group interview5 Interviews, either individual or group interviews, often precede or follow questionnaires to develop ideas or triangulate research findings (Oppenheim, 1992). A focus group interview or "a thinking society in miniature" (Markova, Linell, Grossen, & Orvig, 2007) was, thus, used in this research to develop ideas to prepare a questionnaire in the later stages of the research. By producing qualitative data, focus group interviews allow the researcher not only to learn why an issue is important but also what is salient about it (Litosseliti, 2003). The aim in such interviews is to collect data on group beliefs and norms regarding a particular topic or set of issues through the capture of intra-group interaction (Bloor & Wood, 2006), and explore people's behavior and attitudes on "issues largely taken for granted" (Hesse-Biber & Leavy, 2006). Focus group interviews, as "a method of least resistance," are arguably well-suited for such purposes, as people feel more comfortable to express their attitudes and feeling when they are in a group (see Barbour, 2008). The interview schedule was prepared in the light of research questions of the thesis. Because the questionnaire aimed to tap into the respondents’ attitudes and their linguistic behavior in different domains of home, education, and media, the focus group interview protocol contained general questions germane to these issues. The focus group consisted of six mothers of young children. I managed to recruit the participants from a language school, where parents waited for their children’s class to be over to pick them up. With the consent of authorities of the language school, I was introduced to the parents by one of the assistants. The interview was conducted in one of the free classes and lasted forty-five minutes. Before the interview began, I provided a brief description of the research and reassured the participants of the confidentiality of their identities. The participants felt very comfortable when they were assured that their names were not required. The discussions during the interview were audio-recorded for analysis. The only issue, of which I later became aware, was caused by the room where the interview took place. Because the room was not carpeted, the respondents' voices echoed making it difficult to transcribe the data. However, the issue was not a hindrance to data analysis because a detailed word-for-word transcription was not required for this section of the research. 3.4.6.2 Questionnaire Questionnaires are the most commonly used data collection instruments (Dornyei & Taguchi, 2010). Questionnaires are known for being relatively easy to analyze, but difficult to construct. There are various challenges which the researcher needs to anticipate before he/she uses a questionnaire. Designing questions, the effect of ordering questions, and reliability are the most important issues which are briefly discussed below. 3.4.6.1 Focus group interview5 5 The focus group interview was carried out in the first phase of data collection. However, the focus group interview is discussed in this section since it was used in this thesis as an instrument which assisted with designing the questionnaire. 52 The analysis of the focus group data provided some clues to designing the attitude questionnaire. In terms of the domains of language use, parents explicitly talked about the influence of satellite channels, especially Turkish channels, on their children's linguistic behavior, the impact of Farsi-only education system on their own behavior in the home, and so on. These issues were included in the questionnaire in the form of different questions (multi-item scales). 3.4.6.2.1 Designing questions reliability (Schuman & Presser, 1981). Dornyei & Taguchi, 2010; Oppenheim, 1992; Schleef, 2014), multi-item scales were designed for investigating Azeris' attitudes towards languages in different domains. A multi-item scale is composed of at least four differently-worded questions (items) addressing the same issue (e.g. attitudes towards a language) (Schleef, 2014). One major challenge in making such scales is that different question orders can yield different responses. Although the degree and seriousness to which the impact question orders might have on responses is a topic of debate (see Schuman and Presser, 1981), I tried to reduce the potential effects of the question order by clearly separating the questions addressing the same issue from each other, in the hope that the respondents would not readily associate similar questions. Such an impact, if occurs, can reduce the internal consistency of the questionnaire, i.e. reliability (Schuman & Presser, 1981). 3.4.6.2.2 Reliability The challenges discussed above can bring about a major setback for questionnaires, namely producing a questionnaire with unreliable results. Reliability, i.e. the consistency of the results (Oppenheim, 1992; Nunan, 1992) is considered to be the most significant issue in questionnaires quantitatively analyzed. In such questionnaires, reliability is tested by doing a factor analysis and calculating Cronbach's Alpha using a statistical package such as SPSS. Cronbach's Alpha is an index between .00 and 1 which indicates to what extent a particular item in the questionnaire correlates with other similar items. Cronbach's Alpha more than 0.70 calculated for a questionnaire means the questionnaire enjoys acceptable reliability (Dornyei & Taguchi, 2010). After piloting the questionnaire with a small sample, the data is fed into the program, and Cronbach's Alpha is then calculated. The Cronbach's Alpha for each set of questions is cited where the results are reported (e.g. in chapter seven). 3.4.6.2.1 Designing questions Although it seems a very easy task to write questions for a questionnaire, it in practice turns out to be very tricky (Schleef, 2014). There are a lot of challenges which the researcher should be ready to face and solve. The first is to make a decision on the type of questions to be used in the questionnaire, that is, closed-ended questions and/or open- ended questions. Each type has its own advantages and disadvantages, with the main ones being ease of construction and coding problems. Closed-ended questions are hard to construct but easy to analyze while the open-ended questions are easier to construct but hard to code and analyze. The questionnaire in this research used mainly closed-ended questions with a few open-ended questions. Open ended questions were designed to elicit demographic information making them easy to code and analyze. Another challenge in questionnaire design is the context effects, that is, Another challenge in questionnaire design is the context effects, that is, "differences in people’s responses to attitudinal questions brought about by changes in the form, wording, or sequencing of the questions" (Rimal & Real, 2005). Loaded questions, double-barrel questions, and questions longer than twenty words need to be avoided so as to be able to collect valid data. Dornyei & Taguchi (2010) suggest that questions should address only one issue at a time, and they should not exceed twenty words; otherwise, they may cause confusion and accordingly produce invalid data. Because question wording and the order of questions appearing on a questionnaire can affect participants' responses (Benton & Daly, 1993; Bradburn & Mason, 1964; 53 Dornyei & Taguchi, 2010; Oppenheim, 1992; Schleef, 2014), multi-item scales were designed for investigating Azeris' attitudes towards languages in different domains. A multi-item scale is composed of at least four differently-worded questions (items) addressing the same issue (e.g. attitudes towards a language) (Schleef, 2014). One major challenge in making such scales is that different question orders can yield different responses. Although the degree and seriousness to which the impact question orders might have on responses is a topic of debate (see Schuman and Presser, 1981), I tried to reduce the potential effects of the question order by clearly separating the questions addressing the same issue from each other, in the hope that the respondents would not readily associate similar questions. Such an impact, if occurs, can reduce the internal consistency of the questionnaire, i.e. 3.4.6.2.3 Piloting the questionnaire The questionnaire was ready to be piloted in January 2012. I e-mailed it to a colleague to pilot it with the participants similar to the target participants, that is, parents of young children. Unfortunately I could not undertake the piloting myself due to the long distance and high costs of traveling between New Zealand and Iran. The answers were fed into SPSS for preliminary analysis. The analysis indicated high reliability for most of the domains. A factor analysis was also run to examine the internal consistency of the questions. The analysis of Likert 54 Scale questions yielded satisfactory results. The major issue involved the Semantic Differential questions. Semantic differential questions ask participants to mark on a continuum between two opposing adjectives. The analysis clearly showed participants' inability and/or indifference to understand and answer those questions. Semantic Differential questions were consequently removed from the questionnaire. Some modifications were also made to factual questions at the end of the questionnaire. 3.4.6.2.4 Questionnaire administration Since the questionnaire was to be completed by parents of young children, I contacted some schools and language institutes where young children were studying. Some of those schools and institutes allowed the research to take place in their schools after I discussed my research topic with them. Having received their consent, I gave the questionnaires to teachers working in those schools and institutes. They were well- informed of the procedure. They were asked to provide each child with a questionnaire and ask them to bring back the completed questionnaire in a week. Since an information sheet was available on the questionnaire (see Appendix One), no more explanation was given to children. 150 questionnaires were returned in three weeks, out of which 107 were complete and used in the analysis. The remaining 43 questionnaires were excluded because they were incomplete. In sum, this study adopted a mixed-methods approach to investigate complex social issues, that is language policies, language practices and language shift/maintenance, which is in practice translated into two approaches of qualitative and quantitative measures. The qualitative strand of the research made use of a structured-interview with children, a semi- structured interview with kindergarten authorities, a focus-group interview with six parents of young children, three case studies, and photos of public signage whereas the quantitative strand included an attitude questionnaire. Providing "narrative" data (Plowright, 2011), the qualitative data enabled me to examine "the ‘whole person’ holistically within that person’s natural environment," (Castro et al., 2010) which allowed in-depth analysis of complex human issues. According to Castro et al. (2010, p. 342), qualitative approaches have the capacity to produce "rich detailed accounts of human experiences (emotions, beliefs, and behaviors)." The main challenge in such approaches, however, is ungeneralizability of the findings or what Bloor and Wood (2006) call "the sacrifice of reliability in pursuit of validity," which is offset by using quantitative approaches in mixed methods. Bloor and Wood (2006) argue that in interviews, for 55 example, the researcher sacrifices "standardization and repeatability" between interviews to understand "more fully the social meanings of the respondent’s world" (Bloor & Wood, 2010, p. 104). This thesis has made use of quantitative measure, namely a questionnaire, to counterbalance the weaknesses associated with qualitative approaches. 4.0 Introduction The education system is believed to be one of the strongest policy devices (Shohamy, 2006, 2008), if not the key one (see May, 2008a, 2012b), which has historically played a significant role both in nation-building and language maintenance endeavors (Wright, 2012). The education system as a strong Discourse planning device, i.e. a site where particular attitudes and views about languages and groups are (re)produced, is so effective that some have argued that "schools often leave no room for resistance" (Shohamy, 2009, p. 186). Schools (and preschools and kindergartens, by extension) are said to reflect the values and priorities of the dominant group, and produce the status and power differences between groups (Cummins, 1988, p. 130). The presence or absence of languages, and attitudes towards languages in the education system can then arguably play a significant role in language maintenance/shift processes (Brown, 2012, p. 282; Jones, 2012), especially when those attitudes are shaped at the early stages of the lifespan, making them relatively unlikely to change (Garrett, Coupland, & Williams, 2003, p. 5). This chapter accordingly explores the dynamics of language policy in kindergartens in Tabriz as the first of three domains to be discussed, i.e. the education system (chapter four), linguistic landscapes (chapter five), and broadcast media (chapter six). This chapter presents a picture of language and ethnic minority policy with respect to Azeri education in Tabriz from its official articulation to its local enactment. This chapter presents a picture of language and ethnic minority policy with respect to Azeri education in Tabriz from its official articulation to its local enactment. Using the "policy as text, discourse, and performance" model (Lo Bianco, 2005, 2008c, 2012a) (see chapter two), the chapter first examines the key policy documents, in this case the three Articles from the Iranian Constitution, for mainly one reason, namely the kindergarten principals' reference to language policies outlined in the Iranian Constitution in their interviews. Articles 15, 16, and 19 of the Constitution are macro formal language laws about the official language and minority languages in Iran (policy as text). Although the impact of "language laws and officiality" on language practices is neither "guaranteed nor consistent" (Spolsky, 2004, p. 8), they bestow different functions, statuses, and values on different languages, defining a set of possibilities for languages and their speakers. 3.5 Conclusion This chapter has described the approach and the data collection instruments used in this thesis. It was argued that the most appropriate method to investigate complex sociolinguistic issues is mixed methods, that is, using both quantitative and qualitative approaches. To make this possible, the research assumes a pragmatic and transformative- emancipatory paradigm in using mixed methods. It is pragmatic in that it holds that the compatibility thesis holds true and methods can be and most of the time should be mixed to answer complex social questions; and, it is transformative-emancipatory in that it aims to promote social equity and justice in a language contact situation. Complex social phenomenon such as language policies and language shift require different kinds of methods so as to best understand and make inferences about these complexities. Such social phenomenon cannot be fully understood using either purely quantitative or qualitative techniques as they look into the issues from only one perspective which is oftentimes limited and biased. We need a variety of data sources and analyses to fully understand complex multifaceted institutions or realities. Mixed methods can provide such a toolbox (Teddlie and Tashakkori, 2003). As Bergman (2010) notes, "mixed methods research is eminently suited for exploring variations in the construction of meaning of concepts in relation to how respondents, for instance, make sense of their experiences or report on attitudes in interviews or questionnaires, respectively" (Bergman, 2010, p. 172). The chapter has further described different qualitative and quantitative methods used in this thesis to collect different data sets in different domains and institutions. Structured-interviews with children, semi-structured-interviews with kindergarten authorities, photos of public signage, data from broadcast media, and an attitude questionnaire and the challenges associated with each method were explained in this chapter. The next chapter presents an analysis of interviews with kindergarten authorities regarding language policies in these institutions. 56 Chapter Four: The Education System: A Discourse Planning Mechanism Chapter Four: The Education System: A Discourse Planning Mechanism 4.0 Introduction The legitimation of particular languages can play a significant part in empowering certain groups and disempowering others which might result in acceleration or deceleration of language maintenance/shift among groups. 57 Having introduced these language laws, I will present the data from interviews with kindergarten authorities in Tabriz (policy as discourse) demonstrating what the policy implementers' attitudes and ideologies are on the ground. The analysis shows how macro policies, inferred from the Constitutional Articles, are interpreted and mediated by preschool authorities at the meso level, revealing policy implementers' attitudes and perceptions about languages (Azeri and Farsi). This is important because such an investigation can lead us to "the nature of their language policy" (Spolsky, 2004, p. 8). Finally, discussing 'policy as performance' with respect to the preschools in Tabriz, the role of the education system in planning Discourse(s) is discussed. 6 The English translation of the Iranian Constitution is available at http://www.iranonline.com/iran/iran- info/Government/constitution.html 4.1 Language laws: Policy as text The texts selected for analysis as part of the present study include Articles 15, 16, and 19 of the Iranian Constitution6. These are the written macro policies about ethnic minority languages and linguistic human rights in Iran. They provide a good starting point as they show what status has been legally bestowed upon different languages in the multilingual and multiethnic country of Iran. They can clarify how minority languages are treated, whether they are promoted, tolerated, or suppressed (Sheyholislami, 2012). The right of minority peoples to use and develop their native languages and writing systems has been constitutionally guaranteed in Iran: Article 15: The official language and script of Iran, the lingua franca of its people, is Persian. Official documents, correspondence, and texts, as well as text-books, must be in this language and script. However, the use of regional and tribal languages in the press and mass media, as well as for teaching of their literature in schools, is allowed in addition to Persian. Giving Farsi the role of a lingua franca in Iran, Article 15 acknowledges Iran's multilingual and multiethnic make-up, and suggests that the country needs a common language so that its people can communicate with each other. Farsi is also designated as the only official language of the country giving it a higher status compared to all other languages. Furthermore, the Article legitimates the provision of institutional support for minority languages in the form of using minority languages in the media and the education system. Giving Farsi the role of a lingua franca in Iran, Article 15 acknowledges Iran's multilingual and multiethnic make-up, and suggests that the country needs a common language so that its people can communicate with each other. Farsi is also designated as the only official language of the country giving it a higher status compared to all other languages. Particular attention has been paid to writing in Farsi. The Article stresses that all types of official writing including all correspondences, documents, textbooks, etc. must be 58 carried out in Farsi. One explanation for the emphasis on writing in Farsi can be an attempt to strengthen national identity and unity. As Sebba (2006, p. 100) argues, the selection of a particular orthography over others can be considered a political and ideological process symbolizing battles over aspects of national, regional, and ethnic identity (cf. Rosowsky, 2010). 4.1 Language laws: Policy as text Because Iran is a multiethnic country with different regional ethnic minorities, it could be argued that writing all the official documents, textbooks, forms, etc. in Farsi plays an important role in unifying all the groups. As a result, Iran is represented as one country with one writing system, and perhaps with one language, and one national identity. According to Article 15, teaching minority languages and their presence in the mass media besides Farsi is "allowed." Although the Article does not specify the language of instruction in schools, as Sheyholislam (2012) writes, it can be readily inferred that "when 'text-books' are required to be in Persian then education must be through the medium of Persian" (p. 31). Moreover, the Article does not make any reference to teaching non-Persian languages as a subject. Nor does it provide any provisions about rights of minority people to use their language in administration and public services. Sheyholislam (2012) argues that because a state's administrative work predominantly depends on written texts and Article 15 is very specific that such texts should be in Farsi, the situation for minority languages in Iran is similar to monarchy's era (see chapter one). Farsi is thus implicitly treated as the "ideal" language for education, development, etc. (see Phillipson, 1988, pp. 341-342) for all Iranians regardless of what ethnicities they come from. This may arguably result in a weaker ethnic identity and social integration, and language shift among minorities. Zhou (2000) argues that communities with their own writing systems may have a stronger sense of identity and social integration which may bring about "passive resistance" to the use of non-native scripts in schools and communities. Minority communities with little or no history of L1 writing system usage, by contrast, are more open to an L2 writing system (p. 145). The choice of a certain language, in this case Farsi, as the and not a, medium of instruction or the language of initial literacy for all in a multilingual countries necessarily advantages the speakers of that language over the speakers of other languages (Mangubhai, 2002, p. 492). This particular language of instruction, especially if it plays a role in upward socioeconomic mobility, can consequently endanger minority languages because it is seen as a more (instrumentally) valuable language. 59 Literacy is regarded as one of the most decisive factors influencing the fate of languages (Tacelosky, 2001). 4.1 Language laws: Policy as text Literacy in a minority language is said to provide a range of advantages for minorities. Given the sociopolitical marginalized status of minority languages, Hirvonen (2008, p. 38), for instance, argues that minority children should be educated in their own language because their language does not enjoy the opportunities for use and development in the society. The least benefit of such education for minority people will be having language skills and becoming functionally bilingual which, as Spolsky (2008b, p. 152) notes, can play a role in the mobilization of ethnic movements, preserving the passive knowledge of the language, contributing to the sense of identity and connection to tradition, and providing "a reservoir tappable in the special conditions of successful re- use of the language" (Spolsky, 2008b, p. 158). Literacy potentially provides more uses and functions such as in employment, leisure reading, and use of the language in public space as well as assisting with the construction, maintenance and transmission of ethnic identity (Avni, 2011). Any languages whose speakers lack literacy are considered endangered because lack of literacy or a written standard in a language, e.g. the case of Swiss German vs. High/standard German, is highly likely to prevent the language from spreading to non- oral domains (Grin & Korth, 2005). Reviewing research into minority language education, Mangubhai (2002) also writes that the benefits of literacy in L1 and its positive impact on subsequent literacy in L2 cannot be neglected. As Mangubhai (2002) notes, research has shown no negative impact of literacy in L1 on literacy in L2; rather, the reverse is true. In contexts where minority children are required to begin their literacy in L2, Mangubhai (2002, p. 493) argues that the outcome depends on non-school-based factors such as the particular language and cultural background, and how minorities view themselves and are viewed by the larger society (cf. Driessen, Slik, & Bot, 2002; Kaplan & Baldauf, 1997). Given the importance of literacy, communities that do not receive literacy in their language(s) have been likened to a colonized community. These languages are seen to possess a lower status and noneconomic functions. In such situations, the dominant language becomes the key to "to educational wisdom, employment and wealth" (Baker, 2002, p. 232), and, consequently, "education determines access to influence (Phillipson, 1988, p. 4.1 Language laws: Policy as text 344), and an instrument to maintain the hegemony of the ruling class which is usually done by channeling "youth into status and occupational roles that support the existing power structure" (p. 212). 60 Although Article 15 has defined a desirable environment for languages in Iran, that is acknowledging multilingualism and "allowing" institutional support for minorities in the form of literacy and/or their presence in the media, the extent to which, if any, as well as how this policy is implemented in reality needs to be investigated. Before exploring the implementation of this policy, another Article relevant to minorities in Iran is examined below. Article 19: Article 19: All people of Iran, whatever the ethnic group or tribe to which they belong, enjoy equal rights; and color, race, language, and the like, do not bestow any privilege. Article 19 provides a framework for protecting human and linguistic rights. The Article states that all Iranians should have equal rights, and no discrimination is allowed based on color, race, or language. The Article shows the government's commitment not to make language, race, color, and such factors as a basis for discrimination. As Paul writes, the assertion that in Islam, and thus in the Islamic Republic of Iran, "the question of border, colour, language and race doesn't exist" seemed to be an indispensable constituent of the IRI narrative (Paul, 1999, p. 209). For linguistic rights to be respected, however, Article 19 provides a framework for protecting human and linguistic rights. The Article states that all Iranians should have equal rights, and no discrimination is allowed based on color, race, or language. The Article shows the government's commitment not to make language, race, color, and such factors as a basis for discrimination. As Paul writes, the assertion that in Islam, and thus in the Islamic Republic of Iran, "the question of border, colour, language and race doesn't exist" seemed to be an indispensable constituent of the IRI narrative (Paul, 1999, p. 209). For linguistic rights to be respected, however, governments would need to provide ethnic minorities with facilities, such as text books in minority languages, teacher training centers to train and prepare teachers for teaching those languages, etc. (May, 2007, 2011, 2012b). None of these has been provided for minority languages in Iran leaving this Article of the Constitution neglected (Hassanpour, et al., 2012). 4.1 Language laws: Policy as text As a consequence, this Article can be claimed not to have achieved its apparent goal, i.e. social and linguistic justice based on equal language rights. "The illusion of freedom" created by Article 15 and 19 which ostensibly advocate diversity and multilingualism but take a monolingual approach in practice is seen one of the most influential ways to produce ideological hegemony that will be in the interest of the dominant powers in the society (van Dijk, 2008, p. 11). Article 19 provides a framework for protecting human and linguistic rights. The Article states that all Iranians should have equal rights, and no discrimination is allowed based on color, race, or language. The Article shows the government's commitment not to make language, race, color, and such factors as a basis for discrimination. As Paul writes, the assertion that in Islam, and thus in the Islamic Republic of Iran, "the question of border, colour, language and race doesn't exist" seemed to be an indispensable constituent of the IRI narrative (Paul, 1999, p. 209). For linguistic rights to be respected, however, governments would need to provide ethnic minorities with facilities, such as text books in minority languages, teacher training centers to train and prepare teachers for teaching those languages, etc. (May, 2007, 2011, 2012b). None of these has been provided for minority languages in Iran leaving this Article of the Constitution neglected (Hassanpour, et al., 2012). As a consequence, this Article can be claimed not to have achieved its apparent goal, i.e. social and linguistic justice based on equal language rights. "The illusion of freedom" created by Article 15 and 19 which ostensibly advocate diversity and multilingualism but take a monolingual approach in practice is seen one of the most influential ways to produce ideological hegemony that will be in the interest of the dominant powers in the society (van Dijk, 2008, p. 11). Article 16 is specifically about Arabic which will be discussed very briefly here as it is beyond the scope of this thesis. Article 16: Article 16: Since the language of the Qur'an and Islamic texts and teachings is Arabic, and since Persian literature is thoroughly permeated by this language, it must be taught after elementary level, in all classes of secondary school and in all areas of study. 61 Arabic seems to have gained this special attention because of its association with Islam and the holy book of Qur'an. Given that only two percent of Iranian population speaks Arabic (CIA Factbook), Article 16, rather than promoting a particular minority language, apparently aims to strengthen and increase the liturgical value of Arabic in Iran by providing Iranians with the linguistic skills to enable them to engage with Islamic texts and literature (see Paul, 1999, pp. 210-211). How this policy is implemented and what teachers and students' attitudes are towards Arabic and other languages in schools is beyond the scope of this study and subject to further research. However, there is anecdotal evidence that English is favored far more than Arabic in Iranian schools although English has not been legally supported in the Constitution. In sum, as discussed above, minority linguistic rights are officially guaranteed in the Constitution, while choosing Farsi, the official language of the country, as the language of all official texts including textbooks. To find out how these policies are mediated by the education system, and to what extent this has had an influence on Azeris at the grass roots level, the analysis, as noted in chapter two, should go beyond the written policy. The attitudes, beliefs and ideologies of those involved in policy enactment should be investigated. To this end, the following section presents and discusses the data collected through interviews with pre-school authorities in Tabriz. 4.2 Interviews with kindergarten authorities: Policy as discourse Following Gee (1999, 2011), Mumby and Mease (2011), Alvesson and Karreman (2005), and Fairhurst and Putnam (2004), this thesis, as noted in chapter two, distinguishes between discourse (with a small 'd') and Discourse (with a capital 'D'). (Big 'D') 'D'iscourse refers to "general and enduring systems of thoughts" whereas 'd'iscourse is defined as "talk and text in social practices" (Fairhurst & Putnam, 2004, p. 8). Using this distinction, the following sections examine policy as discourse, and Discourse planning respectively with regard to the education system. Organizations and institutions, such as education systems, are viewed as "discursive constructions." In other words, discourse is seen as "constitutive of organizing" which needs to be considered in investigations (Mumby & Mease, 2011, p. 283; see also Alvesson & Karreman, 2005; Fairhurst & Putnam, 2004). Including and exploring discourse, i.e. discussions about policies, in policy analysis can yield "the precise interpretation of the intended language policy, its level of seriousness or otherwise, covert messages it carries or conceals" (Lo Bianco, 2008c, p. 168). Supplementing formal 62 language policy analyses with other sources of information such as "analysis of the discourses and interests of key players" can improve the critical orientation towards language policies which can help us understand how formal policies are to be "interpreted, evaluated and enacted" (Lo Bianco, 2008c, p. 168). This section presents results of an analysis of ten face-to-face interviews with pre- schools (kindergartens) in Tabriz supplemented with brief observations. Kindergartens rather than primary or secondary schools were selected for two main reasons. Firstly, instruction in these kindergartens and preschools is essentially conducted in the form of oral interactions between teachers and students. Because there are no written texts involved in education at this level, this potentially provides space to defy Farsi-only textbooks and instruction in higher levels. That is, minorities like Azeris can potentially use their language in instruction. Secondly, the majority of kindergartens and preschools in Tabriz accept children as young as one year old. Given the long hours that children stay in these institutes (as long as eight hours), they can play an important role in formation of particular attitudes and linguistic behavior in young children. The main interview questions revolved around the availability of formal policies with respect to using a particular language as the medium of instruction in kindergartens in Tabriz. Kindergarten authority's answer 9 The interviews were conducted in Azeri, and then translated into English. 9 The interviews were conducted in Azeri, and then translated into English. 7 Interview question 8 8 Kindergarten authority's answer 9 4.2 Interviews with kindergarten authorities: Policy as discourse The main following question was about the informal internal policies of those kindergartens regarding the language of instruction if there were no formal policy about the issue of the medium of instruction. The questions were posed in semi-structured interviews which allowed enough flexibility to pose other follow-up questions to probe into the issue to a greater extent while a previously selected set of questions served as "the discursive compass that steered the interviews back on track" whenever there appeared to be a significant digression (Gao & Park, 2012, p. 543) (for more details on methodology, see 3.4.1) Excerpt 1 Excerpt 1 10 Using follow-up questions, it was clarified that a policy-making committee consisted of some of the experienced preschool and kindergarten authorities existed who was responsible for making policies with respect to educational issues. The committee worked under the supervision of the Welfare Organization. [IQ]7 Is there a rule or regulation to tell you what language to use in classes as the medium of instruction? [IQ]7 Is there a rule or regulation to tell you what language to use in classes as the medium of instruction? [KAA]89 No, but preferably, they tell us to use Farsi because the official language is Farsi. We have some children who unfortunately do not know Farsi at all, or of course we cannot blame them. That’s why we have a difficult task in this bilingual area. So it is not practical to use only Farsi at first. [KAA]89 No, but preferably, they tell us to use Farsi because the official language is Farsi. We have some children who unfortunately do not know Farsi at all, or of course we cannot blame them. That’s why we have a difficult task in this bilingual area. So it is not practical to use only Farsi at first. 63 [IQ] So it is in the hand of the management as to what language to use? [KAA] Yes, but we try to see what the need is. If we focus on Farsi-only programs at first, children will lose interest. On the other hand, if we have Azeri-only classes they’ll have trouble understanding the material at schools. You know, children come here with a background in Farsi, they have learned a lot from TV, games and even home; some parents speak Farsi with them. Fortunately, you can hardly find a child who doesn’t know Farsi at all. It is easy to work with such students. They learn fast. They fully understand Farsi in 2 or 3 months. The above excerpt which was repeated almost identically by the nine other kindergarten authorities reveals very useful points about language policies in the education system. Clearly as stated above, there is no written and formal policy as to what language should be used in pre-schools as the medium of instruction. However, the informal policy seems to be using Farsi as the medium of instruction, as is made clear in the first line, i.e. they tell us to use Farsi because the official language is Farsi, 'they' referring to a policy-making committee.10 This statement is repeated in a different form in another kindergarten's manager's responses. He believes that there are rules and regulations which stress that Farsi should be used in classes as the medium of instruction although those rules are not written. Excerpt 2 [IQ] Is there a rule or regulation to tell you what language to use in classes as the medium of instruction? [IQ] Is there a rule or regulation to tell you what language to use in classes as the medium of instruction? [IQ] Is there a rule or regulation to tell you what language to use in classes as the medium of instruction? [KAA] Yes there are rules and regulations although you cannot find them on paper easily. The emphasis is on Farsi, though. In line with the above head teacher's answers, another pre-school head teacher clearly referred to the absence of clear, formal written policies. Excerpt 3 [KAA] There are no explicit rules regarding the language. We ask parents to speak Azeri at home until when the child is three years old. The problem is that sometimes some children do not know Farsi at all and sometimes some children do not know Azeri at all. The private kindergartens are under the authority of an organization called Welfare Organization. The policies are not really clear and when a supervisor visits the kindergarten, he/she pays much attention to simple things like colors of 10 Using follow-up questions, it was clarified that a policy-making committee consisted of some of the experienced preschool and kindergarten authorities existed who was responsible for making policies with respect to educational issues. The committee worked under the supervision of the Welfare Organization. 64 classes rather than quality of education. So what we do is we try to integrate both languages in the first month and then we little by little switch to Farsi-only classes. classes rather than quality of education. So what we do is we try to integrate both languages in the first month and then we little by little switch to Farsi-only classes. The absence of clear rules and paying little or no attention to educational issues has left the fate of languages to market forces. As Fishman (2006) argues, any "no-policy policy" situation, works in favor of the dominant language. The lack of supportive and promotive policies with respect to Azeri seems to have left no choice for the authorities but to creatively modify the unwritten policies favoring Farsi. Because Azeri children will attend the primary school in the following years where the textbooks are in Farsi, kindergartens consider it their first priority to provide the linguistic capacity and skills required to understand those textbooks. As the kindergarten head teacher notes, because children come from different linguistic backgrounds, the teachers have a difficult job. [IQ] Is there a rule or regulation to tell you what language to use in classes as the medium of instruction? They, therefore, use both Azeri and Farsi in the beginning month(s) of the year, and then switch to a Farsi- only policy making the education sector serve as a unifying and nation-building tool. This is indeed what school as an institution normally does; that is, working towards "uniformity and monolingualism in the approved variety associated with literacy" (Spolsky, 2009, p. 91). This strategy is also used to gain and/or secure the Azeri-speaking children's trust as one of the head teachers notes: Excerpt 4 Excerpt 4 [KAA] Psychologically speaking, we need to speak Azeri with the child so he or she could trust us, especially emotionally, except those whose mother tongue is Farsi. The children can speak Farsi well when they finish kindergarten. My classroom observations as well as observations I had during the interviews confirm the teachers and managers' responses. Because I was collecting data near middle of the academic year, as the authorities reported above, they had already switched to Farsi-only policies both in classes and outside the classes in kindergartens. As I entered a class, the teacher asked the students in Farsi to greet me. All the instructions were in Farsi and the children could perform all of them. They could also answer their teacher's questions in Farsi almost flawlessly. The Farsi-only policy was also observed outside the classes. While I was interviewing the manager, a child came in the office and talked about a little problem he had. All the conversation was carried out in Farsi. As one of the authorities noted above, Azeri children can speak Farsi quite well by the end of kindergarten. Of great interest are the attitudes and perceptions of the head teacher in Excerpt 3. She views not knowing Farsi or Azeri at all by the age of five or six as a problem. The line 65 'The problem is that sometimes some children do not know Farsi at all and sometimes some children do not know Azeri at all' implies that the authorities expect and promulgate the view that children should be able to know both Azeri and Farsi by the age they start kindergarten. This can be called 'bilingualism in favor of the dominant language,' as it implies that L2 should be introduced in the domain of home. [IQ] Is there a rule or regulation to tell you what language to use in classes as the medium of instruction? This condition is highly likely to eventually result in language shift from L1 to L2 considering the higher prestige, and instrumental value of L2 (Baker, 1992, 2006; Errihani, 2008; Oliver, Collard, Rochecouste, & Purdie, 2002; Spolsky, 2009). Excerpt 3 also indicates that speaking Azeri up to the age of three and Farsi afterwards with children in the home appears to be the norm expected and advertised in these institutes attempting to reduce or remove the school-home linguistic discontinuity (Edwards & Giles, 1984) which exists in Tabriz. Such attitudes have been attributed to the absence of L1 in the education system. As Spolsky (2009, p. 90) argues, using only the official language with children, and denying, ignoring and punishing the home language of children in schools may persuade the child of his/her "deficiencies" and his/her parents "disadvantaged status" (see also Tacelosky, 2001). These negative attitudes towards the home language are highly likely to make an impact on language choice in favor of the dominant language leading to language shift, loss, or death. Positive/negative attitudes are considered to be playing a significant role in language maintenance/shift (Baker, 1992, 2006; Errihani, 2008; Oliver, et al., 2002), especially when they are shaped at the early stages of lifespan making them relatively unlikely to change (Garrett, et al., 2003, p. 5). Positive attitudes towards Farsi and negative attitudes towards Azeri can be inferred from the managers' responses. Expression of regret by using the word 'unfortunately' (Excerpt 1) for those children who do not know Farsi by the age of 5-6, when they attend kindergartens, clearly shows that the kindergarten authorities expect Azeri children to know some Farsi by that age. The positive attitudes towards knowing Farsi by children are demonstrated in the last lines. Excerpt 5 [KAA] Fortunately, you can hardly find a child who doesn’t know Farsi at all. It is easy to work with such students. They learn fast. They fully understand Farsi in 2 or 3 months.' Positive attitudes towards and pride and joy in the ability of very young Azeri children to speak Farsi was found in the comment of another kindergarten's authority who was also 66 the head of a committee responsible to decide on the teaching material for each year. Using adverbs such as 'beautifully' and 'perfectly' for those children who speak Farsi at the age of four demonstrates her favorable attitudes towards very young Farsi-speaking children. Excerpt 9 Excerpt 9 [T]11 I was living outside Tabriz a few years ago, and wanted to teach at a school. They didn’t give me the first year class because of my Azeri accent. So that’s why I stress the importance of speaking Farsi without accent in classes. [T]11 I was living outside Tabriz a few years ago, and wanted to teach at a school. They didn’t give me the first year class because of my Azeri accent. So that’s why I stress the importance of speaking Farsi without accent in classes. No digression from the standard official language even in the form of an accent seems to be the de facto policy within the kindergartens. The inferiority of a minority accent in the eyes of dominant groups and its impact on successful job application has been indeed documented (Shuy & Fasold, 1973). Speaking "accent-free" Farsi (see Shohamy, 2006, for myth as an LPP mechanism) is regarded so important that one of the authorities brought up the issue in her answer to the question about the policies in her workplace: Excerpt 10 [KAA] We try to speak accent-free Farsi so that students can speak Farsi without accent in the future. The low esteem and stigma attached to Azeri seems to be evident in classes as well. A teacher reported that some children who come from Azeri-only backgrounds and whose parents teach them Azeri words, say for father and mother, rather than the Farsi equivalents, are mocked by other students when they use those Azeri words in class. Excerpt 11 [T] Those children, under the influence of us here, change their words to Farsi ones. Those children find themselves alone to use those Azeri words. They are mocked by other children so they quit using them. The teacher's comment clearly demonstrates that language shift from Azeri to Farsi takes place in these educational institutes. The messages delivered to children and their parents in and through these institutes are about Azeri's low prestige and low instrumental value. The higher instrumental value of Farsi and its tangible economic and social benefits, on the one hand, and the stigma attached to Azeri, on the other, seems to have made the educational institutions and parents support lowering the age for the introduction of Farsi. 11 Teacher [IQ] Is there a rule or regulation to tell you what language to use in classes as the medium of instruction? Excerpt 6 [KAA] If we speak Azeri with children under 3 and then teach them Farsi, they learn Farsi beautifully. I’ve seen children who can speak Farsi perfectly by the age of four. One possible explanation for the managers' positive attitudes towards Farsi seems to be the higher instrumental value of Farsi compared to Azeri as reflected in the following excerpt: Excerpt 7 Excerpt 7 [KAA] We use Farsi because we are thinking of children’s future. The children should be able to speak Farsi so that they can succeed in their future life. [KAA] We use Farsi because we are thinking of children’s future. The children should be able to speak Farsi so that they can succeed in their future life. Farsi as the official language of the country, the language of administration and education, is regarded a language of a higher value which facilitates upward social and economic, and educational mobility. As a consequence, not only the managers but parents also insist that the medium of instruction should be Farsi in the kindergartens: Excerpt 8 Excerpt 8 [KAA] Usually, the parents ask us to use Farsi in the kindergartens because they see that their children have trouble at schools, specially understanding the meanings of the words, as it is with my own child. [KAA] Usually, the parents ask us to use Farsi in the kindergartens because they see that their children have trouble at schools, specially understanding the meanings of the words, as it is with my own child. Stigma attached to speaking Azeri, and even speaking Farsi with an Azeri "accent," also seems be playing a role in shaping positive attitudes towards Farsi and the promulgation of starting speaking Farsi in the home as soon as when children are three years old. One of the teachers working in the kindergarten refers to this issue: Once I myself was mocked because my Azeri accent. Another teacher takes this issue even further and talks about her experience a few years ago when she was rejected for her prized job, teaching the first grade, because of her speaking Farsi with Azeri accent. 67 11 Teacher Excerpt 9 This necessarily means that Farsi should be taught to young children in the home which is highly likely to result in subtractive bilingualism and language shift from Azeri to Farsi (see Ting, 2003). The gradual lowering of the student age for the introduction of L2 (here Farsi) may suggest parents and authorities' tendency to view L2 as equal to L1 which 11 Teacher 68 might have implication for future actions on the part of parents to speak Farsi at home and stop passing on Azeri to the next generation. Parents' attitudes towards Azeri and Farsi will be investigated in detail in chapter seven. In sum, 'no digression from the standard Farsi' seems to be the first priority in pre- schools in Tabriz, be it in the form of speaking a minority language or speaking it with an "accent." Although there might be no rules available on paper, the authorities know that they should prepare Azeri-speaking children for the future school years in which both the teaching material and the medium of instruction is Farsi. Exploring local policy implementers' discourse has demonstrated they hold positive attitudes towards Farsi whereas they are uncommitted, if not negative, to supporting Azeri. Such attitudes mainly seem to have originated from the lack of explicit supportive policies for Azeri within the education system which has resulted in its lower status and instrumental value in relation to Farsi. In this instance, this relates to how the lower public esteem and instrumental value of Azeri in relation to Farsi leads many teachers and principals to value the acquisition of Farsi over Azeri even before primary schools. In other words, the absence of explicit and deliberate supportive policies for Azeri in the education system has resulted in conversion of ideology as default policy among Azeri teachers and principals (see also Fishman, 2006). As Shannon (1999) argues, with no policies to guide schools and teachers differently, their practice will be based on the prevalent ideology in the society, which according to Fishman (2006), always serves the dominant language and its speakers. Language management in Iran does not consider the linguistic background of minority children, and provides no protective and supportive policies (in this case for Azeris) obliging different institutions to promote minority languages. Pre-schools and kindergartens in Tabriz can be said to function based on their own ideologies. Excerpt 9 Those ideologies, as seen above, ultimately favor Farsi, resulting in authorities and teachers' suggestions that parents should speak the dominant language in the home (see excerpt three above). In other words, lack of Farsi knowledge even before kindergarten is viewed as a problem. The following section discusses how such ideologies and values are Discursively constructed through the education system. 69 performance Discourse planning, as defined in chapter two, refers to modifying or reinforcing a particular world-view, attitudes and/or ideologies conducted through state-run domains and institutions which function as a role model for people to follow (c.f. Lo Bianco, 2010a). It is through these domains and institutions that certain messages with respect to languages and their speakers present in societies are delivered which "shape and fix meanings and social realities" (Mumby & Mease, 2011, p. 285). It is argued that these Discourses "order and naturalize the world in particular ways" (Fairhurst & Putnam, 2004, p. 9). The education system, language-in-education planning (acquisition planning) (Cooper, 1989), has historically been a decisive institution playing a role in Discursive construction of what should be seen as a "problem" requiring a particular treatment (Lo Bianco, 2005, p. 256), as well as establishing and/or maintaining social power among elites, nation-building, and the fate of languages. This is usually carried out through curriculum, textbooks, tests, and teachers which influence the minds of students, however, realized so subtly that makes it very difficult to distinguish it from "the indoctrination of ideologies of powerful groups or organizations in society" (van Dijk, 2008, p. 12). Kindergartens and preschools in Tabriz, as "the microcosm of wider social interaction" (Edwards & Giles, 1984, p. 119), can be said to reflect the values and priorities of the dominant group (re)producing the status and power difference between groups (Cummins, 1988, p. 130), making them the sites where Azeri children first encounter "social policy of the broadest kind (cultural pluralism or assimilation for example)" (Edwards & Giles, 1984, p. 120). Preschools and kindergartens can be consequently viewed as planned ideological institutions where particular attitudes and views about languages (Azeri, Farsi) and groups are molded through messages implicitly advertised by authorities. The absence of Azeri from such a domain may arguably help to "perpetuate linguistic decline" (Brown, 2012, p. 282). Such Discursive practices of the education system (van Dijk, 2008) consequently play a significant part in language maintenance/shift (Jones, 2012). The absence of Azeri and lack of supportive policies for Azeri in the education system on the one hand, and using Farsi as the only medium of instruction on the other, communicates explicit and implicit messages to the public about the desired values and 70 priorities within the education system. performance As a result, Azeri kindergarten students' inability to speak Farsi before primary school, and in most cases kindergarten, in Tabriz is Discursively constructed, and consequently viewed, as a 'problem' which requires preschools and Azeri parents' involvement in speaking Farsi with the children in kindergartens as well as in the home. Such Discourse(s) created within the education system therefore constructs and defines "an ideal parent" as the one who speaks Farsi with the child in the home. As Nakagawa (2000) argues, educational policies can Discursively construct certain ways of viewing "ideal parents" and how they should interact with schools, which is usually directed to minorities and lower income families. And, if such parents do not behave the way they should according to the school, their children may receive less attention from the education system (p. 447). The education system in Iran can be arguably claimed to be playing a major role in creating "external push factors" which "direct internal pull factors" (see May, 2008a) among Azeris to conform to the mainstream ideology favoring Farsi. Because the Discursive practices of the education system are closely intertwined with immediate tangible benefits it offers, receiving education in Farsi is associated with upward social mobility. Such tangible economic benefits provided by education in the official language makes it in turn a more valuable and prestigious language. The Discursive construction of the dominant language as the ideal language for economic growth and success may bring about a lower status and value of other languages absent from the education system (Azeri, in this case). The analysis of kindergarten principals' views about policies confirms Spolsky's assertion about policies being covert and unwritten most of the time. Spolsky (2004) writes that language policies, both at national and institutional level, are not formal and written most of the time, so that the nature of such policies should be derived from the "practices and beliefs." Even when there is a written and formal policy, its effect is neither "guaranteed nor consistent" (Spolsky, 2004, p. 8). Covert institutional discriminations of this type, i.e. "linguicism" (Phillipson, 1988, pp. 341-342), are used to "legitimate, effectuate and reproduce an unequal division of power and resources (both material and non-material) between groups which are defined on the basis of language (on the basis of their mother tongue)" (Skutnabb-Kangas, 1988, p. 13). performance The choice of Farsi as the medium of instruction or the language of initial literacy for all in a multilingual nation-state like Iran necessarily advantages the speakers of that language over the speakers of other languages (see Mangubhai, 2002, p. 492). It is through this process that the dominant 71 group/language presents "an idealized image of itself, stigmatizing the dominated group language, and rationalizing the relationship between the two, always to the advantage of the dominant group/language" (Phillipson, 1988, p. 341). The education sector in Iran also makes what Lo Bianco (2008b, p. 122) calls "systematic and repeatable socialization" possible. That is, schools, when used as a nationing tool, are capable of unifying people via formal education over generations so that "the will of the young" is bent to "the will of the nation" (see May, 2008a, p. 171). Given that such policies have historically resulted in the repression and endangerment of regional languages (Oakes, 2011), viewing educational policies, from script choice (Sebba, 2006) to testing (McNamara, 2008; Menken, 2008; Shohamy, 2006, 2008), solely as educational/linguistic issues are, therefore, considered "simplistic." Educational policies have been usually surrounded by "political debates about national identity, dominance, and control by elites in power, power relationships among politicians and civil servants, questions about social order, and the perceived potential subversiveness of language minorities" (Baker, 2002, p. 237). Education systems are viewed as "ideological state apparatuses" (Althusser, 1969) which represents "a deliberate and planned environment" where powerful messages about "prestigious social norms regarding language usage" (Escandell, 2011, p. 326) are delivered to students from local and national authorities (Brown, 2012, p. 282), molding particular attitudes and mentalities (Gao & Park, 2012; Lopez, 2008, p. 50). Such Discursive practices of the education system (van Dijk, 2008) may in turn play a significant part in language maintenance/shift (Jones, 2012). The analysis of policy as text, discourse, and performance (Discourse planning) presented above suggests that minority languages in Iran have been constitutionally legitimated in addition to Farsi. However, they have not been institutionalized, i.e. they have not been through a process by which they come "to be accepted or taken for granted in wide range of social, cultural, and linguistic domains or contexts, both formal and informal" (May, 2008a, p. 6). performance An inevitable corollary of such policies has been the acceptance of Farsi as a key to educational and financial success lowering the status, symbolic and instrumental value of Azeri. In other words, the lack of supportive policies for the institutionalization of Azeri on the one hand, and the intertwined Discursive and pragmatic functions of the education sectors favoring Farsi, on the other, go hand in hand to promote Farsi as the ideal language for socioeconomic and educational development. It is in such circumstances that the prevalent ideologies are converted, and consequently function, as default policies. Such attitudes, beliefs, and ideologies promulgated directly or 72 indirectly in the education sector may function as input, i.e. "a predisposing factor," resulting in particular output (see Baker, 1992, p. 12; Garrett, et al., 2003, p. 67). As the data above illustrated, the particular output in the case of Azeri has been uncommitted, if not negatively-oriented, kindergarten and preschool principals and teachers. Such uncommitted behavior on the part of the education sector with respect to Azeri education can ultimately play a decisive role in Azeri maintenance. 4.4 Conclusion This chapter has shown how macro language policies are interpreted, mediated and enacted in Tabriz with respect to Azeri. Though legally possible, Azeri is taught in schools neither as a subject nor as a medium of instruction resulting in its lower status and value in the eyes of Azeris. Both kindergarten authorities as the policy implementers and parents and children as the policy recipients consider learning Farsi a necessity since Farsi can provide tangible pragmatic benefits for them. The "sink or swim" (May, 2008a) style of education explicitly and implicitly promotes and promulgates the ever-increasing value and status of Farsi resulting in an inclination in Azeris' attitudes in favor of Farsi and against Azeri. It has been argued here that such policies may work in tandem to bring about subtractive bilingualism and finally language shift. Minority people, if they want to cohabitate with a majority group in a society, have little choice but to learn a language of wider communication besides their own language so that they can access their heritage culture and tradition as well as participate in the social, economic, and political life of their country (e.g. see Tacelosky, 2001, p. 52). As a result, bilingualism becomes a necessity for them, not something they themselves have chosen (Skutnabb-Kangas, 1988, p. 10). It is in such settings that the education system becomes a very strong LPP mechanism to impose language policies and turn ideologies into practices through formal education (Shohamy, 2006, 2008). Labelling schooling as "colonialistic," Hopson (2011) similarly avers that "schools and colonialism are inextricably linked", and "schools do not exist without some larger agenda of colonialism and colonization" (p. 212). That is, schools are used as an instrument to maintain hegemony of the ruling class which is usually done by channeling "youth into status and occupational roles that support the existing power structure" (Hopson, 2011, p. 212). In other words, education systems as "the property of states," carry "the imprimatur and conditioning of political systems" (Lo Bianco, 2008b, p. 113). 73 Despite the fact that education systems can be used for grass roots language activism to "negotiate, demand, and introduce alternative language policies" (Shohamy, 2006, p. 76), educational policies are not resisted and contested in Tabriz. Bestowing different statuses and values upon, and providing tangible benefits through certain languages (Farsi in this case), the education system plays a decisive role in the fate of languages. 4.4 Conclusion As May (2008a, p. 153) writes, "the spread of standardized education, the associated literacy demands of the labour force, and the inevitable and wide spread of interaction required in dealing with state agencies" marginalizes other languages which are not widely used in the public realm to the extent that they become "inconsequential." In other words, the education system plays a major role in shaping a certain type of ecology for languages through its Discursive and pragmatic functions which is highly likely to result in language maintenance within dominant communities, and language shift among other smaller' groups. To showcase the impact of the education system on a written domain and examine language policies in another major domain, the next chapter examines the language policies in the domain of the public sphere (linguistic landscape) in Tabriz. The analysis of public versus private signs demonstrates, firstly, to what extent Azeri is supported by policies in that domain, and secondly, the Azeri people's language choice for writing in public signage. It also provides evidence of the degree to which Azeris' literacy has been influenced by the educational policies discussed in this chapter. 74 74 5.1 Linguistic landscapes Research on the linguistic landscape (LL) has constantly grown within the field of language policy and planning since Landry and Bourhis' (1997) systematic investigation of linguistic landscapes in Canada. The linguistic landscape is defined as "the language of public road signs, advertising billboards, street names, place names, commercial shop signs, and public signs on government buildings" (Landry & Bourhis, 1997, p. 25) as well as graffiti, posters, stickers, etc (Kallen, 2010). Language use in public signage, though very chaotic on the surface, is assumed not to be arbitrary and random, but rather systematic. It is believed that the examination of language use in public can reveal various types of information about the LL in general, the messages it could deliver, economy, policy and multilingualism in a particular context (Shohamy & Gorter, 2009). Because the LL offers a space to defy declared policies (Shohamy & Gorter, 2009), it can reflect the formal and explicit language policies on the one hand, and the de facto practices at the grass roots level on the other. Further, the investigation of the questions of "linguistic landscape by whom?" and "linguistic landscape for whom?" may provide a cogent answer to the question of "linguistic landscape quo vadis?" (Backhaus, 2007, p. 2), pointing to the direction the linguistic landscape in a given area is heading and, very likely, to the fate of languages and groups in that area. Because the presence of minority and endangered languages in the linguistic landscape can contribute to their visibility and vitality (Shohamy & Ghazaleh-Mahajneh, 2012), often by impacting the perceptions and attitudes of people (Crystal, 2000; Dailey, Giles, & Jansma, 2005; Landry & Bourhis, 1997), the investigation of the dynamics of language ideologies in the linguistic landscape can serve as "a canvas where the dominant and marginalized discourses on languages are depicted" (Reershemius, 2011, p. 38). Examining the linguistic landscape, thus, "offers a rich domain of 'real life', authentic language in very dynamic and energetic uses" (Shohamy & Gorter, 2009, p. 3). 5.0 Introduction The previous chapter examined the dynamics of implementation of educational policies in light of language rules and regulations stated in the constitution. I argued that the education system in Tabriz serves Farsi by (re)producing Discourses with respect to higher prestige and value of Farsi compared to Azeri. Because there are no proactive policies to support and promote Azeri within the education system, the public ideology that 'Farsi is necessary for upward socioeconomic mobility' functions as the default policy within kindergartens and preschools. This chapter explores language policies and language use in public signage (known as linguistic landscapes) as another domain surrounding the domain of home. It is argued that linguistic landscapes (LL) have the power to affect the perceptions of groups, and these perceptions constitute a potentially important contributing factor to language maintenance/shift in multilingual settings (cf. Atkinson & Kelly-Holmes, 2006; Reershemius, 2011). In this sense, LL can function as a Discourse planning device influencing people's perceptions of the status of different languages, and even affecting their linguistic behavior. The linguistic landscape, therefore, may influence language use (Cenoz & Gorter, 2006, p. 67; Sayer, 2010). The chapter begins with a definition and discussion of linguistic landscapes in the literature. An analysis of the official governmental signage, i.e. top-down LL, is then presented to explore language policies of the state within this domain. Supplementing an analysis of language use on Azeris' private signs with survey data on literacy in Tabriz, the interplay between educational policies discussed in the previous chapter and language use in the LL is discussed. Linguistic landscapes, as "a space for language battles" (Shohamy, 2006, p. 123), are studied here to shed light on mainly three areas: (a) official and private language ideologies (Jaworski & Thurlow, 2010); (b) the interplay between educational policies and language use in the linguistic landscape (Shiohata, 2012); and (c) the impact of official language policies on individuals at the grass roots level (Cenoz & Gorter, 2009). Comparing and contrasting language use in the official governmental signs and private individual signage can illuminate "how authorities wish to portray a local linguistic situation," and whether, if at all, that particular portrayal is accepted by the general population (Puzey, 2012, p. 141). 75 5.2 Language use in the linguistic landscape Language use in the linguistic landscape is believed to be governed by three main rules, namely "write signs in a language you know," "prefer to write signs in the language or languages that intended readers are assumed to read," and "the symbolic value condition" (Spolsky & Cooper, 1991). Because the linguistic landscape is essentially a written domain, the first rule can be considered necessary as without the language knowledge one could not set up a sign in that language, unless the sign owner asks the sign 76 writer or originator to use that language in the sign. Likewise, the second rule, also known as "presumed reader" is applied when some information is supposed to be communicated to a presumed reader. The major function of this kind of signs can be said to be informational (c.f. Landry & Bourhis, 1997). Accordingly, literacy in a language(s) is a prerequisite condition to create signs. Therefore, the interplay between educational policies providing literacy in a language(s) and language choice/use in the LL cannot be neglected. Shiohata (2012), for instance, shows how the introduction of Wolof into the curriculum has promoted its use in the linguistic landscape in Dakar, Senegal. The linguistic proficiency of the sign writer and the potential sign reader are, thus, considered two influential factors which clearly impact language use in the LL. Although literacy in a language, both on the part of sign writers and sign readers, is a prerequisite condition to create signs, language use in the LL is not always governed by writing and reading abilities of individuals. Rather, language is often used symbolically for "political and sociocultural" purposes (Spolsky & Cooper, 1991, p. 84). The symbolic value condition states that "prefer to write signs in your own language or in a language with which you wish to be identified." This rule is often used to explain why dominant languages such as English, French, or Italian are present in non-English, French, or Italian speaking communities. Piller (2001, 2003), for example, argues that using such languages makes the reader activate values such as success, international orientation, modernity, etc. by just recognizing that the message is in English although they cannot understand the message. In these signs, because English is not directly related to the product or service, its use functions as a secondary discourse suggesting the interrelation between the international markets and knowledge of English (Piller, 2001, p. 157). 5.2 Language use in the linguistic landscape In such circumstances, English functions as an index of social stereotypes of modernity, progress, prestige, superior quality or commercial value, and globalization (Kasanga, 2012; Piller, 2003) rather than a "spatial presence" of an "ethnolinguistic community" (Androutsopoulos, 2014, p. 83). On these signs, the linguistic medium rather than the content of signs convey particular meanings (Sayer, 2010). That is, languages such as English with "connotational value" (Cenoz & Gorter, 2006, p. 70) are used to "symbolize foreign taste and manners" rather than index a community speaking those languages (Scollon & Scollon, 2003, p. 118). In other words, in cases where the symbolic value condition applies, "proclaiming ownership is more important than being understood" (Spolsky & Cooper, 1991, p. 85). The political, economic, and social value of a language can, thus, rule out the first and second rules in these signs. 77 Language is used for informational or symbolic purposes in public signage by both official governmental agencies and individuals. These domains of LL are respectively known as top-down and bottom-up signage (Ben-Rafael, Shohamy, Amara, & Trumper, 2006). The top-down and bottom-up domains of linguistic landscapes and de facto language policies with respect to each domain are discussed below. 5.3 Top-down and bottom-up linguistic landscapes Ben-Rafael, Shohamy, Amara, and Trumper's (2006) define top-down linguistic landscapes as those official signs put up by the government or related institutions, such as signs put up by banks, hospitals, insurance companies, and governmental offices, street names and traffic signs. Language use on top-down signage is likely to be constrained and regulated by language policy and planning directives (Kelly-Holmes & Atkinson, 2007, p. 39) although there is no guarantee that all the official signs meet these policies and directives (Puzey, 2012). By contrast, bottom-up linguistic signs are those placed by individuals and private institutions and organizations, commercial enterprises, etc. (Gorter, 2006, p. 3). The main difference between top-down and bottom-up signage lies in the fact that top-down signs are expected to "reflect a general commitment to the dominant culture" and "overt language policies of a given state" while bottom-up signs are "designed much more freely according to individual strategies," manifesting the "covert language policies of a community, the grass roots cultural identity and aspirations of its members" (Ben-Rafael, et al., 2006, pp. 10, 32). Put differently, language choice or "code preference" (Scollon & Scollon, 2003) in official top-down signage may be determined by power relations, The main difference between top-down and bottom-up signage lies in the fact that top-down signs are expected to "reflect a general commitment to the dominant culture" and "overt language policies of a given state" while bottom-up signs are "designed much more freely according to individual strategies," manifesting the "covert language policies of a community, the grass roots cultural identity and aspirations of its members" (Ben-Rafael, et al., 2006, pp. 10, 32). Put differently, language choice or "code preference" (Scollon & Scollon, 2003) in official top-down signage may be determined by power relations, whereas nonofficial bottom-up signage may make use of languages for different purposes such as solidarity (Backhaus, 2006, pp. 62-63). It is the investigation of the discrepancy (or lack thereof) between language use pattern in top-down signs and that of bottom-up signage that provides useful information about ideologies, policies, and aspirations of policy-makers and communities as well as the vitality and power relations between groups and their languages, especially in bi/multilingual contexts (Landry & Bourhis, 1997). whereas nonofficial bottom-up signage may make use of languages for different purposes such as solidarity (Backhaus, 2006, pp. 62-63). 5.3 Top-down and bottom-up linguistic landscapes It is the investigation of the discrepancy (or lack thereof) between language use pattern in top-down signs and that of bottom-up signage that provides useful information about ideologies, policies, and aspirations of policy-makers and communities as well as the vitality and power relations between groups and their languages, especially in bi/multilingual contexts (Landry & Bourhis, 1997). Below, de facto language policies on the top-down and bottom-up domains of linguistic landscapes in Tabriz are explored. In other words, the city of Tabriz is socio- linguistically characterized by identifying the languages its inhabitants know and the languages they claim to use (Spolsky & Cooper, 1991) as well as the reasons behind the 78 current linguistic landscape, throwing light on the ethnolinguistic situation in Tabriz (see Calvet, 2006, p. 41). 5.4 Top-down signs in Tabriz: An indicator of ideologies and a Discourse planning mechanism 553), is not surprising, because, as discussed in the previous chapter, Article 15 of the Iranian Constitution clearly states that all official writing should be in Farsi. As a result, all the official signage in Tabriz is in Farsi indicating its institutional and political power over English, and Azeri, except for a few signs which seem to be an exception to the rule. The presence and dominance of Farsi in the top-down linguistic landscape of Tabriz is highly likely to indicate explicit and implicit language policies and ideologies. As Coupland (2010, p. 96) writes, ideologies related to language policy and planning leave their imprint on the visible environement, allowing the examination of public signage as "the outward evidence of language policy" (Spolsky, 2004, p. 1). "Public sector signage" is therefore viewed as an "institutional articulation of an explicit and prescribed set of legislative and language planning principles." The dominance of Farsi, as the official language of the state, in the linguistic landscape of Tabriz suggests LL is used as a device to index Tabriz as a community where Farsi is spoken. Given the strong links between LL and linguistic identity (Puzey, 2012, p. 141), Farsi in the linguistic landscape of Tabriz seems to be used to connote national unity and national identity. Because the choices of languages in LL may be also motivated by "stereotypes of readers, of what policy-makers think of them as they construct people as lingua persona" (Shohamy & Gorter, 2009, p. 3), the analysis of top-down linguistic landscape can indicate what kind of community is favored and/or aspired to by policy makers. In other words, "LL underscores the ideology’s orientation to future action" (Sloboda, 2009, p. 175). The dominance of Farsi on top-down signage could be, therefore, construed as the policy makers' desire for a community where Farsi is the dominant language which is widely used. The presence of English in the linguistic landscape of Tabriz, however, cannot be construed as an indication of policy-makers' desire to establish a community where English is spoken as it is in stark contradiction with national integrity. Nor can it be an index of its use as a language of wider communication. The presence of English in the top- down domain of the LL can be said to be of symbolic and informational functions (c.f. Landry & Bourhis, 1997). 5.4 Top-down signs in Tabriz: An indicator of ideologies and a Discourse planning mechanism Linguistic landscapes data were collected on three main streets in the downtown area in Tabriz. These streets were chosen for data collection for two main reasons. Firstly, these streets represented a fair picture of the LL of Tabriz. This decision was reached after mapping out different areas of the city. Secondly, there were several schools located on these streets and neighboring areas, providing potential young recipients of Discourses promulgated on the LL, which can have an impact on the new generation's attitudes and perception (for methodological details, see chapter three). Of the total number of 455 signs, defined here as "written text within a spatially definable frame" (Backhaus, 2006, p. 55), 163 signs were identified as top-down. These signs, as noted above, included signs put up by banks, hospitals, insurance companies, and governmental offices, street names and traffic signs. Based on language(s) used on the signs, top-down signage was categorized into five types of signs: a) Farsi only signs, b) Farsi signs with their English translation below them, c) signs with Farsi and Azeri on them, d) Azeri only signs, and e) signs with Azeri and English on them. Language of sign Number of signs (percentage) Farsi only 88/163 (54%) Farsi & English 72 (44%) Azeri & Farsi 1(0.61%) Azeri only 1(0.61%) Azeri & English 1(0.61%) Table 5.1: The number of signs in different languages in top-down signage in Tabriz Language of sign Number of signs (percentage) Farsi only 88/163 (54%) Farsi & English 72 (44%) Azeri & Farsi 1(0.61%) Azeri only 1(0.61%) Azeri & English 1(0.61%) Table 5.1: The number of signs in different languages in top-down signage in Tabriz Table 5.1: The number of signs in different languages in top-down signage in Tabriz As table 5.1 demonstrates, Farsi only signs, and secondly the Farsi and English bilingual signs, dominated top-down signage in the city of Tabriz, while Azeri signs are minimally represented in the linguistic landscape of the city. The dominance of Farsi, presence of English, and absence of Azeri in the top-down signage in Tabriz are respectively discussed. 79 The dominance of Farsi in the official governmental signs, both in frequency of occurrence and the prominence of display - i.e. "numerical superiority" rather than other features of signs such as font size, color, texture, position of text, etc. (Kasanga, 2012, p. 5.4 Top-down signs in Tabriz: An indicator of ideologies and a Discourse planning mechanism As noted above, English holds the status of the international language which connotes modernity, international orientation, globalization, and success. Symbolically, the presence of English in the official and governmental signage helps the state present the country as a modern, successful, and internationally-oriented nation-state. 80 The use of English in the top-down signage can be also construed as informational. That is, English is used to communicate messages to potential tourists from other countries. As Figure 5.1 shows, English is used to provide the sign reader with directions to get to the airport or Azerbaijan Square. Language use in top-down signage seems to be more informational, asking people to do something, giving directions, etc. countries. As Figure 5.1 shows, English is used to provide the sign reader with directions to get to the airport or Azerbaijan Square. Language use in top-down signage seems to be more informational, asking people to do something, giving directions, etc. Figure 5.1: Farsi-English bilingual sign Given the status and the functions of Farsi and English, their presence in the linguistic landscape of Tabriz seems logical. Farsi provides access to information for Azeris and other Iranians visiting the city, and English plays the same role for potential international visitors. However, what is of paramount importance is the absence of Azeri, as a minority language, from the governmental and official signage which can be discussed from two perspectives, namely how the absence of minority languages from LL reflect the relative power and status of different languages in a sociolinguistic context, and how linguistic landscape contributes to the Discursive construction of the sociolinguistic context (Cenoz & Gorter, 2006). Figure 5.1: Farsi-English bilingual sign Figure 5.1: Farsi-English bilingual sign Figure 5.1: Farsi-English bilingual sign Given the status and the functions of Farsi and English, their presence in the linguistic Given the status and the functions of Farsi and English, their presence in the linguistic landscape of Tabriz seems logical. Farsi provides access to information for Azeris and other Iranians visiting the city, and English plays the same role for potential international visitors. 5.4 Top-down signs in Tabriz: An indicator of ideologies and a Discourse planning mechanism However, what is of paramount importance is the absence of Azeri, as a minority language, from the governmental and official signage which can be discussed from two perspectives, namely how the absence of minority languages from LL reflect the relative power and status of different languages in a sociolinguistic context, and how linguistic landscape contributes to the Discursive construction of the sociolinguistic context (Cenoz & Gorter, 2006). The presence and dominance of Farsi over Azeri in the LL of Tabriz mirrors the power relations between the two languages on the one hand, and the lack of supportive policies to make the use of Azeri in the public space possible, on the other. Farsi, as seen 81 in chapter four, has been institutionalized as a norm for writing and reading through the education system. Besides the institutional power, Farsi, as the official language, also enjoys more political power, and higher status and prestige. The absence of Azeri signs consequently suggests its lower status and weaker position in comparison to Farsi. The presence or absence of minority languages in the public space is believed to influence the status and vitality of languages in a given area (Shohamy & Ghazaleh- Mahajneh, 2012) by impacting the perceptions and attitudes of people (Dailey, et al., 2005; Landry & Bourhis, 1997). Crystal (2000), for example, notes that an endangered community's prestige can be increased through its visibility which can be achieved through the use of the language in public signage, implying the acceptability of the language's presence in the wider community (Crystal, 2000, p. 131). Puzey (2012) also considers the promotion of minority languages in the LL to be "a fundamental step towards greater recognition," which often "inspires debates and frequently becomes topos of language activism and campaigning" serving as "a catalyst for challenging negative stereotypes or old prejudices and for other developments of direct benefit to the language" (p. 143). The presence of minority languages in the linguistic landscape can ultimately help to "redress the balance between majority and minority languages" (Puzey, 2012, p. 144). In this sense, linguistic landscapes can serve as "a visible institutional support" (Dailey, et al., 2005, p. 28) for minority languages. The lower status of Azeri is also reflected in the signs on which Azeri appears. 5.4 Top-down signs in Tabriz: An indicator of ideologies and a Discourse planning mechanism One Azeri-Farsi bilingual sign found in the linguistic landscape of Tabriz was a small (perhaps 5cm by 3cm) sign located on some trees along one of the streets where I was collecting data (see Figure 5.2). The sign was placed by the Regional Municipality One, Department of Parks and Greensward (my own translation). The sentence on the top in the white font is in Azeri, saying 'Trees grow in (come from) Heaven' implying trees are a divine blessing, while all the other words below that are in Farsi. Given that the relative size of signs can point to the relative power of languages (Scollon & Scollon, 2003), such signs where Azeri, if at all, appears can connote a small, weak, and minoritized community. Although signs like Figure 5.2 index a community where people can understand both Azeri and Farsi, they simultaneously symbolize a more powerful community who can speak Farsi and a weaker community who can speak Azeri. Taking the social and physical world that surrounds signs, i.e. Tabriz as a diglossic city, into account, it can be argued that the dominance of Farsi and the absence of Azeri together creates "Discourse(s) in place" (Scollon & Scollon, 2003) signaling the relatively weaker 82 position and status of Azeri. It has been argued that such Discourse(s) can function as a contributing factor in the construction of particular ecology for languages affecting the speakers' attitudes and perceptions (Landry & Bourhis, 1997). It is, therefore, suggested here that the top-down domain of linguistic landscape which is governed by the state be considered as a device for Discourse planning (see chapter two). Figure 5.2: Azeri/Farsi bilingual sign (Type C) The absence of Azeri from the top-down LL in Tabriz, as the data demonstrates, suggests the linguistic landscape work against Azeri's status and prestige which may arguably influence Azeri's vitality in Tabriz. Linguistic landscapes, as Landry and Bourhis (1997) contend, have the power to affect the perceptions of groups, and these perceptions constitute a potentially important contributing factor to language maintenance/shift in multilingual settings (cf. Atkinson & Kelly-Holmes, 2006; Reershemius, 2011). In this sense, LL can influence people's perceptions of the status of different languages, and even affect their linguistic behavior (Cenoz & Gorter, 2006, p. 67; Sayer, 2010). The linguistic landscape, therefore, might have an influence on language use (cf. Hornsby, 2008). 5.4 Top-down signs in Tabriz: An indicator of ideologies and a Discourse planning mechanism In sum, the absence of Azeri from the governmental official signage implies that LL, as Shohamy (2006) maintains, serves as one of the LPP devices at the disposal of the state. Tree code Farsi language: "The Department of parks and greensward" Farsi language: Municipality (Region 1) Azeri language: "Trees grow in (come from) Heaven" Farsi language: Tabriz Municipality Figure 5.2: Azeri/Farsi bilingual sign (Type C) Tree code Farsi language: "The Department of parks and greensward" Farsi language: Municipality (Region 1) Azeri language: "Trees grow in (come from) Heaven" Farsi language: Tabriz Municipality Figure 5.2: Azeri/Farsi bilingual sign (Type C) Tree code Farsi language: "The Department of parks and greensward" Farsi language: Municipality (Region 1) Azeri language: "Trees grow in (come from) Heaven" Farsi language: Tabriz Municipality Farsi language: "The Department of parks and greensward" Farsi language: "The Department of parks and greensward" Figure 5.2: Azeri/Farsi bilingual sign (Type C) Figure 5.2: Azeri/Farsi bilingual sign (Type C) The absence of Azeri from the top-down LL in Tabriz, as the data demonstrates, suggests the linguistic landscape work against Azeri's status and prestige which may arguably influence Azeri's vitality in Tabriz. Linguistic landscapes, as Landry and Bourhis (1997) contend, have the power to affect the perceptions of groups, and these perceptions constitute a potentially important contributing factor to language maintenance/shift in multilingual settings (cf. Atkinson & Kelly-Holmes, 2006; Reershemius, 2011). In this sense, LL can influence people's perceptions of the status of different languages, and even affect their linguistic behavior (Cenoz & Gorter, 2006, p. 67; Sayer, 2010). The linguistic landscape, therefore, might have an influence on language use (cf. Hornsby, 2008). In sum, the absence of Azeri from the governmental official signage implies that LL, as Shohamy (2006) maintains, serves as one of the LPP devices at the disposal of the state. 83 One more contributing factor to the strength of this LPP device, i.e. linguistic landscape, is the situation of "no-policy policy" (Fishman, 2006), i.e. a situation where there are no authoritative supportive policies for minority groups on the one hand, and the dominant group distributes "rewards in accord with its interests, values and goals," on the other, creating a situation working against the minority languages. This terminology is extended to the field of linguistic landscape in this study. 5.4 Top-down signs in Tabriz: An indicator of ideologies and a Discourse planning mechanism The no-policy policy situation with respect to LL in Tabriz can be observed not only with regard to presence or absence of languages on the signs (e.g. the absence of Azeri), but also regarding the type, color, and size of the signs (e.g. using different types, and colors for the same sign pointing to the same street). Figures 5.3 and 5.4 are interesting exemplars of a "no-policy policy" situation in Tabriz which provide useful insights on what inferences can be made based on language choice in the public sphere. Figure 5.3, an Azeri only sign, was a street (bridge) name which was written in Azeri. The presence of Azeri only signs in a diglossic city like Tabriz where a language different from the official language of the country is spoken can be taken as a challenge to national identity. The denial of Farsi in such signs placed by governmental institutions could suggest the denial of Iranian identity. On the other hand, the absence of Farsi on these signs makes it difficult, if not impossible, for travelers from other cities to read those signs. Figures 5.3 and 5.4 are interesting exemplars of a "no-policy policy" situation in Tabriz which provide useful insights on what inferences can be made based on language choice in the public sphere. Figure 5.3, an Azeri only sign, was a street (bridge) name which was written in Azeri. The presence of Azeri only signs in a diglossic city like Tabriz where a language different from the official language of the country is spoken can be taken as a challenge to national identity. The denial of Farsi in such signs placed by governmental institutions could suggest the denial of Iranian identity. On the other hand, the absence of Farsi on these signs makes it difficult, if not impossible, for travelers from other cities to read those signs. Figure 5.3: Azeri only sign (Type D) Figure 5.4 also implies a no-policy policy situation. This Azeri-English bilingual street name is an example of "duplicating multilingual writing" where exactly the same text is provided in two different language (Reh, 2004, p. 8). 5.4 Top-down signs in Tabriz: An indicator of ideologies and a Discourse planning mechanism Acknowledging the societal multilingualism in a given area, this practice may be a reaction to technical and/or Azeri language Figure 5.3: Azeri only sign (Type D) Azeri language Figure 5.3: Azeri only sign (Type D) Azeri language Azeri language Figure 5.3: Azeri only sign (Type D) Figure 5.3: Azeri only sign (Type D) Figure 5.4 also implies a no-policy policy situation. This Azeri-English bilingual street name is an example of "duplicating multilingual writing" where exactly the same text is provided in two different language (Reh, 2004, p. 8). Acknowledging the societal multilingualism in a given area, this practice may be a reaction to technical and/or Figure 5.4 also implies a no-policy policy situation. This Azeri-English bilingual street name is an example of "duplicating multilingual writing" where exactly the same text is provided in two different language (Reh, 2004, p. 8). Acknowledging the societal multilingualism in a given area, this practice may be a reaction to technical and/or 84 affective aspects of communication. As Reh (2004, p. 8) argues, duplicating multilingual writing is practiced in situations where all members of the target community cannot be reached using one language, where the sender intends to address a particular group (e.g. trade, tourism), and for educational purposes. These assumptions, however, do not hold true in this case given that Tabriz is diglossic in Azeri and Farsi rather than Azeri and English. Moreover, if the sign initiator intended to reach tourists, the sign should have been in Farsi and English for national and international tourists to understand the sign, i.e. for "cross-cultural consumption" (Sayer, 2010, p. 146). The current sign ignores the national tourists as they are not typically proficient enough to understand contents in English and Azeri. Reh (2004) further argues that duplicating multilingual writing may be used in reaction to affective aspects of communication, that is, a situation where using only one language ensures comprehension of text. In such a situation, using more than one language in signs serves identity purposes and suggests the equality of linguistic and cultural groups in a given area (Reh, 2004, pp. 8-9). This argument cannot hold true in this example either. Firstly, a portion of the population in Tabriz, though small, is from other cities who do not understand Azeri. 5.4 Top-down signs in Tabriz: An indicator of ideologies and a Discourse planning mechanism An analysis of Azeris' language use in the bottom-up signage in Tabriz is presented here to examine Azeris' linguistic behavior in the linguistic landscape in the light of their literacy rate in Azeri. Such an analysis can establish a link between educational policies discussed in the previous chapter and Azeris' linguistic behavior in the linguistic landscape of Tabriz. 5.5 Bottom-up signs in Tabriz: An indicator of language change English translation Azeri language set), more research should be carried out to investigate where these signs originate from, and what ideologies the sign initiators hold. Figure 5.4: Azeri/English sign (Type E) English translation Azeri language Figure 5.4: Azeri/English sign (Type E) English translation Azeri language English translation Figure 5.4: Azeri/English sign (Type E) Figure 5.4: Azeri/English sign (Type E) The analysis of language policies in the top-down domain of linguistic landscapes in Tabriz demonstrates the state's ideologies and attitudes towards languages. Like the education system where there are no supportive policies for Azeri (as discussed in chapter four), the data analysis above suggests the same situation for Azeri in the top-down LL in Tabriz. Below, the dynamics of language use in the bottom-up LL in Tabriz is discussed. As noted above, because the bottom-up domain of public signage provides a potential space for bottom-up language agency and activism, it can show to what extent, if at all, a minority group uses its ethnic heritage language in writing suggesting people's acceptance of or resistance against language policies of the state. An analysis of Azeris' language use in the bottom-up signage in Tabriz is presented here to examine Azeris' linguistic behavior in the linguistic landscape in the light of their literacy rate in Azeri. Such an analysis can establish a link between educational policies discussed in the previous chapter and Azeris' linguistic behavior in the linguistic landscape of Tabriz. 5.4 Top-down signs in Tabriz: An indicator of ideologies and a Discourse planning mechanism Secondly, although the presence of Azeri on this sign may serve identity purposes and mark the Azeri geographical territory (Landry & Bourhis, 1997), it is highly unlikely to show the equality of linguistic and cultural groups given that there are no English-speaking communities in Tabriz. In addition, if such bilingual signs were to suggest equality of linguistic groups, one would expect them to be in Azeri and Farsi firstly, and secondly, the numerical distribution of such signs over the city should have been nearly equal, unlike the present dominance of Farsi only and Farsi/English signs. Thirdly, the types of such signs, i.e. color, size of the font, and languages used on the sign, should have been constant all over the city. The type and color of signs in Figures 5.3 and 5.4 suggest that they have been erected by the same governmental institution. However, different code preference pattern in these two signs (Figures 5.3 and 5.4) suggests a no- policy policy situation which can arguably work against Azeri's vitality in the long run. The absence of Farsi from the top-down governmental signage further creates Discourse(s) implying Azeri nationalism which seems to be paradoxical. It is paradoxical and surprising in that these signs are put up by governmental intuitions. The denial of Farsi on these signs clearly suggests the denial of Iranian identity posing a challenge to the national integrity of the county. Given the small number of these signs (2 signs in this data 85 set), more research should be carried out to investigate where these signs originate from, and what ideologies the sign initiators hold. Figure 5.4: Azeri/English sign (Type E) The analysis of language policies in the top-down domain of linguistic landscapes in Tabriz demonstrates the state's ideologies and attitudes towards languages. Like the education system where there are no supportive policies for Azeri (as discussed in chapter four), the data analysis above suggests the same situation for Azeri in the top-down LL in Tabriz. Below, the dynamics of language use in the bottom-up LL in Tabriz is discussed. As noted above, because the bottom-up domain of public signage provides a potential space for bottom-up language agency and activism, it can show to what extent, if at all, a minority group uses its ethnic heritage language in writing suggesting people's acceptance of or resistance against language policies of the state. 5.5 Bottom-up signs in Tabriz: An indicator of language change Of the total number of 455 signs, 292 signs were identified as bottom-up. Based on the language(s) used on signs, bottom-up signs including signs on shop windows and any signs put up outside shops by the owner, graffiti, and signs on private houses were 86 categorized into five types of signs; a) Farsi only signs, b) Farsi signs with English equivalent, c) English only signs, d) Arabic only signs and, e) Azeri only signs. Language of signs Number of signs (percentage) Farsi only 234 (80%) Farsi & English 49 (17%) Arabic 1(0.34%) English only 7 (2.31%) Azeri only 1(0.34%) Table 5.2: The number of signs in different languages in bottom-up signage in Tabriz Language of signs Number of signs (percentage) Farsi only 234 (80%) Farsi & English 49 (17%) Arabic 1(0.34%) English only 7 (2.31%) Azeri only 1(0.34%) Table 5.2: The number of signs in different languages in bottom-up signage in Tabriz Table 5.2: The number of signs in different languages in bottom-up signage in Tabriz As table 5.2 shows, Type A, i.e. Farsi only signs, constitute the highest number of signs in bottom-up signage, that is, 234 signs out of the total of 292. Type B, i.e. bilingual signs using Farsi and English, occupied the second-most prominent place. These signs had the name of the shop or business written in Farsi with their English translation or transliteration below or next to the Farsi words. The statistics shown in the table above clearly demonstrates the dominance of Farsi and English and the absence of Azeri on the bottom-up LL in the city of Tabriz. Figure 5.5: Farsi-only bottom-up sign Figure 5.5: Farsi-only bottom-up sign 87 87 The absence of Azeri and the dominance of Farsi in the bottom-up LL in Tabriz can be plausibly explained using the first and second rules proposed by Spolsky and Cooper (1991). As discussed above, two major factors influencing the presence/absence of language in the LL can be sign writers and sign readers' ability to read and write in a language(s) (Spolsky & Cooper, 1991, pp. 81-84). 5.5 Bottom-up signs in Tabriz: An indicator of language change The p value for all the tests were calculated at .00, 88 indicating that there is a significant difference between the abilities in two languages in each pair (see table 5.4). Pair Mean Std. Deviation t df Sig. (2-tailed) Speaking Azeri Speaking Farsi 1.523 2.408 6.544 106 .000 Comprehending Azeri Comprehending Farsi .607 1.692 3.714 106 .000 Reading Azeri Reading Farsi -2.953 3.100 -9.854 106 .000 Writing Azeri Writing Farsi -3.766 3.293 -11.832 106 .000 Table 5.4: Paired samples t-test results Table 5.4: Paired samples t-test results Azeri participants' lower literacy compared to Farsi, in the light of Spolsky and Cooper's (1991), first and second rules, can be arguably seen as one of the reasons for the absence of Azeri and the dominance of Farsi in the linguistic landscape of Tabriz. Azeris seem to be either not able to use Azeri in a written domain such as LL, or assume the reader would not be able to comfortably read the Azeri sign. We could then argue that Farsi is apparently used to guarantee a successful communication between the sign writer/owner and the sign reader. As noted above, literacy in a language is not the only factor influencing language use in the LL. As Shohamy (2006, p. 123) argues, although the primary drive for public signage is the market principle, the political and ideological considerations should be taken into account. In other words, language(s) are not always used in the LL to transmit factual information, but for its "connotational value" (Cenoz & Gorter, 2006). That is, literacy in a language and using signs to transmit information is not the only factor playing a role in language choice in the linguistic landscape, and the symbolic use of language(s) in the LL (Landry & Bourhis, 1997) should be also considered. The presence of English in the signage put up by Azeris (49 signs, 17%) in Tabriz where English is not used as a language of wider communication functions as an index of social stereotypes of modernity, progress, and globalization (Piller, 2003, p. 170) rather than a "spatial presence" of an "ethnolinguistic community" (Androutsopoulos, 2014, p. 83). On these signs, the linguistic medium rather than the content of signs convey particular meanings (Sayer, 2010). That is, languages such as English are used to "symbolize foreign taste and manners" rather than index a community speaking those languages (Scollon & Scollon, 2003, p. 118). 5.5 Bottom-up signs in Tabriz: An indicator of language change To address the impact of Azeris' literacy in Azeri and Farsi on their linguistic behavior in the LL, two questions in an attitude questionnaire administered to parents of young children (see chapter three) asked the respondents to rate their abilities in the four skills of speaking, listening, reading and writing in Azeri and Farsi from 1 to 10, one having almost no ability and 10 having the ability of a native speaker's. These questions were designed to examine the impact of the educational policies to promote the official language on the linguistic proficiency of Azeris in Azeri and Farsi. The data analysis yielded the following means for each skill in Azeri and Farsi. Speaking Comprehension Reading Writing Azeri 9.44 9.64 6.33 5.54 Farsi 7.92 9.03 9.28 9.31 Table 5.3: The mean for Azeris' abilities in four skills in Azeri and Farsi Speaking Comprehension Reading Writing Azeri 9.44 9.64 6.33 5.54 Farsi 7.92 9.03 9.28 9.31 Table 5.3: The mean for Azeris' abilities in four skills in Azeri and Farsi Table 5.3: The mean for Azeris' abilities in four skills in Azeri and Farsi As table 5.3 demonstrate, the participants reported they had higher abilities in speaking and listening comprehension of Azeri than reading and writing skills. In addition, the Azeri participants reported a higher ability in reading and writing Farsi compared to their reading and writing ability in Azeri. The discrepancy between Azeri's ability in reading and writing Azeri and Farsi can be plausibly attributed to the Farsi-only education system in Iran, as discussed in chapter four. It appears that the institutionalization of Farsi has converted Azeri into more of an oral language. The presence of some literacy among Azeris, however, could be attributed to the common writing system between Azeri and Farsi, i.e. Perso-Arabic alphabet, as well as a long history of literacy in Tabriz. More in-depth research needs be conducted to find out where and from what activities literacy among Azeris originates. The results of Paired samples t-tests carried out illustrate that the difference between the means shown in tables 5.3 is highly significant. The tests were run on four pairs, namely, speaking Azeri and Farsi, comprehending Azeri and Farsi, reading Azeri and Farsi, and writing Azeri and Farsi. 5.5 Bottom-up signs in Tabriz: An indicator of language change The "iconic" use of English (Sayer, 2010) on the signage erected by private businesses in Tabriz suggests that Azeris use English 89 symbolically to communicate concepts such as modernity, progress, success, and international orientation although they may not be able to read or write English. A parallel can be plausibly drawn between the symbolic use of English as an internationally prestigious language and Farsi as the nationally prestigious language in Tabriz. Farsi, as the official language, which has been institutionalized in other domains, enjoys a higher prestige and status compared to Azeri. Besides Azeris' lower literacy in Azeri and higher literacy in Farsi, as shown above, the dominance of Farsi in the bottom- up signage can be arguably attributed to the higher prestige, status, and value of Farsi as well (see chapter four). By using Farsi in bottom-up signage, Azeri people seem to achieve two main goals. Firstly, they guarantee a successful communication not only with other Azeris but also potential tourists and visitors from other cities of Iran, given the fact that Farsi functions the lingua franca in Iran. Secondly, by using Farsi, they enjoy its higher prestige and status in their business. Like English which symbolizes internationalization and globalization, Farsi too can be said to connote official recognition, success and progress at the national level. Ben-Rafael, et al. (2006) argue that bottom-up signage may manifest covert policies of a community on the one hand, and cultural identity and aspiration of its members, on the other. The dominance of Farsi and the absence of Azeri in locally- produced signs could accordingly suggest that the educational policies of the state to promote Farsi as an institutionalized 'written' language have been successful in the case of Azeri in Tabriz leading to particular de facto policies (Shohamy, 2006). This could be one of the reasons explaining why Azeris, with a lower literacy in their ethnic language, do not use the language in the macro written domain of the public sphere. This in turn has resulted in the absence of manifestations of cultural identity in the linguistic landscape of the city implicitly suggesting Azeris aspirations to be identified with Farsi and the mainstream Farsi-speaking community. Moreover, given that Azeris typically are not proficient users of English, Azeris seem to be using English in the bottom-up LL primarily because of its symbolic value rather than pragmatic and informational. 5.5 Bottom-up signs in Tabriz: An indicator of language change The sign owner, identifying his/her business if not himself/herself with English and the English speaking community, is trying to attribute more prestige to his/her business through English as a global language, bringing to the mind of the audience qualities such as power, prestige, superior quality or commercial value, modernity, and economic value (Kasanga, 2012, p. 560). The data analysis suggests that English in bottom-up private signs in Tabriz fulfils little or no informational function. 90 Rather, the omnipresence of English in LL in Tabriz can be said to mark the emerging process of globalization (Cenoz & Gorter, 2009, p. 57). 5.6 Discussion As such the linguistic landscape can be seen as a language policy mechanism through which particular attitudes and perceptions about languages are instilled in the society. The absence of Azeri from the bottom-up signs in Tabriz, as noted above, can be arguably attributed to educational policies which have resulted in relatively lower literacy in Azeri. Having a weaker ability in reading and writing Azeri compared to Farsi, Azeris seem to feel more comfortable to read and write in Farsi rather than Azeri. As discussed above, another contributing factor for the absence of Azeri from Azeri people's signs can be Azeri's lower status and prestige. While the absence of Azeri from the private signs can be attributed to literacy and prestige issues, the absence of Azeri from the official signs cannot be related to such issues. Rather, the absence of Azeri from the top-down domain of the LL in Tabriz suggests the state's ideologies and explicit policies. It can be argued that there are no explicit supportive and protective policies to promote and promulgate the use of Azeri in the public space which could have been regarded as a type of institutional support. Symbolically, such an absence (Discursively) constructs a weak and marginalized community who speaks Azeri. Furthermore, such policies in the LL can be taken as an attempt to index Tabriz as a city where Azeri is not spoken. To summarize, given that the meaning, value, and power of language(s) can be derived from their presence in space (Jaworski & Thurlow, 2010, p. 10), the data analysis suggests that the presence of Azeri as the local and ethnic language of Azeri people in Tabriz is neither acknowledged nor supported in the top-down LL of Tabriz. This has led to an absence of "metacultural representation and practice - where "metacultural" refers to cultural practices that communicate cultural difference" (Coupland & Garrett, 2010, p. 14), which can be attributed to the impact of macro official laws and regulations within LL and domains outside LL (e.g. the education system). In the case of English, there seems to be one main similarity and one main difference between using English in the official signage and private LL in Tabriz. 5.6 Discussion Language use in the top-down and bottom-up domains of the linguistic landscape of Tabriz was examined above to explore the language policies in the public sphere as well as illuminate the dynamics of grass-roots language use in a written domain in Tabriz. Top- down governmental linguistic tokens, as noted, are very likely to index the overt policies in a state. These tokens, in this sense, become the markers of power and status of groups and their languages. By contrast, bottom-up, individual and locally produced signage may be a manifestation of covert policies of a community on the one hand, and cultural identity and aspirations of its members, on the other. Together, the linguistic landscape analysis is able to provide a window into the effect of globalization and language contact in multilingual settings (Ben-Rafael, et al., 2006). The findings of this chapter can be discussed under the three sections of the dominance of Farsi, presence of English, and absence of Azeri both in the official and private signs. The analysis of the linguistic landscape data illustrated the dominance of Farsi in both governmental and private signs. The dominance of Farsi in the bottom-up signage suggests two points. Firstly, Farsi has been institutionalized as the written language which is apparently more convenient for Azeris to use for writing. This cannot, however, be separated from Azeris' lower literacy in Azeri. Secondly, Farsi enjoys higher status and prestige giving it an added value. As a result, by using Farsi, Azeri people can guarantee successful communication among themselves and with potential visitors from other cities, as well as being identified with speakers of a language of higher status and prestige. The higher status and prestige of Farsi can be extended to top-down domain of signage as well. Symbolically, the dominance of Farsi in official governmental signs not only reflects its institutional and political power over Azeri, but it also contributes to the (Discursive) institutionalization of the official language of the state. Farsi is unsurprisingly the most commonly-used language because it is the official language, the language of education, official transactions, judiciary, a powerful symbol of nationalism and national identity. Farsi's high visibility, thus, can be an indication of its prominent role at the national level, as well as its status and prestige. As Williams's (2008) notes, top-down signs are "the iconographic representation of nationalist symbols in the landscape" (Williams, 2008, p. 91 26). 5.6 Discussion Both the state and Azeri individuals can be said to try to symbolically represent the country as a modern, international, and successful country which reflects the state and Azeris' awareness of the status, prestige, and the associations attached to English. The main difference, however, is that Azeris mostly make use of English in the bottom-up signage only as an iconic language (e.g. in the form of shop names appearing in English). This means that a large portion of the English usage is to activate values attached to English as a highly prestigious international language rather than transmit factual information. By 92 contrast, English is used in the top-down signage to transmit factual information such as directions, requests, etc. (e.g. in the form of traffic signs). In sum, I have argued that language use in the LL is under the constant influence of two forces, namely, the macro official policies and the people's personal choices, desires, and motivations. These forces are realized through writing in public, which is seen as an influential tool to secure institutional authority because it embodies power and resistance, i.e. a tool which can also be turned and used against the powers (Coulmas, 2009, p. 14). The outcome of the "battle" (Shohamy, 2006) in the linguistic landscape between groups and their languages, therefore, would fall between the two extremes of total assimilation into and resistance against the declared policies stated by the dominant group. It can be argued that in total assimilation, one would find almost no difference in languages used in top-down and bottom-up signage in bilingual contexts. By contrast, in total resistance, the difference in languages used in both types of signage would be noticeable. This comparison may also indicate the vitality of languages within a community. The comparison between the top-down and bottom-up LL make-up of the city suggests that Azeri has lost the linguistic landscape "battle" (Shohamy, 2006), leading to a lack of resistance in the bottom-up LL. The analysis also indicates the low, or around zero, vitality of Azeri in Tabriz as a written language, i.e. it is predominantly used as an aural/oral language. 5.6 Discussion The contributing factors, such as the lack of linguistic proficiency in Azeri and higher symbolic value of Farsi and English, has led to the current LL composition of the city, suggesting a total assimilation of Azeris into the dominant culture in the domain of language use in the public space. composition of the city, suggesting a total assimilation of Azeris into the dominant culture in the domain of language use in the public space. Given that "unequal distribution and prominence of languages in the LL stems from differential levels of vitality and stereotypes attached to each" (Kasanga, 2012, p. 565), the LL in Tabriz can be argued to serve as "a mechanism to affect, manipulate and impose de facto language practices in hidden and covert ways" (Shohamy, 2006, p. 111). As argued in chapter two, such manipulations are carried out Discursively creating a particular ecology for languages. That is, particular language use patterns implicitly convey certain messages to audience with respect to what group(s) and language(s) are valued. Moreover, linguistic landscape in Tabriz can be said to run both ahead of and behind the actual (objective or subjective) ethnolinguistic vitality of Azeri (Coupland & Garrett, 2010). It runs behind the actual ethnolinguistic vitality of Azeri because it depicts a loss of writing and reading domain which has already taken place. It also runs ahead of 93 the actual ethnolinguistic vitality in that it is very likely to be an indication of even a greater loss for Azeris in future. It would be also fair to claim that the LL items in Tabriz are not a faithful representative of the people's linguistic repertoire. That is, Azeri in Tabriz is certainly not an extinct language, although it is hard to find Azeri signs, and English is not actively used for communication, although it is actively present in the LL of the city. The current language use pattern in the LL of Tabriz, thus, suggests the people and institutions make use of the linguistic resources in order to symbolically construct a public sphere they desire and aspire to (Ben-Rafael, et al., 2006; Coupland, 2010). It is this gap between the real and complete linguistic repertoire and what is seen in the public use of languages that indicates the actual role of language policy and its scope within a community (Negro, 2009, p. 216). 5.6 Discussion Given that the number and type of multilingual written texts in a particular area is dependent on factors such as "the number of languages present, language policy, the status of speakers, the self-esteem of speakers, the reader-orientation of text suppliers," the numerical distribution and type of these texts on signs can reflect "the social layering within a community" (Reh, 2004, p. 1). As the numerical distribution of languages on the signs in this study suggests, the social layering in Tabriz can be claimed to be as follows. Farsi and Farsi speaking people have been given the highest priority. English and the English speaking communities are seen as the second most important groups. Azeri and Azeri speaking people seem to be considered to be the least significant group in the city, displaying symbolic power of languages (see Rosendal, 2009). 12 Radio Tabriz also airs Azeri and Farsi programs, and thus, can be regarded as a type of institutional support. It has not been included in this thesis for two main reasons, however. Firstly, a focus-group interview with mothers of young children and interviews with children revealed that Radio Tabriz does not have many listeners. Secondly, television is considered a much stronger medium than radio given the captivating visual nature of the television medium (e.g. see Hult, 2010b, p. 161). Chapter Six: The Media: A Discursive Space Chapter Six: The Media: A Discursive Space 6.0 Introduction The analysis of (de facto) policies in the education system and linguistic landscapes in the previous chapters suggested a lack of protective policies for Azeri, thus contributing to Discursively shaping an undesirable ecology for Azeri. As a corollary, Azeri is not regarded as a default choice for writing. This chapter examines de facto policies in the broadcast media, arguably one of the most important types of institutional support for minority groups (Giles, et al., 1977). It is argued that the relative presence of languages in the media can function as a contributing factor in the formation of positive image of languages and their speakers (Hult, 2010b), raising the status of languages not only in the eyes of speakers of the minority languages, but also in the eyes of the speakers of the majority languages (Bell, 2010; de Bres, 2010). This "prestige factor" is considered very important to children within minority communities because "they hold the key to the minority language’s future" (Howel, 1992, p. 217). In the last fifteen years, the Islamic Republic of Iran Broadcasting (IRIB) has established provincial channels which broadcast programs in regional and minority languages. The airtime for programs in the regional language varies from channel to channel. In line with promoting minority languages, Sahand TV, the provincial channel covering East Azerbaijan, but not limited to East Azerbaijan, was established in 2000. The chapter aims to illuminate the functionality of Sahand TV which can be considered, along with Radio Tabriz12, to be a type of institutional support for Azeri provided by the government. Although the central government has taken a step to promote minority languages by establishing provincial channels, exploring language use patterns on the channel (de facto policies) suggests a different scenario. 5.7 Conclusion This chapter has explored de facto language policies in the linguistic landscape in Tabriz to investigate what type(s) of Discourse(s) are mainstreamed within this domain. The chapter has shown how "language shift of formal and written language is caused or consciously facilitated (i.e., by conquest or other major dislocations of the status quo)" (Fishman, 2006, p. 318). The data analysis has suggested that the exclusion of Azeri as the autochthonous language, and the symbolic use of Farsi and English, i.e. "double symbolism" (Atkinson & Kelly-Holmes, 2006, p. 254) in the LL in Tabriz, has led to what Atkinson and Kelly-Holmes (2006, p. 254) call "a double disenfranchisement of the minoritised variety." This seems to have resulted in Azeris' lack of devotion, and perhaps negative attitudes and perceptions, to using their native ethnic language in the LL which is 94 of great importance for Azeri maintenance. Such a double disenfranchisement of Azeri can endanger its vitality and maintenance in Tabriz in the long term. of great importance for Azeri maintenance. Such a double disenfranchisement of Azeri can endanger its vitality and maintenance in Tabriz in the long term. The next chapter investigates the policies with respect to another important domain, i.e. broadcasting media. The data analysis in this chapter shows the only available TV channel for Azeris is in fact used as a vehicle for Discourse planning, i.e. to disseminate and distil particular attitudes in Tabriz. Examining the impact of satellite channels on Azeris in Tabriz, the chapter explores how the lack of attractive programs on Azeri local channel can bring about unintended outcomes. 95 6.1 Media and language policy and planning Language policy and planning processes are closely integrated within the social, cultural, political and economic ecology (Kaplan & Baldauf, 1997, pp. 310-320; Spolsky, 2004, p. ix; 2009, p. 1). Media outlets, both as "a domain" for language use (Fishman, 1974a; 1991; 2001) and a "social institution" (Lundsten, 1999), are considered to be key 96 components of the ecology (Cormack, 2007b; Hult, 2010b), and key agents in the "political economy of language" (Spitulnik, 1999). The significance of the media comes from their potential capacity not to merely "reflect events," but also "create" them. It is argued that this "creative function" of the media is not "spontaneous," but "planned, planted, and incited" (Bell, 2010; Berg, 1972, p. 256). It is maintained that this creative function of the media is Discursive, i.e. the media Discursively constructs "specific language regimes through, for example, the ways in which languages are represented in public discourse and language problems are defined" (Hult, 2010b, p. 160; Lo Bianco, 2005). In other words, as argued in chapter two, media outlets are engaged in what can be labeled Discourse planning (see chapter two). By giving different values and exposure to different linguistic varieties, the media legitimate the socioeconomic and political dominance of some social groups over the others. The media are accordingly regarded as important sites for "ethnolinguistic representation and the production of language ideologies" (Spitulnik, 1999, p. 149), and "discursive space" serving as "a window into contemporary processes of social change" (Heller, 2010, p. 280). The role of the media in both majority and minority language politics, as a domain in which "language conflicts play out" and in which "the linguistic order of a policy is (re)produced," has been well documented (Hult, 2010b, p. 159). Reviewing research carried out in Japan, Cyprus, and Dutch, Flemish, German-Swiss communities, Hult writes that the media, especially radio and television, function as a language policy agent to elevate or to perpetuate the already-elevated status of languages used on those media. Sung-Yul Park (2010) similarly shows how Korean national television channels "rationalize and naturalize ideologies of linguistic nationalism" by not only reaching their audience in the national language but also guiding and "correcting" the language of ordinary citizens (p. 76). 6.1 Media and language policy and planning The choice of linguistic code in television programming, or generally in mass media, is seen as "the communicative space of the nation-state," which conveys meanings about the power relations between linguistic varieties and the speech communities associated with them (Lin, 2009, p. 311). In such circumstances, as Karam (1974) argues, the mass media serve as "agents of conventionalization" providing people with "models of imitation of prestigious spoken and printed usage" (p. 116). As a consequence, media functions as a contributing factor in the "creation and reproduction of sociolinguistic inequalities" by bestowing preference on the dominant language over the others (Lin, 2009, p. 312). 97 To create and (re)produce sociolinguistic inequalities, the media serving dominant groups often attempt to stigmatize minority languages and their speakers, in extreme cases through what Alia and Bull describe as "imput[ing] filth to ethnic minorities" (Alia & Bull, 2005, p. 15). Emphasizing that media are not just "innocent bystanders and neutral observers," but they are "culpable" (p. 15), Alia and Bull report on various cases in which media were used against minorities. They argue that an overemphasis on drunkenness and crime among Australian aboriginals and Maori people in New Zealand in the 1950s, for example, resulted in the projection of a biased image of those peoples. It is in such circumstances that dominant cultures use the media to present and defend images of themselves as "competent and unblemished" at the expense of ethnic minority groups (p. 85). And it is in such circumstances that "minority people find their voices silenced in many spheres" (Alia & Bull, 2005, p. 73). Alia and Bull (2005) and Browne (2007) argue that the minority media can undo such a silence for minority communities and become a voice for the voiceless minorities, contributing to language maintenance endeavors. Based on the relative success of dominant media outlets in the (re)production and dissemination of language ideologies in their interest, the media are consequently considered important for minority communities (Giles, et al., 1977). Bell (2010), for instance, writes that broadcasting media stand second in importance after education for the vitality of Maori in New Zealand. One of the main roles, perhaps the most important one, attributed to ethnic minority media in language maintenance is the status-building nature of the media (Bell, 2010; de Bres, 2010; Giles, et al., 1977; Ó Laoire, 2000). 6.1 Media and language policy and planning Considered as a major institutional support (Giles, et al., 1977), it is believed that the media can raise the status of languages not only in the eyes of speakers of the minority languages, but also in the eyes of the speakers of the majority languages (Bell, 2010; de Bres, 2010). As Bell (2010) puts it, languages which are not used in prestigious domains such as the media are not regarded as having an instrumental value in the wider society which might indicate to speakers that their language has no status and it is not "worth their while to talk it" (p. 12). In other words, "the role of language in broadcasting is circular," i.e. the use of a particular language in broadcasting is an indication of its high status and it in return enhances it status (Bell, 2010, p. 9). The existence of the media in minority languages is, thus, likely to challenge some minorities' perceptions of the languages as old-fashioned, rural and backward. Associating with new media and technology, a minority language might be seen as a contemporary, living tool (Cunliffe, 2007, p. 134). This "prestige factor" is considered very important for the vitality and maintenance of languages because it can influence the 98 attitudes and perceptions of minority communities, especially children. (Howel, 1992, p. 217). By virtue of the fact that minority people see lives like their own on television materialized in and through their ethnic language, they see an extension of domains in which their language is spoken, making them view their language as prestigious and as useful as other "bigger" languages. That is perhaps why Howel (1992) argues that languages depend on broadcasting, along with parents and teachers' diligent efforts to transmit a language to the next generation, for their continuation and survival. Television and radio, among the mass media, are seen as the most influential vehicles for transmitting languages and national and popular cultures to the next generation (Howel, 1992, p. 217). It is believed that this might affect the language of the home in the long run (Ní Neachtain, 2000; Ó Laoire, 2000), making parents and children more loyal and committed to their ethnic language. Media, particularly broadcasting media, are also regarded as key elements of "empowerment" of a minority language, i.e. "an expression of the degree to which a minority language is invested legally, economically and socially.... 6.1 Media and language policy and planning for an effective struggle against competing majority languages" (Aitchison & Carter, 1997, p. 357). Summarizing the functions of broadcasting media into the five categories "communicative, cultural, economic, status and linguistic," Jones (2007) similarly argues that the media are vital for the well-being of a community. According to Jones, these functions may lead to higher status of that language, its added use and usefulness in the community, a richer linguistic repertoire of the community, stronger collective identity of the community and its economic development (Jones, 2007, pp. 190-191). Power to control agendas in public discussion and access to opinion-forming sources to promote language interests are considered necessary elements of empowerment which can ease intergenerational transmission to a great extent (Aitchison & Carter, 1997). Because the media, especially television, represents a high status domain, the ways in which languages are used in the media can convey the comparative importance of languages in a given society (Cooper, 1989, p. 32). Media, as a strong status-building tool and institutional support for minorities, is, therefore, seen as an instrument for minority empowerment (Downin, 1992, p. 256). The presence of ethnic minority groups and their languages in the media can also benefit those marginalized groups and their languages in the political arena. Because the media makes it possible for some sense of public sphere to develop, it is impossible for a community to develop politically without having access to and being present on the media 99 (Cormack, 2007b). Furthermore, the presence of a minority language in the areas of public discourse, i.e. mass media, prevents its exclusion from being viewed as "natural and inevitable" (Tollefson, 1991, p. 12). The presence of minority groups and their languages on the media may enable them to stand up to other neighboring communities, which can serve as a contributing factor to language maintenance. This is usually achieved by developing "critical mass," a necessary condition to preserve languages, leading to a stronger cultural identity (Alia & Bull, 2005, p. 115). As Hult (2010b) notes, the relative presence or absence of languages in the public sphere can function as a contributing factor in the formation of the image of languages and their speakers within "discourses of nationality" (Hult, 2010b, p. 160). 6.1 Media and language policy and planning Despite the media being used as a mechanism to diffuse and promote language ideologies (Shohamy, 2006; Stuart-Smith, 2006), reliance on the media to support minority languages has been critiqued, leading to skepticism about the benefits of the media for minority groups and their languages. Referring to the impact of the media on encouraging minority communities to shift to dominant languages, Spolsky (2008b, p. 152) expresses doubts about the role of the media in language maintenance or reversing language shift. Posing the question "Can the instrument of defeat be turned into a method of defence?" Spolsky argues that the reliance on the media, television in particular, in language maintenance or reversing language shift seems to come from the influential role of the media in language shift processes across the globe. Similarly, Fishman (2001), labeling the enthusiasm of minority media activists as "fetish," expresses skepticism with respect to the role of the media in reversing language shift. He maintains that lower-scale face-to-face interactions can never be solely seen as a by-product of higher- scale domains and institutions such as the media and education system (Fishman, 1991, p. 4). That is, the assumption of the existence of some sort of a "domino effect" between domains, i.e. "once a (high-status) domain is lost to a superposed language, others are sure to follow," does not hold true (Boyd, 2011, p. 30). These critiques seem to originate from the complexity of the issue of language maintenance on the one hand, and the inability to isolate the influence of broadcasting from other social factors at work (Bell, 2010; Browne, 1996; Moring, 2007), on the other. This has resulted in a paucity of empirical evidence showing the impact of broadcasting, if any, on language maintenance. As a result of the failure to establish such a cause-and- effect relation between media and behavior, Cormack (2007b, p. 63) argues that validating 100 such a relation is likely to be "gross simplification and distortion" (cf. Emmett, 1966; Stuart-Smith, 2006). Although arguments made by Spolsky, Fishman, and Boyd about the impact of the media on language maintenance appear logical, they cannot be necessarily understood as undermining the role of the media in language maintenance and/or reversing language shift processes. Spolsky's question, rather than being a wholesale denial of the role of the media, suggests one important point; that is, the media has served the dominant group as an instrument of defeat. 6.1 Media and language policy and planning However, its service for minority communities as a method of defense remains a topic of debate. This implies that not all the minority media work in favor of ethnic minority groups and their languages. As Riggins argues, mere allocation of time, money and broadcasting space to minorities should not be taken as "spontaneous gestures of goodwill". A state might have "inconsistent policies promoting minority media while simultaneously following policies of containment and repression" (Riggins, 1992a, p. 8). Then, as Heller (2010, p. 278) writes, "it matters what relationship specific media spaces bear to the State, and what kinds of regulation may operate them." The same argument applies to assertions made by Fishman and Boyd. The key words in their discussion are "solely" and "sure" and assuming total dependence on the media to maintain languages. Although Fishman (1991) pointedly stresses that the media can never replace face-to-face communication, intergenerational transmission in particular (see also Ó Laoire, 2000), he implicitly agrees with the importance of the media in the fate of languages by placing the media in the top two levels of the Graded Intergenerational Disruption Scale (GIDS). It is certainly unrealistic to assume an ineluctable cause and effect relation between the media and language behavior. Yet, the part the media play in diffusing linguistic norms and values cannot be neglected in sociolinguistic studies (Stuart- Smith, 2006). The debate between those who believe in the impact of the media on behavior and perceptions (e.g. Korhonen & Lahikainen, 2008; Muto, 2004), and by extension on language maintenance and ethnic identity (Baetens Beardsmore & Van Beeck, 1984; Bell, 2010; Cormack, 2007b; Howel, 1992), and those critiquing the existence of such an effect (e.g. Cormack, 2007b), has made some take a different approach to investigate about the nature of media. These scholars argue that talking about the "effects" of media on behavior might be a mistake and "barking up the wrong tree" (Heller, 2010, p. 279; Riggins, 1992a). Instead, the media is framed as "discursive space" whose regulators, participants and consumers should be identified. And, issues such as "who controls it, what kinds of 101 interest they may have, the way they do it, and what consequences this may have for ranges of speakers who control diverse arrays of linguistic resources" should be investigated (Heller, 2010, p. 278). 6.1 Media and language policy and planning In other words, the media are regarded as sites where language ideologies are constructed, reproduced, contested and modified (Heller, 2010). In this sense, the media can function as space for minorities to voice themselves, challenging the legitimating ideologies which are produced on other media outlets, ultimately contributing to language maintenance endeavors (Alia & Bull, 2005; Browne, 2007). Discourse(s) the media create through manipulating the presence/absence of languages in their programs can have clear implications for status planning and prestige planning for those languages and their speakers. The following sections accordingly examine language choice pattern in relation to various programs broadcast on Sahand TV, indicating the types of Discourse(s) (re)produced and disseminated on this medium. The analysis aims to shed light on underlying ideologies as well as the nature of the medium, showing to what extent, if at all, it is a tool for "cultural preservation" or it contributes to the "assimilation of ethnic minority audiences to the dominant culture" (see also Watson, 1996; Williams, 2007). 13 Sahand TV broadcast 24 hours a day at the moment. However, the programs broadcast after 12 midnight are the reruns of the previous day. 6.2 Representation of languages on Sahand TV During phase one of the data collection between January 2011 and June 2011, During phase one of the data collection between January 2011 and June 2011, Sahand TV programs were recorded for analysis. Like linguistic landscapes data collection (see chapter three and five), the main methodological issue in media research is sampling data from the amount of data available for collection and analysis. As a result, a constructed week was used as the sampling method to get a fair representation of programs (see Hester & Dougall, 2007, for the efficiency of constructed week sampling). Using this method was very important because the type of programs vary on days associated with certain religious and cultural events and ceremonies. The constructed week sampling method involved selecting one day in each week for recording for the period of seven weeks when the constructed week was complete. Each recording took around 12 hours because Sahand TV broadcasting time was between 12 pm and 12 midnight at the time of data collection13. Two main methodological issues arose in collecting this data set, namely categorizing programs based on the language in which they were aired, and how to deal 102 with code-switching, if any. Categorizing programs based on the languages in which they were broadcast was not a difficult undertaking. Programs aired in Arabic were essentially limited to prayers time which included broadcasting verses of Qur'an and praying. No code-switching was observed in these programs. It is worth noting though that the translation of the Qur'anic verses appearing on the screen was all in Farsi. This again reinforces the argument made in the previous chapters that Azeri is not a default choice when it comes to writing. Code-switching was not observed in Farsi-programs either, making their categorization as Farsi programs unchallenging. These programs were either imported from other Farsi channels or produced in Farsi in Tabriz by Azeris themselves for a special audience (see below). Code-switching from Azeri to Farsi is now a common phenomenon (Hawes & Mirvahedi, 2013; Holmes, 2013). Given that Azeri has been overwhelmed by Farsi in different domains and institutions over the last one hundred years (see chapter one), Azeri includes a wide range of Farsi vocabulary, idioms and expressions realized in the form of inter- and intra-sentential code-switching . Azeri programs broadcast on Sahand TV were not an exception. 6.2 Representation of languages on Sahand TV The language of the program Duration in minute Duration in percentage Azeri 2237 min 47.30 % Farsi 2231 min 47.15 % Arabic 263 min 5.55 % Total 4731 min 100 % Table 6.2: The mean for airtime for Arabic, Azeri, and Farsi programs in seven days The language of the program Duration in minute Duration in percentage Azeri 2237 min 47.30 % Farsi 2231 min 47.15 % Arabic 263 min 5.55 % Total 4731 min 100 % Table 6.2: The mean for airtime for Arabic, Azeri, and Farsi programs in seven days Table 6.2: The mean for airtime for Arabic, Azeri, and Farsi programs in seven days Bell (2010) and Georgiou (2010) argue that broadcast media not only mirror the society's norms but also have the potential to lead. Investigation of broadcast media can then yield two types of information, namely what are the norms and values in the society, and what are the policy makers seeking to achieve through media. In other words, the analysis of programs broadcast on a channel can both reflect existing norms and values in the society as well as (re)producing those norms and values. This (re)production of norms and values, as argued in this chapter and chapter two, is conducted by providing role-models for the audience to follow, i.e. Discourse planning. Bell (2010) and Georgiou (2010) argue that broadcast media not only mirror the society's norms but also have the potential to lead. Investigation of broadcast media can then yield two types of information, namely what are the norms and values in the society, and what are the policy makers seeking to achieve through media. In other words, the analysis of programs broadcast on a channel can both reflect existing norms and values in the society as well as (re)producing those norms and values. This (re)production of norms and values, as argued in this chapter and chapter two, is conducted by providing role-models for the audience to follow, i.e. Discourse planning. The data set presented in tables 6.1 and 6.2 above provides some evidence of the current linguistic order, i.e. bilingualism, in Tabriz on the one hand, and the role Sahand TV might play in Discourse planning on the other, i.e. it can be considered a tool to "(re)produce the current linguistic order" (Hult, 2010b, p. 172). 6.2 Representation of languages on Sahand TV Programs were labeled as Azeri in the table above based on the main language in which they were aired. The writing appearing on the screen was again overwhelmingly in Farsi except for names of a few programs. Inter- and intra-sentential code-switching incidents were not taken as an influential factor affecting the analysis. A separate research project is required to explore the degree to which as well as what type of code-switching takes place on Sahand TV. Thursday April 21, 2011 Friday April 29, 2011 Saturday May 7, 2011 Sunday May 15, 2011 Monday May 23, 2011 Tuesday May 31, 2011 Wednesday June 8, 2011 Azeri 40.18 % 288 min 55.94% 391 min 37.27 % 265 min 55.15% 423 min 45.15% 298 min 35.82 % 245 min 55.62% 327 min Farsi 53.55% 384 min 38.91% 272 min 58.09% 413 min 39.90% 306 min 48.63% 321 min 58.33% 399 min 39.28% 231 min Arabic 6.27 % 45 min 5.15% 36 min 4.64% 33 min 4.95% 38 min 6.22% 41 min 5.85% 40 min 5.10% 30 min Table 6.1: Airtime for Arabic, Azeri, and Farsi programs Table 6.1 clearly shows that Azeri and Farsi programs were each allocated the most airtime every day. However, there seems to be no particular pattern. Azeri programs dominated Sahand TV airtime on some days and Farsi programs dominated on others. Arabic programs, by contrast, were allocated the least airtime presumably because Arabic 103 is not a language of wider communication in Tabriz. Given that Arabic, as the language of Islam, is only used for religious purposes, it seems likely that Arabic on Sahand TV has a symbolic function (c.f. Paul, 1999). By and large, as table 6.2 shows, nearly half of the airtime over the constructed week was allocated to each of Azeri and Farsi program categories. 6.2 Representation of languages on Sahand TV Sahand TV's programming can, therefore, be construed as a kind of mirror which reflects policy-making body's potential desire and intention to establish a bilingual society in Tabriz, and perhaps in other Azeri-speaking cities. Given that Azeri is not the language of institutions (at least in the written domain as discussed in previous chapters), Sahand TV's programs can be interpreted as an attempt to depict and potentially perpetuate a diglossic context. It is argued that bilingualism and a diglossic society can be a way to maintain linguistic diversity (Fishman, 1972b; O'Connell, 2007). Sahand TV's attempt to perpetuate such a context for Azeri can be then considered to be a useful institutional support for 104 Azeri. However, the analysis presented above provokes two further questions about Sahand TV, and thus, relying solely on the analysis above would be simplistic and misleading. Considering that there are many Farsi-only (national and international) television channels available in Tabriz, why should Farsi programs be broadcast on Sahand TV? And what might be the reasons and motivations for such programs? Although using Farsi in some programs seems inevitable, such as when a national event is going on in Tabriz which needs to be reported to the whole country, allocating equal amounts of airtime to Azeri and Farsi on Azeris' local channel does not appear to be without intention. The following section discusses the underlying ideologies and the type of Discourse(s) propagated on the medium by exploring "what communities are imagined, in the service of what sets of interests? Who is constructed as a producer of discourse" (Heller, 2010, p. 279). 6.3 Representations of communities on Sahand TV Producing programs in Farsi, particularly for children and teenagers, reinforces the idea that the policy-making body may, consciously or unconsciously (see Lo Bianco, 2005, p. 262), intend to promulgate the Discourse of uniformity by, for instance, encouraging the young generation to learn Farsi as its first language. In addition, movies, documentaries and scientific programs, usually foreign ones dubbed in Farsi, imply the higher status and usefulness of Farsi to the Azeri viewer whereas shows about villages and small towns broadcast in Azeri may deliver the message that Azeri is useless, backward and old-fashioned, and thus is a hindrance for (young) Reflecting on the types of programs associated with the two languages, i.e. Azeri and Farsi, suggests that Sahand TV seems to be engaged in creating a particular Discourse about the two languages and their speakers. Producing programs in Farsi, particularly for children and teenagers, reinforces the idea that the policy-making body may, consciously or unconsciously (see Lo Bianco, 2005, p. 262), intend to promulgate the Discourse of uniformity by, for instance, encouraging the young generation to learn Farsi as its first language. In addition, movies, documentaries and scientific programs, usually foreign ones dubbed in Farsi, imply the higher status and usefulness of Farsi to the Azeri viewer whereas shows about villages and small towns broadcast in Azeri may deliver the message that Azeri is useless, backward and old-fashioned, and thus is a hindrance for (young) Azeris' upward socioeconomic mobility. As a response to Fairclough's questions, it can be arguably claimed that Sahand TV programs in Azeri and Farsi seem to serve Farsi and the Farsi-speaking communities by foregrounding Farsi as the language of science, economy, entertainment, and more importantly, the language of the young generation. The interests behind the formulation and projection of such images of Farsi versus Azeri and the associated communities could be seen to be the state's attempts to encourage uniformity among Azeris. There seems to be interrelationships between the economics and politics of the media and Discourses disseminated on these media outlets on the one hand, and their usefulness for minority groups, on the other. Fairclough (1995, pp. 42-43) argues that the "economics" and "patterns of ownership" influence the type of Discourse (re)produced and propagated on the media. Building upon this argument, Caspi and Elias (2011) distinguish between three types of minority media, namely media-about, media-for, and media-by minority (see table 6.4). 6.3 Representations of communities on Sahand TV The analysis of the data set presented in table 6.3 is based on Fairclough's (1995) argument that media "representations involve particular points of view, values and goals." He writes that in media analysis, representations should be compared and evaluated "in terms of what they include and what they exclude, what they foreground and what they background, where they come from and what factors and interests influence their formulation and projection" (p. 47). Airwaves have been an important site of "contact and contestation," not only between languages but also between identities, and discourses (Garrett, 2007, p. 141). Such social identities (e.g. gender, class, ethnicity, etc.) of the media audience are constructed through the choice of topic, code, register, and style (Spitulnik, 1999). The type of Discourse(s) the provincial channel of Sahand TV seems to be trying to plan, i.e. a diglossic society, can be challenged given the following data on the types of programs and the languages in which they are aired. Comparing and contrasting types of programs broadcast in Azeri and Farsi can accordingly yield insightful results about the type of Discourse which is actually promulgated on Sahand TV. 105 Programs in Azeri Programs in Farsi  programs about the province and its towns, and villages  quiz show  eleven o'clock news in the evening  religious programs  Azeri music  sports  some economic, political, economic forums.  movies, TV series  documentaries and scientific programs  five o'clock news  children and teenagers' programs  Farsi music  sports  commercials Table 6.3: Types of programs broadcast in Azeri and Farsi on Sahand TV Programs in Azeri Programs in Farsi  programs about the province and its towns, and villages  quiz show  eleven o'clock news in the evening  religious programs  Azeri music  sports  some economic, political, economic forums.  movies, TV series  documentaries and scientific programs  five o'clock news  children and teenagers' programs  Farsi music  sports  commercials Table 6.3: Types of programs broadcast in Azeri and Farsi on Sahand TV Table 6.3: Types of programs broadcast in Azeri and Farsi on Sahand TV Reflecting on the types of programs associated with the two languages, i.e. Azeri and Farsi, suggests that Sahand TV seems to be engaged in creating a particular Discourse about the two languages and their speakers. 6.3 Representations of communities on Sahand TV Caspi and Elias (2011, p. 67) suggest twelve criteria under three 106 main headings, initiative and design, functioning, and control. They maintain that a close analysis of these twelve criteria, i.e. initiative, ownership and financing, personnel, management, agenda, attitudes towards country of origin, attitudes towards majority, reference group, minority access and representation, political, economic and public control, can illuminate the true nature of minority media. In other words, such an analysis can throw light on where a minority medium may be located between the two poles of media-for and media-by minority, raising our attention to minority media leaders' motivation and interests and their ability to guarantee "the minority's self-expression and empowerment" (Caspi & Elias, 2011, p. 62). They argue that the most useful and effective media outlets for a minority group would be those initiated, funded and governed 'by' the minority group itself. Media-about Media-for Media-by Medium's language Majority language Minority language (or a combination of both languages) Minority language Medium oriented towards Majority Minority Minority Medium mostly serves Majority Primarily majority, but also minority to some extent Minority Minority representation in the medium Under-representation/ frequently distorted and stereotyped representation Compensative representation Compensative representation Table 6.4: Mainstream media (media-about) versus minority media, taken from Caspi and Elias (2011) In the same vein, some scholars consider it necessary for minority communities to exercise full control over financing, management, and program production of their own media, allowing programs to be designed in response to the ethnic, cultural, and informational needs of the community (Cormack, 2007a; Riggins, 1992b). Moring (2007) similarly argues that a primary condition for minority media to succeed in their mission, e.g. empowerment, which might lead to better language maintenance and/or reversing language shift, is their "institutional completeness." That is, they should be fully developed and able to work independently, as much as possible, from the dominant majority group. Moring (2007) writes that the institutional completeness of a medium is likely to assist with the "functional completeness" of a language, i.e. normalization of its use in the society, which provides a situation where a minority group can live "in" and "through" their language if they choose to do so (Moring, 2007, p. 6.3 Representations of communities on Sahand TV 18).This seems idealistic and difficult to achieve in reality because minority communities are often economically and politically in a lower In the same vein, some scholars consider it necessary for minority communities to exercise full control over financing, management, and program production of their own media, allowing programs to be designed in response to the ethnic, cultural, and informational needs of the community (Cormack, 2007a; Riggins, 1992b). Moring (2007) similarly argues that a primary condition for minority media to succeed in their mission, e.g. empowerment, which might lead to better language maintenance and/or reversing language shift, is their "institutional completeness." That is, they should be fully developed and able to work independently as much as possible from the dominant majority group Moring 107 position, and consequently, in need of help from the dominant group. However, dominant groups may ideally provide adequate latitude and autonomy so that minority communities can have their desired media outlets. Yet, as Guyot (2007) puts it, few countries give full autonomy to their regional and local channels. The Islamic Republic of Iran Broadcasting (IRIB) is a corporation which has been initiated and governed by the central government. The head of the IRIB is appointed directly by the Supreme leader, and the functioning of the IRIB is supervised by a council (consisting of eight members, two representatives appointed by the President, the head of the Judiciary Branch, the Islamic Consultative Assembly, and the Iranian Parliament). Given the absence of private-sector media in Iran, broadcasting media in Iran can be arguably said to be controlled and governed by the central government. Sahand TV along with all the other provincial channels as branches of the IRIB was initiated, and is owned, financed, and controlled by the IRIB. Therefore, factors mentioned by Caspi and Elias such as "initiative, ownership and financing, personnel, management, agenda, attitudes towards the majority, political control, economic control and public control, etc." (Caspi & Elias, 2011) are governed and/or supervised by the government. As a result, Sahand TV can be said to be a channel for minority because the language of the medium is a combination of minority and majority languages (see also Browne, 1996), medium is mostly oriented towards the minority although it primarily serves the majority group, and minority's representation is of compensative nature (see Caspi & Elias, 2011, p. 64). 6.3 Representations of communities on Sahand TV It is therefore not surprising that the Farsi and Farsi-speaking community are held in high regard. The data analysis presented above suggests that Sahand TV suffers from a lack of "institutional completeness" (Moring, 2007) expecting to receive directives from the IRIB headquarter which has led to its current programming. As argued above, it has been suggested that the institutional incompleteness of a medium can result in the functional incompleteness of a minority language. That is, the media can influence minority people's attitudes and perceptions in a way that the people "prefer" dominant language(s) in the long run. The impact of institutional incompleteness of Sahand TV on Azeris is discussed in chapter seven. In sum, Azeri people seem to have little or no say regarding what programs should be produced and broadcast on Sahand TV because Sahand TV is owned, financed, and controlled by the majority group. Sahand TV, as a medium for minority, has the potential to be used as a manipulative tool for assimilationist purposes by the dominant group as it is 108 the dominant group who owns, manages, and controls the medium. By controlling the media agenda, financial issues and budget, personnel recruitment, etc. media-for minority types can be used as a language policy agent in the hands of the powerful dominant group (Caspi & Elias, 2011). This seems to be achieved surreptitiously by giving just enough access to "lull indigenous groups and other minorities into feeling that they had an effective means to make their voices heard" (Browne, 1996, p. 235). Such policies might be referred to as "new assimilationism," because they are disguised as multiculturalism (Riggins, 1992a, p. 9), or be likened to the neutron bomb which destroys the soul of the people but leave them physically intact (Rosemarie Kuptana's speech cited in Alia and Bull (2005)). Utilizing such assimilationist policies through media help the state "to cultivate a loyal and quiescent population at a relatively low cost and thus minimize the need for the expensive application of state power" (Hoddie, 2006 p. 5). 6.4 Conclusion The aim of this chapter was to examine the "politics of mass mediation" (Milani & Johnson, 2010, p. 4) of Sahand TV as a type of institutional support provided by the government. The question was discussed primarily from one perspective, namely the language choice and the type of programs broadcast on Sahand TV in Azeri and Farsi, and how that shapes the medium's Discursive practices. The analysis has shown how Sahand TV's institutional incompleteness is realized in practice, and what types of language attitudes and ideologies are propagated, establishing particular relationships between media content and language choices by using minority and majority language in a particular way. Given that central to the investigation of language use on the media as "discursive space" (Heller, 2010; Milani & Johnson, 2010) is the importance placed on ideology (O’Keeffe, 2011), the analysis of airtime and the type of programs broadcast in Azeri and Farsi has shed light on how Sahand TV contributes to the Discursive formation of particular ecology based on dominant group's ideology, which may have implications for the status of Azeri and Azeri maintenance in Tabriz. The analysis above suggests that the politics and economics of Sahand TV influencing the quantity, quality, and type of programs broadcast in Azeri and Farsi play a principal role in Sahand TV's functionality. Because Sahand TV is owned, financed, and controlled by the majority group (the central government), it broadcasts programs in both Azeri and Farsi serving both majority and minority groups, making it a medium-for minority. As a consequence, Sahand TV seems to be implicitly promulgating an 109 assimilationist Discourse. Doing that, it can be arguably claimed that Sahand TV does not contribute to the formation and/or maintenance of desirable language ecology for Azeri. Rather, the Discourse of assimilation can potentially result in lowering the status of Azeri, accelerating the language shift process provided that it attracts enough audience. Chapters four, five, and six have explored the language policies and discursive and/or linguistic practices in the domains of education, linguistic landscape, and the media. As argued in chapter two, these domains surrounding the domain of home each contribute to the formation of particular language ecology in Tabriz. In the following chapter, Azeri parents and children's behavior and the dynamics of family language policy in the domain of home with respect to Azeri intergenerational transmission are examined. Chapter Seven: Home: A pivotal domain Chapter Seven: Home: A pivotal domain 7.0 Introduction Chapters four, five, and six analyzed language policies within the education system, linguistic landscape and broadcast media respectively. The analyses examined top- down pressures on Azeri in Tabriz, providing evidence of how language policies in macro domains in Tabriz contribute to the formation of an unsupportive ecology for Azeri. This chapter explores grass roots attitudes and behavior to investigate how language choice in the home is influenced by the ecology surrounding home. Both "external push factors," formed in the language ecology, and "internal pull factors" present in the community are said to be involved in language maintenance/shift processes, with the former usually directing the latter (May, 2008a, p. 146). Examining attitudes and practices at micro and grass roots level in relation to language policies at the macro level thus becomes an essential feature of an in-depth investigation (Liddicoat & Baldauf, 2008). The analysis of these data sets can shed light on the extent to which bilingualism in Azeri and Farsi in Tabriz is stable. The chapter begins by defining family language policy and rationalizing the investigation of family language policy in relation to pressures from the ecology surrounding the home. Azeri parents and children's attitudes and behavior with respect to language(s) in the domain of home are then analyzed. Finally, the participants' attitudes towards educational policies and broadcast media are discussed. 6.4 Conclusion Azeris' attitudes and responses towards educational policies, and media are explored to provide possible explanations for current family language policies. Chapter seven demonstrates how and to what extent parents and children's linguistic choice in the home is influenced by external domains. 110 7.1 Family language policy Family language policy, defined as "explicit and overt planning in relation to language use within the home among family members" (King, Fogle, & Logan-Terry, 2008, p. 907), is an important area of investigation in language maintenance studies. It has been persuasively argued that the domain of home is the most important domain for language maintenance because it is the site for establishing "the bedrock of language maintenance" (King, et al., 2008, p. 917), i.e. intergenerational transmission of heritage language, culture and values (Fishman, 1991, 2001). Favorable family language policy towards language(s) can determine, and potentially guarantee, their vitality within a community to a great extent. By contrast, negative attitudes may be a prime cause of intergenerational discontinuity of a language (Baker, 2006). As a result, some have argued that "all meaningful language policy is ultimately played out in the home" (Caldas, 2012, 111 p. 351), and policies can be said to have succeeded if favorable attitudes and perceptions about the use of the dominant language in the home are formed. The case of Irish illustrates the point very well. Irish has been seriously overwhelmed by English for over one hundred years. Despite Irish being designated as the first national language, and the state's constant intervention in all macro domains such as the education sector, the media, public sphere, etc., healthy intergenerational continuity for Irish is still disrupted and children and parents increasingly learn and use English (Spolsky, 2009). In Lo Bianco's (2012a, p. 518) words, "Ireland represents a case of failure and success, conquering all areas of formal legal recognition but marked by relative neglect of domain [sic] normalization." The language contact situation in Ireland indicates that the absence of a favorable family language policy may affect language maintenance efforts. Fishman (1991, 2001) consequently argues that most emphasis should be placed on face-to-face micro-scale interactions in the domain of home and neighborhood, and language policies in macro domains such as education systems, media, etc. should not be taken as a contributing factor to language vitality per se. Such a situation, i.e. attempting to control the language of education, the mass media, workplace, etc. and neglecting the very intimate intergenerational transmission of language, has been likened to "constantly blowing air into a tire that has a puncture" (Fishman, 1991, p. xii). 7.1 Family language policy That is, all language maintenance endeavors which do not lead to favorable family language policy in the home are seen to be in vain. The case of language shift in Ireland and Fishman's argument, however, does not seem to suggest that family language policy in the home and family members' attitudes and practices are not influenced by the outside society. Nor does it seem to necessarily mean that the loss of external macro domains will necessarily result in language shift in the domain of home (Boyd, 2011). Rather, the language shift from Irish to English clearly shows that home is the institution which plays the most significant role in language maintenance, and when a language is not maintained in the home, safeguarding a language in other macro domains may not result in language maintenance. In other words, when stable bilingualism is disrupted in a sense that families favor a dominant language in the home, it is not easy to maintain an ethnic heritage language only through protective policies in the education system, media, etc. Furthermore, the case of Irish illustrates Liddicoat and Baldauf's (2008) point very well; ultimately, it is people at the grass roots level that determine to what extent particular policies have succeeded in achieving their goals. Consequently, examining the dynamics of family language policy in the home in 112 relation to language policies at the macro level seems to be necessary. Such an inquiry can provide a window into language ideologies and attitudes of family members, reflecting attitudes and values at the societal level (King, et al., 2008). Investigating language attitudes and ideologies within the family unit is important because home is a domain where language ideologies are both "formed and enacted," and it is within the family unit that "dominant ideologies intersect and compete with local or individual views on language and parenting" (King, et al., 2008, p. 907). Home can, thus, be seen as a "battlefield" (Calvet, 1998) where ideologies compete for attention, and a site which both "records and reflects" multilingualism and language contacts found in multilingual societies. As a result, the behavior and attitudes of the members of families, i.e. children and parents, can function as a "tiny social barometer" which is sensitive to the pressures outside home (Harrison, 2007, p. 8). 7.1 Family language policy Studying this barometer is believed to shed light on the extent to which family language policies are influenced by external forces, whether such external policies are contested or endorsed, and which language(s) family language policies are supporting. 14 A Cronbach's Alpha for a group of questions above .70 shows acceptable reliability (see chapter three 7.2 Azeri parents' attitudes towards language use in the home An attitude questionnaire administered to 150 Azeri parents of young children (see Appendix One), and 50 face-to-face interviews with children (see Appendix Two) are drawn upon here to investigate family language policy among Azeris. The data from the questionnaires was collected through seven-point Likert Scale questions, and then analyzed quantitatively using SPSS software. The questions revolved around Azeri parents' attitudes towards Azeri and Farsi in the home, their attitudes towards educational policies and the media available to them. The questions were designed based on a focus- group interview I had undertaken with six mothers of young children (see chapter three). Eight questions on the questionnaire addressed Azeri parents' attitudes with respect to Azeri and Farsi use in the home. A factor analysis of those questions yielded two distinct factors. A scaled formed using questions 10, 40, and 41 with the Cronbach's Alpha of 0.66 shows Azeri parents' attitudes towards Azeri in the home. Another scale formed using questions 1, 17, 22, and 30 with the Cronbach's Alpha14 of 0.83 shows Azeri parents' attitudes towards using Farsi in the home. Table 7.1 below lists the mean for the questions addressing attitudes towards Farsi in the home. Given that 3.50 is the midpoint on the agreement/disagreement continuum (1 113 indicating total agreement, and 7 total disagreement), the mean 3.96 indicates that the participants in the study are moderately inclined towards disagreeing with the use of Farsi in the home. They do not have strong views against using Farsi in the home, however. Farsi, as the official language of the state, seems to have gained a foothold in the domain of home in Tabriz. These relatively moderate views about the presence or absence of Farsi in the home among Azeris can be attributed to external influences coming from state-run institutions and domains such as the education system, the media, and so on, which has give Farsi more instrumental value compared to Azeri. indicating total agreement, and 7 total disagreement), the mean 3.96 indicates that the participants in the study are moderately inclined towards disagreeing with the use of Farsi in the home. They do not have strong views against using Farsi in the home, however. in the home. They do not have strong views against using Farsi in the home, however. Farsi, as the official language of the state, seems to have gained a foothold in the domain of home in Tabriz. 7.2 Azeri parents' attitudes towards language use in the home These relatively moderate views about the presence or absence of Farsi in the home among Azeris can be attributed to external influences coming from state-run institutions and domains such as the education system, the media, and so on, which has give Farsi more instrumental value compared to Azeri. N Minimum Maximum Mean Std. Deviation Farsi_at_Home 101 1.00 7.00 3.9624 1.45374 Valid N (listwise) 101 Table 7.1: The mean for attitudes towards Farsi in the home N Minimum Maximum Mean Std. Deviation Farsi_at_Home 101 1.00 7.00 3.9624 1.45374 Valid N (listwise) 101 Table 7.1: The mean for attitudes towards Farsi in the home Table 7.2 below shows Azeri parents' attitudes towards using Azeri in the home. As shown below, Azeri parents participating in the research do not hold strong views about using Azeri in the home. The mean (3.11) shows the respondents reported relatively moderate attitudes towards using Azeri in the home. Given the role of Azeri in Tabriz, i.e. the ethnic language spoken daily in Tabriz, it is surprising that Azeri parents do not express stronger attachment to Azeri. The results of a paired-samples T test between attitudes towards Azeri (M=3.11; SD= 1.40) and attitudes towards Farsi (M= 3.96; SD= 1.45) shows the difference is significant (t (100) = 3.20, p =.02), meaning that Azeris report stronger attitudes towards using Azeri than Farsi in the home. Yet, such relatively weak attitudes with respect to Azeri use and moderately positive attitudes towards Farsi in the home are likely to affect language use and family language policy in the home resulting in language shift from Azeri to Farsi in the long run. N Minimum Maximum Mean Std. Deviation Azeri_at_Home 107 1.00 7.00 3.1184 1.40324 Valid N (listwise) 107 Table 7.2: The mean for "attitudes towards Azeri in the home Table 7.2: The mean for "attitudes towards Azeri in the home The following section explores Azeri children's reported linguistic choice when they speak with their father, mother, and siblings. The data analysis provides more evidence regarding the presence of Farsi in the home among Azeris in Tabriz. 114 7.3 Azeri children's linguistic choices in the home Children's significant role in influencing language practices in the home has been acknowledged in the literature. Because children are sensitive to "the disfavored status of their elders’ language" (Harrison, 2007, p. 8), they may choose the more prestigious and dominant language under the influence of the education sector, the media, and the public sphere (Spolsky, 2009). Influenced by language policies outside home as well as family language policies, children, depending on a variety of considerations, "make decisions, conscious or not, as to the language(s) they want to use at home, with their peers and in the public domain" (McCarty, Romero-Little, Warhol, & Zepeda, 2009; Shohamy, 2006, p. 48). Such linguistic choices among children in the home are highly likely to disrupt the intergenerational transmission of weaker languages. This section explores the dynamics of family language policy among Azeris from the children's perspective. Fifty children were interviewed about what language(s) they use with different members of the family. The reported data collected from the interviews suggests that Azeri is not the only language spoken in the home. Children’s language use at home demonstrates that Azeri is used inside the domain of home as the dominant language between children and their father. Yet, other languages, Farsi being the most dominant one, are also used in the domain of home in Tabriz. 115 Figure 7.1: Language used with the father at home As shown in Figure 7.1, Azeri is the dominant language reported in the home between the child and the father. Thirty nine children (78%) report that they use Azeri to interact with their father. Farsi stands second with seven children speaking Farsi at home with their 39 7 2 1 1 0 5 10 15 20 25 30 35 40 45 Azeri Farsi Azeri & Farsi Azeri & Turkish Azeri, Farsi and Turkish Figure 7.1: Language used with the father at home 39 7 2 1 1 0 5 10 15 20 25 30 35 40 45 Azeri Farsi Azeri & Farsi Azeri & Turkish Azeri, Farsi and Turkish 45 Figure 7.1: Language used with the father at home As shown in Figure 7.1, Azeri is the dominant language reported in the home between the child and the father. Thirty nine children (78%) report that they use Azeri to interact with their father. Farsi stands second with seven children speaking Farsi at home with their 115 father. 7.3 Azeri children's linguistic choices in the home Farsi, as in the case of parents, stands in the second place with 9 children using it. Azeri and Farsi with three children using it, Turkish with one child using it with the sibling, and Farsi and Turkish with one child using it take the next places. The presence of Farsi is obvious in the home. Although the presence of Farsi in the home might not be strong enough to suggest imminent large-scale language shift from Azeri to Farsi, the analysis could indicate incipient stages of disruption of stable bilingualism in Tabriz. 33 11 5 1 0 5 10 15 20 25 30 35 Azeri Farsi Azeri & Farsi Azeri, Farsi, and Turkish 24 25 30 Figure 7.2: Language used with the mother at home 33 11 5 1 0 5 10 15 20 25 30 35 Azeri Farsi Azeri & Farsi Azeri, Farsi, and Turkish Figure 7.2: Language used with the mother at home 33 11 5 1 0 5 10 15 20 25 30 35 Azeri Farsi Azeri & Farsi Azeri, Farsi, and Turkish 35 Figure 7.2: Language used with the mother at home Figure 7.2: Language used with the mother at home Azeri is also reported as the dominant language used between the siblings at home. Twelve children out of 50 were only children. Twenty four out of the 38 remaining children report they use only Azeri while interacting with their siblings. Farsi, as in the case of parents, stands in the second place with 9 children using it. Azeri and Farsi with three children using it, Turkish with one child using it with the sibling, and Farsi and Turkish with one child using it take the next places. The presence of Farsi is obvious in the home. Although the presence of Farsi in the home might not be strong enough to suggest imminent large-scale language shift from Azeri to Farsi, the analysis could indicate incipient stages of disruption of stable bilingualism in Tabriz. 7.3 Azeri children's linguistic choices in the home Only two children report using both Azeri and Farsi within the family unit. One child uses Azeri and Turkish, and one (2%) uses Azeri, Farsi and Turkish. The situation is slightly different when it comes to the female parent, i.e. mother. Thirty three children speak Azeri with their mother at home. Eleven report that they speak Farsi at home when they talk with their mother. Five use both languages and only one child uses the three languages. This slight difference might be due to the impact of gender on language choice (see Figure 7.2). There is a body of research which suggests women tend to choose the dominant language more often than men, and consequently lead in linguistic change (e.g. Aikio, 1992; Cavanaugh, 2006; Gal, 1978; Holmes, 1993; Mukherjee, 2003; Roman, Juhasz, & Miller, 1994; Yu-Hsiu Lee, 2013). Such a choice has been assumed to be because of women's lower social status as well as their sensitivity to the socioeconomic advantages of learning and using the dominant language. The studies argue that language choice and shift does not take place in a vacuum, and thus, cannot be separated from socioeconomic and cultural contextual factors. The strategic choice of a particular language is usually carried out to achieve socioeconomic gains and acquire symbolic capital (Smith-Hefner, 2009). Aikio (1992) argues that "the belief that use of the majority language can free one from the lower status associated with the minority language may provide sufficient motivation for the switch" (p. 44). The fact that Azeri mothers' interaction with their children tends to be in Farsi, as compared to Father-child interaction, could suggest that women attempt to seek more socioeconomic gain through speaking Farsi not only for themselves, but also for their children. Having realized the transition of their children from an Azeri environment of the home into Farsi-only schools, women might choose to speak Farsi in the home to ease their children's integration into the education system and guarantee higher success and socioeconomic mobility for their children. 116 Figure 7.2: Language used with the mother at home Azeri is also reported as the dominant language used between the siblings at home. Twelve children out of 50 were only children. Twenty four out of the 38 remaining children report they use only Azeri while interacting with their siblings. 7.3 Azeri children's linguistic choices in the home 117 Figure 7.3: Language used with siblings at home 24 9 3 1 1 12 0 5 10 15 20 25 30 Azeri Farsi Azeri & Farsi Turkish Farsi & Turkish No Siblings Figure 7.3: Language used with siblings at home 24 9 3 1 1 12 0 5 10 15 20 25 30 Azeri Farsi Azeri & Farsi Turkish Farsi & Turkish No Siblings Figure 7.3: Language used with siblings at home 24 9 3 1 1 12 0 5 10 15 20 25 30 Azeri Farsi Azeri & Farsi Turkish Farsi & Turkish No Siblings 30 30 Figure 7.3: Language used with siblings at home Figure 7.3: Language used with siblings at home 117 Uncommitted attitudes towards using Azeri in the home revealed through questionnaires from parents on the one hand, and the presence of Farsi in the home making children bilingual as young as four, on the other, could suggest that stable bilingualism in Azeri and Farsi is not the norm. Stable bilingualism, i.e. a situation where domains of language use are functionally separate for each language, is viewed as a key factor assisting with maintaining sociolinguistic pattern of a society (Fishman, 1972b). It has been suggested that if the functional differentiation of languages is disrupted, leading to one language losing its specific domain of use, it is likely that another language may displace it or a new type of functional differentiation of the languages may be arrived at. This is said to take place when the salience of the language erodes for the group (Giles & Johnson, 1981). The analysis of the data from the attitude questionnaires and case studies could be taken as an alarming sign for Azeris suggesting the erosion of the salience of Azeri for Azeris. In other words, home as the last and first resort for language maintenance has been infiltrated by Farsi, threatening healthy intergenerational transmission of Azeri. The presence of Farsi in the domain of home can be seen as a threat rather than an opportunity here given that Farsi is of greater instrumental value and has been institutionalized and legitimized in the macro domains of education, media, public sphere, etc. 7.3 Azeri children's linguistic choices in the home Early bilingualism in the home could also threaten Azeri because, as the questionnaire data suggested, the parents' attitudes towards using Azeri in the home do not seem to be strong enough to guarantee healthy intergenerational transmission for Azeri in the long term. 7.4 Case studies Yu (2010) argues that parents' reported language beliefs should not be taken for granted. Rather, language beliefs need to be explored in practice to examine whether they endorse language behavior. Three children were accordingly observed to explore parents and children's (linguistic) behavior and attitudes in the home. Woodside's (2010, p. 1) definition of a case study as "an inquiry that focuses on describing understanding Yu (2010) argues that parents' reported language beliefs should not be taken for granted. Rather, language beliefs need to be explored in practice to examine whether they endorse language behavior. Three children were accordingly observed to explore parents and children's (linguistic) behavior and attitudes in the home. Woodside's (2010, p. 1) definition of a case study as "an inquiry that focuses on describing, understanding, predicting, and/or controlling the individual (i.e., process, animal, person, household, organization group, industry, culture, or nationality)" is applied (see also Duff, 2008; Yu, 2010). The three case studies were carried out to "describe" what a typical Azeri family language policy might be like, to "explain" and "understand in depth" why these parents and children behave as they do, and "predict" what might happen to Azeri in the long term. The case studies were chosen using "a friend of a friend approach" (Milroy, 1980). This approach provided prior familiarity and helped establish better rapport with the children. 118 The data analysis below suggests to what extent findings from attitude questionnaire are consistent with actual practices in the home, while illuminating the influence of these macro domains on families at the grass roots level. The data analysis below suggests to what extent findings from attitude questionnaire are consistent with actual practices in the home, while illuminating the influence of these macro domains on families at the grass roots level. The children observed in this study are given the pseudonyms, Susan, Anna, and Mary. Each case was observed during three days for approximately 2 hours per session (see chapter three). Each case highlights particular pressure from the ecology outside the home on language attitudes and behavior of family members, namely children and their parents. Case One: Susan The first child observed, called "Susan" for the purpose of this study, is a four- year-old girl (born in Tabriz to Azeri parents) who comes from a middle-class Azeri family. Both her parents hold BAs and work outside the home. Despite the fact that the parents' first language is Azeri, because of their level of education, near-native knowledge of Farsi can be assumed for them. According to ethnographic interviews I undertook with her parents, they have spoken Farsi with her since her birth in the hope that she will not have problems at school later on. All relatives, except for her grandparents who cannot speak Farsi, have been requested by the parents to speak Farsi with Susan. Susan's case clearly illustrates how particular educational policies of states can create and impose particular Discourses, i.e. certain ways of seeing the world (Gee, 2011), on people. Susan's parents' attitudes towards Farsi suggest that they see lack of Farsi knowledge before primary school as a problem. In other words, low or lack of proficiency in Farsi among Azeri children has been Discursively constructed as a 'problem,' which seems to have resulted in viewing learning Farsi as a necessity at an early age in Susan's family. As I argued in chapter four, the implementation of Farsi-only educational policies in Tabriz indoctrinates certain language ideologies. Susan's parents' decision to speak Farsi with her reflects their concern about their child's future at school, and perhaps more generally about her upward socioeconomic mobility. As Spolsky (2011) points out, this is a common trend among minority communities. Parents often introduce the language of school to the domain of home before children begin their formal education in order to "ease their children's integration into school" (Spolsky, 2011, p. 153). However, because both Susan’s parents go to work every day, the child is left with her grandmother. As a result, Susan spends around eleven hours with her grandmother 119 (from 7am to 6 pm) everyday15. Susan's exposure to Azeri from her grandmother and to Farsi from her parents and relatives has resulted in the development of features found in bilingual individuals, e.g. code-switching, at the age of four. Code-switching or code-mixing, i.e. "an alternation of languages within the same discourse or speech act," is seen as a major process that individuals face when they simultaneously learn two or more languages (Grim, 2008, p. 189). 15 The observation took place at the grandmother's home. 16 Please note that Azeri and Persian both use Perso-Arabic alphabet, and are written from right to left. Azeri words and their English equivalents are underlined here. 15 The observation took place at the grandmother's home. 16 15 The observation took place at the grandmother's home. 16 Please note that Azeri and Persian both use Perso-Arabic alphabet, and are written from right to left. Azeri 15 The observation took place at the grandmother's home. 16 l h A i d i b h A bi l h b d i f i h l f A i p g 16 Please note that Azeri and Persian both use Perso-Arabic alphabet, and are written from right to left. Azeri words and their English equivalents are underlined here. Case One: Susan Both intersentential and intrasentential instances of code-switching were observed in Susan's daily speech. It was observed that Susan switched to Farsi when she was addressed in Farsi, and she answered in Azeri when people around her spoke to her in Azeri. It is argued that children's code- switching is similar to that of adults, both being influenced by social, topical, situational, and psychological factors (Bentahila & Davies, 1995; Byers-Heinlein, 2013; Gort, 2012; Grim, 2008). Susan seems to posses this capacity enabling her to switch appropriately to Farsi and Azeri based on different situations. Intrasentential code-switching was also observed. Susan made use of vocabulary and grammatical structures from both languages in her sentences. 1. .رو نشونت بدم 16گاریشقا میخوام show you the ant I want I WANT TO SHOW YOU THE ANT. 15 The observation took place at the grandmother's home. 16 1. .رو نشونت بدم 16گاریشقا میخوام show you the ant I want I WANT TO SHOW YOU THE ANT. 2. کردی میرم به the garden to I'm going I'M GOING TO THE GARDEN. 3. میخوام سو I want water I WANT WATER. 4. پیسدی دختر بی ادب is bad impolite girl THE IMPOLITE GIRL IS BAD. 5. میباره قار Is falling snow SNOW IS FALLING. show you the ant I want 2. کردی میرم به the garden to I'm going I'M GOING TO THE GARDEN. 3. میخوام سو I want water I WANT WATER. 120 These instances of code-switching illustrate Susan's tendency to use and integrate different elements of Azeri and Farsi. Moreover, she seems to be proficient in listening and comprehending Azeri and Farsi. This can be seen in her reactions while watching Farsi programs when she sometimes comments on a scene. For example, in a cartoon where a little girl was annoying her friends, Susan suddenly said in Farsi: "حاال گرگه میاد میخوردش", "Now the wolf will come and eat her!" In another (rare) case, when she was watching a cartoon with her grandmother, the grandmother made a comment on the cartoon which suggested she had not understood the Farsi. Susan translated the character’s words into Azeri and corrected her grandmother. Stuart-Smith (2006, p. 143) argues that television, despite the ongoing debates about its impact on behavior (see chapter six), may be a contributory factor in language change for "certain individuals under specific circumstances" if they show a vicarious experience with television. Susan's experience with television seems to be vicarious in the sense that she "interacts" with characters she sees in the cartoons. The circumstances for her seem to be suitable as she regularly receives and learns Farsi input from her parents. It could be arguably said that Susan's linguistic repertoire is constantly influenced by television. Given that she views exclusively Farsi channels, her linguistic abilities in Farsi could be predicted to improve as she grows up. Susan's case shows that her parents' positive attitudes towards Farsi and its role in their child's future have resulted in her exposure to Farsi since she was born. Such positive attitudes are likely to be formed by expectations that macro domains and institutions, or by what Fishman's (2006) calls "the reward system," create and foster in the society. As discussed in chapter four, education is delivered exclusively in Farsi in Iran. One side- effect of such policies for Azeri families seems to be that Azeri parents become concerned about their children's low proficiency in Farsi, and consequently, their social and economic upward mobility. Such an external push factor could encourage Azeri families to set family language policies which favor Farsi over Azeri in the home. 1. .رو نشونت بدم 16گاریشقا میخوام show you the ant I want I WANT TO SHOW YOU THE ANT. 2. کردی میرم به the garden to I'm going I'M GOING TO THE GARDEN. 3. میخوام سو I want water I WANT WATER. 4. پیسدی دختر بی ادب is bad impolite girl THE IMPOLITE GIRL IS BAD. 5. میباره قار Is falling snow SNOW IS FALLING. As Pakir (2003) argues about her findings, "the desired outcome of a centralized education system was achieved through the population's pragmatic choices" (p. 276). Susan's parents' decision to speak Farsi with her in the home reflects this issue. 121 Case Two: Anna The second child, "Anna," can be compared and contrasted with Susan. Anna is also four. However, Anna's mother is a homemaker. She holds an associate degree. Although her mother sometimes speaks Farsi with her, the majority of interactions are in Azeri. She sometimes reads Farsi material to Anna, and tells her stories in Farsi. Unlike Susan whose parents were both Azeri-speakers, Anna's father is originally from Tehran. He is a university student and self-employed. Although his first language is Farsi, he knows and uses Azeri in the home. Anna's father speaks Azeri with her and she answers in Azeri. Azeri seems to be particularly used to express feelings and emotions, such as reprimanding the child. In informal conversations I had with the mother, I realized that she spoke Azeri because she believed Azeri was hard and children could not learn it if they did not speak it in the home. The mother also noted that she herself did not know Azeri stories or songs. She also maintained that Farsi could be learned through exposure to Farsi on television. As a result, Anna was exposed to Farsi channels on a regular basis. The Discourse of 'learning Farsi at an early age being a necessity' for children seems to have influenced Anna's parents to a lesser degree compared to Susan's parents. In spite of the fact that both parents have decided to speak Azeri with Anna, she is also able to speak Farsi. Her proficiency in Farsi seems to come from mainly two sources, i.e. The Discourse of 'learning Farsi at an early age being a necessity' for children seems to have influenced Anna's parents to a lesser degree compared to Susan's parents. In spite of the fact that both parents have decided to speak Azeri with Anna, she is also able to speak Farsi. Her proficiency in Farsi seems to come from mainly two sources, i.e. television and her grandfather. In contrast to Susan's grandmother who was the main source for Susan's linguistic competence in Azeri, Anna's grandfather and perhaps other relatives from Tehran seem to be a reason for Anna's ability to speak Farsi. 1. .رو نشونت بدم 16گاریشقا میخوام show you the ant I want I WANT TO SHOW YOU THE ANT. 2. کردی میرم به the garden to I'm going I'M GOING TO THE GARDEN. 3. میخوام سو I want water I WANT WATER. 4. پیسدی دختر بی ادب is bad impolite girl THE IMPOLITE GIRL IS BAD. 5. میباره قار Is falling snow SNOW IS FALLING. As reported by the mother, they did not want Anna to learn and speak Farsi as another home language. However, because her grandfather is from Tehran and speaks Farsi with Anna whenever they meet at a party or on the phone, she started learning Farsi from him as well as from television and cartoons as well. This has resulted in Anna's competence to use both languages at the age of four. Like Susan, code-switching was also noted in Anna's daily speech. At a party, depending on her interlocutor, situational code-switching was observed in a sense that if a child spoke Farsi with her, she answered back in Farsi successfully. And, if she was addressed in Azeri, she was competent to converse in Azeri. As Gort (2012) argues, research findings show that young bilinguals use their developing languages appropriately with different interlocutors to achieve a wide variety of stylistic purposes and situational 122 demands. This phenomenon was also observed when Anna used Farsi when she wanted to ask for something or when she sought her parents' affection. These case studies highlight the role of grandparents and intermarriages in language maintenance/shift (see Sofu, 2009). Susan and Anna's cases show how grandparents can play a role in children's linguistic repertoire formation. Without her grandparents' presence, Susan would have been put into a kindergarten while her parents are out. As a result, she would have been exposed to even more Farsi given that the medium of instruction in kindergartens is Farsi (see chapter 4). By contrast, intermarriage between Anna's mother, who is Azeri, and her father, who is a Farsi-speaker, resulting in the communication between Anna and her grandfather has encouraged her to learn Farsi at a very young age although her parents may not want her to learn and use Farsi at that age. Susan's case also illustrates another important point. Susan's grandparents' low proficiency in Farsi compared to her own proficiency in Farsi at a very young age also suggests a change in linguistic behavior of Azeris. A comparison between Susan's grandparents' low or lack of proficiency in Farsi (monolingualism in Azeri) and the younger generation's early bilingualism in Azeri and Farsi indicates a linguistic change in Tabriz. This shift in linguistic behavior in the current generation could suggest that language attrition might occur in the long run as Azeri is increasingly overwhelmed in different domains (see chapter four, five, and six). 1. .رو نشونت بدم 16گاریشقا میخوام show you the ant I want I WANT TO SHOW YOU THE ANT. 2. کردی میرم به the garden to I'm going I'M GOING TO THE GARDEN. 3. میخوام سو I want water I WANT WATER. 4. پیسدی دختر بی ادب is bad impolite girl THE IMPOLITE GIRL IS BAD. 5. میباره قار Is falling snow SNOW IS FALLING. In other words, Mary's case indicates how Farsi from the ecology outside the home formed by language policies in the macro domains can enter home. Whether or not parents speak Farsi with their children, children learn Farsi when, and most of the time before, they enter pre-schools. The case studies explored here as well as the Farsi-only educational policies discussed in chapter four suggest that by the age of seven when children start primary school, most children typically become competent speakers of Farsi. Cases like Mary's clearly demonstrate how Farsi-only education can influence the linguistic repertoire of Azeri children leading to the loss of items of lexicon and idioms. Such a loss of lexicon and idioms can be taken as a marker of language shift from Azeri to Farsi although it seems to be at an incipient stage at the moment (Schmid, 2011). Media and the education system seem to play a part in the formation of Mary's linguistic repertoire. Children repeating sentences they hear on different shows on television and/or in classroom can be interpreted as a sign of the impact of broadcasting media and the education system on children. Webb (2011), for example, argues that television is a "valuable source for language learning" as it provides "authentic aural input." He further writes that television viewing can result in incidental learning of linguistic features. Given that Mary has been exposed to Azeri from her parents since birth, her Farsi sentences and code-switching can be taken as the influence of the media and/or education system on her in the domain of home. In other words, Mary's case indicates how Farsi from the ecology outside the home formed by language policies in the macro domains can enter home. Whether or not parents speak Farsi with their children, The brief, but yet informative, case studies presented above aim to illuminate the dynamics of family language policy in Tabriz. Despite the differences observed, there is a common thread in all the cases explored. That is, Azeri and Farsi are present in the domain of home in Tabriz. The presence of Farsi in the home seems to be a result of either parents' conscious decision-making, primarily affected by pragmatic considerations, or the pressures which have been created and imposed through macro domains such as the education system, media, public sphere, etc. Language dynamics, as Edwards (2010, p. 1. .رو نشونت بدم 16گاریشقا میخوام show you the ant I want I WANT TO SHOW YOU THE ANT. 2. کردی میرم به the garden to I'm going I'M GOING TO THE GARDEN. 3. میخوام سو I want water I WANT WATER. 4. پیسدی دختر بی ادب is bad impolite girl THE IMPOLITE GIRL IS BAD. 5. میباره قار Is falling snow SNOW IS FALLING. Case Three: Mary The third child, here called "Mary," is six. Mary's mother is a homemaker. She has finished high school and did not continue her studies at university level. She speaks Azeri with her daughter most of the time. Mary's father is a bank clerk. He holds a BA degree. He also speaks Azeri with his daughter. The majority of oral communication in the family is conducted in Azeri. However, reading and writing, if any, is done in Farsi. There is a big difference between Mary and the previous cases discussed above. Because Mary is six years old and she is expected to begin primary school the following year, she attends preschool (kindergarten) where the main language is Farsi, as she reported. This means that she is regularly exposed to Farsi every day. She also goes to an English class. She is exposed to television programs around 2 hours a day. She exclusively watches Farsi programs on television. Although the main language in the home is Azeri, Mary uses Farsi words in her Azeri sentences. The Farsi equivalent for the basic lexical item "apple," for example, 123 rather than the Azeri word, seems to be her first choice as she used it several times. She sometimes uses full Farsi sentences as well. For example, when she went to answer the door, she usedکیه؟ in Farsi, meaning "who is it?" and would on occasions repeat some Farsi sentences from cartoons or movies. Having been exposed to both Azeri and Farsi, she can hold conversations in both languages quite well. Media and the education system seem to play a part in the formation of Mary's linguistic repertoire. Children repeating sentences they hear on different shows on television and/or in classroom can be interpreted as a sign of the impact of broadcasting media and the education system on children. Webb (2011), for example, argues that television is a "valuable source for language learning" as it provides "authentic aural input." He further writes that television viewing can result in incidental learning of linguistic features. Given that Mary has been exposed to Azeri from her parents since birth, her Farsi sentences and code-switching can be taken as the influence of the media and/or education system on her in the domain of home. 1. .رو نشونت بدم 16گاریشقا میخوام show you the ant I want I WANT TO SHOW YOU THE ANT. 2. کردی میرم به the garden to I'm going I'M GOING TO THE GARDEN. 3. میخوام سو I want water I WANT WATER. 4. پیسدی دختر بی ادب is bad impolite girl THE IMPOLITE GIRL IS BAD. 5. میباره قار Is falling snow SNOW IS FALLING. 40) notes, reflect "pragmatic desires for social mobility and an improved standard of living." In other words, the reasons why some languages are not transmitted to the next generation are related to "assessments of the likely utility of competing varieties" (Edwards, 2010, p. 40). 124 As argued in chapter two, the implementation of language policies in these domains create and disseminate particular ways of seeing languages and their value and usefulness. The findings from these brief case studies suggest that a desire for educational and As argued in chapter two, the implementation of language policies in these domains create and disseminate particular ways of seeing languages and their value and usefulness. The findings from these brief case studies suggest that a desire for educational and socioeconomic mobility is associated with learning and knowing Farsi. Farsi either finds its way into the domain of home through the media and/or intermarriages, etc., or parents under outside pressures decide to speak Farsi in the home. There is a possibility that, in line with Grin and Korth's (2005) argument about the presence of English in Switzerland, the presence of Farsi in the home turns into the "first" language of the home, and "first" might mean "main," and that "main" will mean "only" in the long run. It is also likely that the excessive presence of Farsi in the social and educational domains may override Azeri's domains, and functions, undermining its vitality. The disruption of stable bilingualism can ultimately lead to language shift and language loss (Fishman, 1972b). The two sets of data, data from the attitude questionnaire and case studies suggest two important points about the dynamics of family language policies. Firstly, Farsi has entered the ecology of the home domain in many families in Tabriz through one means or another. Secondly, the presence of Farsi in the home seems to be associated with pressures from the ecology surrounding the home, particularly concerns that the education system create and expect families to take measures. To shed more light on the link between the domain of home and the education system as well as the media, Azeri parents' attitude regarding the educational policies and broadcast media are explored in the next section. This can provide more evidence for current family language policies among the participating Azeris. 7.5 Azeris' attitudes towards educational policies Chapter four examined the educational policies in pre-schools in Tabriz as well as the three relevant Articles in the Constitution. As mentioned in chapter four, Farsi-only educational policies along with a higher perceived instrumental value of Farsi in the country, and the absence of Azeri in the education sector are the three main factors which seem to have led to kindergartens' authorities and teachers' positive attitudes towards Farsi, and uncommitted attitudes and behavior with respect to Azeri education. As a result, preschool authorities make sure that Azeri children know Farsi before they enter primary schools. They also suggest parents speak Farsi with children at home after the age of three. This section examines the attitudes of policy consumers, i.e. Azeris in general, with respect to possible future Azeri education in schools in Tabriz both as a subject and a 125 medium of instruction. The analysis of the attitudes of policy consumers can (indirectly) show to what extent, if at all, current educational policies are endorsed or contested by Azeris. Ten of 41 questions in the attitude questionnaire described above were designed to investigate Azeri parents' attitudes towards teaching Azeri as a subject as well as using Azeri as a medium of instruction in schools in Tabriz. A factor analysis showed that two questions, namely questions 12 and 35 (see Appendix 1), had a negative correlation with other questions. They were consequently removed from the final analysis. The Chronbach's Alpha for the remaining eight questions was calculated at 0.90 showing the high reliability of these questions. The eight questions addressing Azeri education formed a scale which was labeled as 'attitudes towards Azeri education.' A number between one and seven, one being Strongly Agree and seven being Strongly Disagree, respectively, shows to what extent the participants agree/disagree with the presence of Azeri in schools. The primary analysis of the data revealed that Azeri parents' attitudes about the idea of introducing Azeri into the school system in Tabriz were neither strongly positive nor negative. The mean 3.47 (see table 7.3) suggests that Azeri parents seem to be irresolute with respect to having Azeri in the education system. Given the position and value of Azeri and Farsi for Azeri people, Azeri parents participating in the research seem not to be able to make a firm decision as to whether to support Azeri education or not. N Mean Std. 7.5 Azeris' attitudes towards educational policies Deviation Skewness Kurtosis Statistic Statistic Statistic Statistic Std. Error Statistic Std. Error School_Attitudes MEAN 110 3.4750 .56585 .520 .230 .665 .457 Valid N (listwise) 110 Table 7.3: Attitudes towards Azeri education N Mean Std. Deviation Skewness Kurtosis Statistic Statistic Statistic Statistic Std. Error Statistic Std. Error School_Attitudes MEAN 110 3.4750 .56585 .520 .230 .665 .457 Valid N (listwise) 110 Table 7.3: Attitudes towards Azeri education Table 7.3: Attitudes towards Azeri education The analysis of some of the questions separately, however, sheds more light on the issue, revealing latent attitudes. Examining two sets of questions addressing the presence of Azeri as a subject and a medium of instruction in schools suggests that the respondents seem to support the presence of Azeri in the education system as a subject much more strongly than Azeri as a medium of instruction. Questions number two and twenty three addressed the issue of Azeri as a subject in the education system: 126 2) I wish there were schools which had language courses on Azeri. Answers 1 2 3 4 5 6 7 Frequency 39 26 19 10 4 5 4 Valid percent 36.4 24.3 17.8 9.3 3.7 4.7 3.7 Table 7.4: Results for question number 2 23) I would send my child to a school where there was a course on Azeri. 23) I would send my child to a school where there was a course on Azeri. Answers 1 2 3 4 5 6 7 Frequency 25 11 14 30 9 11 6 Valid percent 23.6 10.4 13.2 28.3 8.5 10.7 5.7 Table 7.5: Results for question number 23 s the tables 7.4 and 7.5 demonstrate, answers one, two and three equivalent to Strongly Agree, Agree, and Somewhat Agree, score higher in total than questions five, six and seven indicating that the majority of the participants agree with having Azeri as a course in schools. It is worth noting, however, that, as comparing Table 7.4 and 7.5 results demonstrates, not all the 78% of the people agreeing with having a course on Azeri may send their children to such schools. Table 7.5 clearly shows that only 46% reported they would send their children to schools where there was a course on Azeri. Moreover, 28% checked the midpoint, suggesting ambivalence towards the issue of having schools where Azeri were taught as a subject. 7.5 Azeris' attitudes towards educational policies It clearly shows that eighty six percent of the participants agreed that 'it would be great if Azeri people could read and write.' The analysis of the questions addressing Azeri education as a subject and as a medium of instruction, respectively, indicates that Azeri parents participating in the research support having a course on Azeri more strongly than having Azeri as the medium of instruction. One explanation for such attitudes may be the parents' concern about their children's future. We could assume that the respondents seem to believe that studying all subjects in Azeri could lower their children's proficiency in Farsi which could in return slows down the upward social and economic mobility. On the flipside, the parents are positive about being able to read and write in Azeri although, as discussed above, they may not send their children to Azeri-medium schools. The table 7.8 illustrates the presence of such attitudes among Azeris. It clearly shows that eighty six percent of the participants agreed that 'it would be great if Azeri people could read and write.' 29) It would be great if Azeri people could read and write Azeri. would be great if Azeri people could read and write Azeri. Answers 1 2 3 4 5 6 7 Frequency 43 35 15 9 2 1 2 Valid percent 40.2 32.7 14.0 8.4 1.9 .9 1.9 Table 7.8: Results for question number 29 zeri parents' preference for using Farsi as a medium of instruction, and potentially havin Azeri parents' preference for using Farsi as a medium of instruction, and potentially having Azeri as a course, may also come from naive and wishful thinking that Azeri, as their mother tongue, will be spoken forever. A question was designed to explore what Azeri parents think about the future of Azeri in Tabriz. They were asked to answer how long they predict Azeri would be spoken in Tabriz, and why. They were asked to choose one of the following: 50-100 years, 100-200 years, more than 200 years, and forever. Eighty four percent of the parents believed that Azeri would be used in Tabriz 'forever.' The other participants (16%) believed that Azeri would disappear sooner or later. Two main reasons stated by the participants to support their ideas were its status as a mother tongue and language of tradition. 7.5 Azeris' attitudes towards educational policies By contrast, the analysis of question 18 which addresses the issue of using Azeri as a medium of instruction in schools indicates that fifty percent of parents would ideally like to have Azeri as a medium of instruction in schools. 18) I wish there were schools where all subjects were taught in Azeri. Answers 1 2 3 4 5 6 7 Frequency 24 14 16 17 13 16 6 Valid percent 22.6 13.2 15.1 16.0 12.3 15.1 5.7 Table 7.6: Results for question number 18 The table 7.6 shows that nearly half of the participating parents wish there were school The table 7.6 shows that nearly half of the participating parents wish there were schools where the children could study all subjects in Azeri. They wish Azeri was able to be used as a medium of instruction. However, investigation of the behavioral aspect of attitudes (see chapter three), data from question 33 in particular, suggests that the parents' wish is not likely to ever materialize as their responses indicate they would still be unwilling to send their children to Azeri-medium schools. As the table 7.7 shows, nearly sixty percent of the participants disagree with sending their children to Azeri-medium schools. 127 33) I would send my child to a school where they taught all subjects in Azeri. 33) I would send my child to a school where they taught all subjects in Azeri. Answers 1 2 3 4 5 6 7 Frequency 17 5 5 12 15 39 13 Valid percent 16.0 4.7 4.7 11.3 14.2 36.8 12.3 Table 7.7: Results for question number 33 The analysis of the questions addressing Azeri education as a subject and as a medium of instruction, respectively, indicates that Azeri parents participating in the research support having a course on Azeri more strongly than having Azeri as the medium of instruction. One explanation for such attitudes may be the parents' concern about their children's future. We could assume that the respondents seem to believe that studying all subjects in Azeri could lower their children's proficiency in Farsi which could in return slows down the upward social and economic mobility. On the flipside, the parents are positive about being able to read and write in Azeri although, as discussed above, they may not send their children to Azeri-medium schools. The table 7.8 illustrates the presence of such attitudes among Azeris. 7.5 Azeris' attitudes towards educational policies They stated that because Azeri is their mother tongue, it will be passed on to the next generation 'in any case,' apparently without being aware of the likely influence of their own attitudes and behavior. Sallabank's (2012) research into processes of 128 language loss shows that speakers do not necessarily realize that if they do not speak a language with their children, the children will not learn it. It is perhaps why Calvet (1998) criticizes using the term 'mother tongue' as it implies that the language children learn is the language they "inherit" from their mother, and in some cases from their father. The term connotes and assumes an automatic maintenance of mother tongues. The majority of Azeri parents taking part in the research seem to take it for granted that Azeri, as a 'mother tongue', will be spoken in the city for ever (see also Spolsky, 2004, p. 5). By contrast, the second group believing in Azeri language attrition and potential death (16%) mentioned reasons such as the impact of the education system and the media resulting in lexical and structural attrition. They stated that Azeri was losing many items of its lexicon as well as idioms and structures to Farsi because it was overwhelmed in the domains of education and media (see chapters four and six). In sum, the analysis of parents' attitudes regarding educational policies suggests that most endorse the current policy, i.e. having Farsi as the only medium of instruction. Although the majority of those responding would ideally like to be able to read and write in Azeri, they do not seem to be interested in having Azeri as a medium of instruction in the education system. As discussed in chapter six, Sahand TV, established, owned, and funded by the government for Azeris, broadcasts programs in Azeri and Farsi. I argued that Sahand TV’s institutional incompleteness is highly likely to result in the functional incompleteness of Azeri in Tabriz; i.e. Azeris may be influenced by Discourses disseminated through this channel. The case studies presented earlier in the chapter showed that children nearly exclusively watch the Farsi channel. The following section examines Azeris' attitudes and behavior with respect to the broadcasting media. 7.6 Azeri children's attitudes and behavior towards television channels I presented data about Sahand TV, the local channel for Azeris in Tabriz, in the previous chapter. As I argued, the analysis of the type of programs broadcast on Sahand TV, and examining the language in which such programs are broadcast, demonstrated two points. Firstly, Azeri and Farsi enjoy an equal share of airtime on this channel. Secondly, Farsi shows broadcast on Sahand TV, as demonstrated in chapter six, are aimed at Azeri children and youth, implying the promulgation of a particular Discourse which produces learning and using Farsi. 129 Liddicoat and Baldauf (2008) argue that policies do not succeed in achieving their goals without people's engagement. The analysis of data about grass-roots attitudes and behavior thus seems worthwhile. This section examines Azeris' reported attitudes and behavior with respect to the broadcasting media available to them. Azeri children's attitudes and behavior are explored here first because children are active users of television and able to learn, e.g. vocabulary, from television (Kirkorian, Wartella, & Anderson, 2008; Kunkel, 1998; Madsen, 1973). As explored in the case studies, their attitudes and behavior with respect to viewing television may consequently have an impact on language maintenance or shift (e.g. Hourigan, 2007). One part of the questions in the 50 interviews with children were specifically designed to learn about Azeri children's attitudes towards different languages in which programs were aired on different channels, and the possible reasons for such attitudes (see Appendix Two). One question designed to explore the young Azeri children's attitudes towards programs broadcast in different languages was which cartoon they would choose if they had the option to choose that cartoon in Azeri, Farsi or Turkish. Turkish was included in the question as I found out during the pilot study that Azeri families also watch Turkish satellite channels broadcast from Turkey (see chapter three for details). Moreover, some of the children reported they knew how to speak Turkish when they were asked how many languages they knew. This question was designed to indirectly examine the children's attitudes towards languages. As the data below shows, such attitudes towards the three languages present on the media tend to favor dominant languages such as Farsi and Turkish. 7.6 Azeri children's attitudes and behavior towards television channels 130 Figure 7.4: Azeri children’s preference to watch cartoons in different languages 4% F/T 16% Turkish 16% Azeri 64% Farsi Cartoon in Farsi/Turkish Cartoon inTurkish Cartoon in Azeri Cartoon in Farsi Figure 7.4: Azeri children’s preference to watch cartoons in different languages As Figure 7.4 clearly demonstrates, 64% of Azeri children reported they preferred to watch cartoons in Farsi, the official language of the country whereas only 16% of the participants preferred Azeri and Turkish. Interestingly, two of the children favored either Farsi or Turkish cartoons, but not Azeri ones, suggesting that Azeri has no place in the media for two of the children interviewed in this study 4% F/T 16% Turkish 16% Azeri 64% Farsi Cartoon in Farsi/Turkish Cartoon inTurkish Cartoon in Azeri Cartoon in Farsi Figure 7.4: Azeri children’s preference to watch cartoons in different languages As Figure 7.4 clearly demonstrates, 64% of Azeri children reported they preferred to watch cartoons in Farsi, the official language of the country whereas only 16% of the participants preferred Azeri and Turkish. Interestingly, two of the children favored either Farsi or Turkish cartoons, but not Azeri ones, suggesting that Azeri has no place in the media for two of the children interviewed in this study. As Figure 7.4 clearly demonstrates, 64% of Azeri children reported they preferred to watch cartoons in Farsi, the official language of the country whereas only 16% of the participants preferred Azeri and Turkish. Interestingly, two of the children favored either Farsi or Turkish cartoons, but not Azeri ones, suggesting that Azeri has no place in the media for two of the children interviewed in this study. A direct question was also posed to children with respect to their actual behavior towards viewing Sahand TV. Figure 7.5 clearly shows that only nine out of 50 children reported they watched Sahand TV on a regular basis. 21 of the children said they did not watch Sahand TV at all, and 20 of the participants reported they watched Sahand TV only a little. It is worth noting that watching Sahand TV would not necessarily mean watching Azeri programs and becoming exposed to Azeri given that nearly half of the programs on Sahand TV are aired in Farsi, especially children's programs (as mentioned in chapter six). 7.6 Azeri children's attitudes and behavior towards television channels 131 Figure 7.5: The number of children watching Sahand TV (in number) To corroborate the data from children's interview, parents were also asked to report on their children's behavior towards different channels in the questionnaire and to state which channel(s) their child watched most often. The following data (see Figure 7.6) provide useful information with respect to the children's reported behavior according to the participating parents. In other words, the data below show how their positive attitudes towards Farsi and Turkish, as suggested above in the case of cartoon preference, were actually realized in their viewing behavior as reported by their parents. The parents' responses correspond to the children's reports on their attitudes and behavior with respect to different channels and their respective language. 0 5 10 15 20 25 Watch Sahand TV Do not watch Sahand TV Watch Sahand TV a little 9 T h e n u m b e r o f c h i l d r e n 21 The num ber of chil dren watc hing Saha nd TV (in num ber) 20 The num ber of chil dren watc hing Saha nd TV (in num ber) Figure 7.5: The number of children watching Sahand TV (in number) 0 5 10 15 20 25 Watch Sahand TV Do not watch Sahand TV Watch Sahand TV a little 9 T h e n u m b 21 The num ber of chil dren watc hing Saha nd TV (in num ber) 20 The num ber of chil dren watc hing Saha nd TV (in num ber) Figure 7.5: The number of children watching Sahand TV (in number) m b ber) g g ( ) To corroborate the data from children's interview, parents were also asked to report on their children's behavior towards different channels in the questionnaire and to state which channel(s) their child watched most often. The following data (see Figure 7.6) provide useful information with respect to the children's reported behavior according to the participating parents. In other words, the data below show how their positive attitudes towards Farsi and Turkish, as suggested above in the case of cartoon preference, were actually realized in their viewing behavior as reported by their parents. The parents' responses correspond to the children's reports on their attitudes and behavior with respect to different channels and their respective language. 7.6 Azeri children's attitudes and behavior towards television channels Such positive attitudes are highly likely to be formed under the influence of the education system, media, linguistic landscapes and other macro domains, or in Spolsky's (2009) words by the ecology outside home. There is a real danger, as Bell (2010) argues, that exposure on a daily basis to media may implicitly signal the death or survival of a language as full vital language. Three points can be made about the data presented above. Firstly, as Browne (2007) notes, many minority media offer few or no engaging programs for children and teenagers to encourage them to use the language. Sahand TV does not seem to be an exception. Farsi seems to have become the main and default language choice for watching television for Azeri children. This attitude is very likely due to the children's exposure to high-quality Farsi programs on television, and perhaps the impact of the Farsi-only education sector. The children interviewed mentioned various reasons for their lower interest in Sahand TV and Azeri programs, and positive attitudes towards Farsi and Farsi programs. They mentioned reasons such as a weaker ability to understand Azeri, being more accustomed to Farsi, liking the way Farsi is spoken, wanting to learn more Farsi, Farsi being a beautiful language, Farsi showing a higher class of the speaker, and so on, to justify their preference for Farsi. Such positive attitudes are highly likely to be formed under the influence of the education system, media, linguistic landscapes and other macro domains, or in Spolsky's (2009) words by the ecology outside home. There is a real danger, as Bell (2010) argues, that exposure on a daily basis to media may implicitly signal the death or survival of a language as full vital language. Three points can be made about the data presented above. Firstly, as Browne (2007) notes, many minority media offer few or no engaging programs for children and teenagers to encourage them to use the language. Sahand TV does not seem to be an exception. Farsi seems to have become the main and default language choice for watching television for Azeri children. This attitude is very likely due to the children's exposure to high-quality Farsi programs on television, and perhaps the impact of the Farsi-only education sector. Secondly, the presence of Turkish in the children's repertoire and their positive attitudes towards Turkish are of great interest. 7.6 Azeri children's attitudes and behavior towards television channels b e r o f c h i l d r e n To corroborate the data from children's interview, parents were also asked to report on their children's behavior towards different channels in the questionnaire and to state which channel(s) their child watched most often. The following data (see Figure 7.6) provide useful information with respect to the children's reported behavior according to the participating parents. In other words, the data below show how their positive attitudes towards Farsi and Turkish, as suggested above in the case of cartoon preference, were actually realized in their viewing behavior as reported by their parents. The parents' responses correspond to the children's reports on their attitudes and behavior with respect to different channels and their respective language. e r o f c h i l d r e n Figure 7.6: Channels children watch most often (in percentage) 50.9 35.8 11.3 1.9 National channels Turkish satellite channels Farsi satellite channels Sahand TV w a t c h i n g S a h a n d T V Figure 7.6: Channels children watch most often (in percentage) 50.9 35.8 11.3 1.9 National channels Turkish satellite channels Farsi satellite channels Sahand TV w a t c h i n g S a h a n d T V Figure 7.6: Channels children watch most often (in percentage) T 132 Three points can be made about the data presented above. Firstly, as Browne (2007) notes, many minority media offer few or no engaging programs for children and teenagers to encourage them to use the language. Sahand TV does not seem to be an exception. Farsi seems to have become the main and default language choice for watching television for Azeri children. This attitude is very likely due to the children's exposure to high-quality Farsi programs on television, and perhaps the impact of the Farsi-only education sector. The children interviewed mentioned various reasons for their lower interest in Sahand TV and Azeri programs, and positive attitudes towards Farsi and Farsi programs. They mentioned reasons such as a weaker ability to understand Azeri, being more accustomed to Farsi, liking the way Farsi is spoken, wanting to learn more Farsi, Farsi being a beautiful language, Farsi showing a higher class of the speaker, and so on, to justify their preference for Farsi. 7.6 Azeri children's attitudes and behavior towards television channels The main reason mentioned by children for their preference for Turkish channels was the better quality of programs, in this case the children's programs, broadcast on Turkish satellite channels. The children found Turkish channels' shows far more appealing that those of Sahand TV. Labelling some of the Iranian and Sahand TV's children's programs as "silly," a seven-year-old boy, for instance, enthusiastically stated that those programs compared to Turkish shows are meaningless and not worth their while to watch. Other children mentioned reasons such as having always watched Turkish programs, being used to Turkish programs, and knowing Turkish better for their preference for Turkish shows. The third point about the data presented above is the low level of positive attitudes towards Azeri among Azeri children. Producing high-quality programs, Farsi and Turkish shows have replaced Azeri ones. This appears to be one of the reasons for children's weaker preference for Azeri and Azeri cartoons. The young children participating in the interviews also mentioned that Azeri is a hard language in their opinion and that they cannot understand standard Azeri (used on the news at 11:00 pm). Having a weaker preference for the Azeri channel also seems to be the case for Azeri people, in general. The data below show the same phenomenon for parents as well. Azeri parents' attitudes and 133 behavior regarding television viewing is investigated. Interestingly, the analysis corroborates the data collected from children although the participating children and parents were not necessarily related. 17 A Cronbach's Alpha for a group of questions lower than .60 shows low (unacceptable) reliability. 7.7 Azeri parents' attitudes and behavior towards television channels As concluded in chapter six, Sahand TV as one of the main institutional supports seems to be only a symbolic gesture by the government for showing their support and care for Azeri. This can be said to be a characteristic of all minority channels which are "for" minorities rather than "by" minorities (Caspi & Elias, 2011). The analyses presented on the educational policies, broadcast media and linguistic landscapes in Tabriz, in chapter four, five, and six, respectively, as well as the Azeris' responses regarding language policies on those domains clearly demonstrate that Azeri does not enjoy much institutional support from the central government. Azeri is not 49.1 33 13.2 4.7 0 10 20 30 40 50 60 National channels Turkish satellite channels Farsi satellite channels Sahand TV Figure 7.7: Channels parents watch most often (in percentage) 49.1 33 13.2 4.7 0 10 20 30 40 50 60 National channels Turkish satellite channels Farsi satellite channels Sahand TV Figure 7.7: Channels parents watch most often (in percentage) 49.1 33 13.2 4.7 0 10 20 30 40 50 60 National channels Turkish satellite channels Farsi satellite channels Sahand TV 60 Figure 7.7: Channels parents watch most often (in percentage) Considering the data above, it can be arguably claimed that Sahand TV has lost the battle to its Farsi and Turkish competitors in attracting viewers. In other words, Azeris do not demonstrate a preference for Sahand TV because it is not "institutionally complete and qualitatively competitive" (Moring, 2007, p. 29). A low level of institutional completeness, i.e. the inability to produce high-quality and attractive programs for people, especially children and teenagers in the minority language (see Cheval, 1992, p. 193), may lead to negative results increasing "the tendency towards a complementary use of media in the minority language, lowering the level of functional completeness of these media" (Moring, 2007, p. 26). As concluded in chapter six, Sahand TV as one of the main institutional supports seems to be only a symbolic gesture by the government for showing their support and care for Azeri. This can be said to be a characteristic of all minority channels which are "for" minorities rather than "by" minorities (Caspi & Elias, 2011). 7.7 Azeri parents' attitudes and behavior towards television channels To investigate the parents' attitudes towards Sahand TV and Farsi channels, six Likert scale questions were designed. A factor analysis demonstrated two distinct underlying factors, labelled as 'attitudes towards Sahand TV,' and 'attitudes towards Farsi channels.' To increase the reliability index (Cronbach's Alpha) from .428 to .67217, question number 28 because of a negative correlation was removed from the analysis. The means (M) for attitudes towards Sahand TV and Farsi channels were respectively calculated at 3.97 and 3.64, on a Likert scale of 1 to 7. The data show Azeri parents' relatively negative attitudes towards Sahand TV compared to Farsi channels. A paired-samples T test was conducted to compare the attitudes towards Sahand TV and Farsi channels, to determine if the difference between the two factors is significant. There was a significant difference in the scores for attitudes towards Sahand TV (M=3.97, SD=1.57) and attitudes towards Farsi channels (M=3.64, SD=.79), conditions; t (103) = 2.17, p = .032. This provides evidence that Azeri parents hold negative attitudes towards Sahand TV compared with Farsi channels. In other words, they prefer Farsi channels to Sahand TV. Figure 7.7 also provides more evidence reinforcing the view that the participants do not watch Sahand TV as much as they watch other national channels broadcast in Farsi and/or Turkish satellite channels. 49.1 % of the participants reported that they most often watched national Farsi channels. Turkish shows formed the second commonly-watched programs among Azeris in Tabriz with 33% of the participants watching those programs. 13.2% of the respondents reported they watch Farsi satellite channels which are broadcast from countries outside Iran. Only 4.7% of the parents said they watched Sahand TV. 134 Figure 7.7: Channels parents watch most often (in percentage) Considering the data above, it can be arguably claimed that Sahand TV has lost the battle to its Farsi and Turkish competitors in attracting viewers. In other words, Azeris do not demonstrate a preference for Sahand TV because it is not "institutionally complete and qualitatively competitive" (Moring, 2007, p. 29). A low level of institutional completeness, i.e. the inability to produce high-quality and attractive programs for people, especially children and teenagers in the minority language (see Cheval, 1992, p. 193), may lead to negative results increasing "the tendency towards a complementary use of media in the minority language, lowering the level of functional completeness of these media" (Moring, 2007, p. 26). 7.8 Discussion Empirical research in multilingual settings demonstrates that the domain of home and interactions among family members do not take place in a vacuum, but rather is influenced by ecology outside home. Family language policy is said to be under constant influence by the pressures created mainly by external macro domains such as education systems and the media (broadcasting media in particular), as well as children's relay role between home and outside domains (e.g. McCarty, et al., 2009; Shohamy, 2006; Spolsky, 2009). Calvet (1998), for instance, reports the findings of a study on the Wolof language in Senegal, clearly displaying the impact of the outside world on the decisions made inside the home by family members. The enquiry showed that Wolof, a dominant language in society, was spoken as a first language in the home more by children whose parents did not speak it as a first language. Based on the results, the researchers concluded that "It is not, therefore, the family that has most influence on pupils, but the milieu." They call such a phenomenon "a case of social assimilation" (Calvet, 1998, p. 68). Spolsky (2009) similarly writes that although the domain of family like other domains has its own policy, not all features within the home are managed internally. Rather, language management in the family is only partly under the control of family members. The family language policy is influenced by "the sociolinguistic ecology inside and outside the home and by the parents' beliefs about the best strategy" (Spolsky, 2009, p. 18). Pakir's (2003) analysis also indicates that parents as the "invisible planners," recognize the advantages of the dominant language for their children, e.g. enhanced employability and upward social mobility. They consequently endorse macro policies in other domains such as education systems in favor of the dominant language, usually resulting in their choice of the dominant language to speak with their children. Some have taken such a stance on the home-society relation even further and maintained that family language policy is not often "consciously planned" but rather has been "predetermined by history and circumstances beyond the family's control" (Caldas, 2012, p. 351; Lane, 2010). The analysis of Azeri children and parents' attitudes towards language policies in the education system and broadcast media, and the examination of the dynamics of family language policy in the home provided insightful findings. 7.7 Azeri parents' attitudes and behavior towards television channels The analyses presented on the educational policies, broadcast media and linguistic landscapes in Tabriz, in chapter four, five, and six, respectively, as well as the Azeris' responses regarding language policies on those domains clearly demonstrate that Azeri does not enjoy much institutional support from the central government. Azeri is not supported in the education sector resulting in a decrease in Azeris' literacy rate in Azeri (see chapter 5). Azeri is not present in written domains such as the linguistic landscape of the city despite the fact that it has been historically a written language for centuries and there is a rich literature in libraries. Azeri does not receive much support even as an oral language. The way Sahand TV functions has led to Azeris turning to Farsi and Turkish 135 channels. Considering such circumstances, it can be said that Azeri has lost its hold on some of the most important formal domains and institutions. 7.8 Discussion Firstly, Azeri parents participating in the research reported moderate attitudes towards potential presence of 136 Azeri in the education system. Although the majority of the participants would like to be able to read and write in their ethnic language, i.e. Azeri, they do seem to not to favor Azeri-medium schools. Endorsing Farsi-only educational policies can be a contributing factor to encourage parents to introduce Farsi in the home before school to ease their children's integration into the mainstream education system. As the three case studies suggested, Farsi has been Discursively constructed as a necessity for Azeri children, motivating parents to speak Farsi with children before primary school begins. Secondly, the participating Azeri children and parents' attitudes and behavior revealed that Sahand TV has not been very successful in attracting a strong audience. As a result of Sahand TV not being qualitatively competitive (Moring, 2007), the participants reported that they watched other national channels in Farsi and/or Turkish channels brought to them through satellite. Turkey, as a regional power, has increasingly become integrated with the West through membership in organizations such as NATO, the Council of Europe, and the G-20 major economies. Given its great geostrategic and geopolitical importance, e.g. being at the crossroads of Asia and Europe, it is seen as a country with a significant economic, cultural, and political potential (Bechev, 2011). Offering attractive job opportunities, especially in the form of trade and business, Turkey has become a gateway to the world of modernity and success, especially for the people with the potential to learn Turkish. One possible explanation for Azeris' interest in Turkish could therefore be the socioeconomic potential Turkey has to offer. The economic gain one can receive through learning Turkish on the one hand, such as discounts in university fees for those who know Turkish, and the mutual intelligibility of the two languages of Azeri and Turkish on the other (Boeschoten, 1998), seem to have affected parents and children's attitudes and preferences towards Turkish, encouraging people to watch Turkish television programs. Having recognized this potential, Azeris seem to be keen to learn Turkish in order to be able to take this opportunity. They apparently see Turkish in addition to Farsi as a tool for upward social mobility. A second possible explanation for Azeris' interest in Farsi and Turkish channels could be their identification with Iranian and broader Turkic identity. 7.8 Discussion Association with the national Iranian identity and identification with the larger Turkic community seem to have attracted Azeris to viewing Farsi and Turkish channels. Farsi channels and Farsi programs on Sahand TV, as I argued in chapter six, may exist to amplify the national Iranian identity (for the role of state-governed media, see Isaacs-Martin, 2008), while one goal of Turkish satellite channels might be to bring all Turks together, in line with pan-Turkic movement, 137 by imposing a larger Turkic identity across the borders (Poulton, 1999). Although a typical Azeri may identify him/herself equally with Azeri and Iranian identity, there might exist some who believe in and identify him/herself only with either national and Iranian identity or broader Turkic identity, denying his/her local Azeri identity (cf. e.g. Hawes & Mirvahedi, 2013; Paul, 1999; Tavakoli-Targhi, 2009). The issue of regional identity in Azerbaijan has been a thorny historical issue (Daniel, 2001, p. 7). More in-depth research is required to investigate what portion of, and to what extent, Azeris identify themselves as Azeri and/or Turks, and/or Iranian. Owing to the "transnational and frontier-crossing" nature of satellite channels, governments cannot exercise their control over the new media (Collins, 2002; Debate, 2000; Sakr, 1999). Due to its low-quality programs, which can be clearly inferred by its failure to attract a noticeable audience, and the availability of satellite channels, Turkish channels in particular, Sahand TV has failed to attract a strong audience. The examination of the reported behavior with respect to viewing broadcast media in Tabriz has revealed the existence of a tendency towards Turkish channels, and perhaps Turkish identity among Azeris. In sum, the analysis of family language policy in the home, in the form of attitudes towards languages and linguistic behavior, illuminated that stable bilingualism between Azeri and Farsi has started to fade. The responding parents reported only moderately positive attitudes towards using Azeri in the home. Moreover, they did not report strong negative attitudes towards Farsi in the home. As the case studies illustrated, Farsi is in fact present in Azeris' houses through the influence of broadcast media, the education system, and intermarriages. Kindergarten or primary school students play a significant role in this process. As Spolsky (2009) argues, parents lose control over family language policy to a great extent as soon as the domain of home opens to the outside pressures of school, peers, etc. 7.8 Discussion As Fishman (1974b) eloquently writes Language planning provides populations with a new name, with a new mission - and, as a result, with the drive and dignity that makes new schools, new factories, new homes, and new diets not only acceptable but also necessary goals to work for and fight for. It is not important that the variety being produced is increasingly unlike anyone's real mother tongue or grandmother tongue. It is only important that it represents a legitimization of a new identity and a new power with which new authorities and new masses are consensually related to each other to the point of believing that they have been so related (Fishman, 1974b, p. 89). Language planning provides populations with a new name, with a new mission - and, as a result, with the drive and dignity that makes new schools, new factories, new homes, and new diets not only acceptable but also necessary goals to work for and fight for. It is not important that the variety being produced is increasingly unlike anyone's real mother tongue or grandmother tongue. It is only important that it represents a legitimization of a new identity and a new power with which new authorities and new masses are consensually related to each other to the point of believing that they have been so related (Fishman, 1974b, p. 89). 138 The presence of the dominant official language, Farsi, in the home as well as relatively positive attitudes towards its use in the home can be taken as a sign of the success of language policies of the state to promote Farsi (Baker, 2006; Caldas, 2012), and the national identity (see Anderson, 1983; Billig, 1995; May, 2006). This has strong implications for Azeri maintenance in Iran. The presence of the dominant official language, Farsi, in the home as well as relatively positive attitudes towards its use in the home can be taken as a sign of the success of language policies of the state to promote Farsi (Baker, 2006; Caldas, 2012), and the national identity (see Anderson, 1983; Billig, 1995; May, 2006). This has strong implications for Azeri maintenance in Iran. 8.0 Introduction This research has demonstrated the dynamic interplay between macro language policies of a nation-state (de jure policies), and grass-roots practices among a minority group (de facto policies) in an under-researched region, i.e. Azeris in Tabriz, Iran, with a particular focus on how those grassroots level attitudes and practices influence Azeri intergenerational transmission. Taking an ecological approach to language maintenance/shift processes and language policy and planning activities, this thesis has illustrated how the ecology shaped by LPP activities and Discourses of the dominant group affects family language policy among Azeris. This final chapter synthesizes the contribution of this research to our understanding of the relation between language policies at national level and language shift among minority groups. The chapter begins with a summary of theoretical frameworks used in the thesis, discussing the contribution of this study to each of those theoretical frameworks. The findings of each chapter of the thesis are then presented. Finally, the areas for further research are discussed. Chapter Eight: Conclusion Chapter Eight: Conclusion 7.9 Conclusion This chapter has examined Azeris' attitudes and practices with respect to using languages in the home as well as language policies in the macro domains of the education sector, and the broadcasting media in the way that they can be seen to influence language choice in the home domain. Although one might take daily usage of Azeri and the size of Azeri-speaking population in Tabriz as evidence that Azeri is not overwhelmed by Farsi, this thesis proposes a different scenario. The data analyzed in this chapter shows that Azeri parents and children use Farsi and Turkish along with Azeri to different extents in the domain of home. Azeri may not be endangered at the moment, but the intergenerational continuity of Azeri appears to be disrupted in some families. Given the lack of supportive and protective policies with respect to Azeri use in macro domains and institutions, and Azeris’ linguistic attitudes and behavior, a warning bell should be sounded about the future of Azeri in Tabriz. Having discussed language policies in the macro domains as well as attitudes at the grass roots level, the next and final chapter of this thesis synthesizes the findings of this thesis and reflects on areas for further research. 139 8.1 Theoretical frameworks Language maintenance is generally viewed as the responsibility and task of a community and of the family (Fishman, 1991; Yu, 2010). Without parents' commitment to pass on their ethnic language to their children, language maintenance efforts and/or reversing language shift endeavors will not succeed in achieving their goals (Grenoble, 2011). However, despite parents' primary and principal role in language maintenance in the home, it is argued that their decisions, choice and agency within the family unit are constrained by ecological factors to the extent that they do not always have a "free" choice (Lane, 2010; Sicoli, 2011; Spolsky, 2009). The family unit in which decisions are made in relation to languages is under pressure from the ecology outside the home which is shaped by macro domains and institutions such as education systems, media, language use in public sphere, etc. Each domain and institution is said to creates its own pressure towards language maintenance/shift (Spolsky, 2011, p. 149). This thesis has integrated a number of theoretical frameworks and models to unravel the question of "what is going on?" (Wolcott, 2008) both within the family unit and the ecology surrounding it. The ecology of language paradigm was used as an 140 overarching framework to acknowledge that language maintenance/shift does not occur in a vacuum. Rather, languages require a sustainable and complex supportive system. Using the Greek root for ecology, i.e. oikos meaning home, I argued that languages need an oikos, i.e. "a complex ecological support system," rather than only home, i.e. the family unit, for their sustained well-being (c.f. Mühlhäusler, 1996, 1992). This paradigm was critiqued in this thesis, however, because its "green" approach towards linguistic issues, does not take adequate account of power relations between groups (Edwards, 2010). The contribution of this thesis to the ecology of language paradigm is to explain how changes in language ecologies, for good or ill, can be made through LPP processes. Two frameworks proposed by Shohamy (2006), and Giles, Bourhis, and Taylor (1977) were utilized to explore the political and ideological nature of manipulations of language ecologies. Using these two models, the dual role of state-run institutions and domains was identified. Shohamy (2006) argues for the recognition and realization of how the education system, media, language rules and regulations, public signage, etc. work in favor of the dominant language. 8.1 Theoretical frameworks Labeled as policy devices and mechanisms, Shohamy argues that these devices are used to manipulate peoples, turning particular ideologies into practices. Such institutions and domains have traditionally been seen as "institutional support" which can play a determining role in the fate of languages (Giles, et al., 1977). What this research has contributed to these frameworks is further attention to, firstly, how these domains and institutions mediate between ideology and practice, and, secondly, to consider who governs those domains and institutions and in whose favor they are utilized. Thirdly, having applied a developed version of Lo Bianco's "policy as text, discourse, and performance" model (Lo Bianco, 2005, 2008c, 2010b, 2012a), the Discursive function of such domains has been stressed in this thesis, suggesting policy often works in a more covert manner. In other words, policy uses people's inattention to and unawareness of its tools and devices and how they operate to its advantage, normalizing certain power relations in the society. Two frameworks proposed by Shohamy (2006), and Giles, Bourhis, and Taylor (1977) were utilized to explore the political and ideological nature of manipulations of language ecologies. Using these two models, the dual role of state-run institutions and domains was identified. Shohamy (2006) argues for the recognition and realization of how the education system, media, language rules and regulations, public signage, etc. work in favor of the dominant language. Labeled as policy devices and mechanisms, Shohamy argues that these devices are used to manipulate peoples, turning particular ideologies into practices. Such institutions and domains have traditionally been seen as "institutional support" which can play a determining role in the fate of languages (Giles, et al., 1977). Lo Bianco's model emphasizes policy as not only the policy texts, but also what precedes and/or follows those texts in the form of discussions and debates, as well as how policy is performed. The analysis of this discursive aspect of policy shows how policies are altered or tailored to achieve certain goals and meet particular needs. This suggests and stresses that the discourse of policy makers, policy implementers, and policy consumers are as important as, if not more important than, the policy texts. 141 The contribution of this research to Lo Bianco's model is distinguishing between and including both small 'd' discourse and big 'D' Discourse in policy analysis. I have argued that policy, besides texts and discourses, can also exist as Discourses, i.e. 8.1 Theoretical frameworks "ways of combining and integrating language, actions, interactions, ways of thinking, believing, valuing, and using various symbols, tools, and objects to enact a particular sort of socially recognizable identity" (Gee, 2011, p. 29). Given that Discourses are embedded in a variety of institutions (Fairclough, 2011; Gee, 2011; van Dijk, 2008), I have demonstrated that implementation of particular policies within the domains and institutions such as the education system, media, and linguistic landscape can create, promulgate, and disseminate particular Discourses about the value and position of groups and their languages in the society. In a sense, performing policies in and through macro domains and institutions, dominant groups can plan favorable Discourses to normalize certain power relations. The performative dimension of policy proposed by Lo Bianco primarily refers to policy implementation which is closely associated with my definition of Discourse planning. This research has used 'Discourse planning' to specifically refer to the performance of state-run institutions and domains which act as a role model for people to follow. It has been suggested in the study that the performance aspect of policy at this level is Discursive, and the type of Discourse promoted and promulgated in such domains and institutions is most often governed by the wishes and intentions of those in power. Policy as performance can be also defined as the practices of policy consumers. This definition corresponds to Spolsky's (2004, 2012) notion of practices in his model, i.e. the linguistic behavior of people at the grass roots level. Having developed and presented a h i d l i h I h d h li i h The performative dimension of policy proposed by Lo Bianco primarily refers to policy implementation which is closely associated with my definition of Discourse planning. This research has used 'Discourse planning' to specifically refer to the performance of state-run institutions and domains which act as a role model for people to follow. It has been suggested in the study that the performance aspect of policy at this level is Discursive, and the type of Discourse promoted and promulgated in such domains and institutions is most often governed by the wishes and intentions of those in power. Policy as performance can be also defined as the practices of policy consumers. This definition corresponds to Spolsky's (2004, 2012) notion of practices in his model, i.e. the linguistic behavior of people at the grass roots level. 8.1 Theoretical frameworks Having developed and presented a comprehensive model in chapter two, I have argued that policy, either as texts, discourses/Discourses, or performance, or in all its forms together, are in fact interventions into language ecologies with strong implications for language maintenance/shift processes. It can be then argued that language maintenance/shift or reversing language shift can be the outcome or result of specific language policies (García, 2012, p. 81). In sum, this research has shown the complex interplay between the layers of language policy and planning, and how and through what mechanisms those LPP decisions are turned into practices, which might result in particular family language policies in the domain of home. In other words, this thesis has demonstrated that the domain of home and the interactions between family members in the home are under the constant influence of outside ecology. "Language minorization" can be then said to reflect "social and political inequalities" (Sallabank, 2012, p. 122). Home has been viewed as the most important 142 domain where languages are maintained. As a result, the mere reliance on the macro domains to promote intergenerational transmission within communities while ignoring the role of family has been likened to blowing air into a tire which has a puncture (Fishman, 1991, p. xii). What I have argued in this thesis is that arguments made by Fishman and other scholars should not be construed as indicating the insignificance of those macro domains and their impact on language maintenance. If the mere reliance on the macro domains to maintain a language is like blowing air into a flat tire, the mere reliance on the domain of home to save or maintain a language can be likened to nails on the road which may cause a puncture in the tire. That is, it is wishful thinking that languages can be maintained or saved only in the home without being recognized and used in macro domains. The findings of the study are synthesized below, discussing each chapter's contribution to this research. Chapter three discussed the methodologies used in this research. Chapter four, five, and six, explored de facto policies in the education system, linguistic landscape and the broadcasting media which constitute the ecology surrounding the domain of home. Azeri parents' attitudes and ideologies were discussed in chapter seven. 8.2 Synthesis of findings The main reason for the research design used in this study was to both describe the linguistic situation in Tabriz and possibly explain why Azeri is being increasingly overwhelmed by Farsi. To do so, as described in chapter three, data was collected in two phases. Phase one of the data collection included interviews with children, authorities of ten kindergartens, a focus-group interview with six mothers of young children, and photos of public signs. A large part of the data collected in phase one was used to explore the ecology external to the domain of home. Using an attitude questionnaire, phase two specifically investigated Azeri parents' attitudes and ideologies towards the two languages, Azeri and Farsi, with respect to their use in the domain of home, education system and the broadcasting media. Analysis of language policies in the three selected macro domains constituting the ecology outside home suggested that Azeri was being increasingly overwhelmed by Farsi. The examination of language rules and regulations in the Iranian Constitution as well as the de facto educational policies with respect to Azeri in pre-schools and kindergartens demonstrated in chapter four how a "sink or swim" (May, 2008a) type of education system 143 serves the dominant language, Farsi. The examination of ten kindergarten and preschool authorities' discourse suggests that they consider Farsi to be a necessity for upward social, educational and economic mobility. By contrast, Azeri is naively seen as a language which will be learned "anyways" as it holds the status of mother tongue. Farsi-only education system as well as the compulsory use of Farsi in other administrative domains and institutions has boosted the instrumental value of the language making Azeris (and perhaps all Iranians) learn Farsi. As a corollary to this, literacy in Azeri has dramatically decreased which is clearly reflected in domains where writing is required, e.g. public signage. One of the domains which mirrors the dynamics of an ethnolinguistic situation is its linguistic landscapes, i.e. public signage (Calvet, 2006). Chapter five investigated the linguistic landscape in Tabriz to showcase Azeri's absence in public signage. The analysis of data, i.e. language use patterns in public signage, in this chapter demonstrates not only Azeri is absent in governmental signage, connoting that Azeri is not officially supported in this domain, but also it is absent on signage erected by the private sector. The absolute absence of Azeri invites a range of interpretations. 8.2 Synthesis of findings The questionnaire data on the parents' attitudes was supplemented by observations conducted over a week in three different homes. The observations were conducted to find out to what extent the parents' reported attitudes and ideologies turn into practice on the ground. The analysis of the data suggests that Azeri parents who participated in the research do not hold strong views about Azeri and speaking it in the home. On the other hand, they hold relatively positive attitudes towards Farsi. The observations also indicate that Azeri children are exposed to Farsi through television, To find out how policies in the macro domains discussed above impact family language policies in the domain of home, chapter seven explored Azeri parents' attitudes and ideologies concerning the use of Azeri and/or Farsi in the home using a large-scale questionnaire. The questionnaire data on the parents' attitudes was supplemented by observations conducted over a week in three different homes. The observations were conducted to find out to what extent the parents' reported attitudes and ideologies turn into practice on the ground. The analysis of the data suggests that Azeri parents who participated in the research do not hold strong views about Azeri and speaking it in the home. On the other hand, they hold relatively positive attitudes towards Farsi. The observations also indicate that Azeri children are exposed to Farsi through television, broadcasting media in general, and, parents are aware of Farsi's higher instrumental value, leading them to speak Farsi to children even before they start primary school. The questionnaire data analyzed in chapter seven also demonstrated the dynamics and challenges minority media may face in the modern era. The presence and availability of satellite channels to minorities has made it possible for them to view channels broadcast from other cities and countries, imposing stiff competition among channels to attract audience. The analysis of Azeris' reported data shows Sahand TV has lost the battle to Farsi channels and Turkish channels broadcast through satellite, making Sahand TV as merely a symbolic institutional support with only 1% viewers. This might lead Azeris to shift to 'bigger languages' in the long run. As Grenoble (2011, p. 35) notes, broadcast media diminish "the last sanctuary for the local language" by bringing languages of wider communication into the home, making the use of the local language increasingly limited. 8.2 Synthesis of findings Firstly, the absence of Azeri on public signage as the marker of geographical territories (Landry & Bourhis, 1997) implies that uniformity rather than diversity is encouraged from above, i.e. the governmental policies. As a consequence, an uninformed person who does not speak the language may walk through Tabriz without realizing that a language different from Farsi is spoken in the city. Secondly, given the Discursive function of linguistic landscapes which can be used in status building, the absence of Azeri suggests that Azeri is not of much instrumental or communicative value. As a result, Azeri is not used for communication purposes in writing on public signage although it is a written language with books available in the library. Thirdly, being viewed as only an oral language, as the linguistic landscape data analysis suggests, Azeri is highly likely to be pushed into only oral domains which in turn might endanger Azeri to a greater extent in future. Chapter six focused on the local TV channel available to Azeris in Tabriz, Sahand TV, as a type of institutional support. Established in 2000, Sahand TV presently broadcasts 24 hours of programs seven days a week. Drawing on an analysis of data collected over a constructed week from programs broadcast on Sahand TV and data collected through an attitude questionnaire, chapter six shows, firstly, that the airtime for Farsi and Azeri programs is nearly equally divided, with some Arabic used on the channel as the liturgical language. Secondly, a deeper analysis into the programs broadcast on Sahand TV suggests Sahand TV's role in Discourse planning. Airing certain programs in Farsi, such as those 144 aimed at children and teenagers, and science/technology related programs, and other programs in Azeri, such as shows related to Tabriz and surrounding towns and villages, Sahand TV seems to be playing a role in Discourse planning. The performance of policies on Sahand TV, in line with the education system and linguistic landscape, can be referred to as a type of public Discourse which not only reflects Azeri's lower status and value compared to Farsi but also conveys this message to its audience. To find out how policies in the macro domains discussed above impact family language policies in the domain of home, chapter seven explored Azeri parents' attitudes and ideologies concerning the use of Azeri and/or Farsi in the home using a large-scale questionnaire. 8.2 Synthesis of findings The analysis suggested that the participating parents believe that children who learn Farsi before school have less difficulty at school, easing their integration into the education system. However, surprisingly, the participants did not show much cognizance about the threats which might endanger Azeri in the future even if they do not transfer the language to the next generation. The major reason for this seemed to be Azeri's status as the "mother broadcasting media in general, and, parents are aware of Farsi's higher instrumental value, leading them to speak Farsi to children even before they start primary school. The questionnaire data analyzed in chapter seven also demonstrated the dynamics and challenges minority media may face in the modern era. The presence and availability of satellite channels to minorities has made it possible for them to view channels broadcast from other cities and countries, imposing stiff competition among channels to attract audience. The analysis of Azeris' reported data shows Sahand TV has lost the battle to Farsi channels and Turkish channels broadcast through satellite, making Sahand TV as merely a symbolic institutional support with only 1% viewers. This might lead Azeris to shift to 'bigger languages' in the long run. As Grenoble (2011, p. 35) notes, broadcast media diminish "the last sanctuary for the local language" by bringing languages of wider communication into the home, making the use of the local language increasingly limited. The analysis suggested that the participating parents believe that children who learn Farsi before school have less difficulty at school, easing their integration into the education system. However, surprisingly, the participants did not show much cognizance about the threats which might endanger Azeri in the future even if they do not transfer the language to the next generation. The major reason for this seemed to be Azeri's status as the "mother The analysis suggested that the participating parents believe that children who learn Farsi before school have less difficulty at school, easing their integration into the education system. However, surprisingly, the participants did not show much cognizance about the threats which might endanger Azeri in the future even if they do not transfer the language to the next generation. The major reason for this seemed to be Azeri's status as the "mother 145 tongue" (see Calvet, 1998), with 84% of the parents believing that Azeri will be spoken in the city forever in any circumstances. 8.2 Synthesis of findings Overall, this thesis has investigated how home as the domain where the major part of intergenerational transmission takes place comes under the influence of pressures from external domains and institutions which form the ecology surrounding home. Furthermore, the thesis has shown those domains and institutions which form the external ecology of the home are governed and controlled by LPP decisions and processes, giving them the status of LPP devices and mechanisms in Shohamy's (2006) terms. Given the Discursive functions of the LPP devices, one may say that LPP processes in this era unlike the past, where aboriginal and indigenous people, for instance, were forced to behave in a certain way (e.g. Alia & Bull, 2005), work through persuasion rather than coercion. As van Dijk (2008, p. 14) puts it, "one needs no coercion if one persuades, seduces, indoctrinates or manipulates people." Such persuasion most often takes place through restrictions defined and brought about by domains and institutions run by the dominant group/people. It seems fair to say that because governments do not and/or cannot access the domain of home directly, what they can do to affect their citizens' linguistic attitudes and behavior is to plan a particular ecology outside the home. This planning, as I have argued in this thesis, can be described as primarily Discursive. Language, and thus its vitality and endangerment, is deeply embedded in a linguistic ecology made up of a complex nexus of components and elements. Efforts towards language maintenance or reversing language shift cannot succeed until they also attend to those elements of the linguistic ecology which are themselves factors in language loss (Grenoble, 2011, p. 44). Accordingly, if any improvement is to be achieved in making the linguistic ecology more desirable for Azeri in Tabriz, 'counter-planning' should be carried out to improve the situation in these three domains for Azeri. I propose the term 'counter-planning' to characterize activities which aim to return the ecology of a language to a desirable and healthy state through LPP decisions and processes. To do so, further research is required in these domains to find out how the linguistic ecology can be improved for Azeri in Tabriz. The following section explores the areas in which further research can be carried out. 8.3 Areas for further research As mentioned above, the case of Azeri in Tabriz is under-researched. Four areas touched upon in this thesis can be consequently researched in more depth. Further research 146 can be undertaken in the domain of the education sector. It would be intriguing to investigate how Azeri children switch to Farsi-only education in primary schools. More research is required to examine student-student and student-teacher interactions inside the classroom in primary schools. This can indicate to what extent Azeri children use the Farsi they have learned in kindergartens. Exploring teachers and authorities' ideologies and attitudes towards languages, as those who are involved in policy implementation, would be also of paramount significance. Such studies could show to what extent Azeri teachers and authorities in Tabriz are willing to be involved in Azeri education if proactive policies existed. Further studies can be undertaken on linguistic landscape and language use in Tabriz. Research into the ideologies and attitudes of the private sector could be conducted, for example through in-depth interviews with shopkeepers and so on, to find out in detail why Azeris choose Farsi and English on their public signage rather than Azeri. Such an investigation could examine whether there is a relation between the absence of Azeri on private signage and low literacy in Azeri (c.f.Shiohata, 2012). The domain of broadcasting media could be also examined in more depth by investigating the attitudes and ideologies of the authorities involved in the broadcasting media in Tabriz. Such an inquiry could explore to what extent authorities in Tabriz have the latitude to prepare Azeri programs, and to what extent the policies in this institution are dictated by the central government. The findings of such a study could suggest how, if at all, Sahand TV is used as a tool for Discourse planning. More qualitative research is required to examine family language policies in the domain of the home. Detailed observations need to be made to examine how language shift takes place in interaction between parents and children in the form of code-switching and code-mixing (c.f. Nercissians, 2001). Such interactions at the grass roots level may demonstrate how Azeri children, influenced by ecology outside the home, switch to Farsi even before they enter Farsi-medium schools. Another more important research topic which emerged from this research is ethnic and national identity and how attachment to those two identities is managed. 8.4 Conclusion This thesis has attempted to expand language maintenance/shift and language policy and planning frameworks theoretically as well as empirically (Shohamy, 2009). The study is a response to Shohamy's critique of the field that "personal experiences have rarely been viewed as part of the domain of language policy, since policies are generally created from the top down, often to meet ideological or political agendas" (Shohamy, 2009, p. 185). This research also extends language maintenance/shift research by investigating the interplay between the LPP activities of nation-states influencing the ecology outside home and the dynamics of family language policy within the ecology inside home. The thesis emphasizes that parents, though acting ostensibly as free agents, do not always have a free choice owing to socioeconomic and political constraints. The study has attempted to both describe the language-contact situation in Tabriz by attending to top-down policies, and illuminate how Azeris respond to those policies by exploring their attitudes and ideologies towards language use in a number of domains. The analysis of de facto policies presented in this research can "reflect and influence ways of thinking about language policies" (Shohamy, 2009, p. 188). This research has explained the issue of how, if at all, Azeri is being transmitted to the next generation. Such a process which mainly takes place in the home is constantly influenced by the ecology in which language contact occurs. An examination of the various top-down pressures on the de facto policy in Tabriz suggests that firstly, Azeri is being increasingly overwhelmed by Farsi in macro domains such as the education sector, linguistic landscape, and the media, and secondly, Discourses created through such domains and institutions pointing to the higher value of Farsi seem to have led to particular attitudes and ideologies towards Azeri and Farsi. Such attitudes and ideologies, as I have argued throughout the thesis, may endanger Azeri in future. Finally, I have attempted to sound a warning bell for Azeris, and potentially for other minorities in Iran, to become more cognizant of LPP processes which may work against their language resulting in language attrition/loss. Although all languages in Iran are officially recognized, in practice they are not supported in macro institutions and domains creating undesirable ecology for minorities. Owing to the lack of proactive policies obliging protection of minority languages, minority languages in Iran face an uncertain destiny. 8.3 Areas for further research There is anecdotal evidence that many Azeris regard themselves as Azeri even if they do not know or speak Azeri because of immigration to other cities or language shift. Furthermore, most Azeris consider themselves as both Azeri and Iranian. Detailed research is required to probe into this issue as it could have a major influence on the fate of Azeri. 147 8.4 Conclusion It is in this situation that government's inaction can be viewed as an action which influences the fate of languages. To maintain and foster minority languages 148 in Iran, more diversity-oriented endeavors as well as proactive and obligatory policies on the part of the government are consequently required. 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As part of this degree, I am undertaking a research project leading to a thesis. This project is examining the language policies, language practices and language vitality of Azeri in Tabriz. The University requires that ethics approval be obtained for research involving human participants. This project has obtained Human Ethics Committee approval. I would like to invite you as a parent of a young child to participate in this research. If you agree, I would like you to complete the questionnaire which has been brought to you by your child. I would be very grateful for your assistance in answering some questions about different language-related issues in Tabriz. This is not a test, so there are no right or wrong answers and you do not even need to write your name on it. I am only interested in your personal opinion. Please give your answers thoroughly and sincerely, as only this will guarantee the success of this investigation. Responses collected will form the basis of my research project and will be put into a written report on an anonymous basis. It will not be possible for you to be identified personally. All material collected will be kept confidential. The names of the participants will not be mentioned during the data collection, and no other person besides me and my supervisors, Professor Janet Holmes and Dr Meredith Marra, will see the data. The thesis will be submitted to the School of Linguistics and Applied Language Studies and deposited in the University Library. It is intended that one or more articles will also be submitted for publication in scholarly journals. If you have any questions or would like to receive further information about the project, p contact me or my supervisors through the following e-mail addresses. Seyedhadi.Mirvahedi@vuw.ac.nz Janet.Holmes@vuw.ac.nz Meredith.Marra@vuw..ac.nz To complete this questionnaire, please consider the following definitions: To complete this questionnaire, please consider the following definitions: 1) Azeri: the language which is spoken in Tabriz 2) Turkish: the language spoken in Turkey. 3) Farsi: the official language of Iran. 4) Sahand TV: the local channel of Tabriz. Wolcott, H. F. (2008). Ethnography: A way of seeing. Lanham: Altamira. Woodside-Jiron, H. (2011). Language, power, and participation: Using critical discourse analysis to make sense of public policy. In R. Rogers (Ed.), An introduction to critical discourse analysis in education (pp. 154-182). New York: Routledge. Woodside, A. G. (2010). Case study research: Theory, methods, practice. Bradford: Emerald. Wright, S. (2012). Language policy, the nation and nationalism. In B. Spolsky (Ed.), TheCambridge handbook of language policy (pp. 59-78). Cambridge: Cambridge University Press. Wurm, S. A. (Ed.). (2001). Atlas of the world’s languages in danger of disappearing. Paris: Unesco Publishing. Yu-Hsiu Lee, H. (2013). Bargirl style of language choice and shift: A tale from the land of smile. Theory and Practice in Language Studies, 3(3), 411-422. Yu, S. (2010). How much does parental language behaviour reflect their language beliefs in language maintenance? Journal of Asian Pacific Communication 20(1), 1-22. doi: 10.1075/japc.20.1.01yu Zhou, M. (2000). Language policy and illiteracy in ethnic minority communities in China. Journal of Multilingual and Multicultural Development, 21(2), 129-148. 181 Information Sheet for Parents ) National channels: Channels which are broadcast nationwide, such as channels 1,2,3,4 5) National channels: Channels which are broadcast nationwide, such as channe 5) National channels: Channels which are br If you are willing to complete the questionnaire, please do so and return it by asking your child to hand it in to his/her teacher. It will take 10-20 minutes to complete this questionnaire. Thank you very much for your cooperation. Seyed Hadi Mirvahedi Linguistics and Applied Language Studies, Victoria University of Wellington, New Zealand Instruction: Please read each question below carefully and check only one box in the table. The questions ask you to rate your agreement or disagreement with a statement from "strongly agree" to "strongly disagree". Please note that St/Ag = Strongly Agree, Ag = Agree, So/Ag = Somewhat Agree, NAg/Dis = Neither Agree Nor Disagree, So/Dis = Somewhat disagree, Dis = Disagree, St/Dis = Strongly disagree. For example: Question St/Ag Ag So/A g N Ag/Dis So/Dis Dis St/Dis Snakes are more dangerous than spiders. × This shows that the respondent agrees that snakes are more dangerous than spiders. Questions St/Ag Ag So/Ag N Ag/Dis So/Dis Dis St/Dis 1 People should speak Farsi with their children at home. 2 I wish there were schools which had language courses on Azeri. 3 Watching Sahand TV is a waste of time. 4 To be a true Azeri, people must speak Azeri. 5 People need Farsi in Tabriz to get a good job 6 There would be no benefit for my child to learn Azeri literature, reading and writing. 7 I like the programs on Sahand TV. 8 To be a true Iranian, people must speak Farsi. 9 It would be a loss if we don’t know Farsi and the Iranian culture. 10 Children who learn only Azeri at home are very intelligent. 11 I like watching Farsi TV channels. 12 Farsi should be taught in schools. 13 Without Azeri, people would lose their Azeri identity. 14 One can live comfortably in Tabriz without Farsi. This shows that the respondent agrees that snakes are more dangerous than spiders. 183 184 Questions St/Ag Ag So/Ag N Ag/Dis So/Dis Dis St/Dis 15 Although I am an Azeri, I’m primarily an Iranian. 16 Azeri has no place in the modern world. 17 I feel proud (like it) when my child speaks Farsi. 18 I wish there were schools where all subjects were taught in Azeri. 19 The programs on Sahand TV are not interesting. 20 Without Farsi, people would lose their Iranian identity. 21 Speaking Farsi is associated with high social class. 22 Children who learn Farsi at home are very intelligent. 23 I would send my child to a school where there was a course on Azeri. 24 There’s no relationship between Azeri identity and the Azeri language. 25 Speaking Azeri is associated with low social class. Instruction: 26 You can be identified as Iranian without being able to speak Farsi. 27 There’s no relationship between Iranian identity and the Farsi language. 28 Farsi programs are much better than Sahand TV programs. 22 It would be great if Azeri people could read and write Azeri. 30 I will stop my child if he/she uses Farsi at home. 31 You cannot be successful if you know only Azeri. 32 Though I am Iranian, I’m primarily an Azeri. 33 I would send my child to a school where they taught all subjects in Azeri. 34 I hate it when my child speaks Farsi at home. 35 I’m happy that children learn Farsi at school 36 I prefer watching Sahand TV to watching Farsi TV channels. 37 Knowing Farsi makes our life in Tabriz easier. 38 Teaching children Azeri at school would be a waste of time. 32 I would not send my child to school where they taught all subjects in Azeri. 184 Questions St/Ag Ag So/Ag N Ag/Dis So/Dis Dis St/Dis 40 I do not care if my children or grandchildren cannot speak Azeri. 41 Azeri people should speak Azeri at home. Part II Personal Details: Please answer the following questions about yourself. As your name is not asked for in this research, the information will be anonymous and will be also treated confidentially. What’s the main street or suburb you are living in? ………………………………………….. (Please do not provide the detailed address) ……………………………………………………  Sex: Male □ Female □  Age: 25-35 □ 36-45 □ over 45 □ 1. Your education level: ……………………………………………………………………. 2. Your job: …………………………………………………………………………………. 3. Your spouse’s education level:……………………………………………………………. 4. Your spouse’s job: ………………………………………………………………………… 5. What language(s) do you most often speak with your spouse at home? .............................. 6. What language(s) do you most often speak with your child at home?.................................. 7. Which channel(s) do you watch most? a) Iranian national channels (1,2,3,4) □ c) Farsi satellite channels □ b) Turkish satellite channels □ d) Sahand TV (Tabriz channel) □ Part II Personal Details: Personal Details: Please answer the following questions about yourself. As your name is not asked for in this research, the information will be anonymous and will be also treated confidentially. Male □ Female □ 1. Your education level: ……………………………………………………………………. 2. Your job: …………………………………………………………………………………. 3. Your spouse’s education level:……………………………………………………………. 4. Your spouse’s job: ………………………………………………………………………… 5. What language(s) do you most often speak with your spouse at home? .............................. 6. What language(s) do you most often speak with your child at home?.................................. 7. Which channel(s) do you watch most? a) Iranian national channels (1,2,3,4) □ c) Farsi satellite channels □ b) Turkish satellite channels □ d) Sahand TV (Tabriz channel) □ 185 Other (please specify): 8. Which channel(s) does your child watch most? a) Iranian national channels (1,2,3,4) □ c) Farsi satellite channels □ b) Turkish satellite channels □ d) Sahand TV (Tabriz channel) □ Other (please specify): ………………………………………………………………………. b) Turkish satellite channels □ d) Sahand TV (Tabriz channel) □ Other (please specify): ………………………………………………………………………. 9. Which one describes you best? a) I’m a native speaker of Azeri □ b) I’m a native speaker of Farsi □ c) I’ve learned Azeri as a second language □ 10. By circling the number, please rate your proficiency to speak, comprehend, read, and write Azeri below. (1= almost no proficiency; 10= native proficiency) Speaking: 1 2 3 4 5 6 7 8 9 10 Comprehension: 1 2 3 4 5 6 7 8 9 10 Reading: 1 2 3 4 5 6 7 8 9 10 Writing: 1 2 3 4 5 6 7 8 9 10 11. By circling the number, please rate your proficiency to speak, comprehend, read, and write Farsi below. (1= almost no proficiency; 10= native proficiency) Speaking: 1 2 3 4 5 6 7 8 9 10 Comprehension: 1 2 3 4 5 6 7 8 9 10 Reading: 1 2 3 4 5 6 7 8 9 10 Writing: 1 2 3 4 5 6 7 8 9 10 186 12. I’m learning (have learned) Turkish by watching Turkish satellite channels. True □ False □ 13. My child is leaning (has leaned) Turkish by watching Turkish satellite channels. True □ False □ 14. My child is learning (has learned) Azeri by watching Azeri channels (Sahand TV or Azeri satellite channels) True □ False □ 15. I spoke Farsi with my child before he/she went to school. True □ False □ 16. I encouraged my child to speak Farsi before he/she went to school. True □ False □ 17. How long do you predict Azeri will be used in Tabriz? …Thank you... Male □ Female □ 50-100 years □ 100-200 years □ More than 200 years □ For ever □ Why do you predict so? ……………………………………………………………………………………………… ……………………………………………………………………………………………… ……………………………………………………………………………………………… ……………………………………………………………………………………………… ……………………………………………………………………………………………… ……………………………………………………………………………………………… ……………………………………………………………………………………………… ……………………………………………………………………………………………… Please write down any comments about the questionnaire or the research: ------------------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------- --------------------------------------- --------------------------------------------------------------------- 12. I’m learning (have learned) Turkish by watching Turkish satellite channels. True □ False □ 13. My child is leaning (has leaned) Turkish by watching Turkish satellite channels. True □ False □ 14. My child is learning (has learned) Azeri by watching Azeri channels (Sahand TV or Azeri satellite channels) True □ False □ 15. I spoke Farsi with my child before he/she went to school. True □ False □ 16. I encouraged my child to speak Farsi before he/she went to school. True □ False □ 187 برگه اطالعات براي والدين من دانشجوی مقطع دکترا در دانشگاه ویکتوریای ویلینگتون نیوزلند هستم و این تحقیق بخشی از مطالعات این مقطع و با هدف انجام رساله دکترا اجرا می شود .اين پروژه سیاست گذاري ها و خط ،مشي هاي زباني رفتارهاي زباني و درجه حیات زبان آذري در تبريز را مي آزمايد .دانشگاه الزم مي داند براي تحقیقي كه با مشاركت كنندگان انساني در ارتباط است، موافقت و تايید اخالقي بگیرد. من دانشجوی مقطع دکترا در دانشگاه ویکتوریای ویلینگتون نیوزلند هستم و این تحقیق بخشی از مطالعات این مقطع و با هدف انجام رساله دکترا اجرا می شود .اين پروژه سیاست گذاري ها و خط ،مشي هاي زباني رفتارهاي زباني و درجه حیات زبان آذري در تبريز را مي آزمايد .دانشگاه الزم مي داند براي تحقیقي كه با مشاركت كنندگان انساني در ارتباط است، موافقت و تايید اخالقي بگیرد. مايلم از شما به عنوان پدر يا مادر يك كودك دعوت به عمل بیاورم كه در اين تحقیق مشاركت نمايید . در صورت تمايل، از شما دعوت می شود پرسشنامه اي كه فرزندتان به همراه آورده است را تكمیل كنید . من از همكاري و مساعدت شما در پاسخ گويي به این سؤاالت که درباره موارد مرتبط با تفاوت ه اي زباني در تبريز است ، بسیار سپاسگزار خواهم بود . اين يك آزمون نیست و هیچ پاسخ درست يا غلطي وجود ندارد و حتي نیازي نیست كه شما نام خود را بر روي آن بنويسید . من تنها مايل به دانستن ديدگاه و نظر شخصي شما هستم .خواهشمند است به طور كامل و صادقانه به سؤاالت پاسخ دهید . تنها در اين صورت است كه موفقیت اين تحقیق تضمین خواهد شد . اطالعات جمع آوري شده مبنای پروژه تحقیقاتي من را شكل خواهد داد و نتایج بدون ذكر نام در قالب گزارشي مكتوب ارائه خواهد شد .ممكن نخواهد بود كه شما شخصاً شناسايي شويد .تمام اطالعات جمع آوري شده محرمانه نگه داري خواهد شد .اسامي شركت كنندگان در طول تحقیق ذكر نخواهد شد و هیچ كس به جز من و اساتید راهنمايم پروفسور ج ن ت هولمز و دكتر مردي ث مارا، اطالعات(داده ها )را نخواهند ديد . رساله دكترا به دانشكده مطالعات زبانشناسي و زبانشناسي كاربردي ارائه شده و در كتابخانه دانشگاه نگه داري خواهد شد. همچنین قرار است يك يا چند مقاله براي انتشار در نشريات علمي ارائه شود . برگه اطالعات براي والدين ،اگر سؤالي داشته يا مايل به دريافت اطالعات بیشتر در خصوص پروژه هستید می توانید از طريق ايمیل هاي زير با من يا اساتید راهنماي من تماس بگیريد: مشاركت كنندگان انساني در ارتباط است، موافقت و تايید اخالقي بگیرد. مايلم از شما به عنوان پدر يا مادر يك كودك دعوت به عمل بیاورم كه در اين تحقیق مشاركت نمايید . در صورت تمايل، از شما دعوت می شود پرسشنامه اي كه فرزندتان به همراه آورده است را تكمیل كنید . من از همكاري و مساعدت شما در پاسخ گويي به این سؤاالت که درباره موارد مرتبط با تفاوت ه اي زباني در تبريز است ، بسیار سپاسگزار خواهم بود . اين يك آزمون نیست و هیچ پاسخ درست يا غلطي وجود ندارد و حتي نیازي نیست كه شما نام خود را بر روي آن بنويسید . من تنها مايل به دانستن ديدگاه و نظر شخصي شما هستم .خواهشمند است به طور كامل و صادقانه به سؤاالت پاسخ دهید . تنها در اين صورت است كه موفقیت اين تحقیق تضمین خواهد شد . اطالعات جمع آوري شده مبنای پروژه تحقیقاتي من را شكل خواهد داد و نتایج بدون ذكر نام در قالب گزارشي مكتوب ارائه خواهد شد .ممكن نخواهد بود كه شما شخصاً شناسايي شويد .تمام اطالعات جمع آوري شده محرمانه نگه داري خواهد شد .اسامي شركت كنندگان در طول تحقیق ذكر نخواهد شد و هیچ كس به جز من و اساتید راهنمايم پروفسور ج ن ت هولمز و دكتر مردي ث مارا، اطالعات(داده ها )را نخواهند ديد . رساله دكترا به دانشكده مطالعات زبانشناسي و زبانشناسي كاربردي ارائه شده و در كتابخانه دانشگاه نگه داري خواهد شد. همچنین قرار است يك يا چند مقاله براي انتشار در نشريات علمي ارائه شود . ا Seyedhadi.Mirvahedi@vuw.ac.nz Janet.Holmes@vuw.ac.nz Meredith.Marra@vuw..ac.nz Seyedhadi.Mirvahedi@vuw.ac.nz Janet.Holmes@vuw.ac.nz Meredith.Marra@vuw..ac.nz براي تكمیل اين پرسشنامه، لطفاً تعاري ف زير را در نظر بگیريد: 1 ) آذري :زباني كه در تبريز بدان سخن گفته مي شود 2 ) تركي :زباني كه در تركیه بدان سخن گفته مي شود 3 ) فارسي :زبان رسمي ايران 4 ) تلويزيون سهند :شبكه محلي) استانی) تبريز 5 ) شبكه هاي ملي :شبكه هايي كه در كل كشور پخش مي شود، مانند شبكه هاي1 ، 2 ، 3 ، 4 ًاگر مايل به تكمیل اين پرسشنامه هستید، لطفا به سواالت پاسخ دهید و از فرزندتان بخواهید كه آن را به معلمش تحويل دهد . پر كردن اين پرسشنامه11 تا21 دقیقه زمان خواهد برد. از همكاري شما بسیار سپاسگزارم. برگه اطالعات براي والدين سید هادي میرواحدي دانشكده مطالعات زبانشناسي و زبانشناسي كاربردي، دانشگاه ويكتورياي ولینگتون، نیوزيلند سید هادي میرواحدي دانشكده مطالعات زبانشناسي و زبانشناسي كاربردي، دانشگاه ويكتورياي ولینگتون، نیوزيلند 188 بخش اول: دستورالعمل: لطفاً سؤاالت زیر را با دقت بخوانید و تنها یك گزینه را در جدول عالمت بزنید . لطفا در عبارات زیر موافقت یا مخالفت خود را با عبارت مطرح شده از بسیار موافق تا بسیار مخالف درجه بندي كنید . به عنوان مثال: عالمت نشان مي دهد كه پاسخ دهنده با این عبارت كه مارها از عنكبوت ها خطرناك ترند موافق است. سؤاالت بسیار موافق موافق تا حدي موافق بي نظر تا حدي مخالف مخالف بسیار مخالف 1 افراد باید با فرزندانشان در خانه فارسي صحبت كنند. 2 اي كاش مدارسي وجود داشت كه دروس زبان آذري در آن تدریس مي شد. 3 تماشاي تلویزیون سهند اتالف وقت است. 4 براي یك آذري واقعي بودن، باید آذري صحبت كرد. 5 افراد براي پیدا كردن یك شغل خوب در تبریز به زبان فارسي نیاز دارند. 6 براي فرزند من سودي نخواهد داشت كه خواندن، نوشتن و ادبیات آذري را بیاموزد. 7 من برنامه هاي تلویزیون سهند را دوست دارم. 8 ،براي یك ایراني واقعي بودن افراد باید فارسي صحبت كنند. 9 این یك زیان و فقدان خواهد بود اگر ما زبان فارسي و فرهنگ ایراني را نشناسیم. 11 كودكاني كه در خانه فقط آذري مي آموزند، خیلي باهوش هستند. سؤاالت بسیار موافق موافق تا حدي موافق بي نظر ( نه موافق و نه مخالف) تا حدي مخالف مخالف بسیار مخالف مارها از عنكبوت ها خطرناك ترند. × لطفاً سؤاالت زیر را با دقت بخوانید و تنها یك گزینه را در جدول عالمت بزنید . لطفا در عبارات زیر موافقت یا مخالفت خود را با عبارت مطرح شده از بسیار موافق تا بسیار مخالف درجه بندي كنید . عالمت نشان مي دهد كه پاسخ دهنده با این عبارت كه مارها از عنكبوت ها خطرناك ترند موافق است. سؤاالت بسیار موافق موافق تا حدي موافق بي نظر ( نه موافق و نه مخالف) تا حدي مخالف مخالف بسیار مخالف مارها از عنكبوت ها خطرناك ترند. × سؤاالت بسیار موافق موافق تا حدي موافق بي نظر تا حدي مخالف مخالف بسیار مخالف 1 افراد باید با فرزندانشان در خانه فارسي صحبت كنند. 2 اي كاش مدارسي وجود داشت كه دروس زبان آذري در آن تدریس مي شد. 3 تماشاي تلویزیون سهند اتالف وقت است. 4 براي یك آذري واقعي بودن، باید آذري صحبت كرد. برگه اطالعات براي والدين 5 افراد براي پیدا كردن یك شغل خوب در تبریز به زبان فارسي نیاز دارند. 6 براي فرزند من سودي نخواهد داشت كه خواندن، نوشتن و ادبیات آذري را بیاموزد. 7 من برنامه هاي تلویزیون سهند را دوست دارم. 8 ،براي یك ایراني واقعي بودن افراد باید فارسي صحبت كنند. 9 این یك زیان و فقدان خواهد بود اگر ما زبان فارسي و فرهنگ ایراني را نشناسیم. 11 كودكاني كه در خانه فقط آذري مي آموزند، خیلي باهوش هستند. سؤاالت بسیار موافق موافق تا حدي موافق بي نظر تا حدي مخالف مخالف بسیار مخالف 1 افراد باید با فرزندانشان در خانه فارسي صحبت كنند. 2 اي كاش مدارسي وجود داشت كه دروس زبان آذري در آن تدریس مي شد. 3 تماشاي تلویزیون سهند اتالف وقت است. 4 براي یك آذري واقعي بودن، باید آذري صحبت كرد. 5 افراد براي پیدا كردن یك شغل خوب در تبریز به زبان فارسي نیاز دارند. 6 براي فرزند من سودي نخواهد داشت كه خواندن، نوشتن و ادبیات آذري را بیاموزد. 7 من برنامه هاي تلویزیون سهند را دوست دارم. 8 ،براي یك ایراني واقعي بودن افراد باید فارسي صحبت كنند. 9 این یك زیان و فقدان خواهد بود اگر ما زبان فارسي و فرهنگ ایراني را نشناسیم. 11 كودكاني كه در خانه فقط آذري مي آموزند، خیلي باهوش هستند. 189 190 سؤاالت بسیار موافق موافق تا حدي موافق بي نظر تا حدي مخالف مخالف بسیار مخالف 11 من تماشاي شبكه هاي تلویزیون فارسي را دوست دارم. 12 زبان فارسي باید در مدارس آموزش داده شود. 13 بدون زبان آذري، افراد هویت آذري خود را از دست مي دهند. 14 افراد مي توانند در تبریز به راحتي زندگي كنند بدون آن كه زبان فارسي بدانند. 15 اگرچه من یك آذري هستم، اما پیش از آن یك ایراني ام. 16 زبان آذري کاربردی در جهان كنوني مدرن ندارد. 17 من احساس غرور مي كنم( دوست دارم ) وقتي فرزندم فارسي حرف مي زند. 18 اي كاش مدارسي وجود داشت كه در آن همه دروس به زبان آذري تدریس مي شد . 19 برنامه هاي شبكه سهند جذاب نیست. 21 بدون فارسي، ایرانیان هویت ایرانیش ا ن را از دست مي دهند. 21 به زبان فارسي حرف زدن به منزله باكالس بودن است. 22 كودكاني كه در خانه فارسي مي آموزند خیلي باهوش هستند. 23 من فرزندم را به مدرسه اي خواهم فرستاد كه درسي براي آموزش زبان آذري داشته باشد. 3 . سطح(میزان )تحصیالت همسر شما: برگه اطالعات براي والدين 24 هیچ رابطه اي بین هویت آذری و زبان آذری وجود ندارد. 25 به زبان آذري حرف زدن به منزله بي كالس بودن است. 26 شما مي توانید یك ایراني شناخته شوید، بدون آنكه قادر باشید به زبان فارسي صحبت كنید. 190 191 سؤاالت بسیار موافق موافق تا حدي موافق بي نظر تا حدي مخالف مخالف بسیار مخالف 27 هیچ رابطه اي بین هویت ایراني و زبان فارسي وجود ندارد. 28 برنامه هاي فارسي خیلي بهتر از برنامه هاي تلویزیون سهند است. 29 خیلي خوب خواهد بود اگر افراد آذري بتوانند به آذري بخوانند و بنویسند. 31 من جلوي فرزندم را مي گیرم اگر در خانه فارسي صحبت كند. 31 شما نمي توانید موفق شوید اگر فقط زبان آذري بدانید. 32 اگرچه من یك ایراني هستم، اما پیش از آن یك آذري ام. 33 من فرزندم را به مدرسه اي مي فرستم كه در آن همه دروس به زبان آذري تدریس شود. 34 من از اینكه فرزندم در خانه فارسي صحبت كند متنفرم. 35 من خوشحالم كه كودكان در مدرسه فارسي مي آموزند. 36 من ترجیح مي دهم به جاي شبكه هاي تلویزیون فارسي، شبكه سهند ببینم. 37 دانستن زبان فارسي زندگي ما را در تبریز آسان تر مي كند. 38 آموزش زبان آذري به كودكان در مدارس اتالف وقت خواهد بود. 39 من فرزندم را به مدرسه اي كه همه دروس در آن به زبان آذري تدریس مي شود نخواهم فرستاد . 40 برایم اهمیتي ندارد اگر فرزندان و نوه هایم نتوانند آذري صحبت كنند. 41 آذري ها(آذري زبان ها ) باید در خانه آذري صحبت كنند. 191 بخش دوم: مشخصات فردي: لطفاً به سؤاالت زیر درباره خود پاسخ دهید .از آنجا كه نام شما براي این تحقیق سؤال نشده است اطالعات بي نام بوده و همچین به صورت محرمانه نگهداري خواهد شد. نام خیابان اصلي یا محله اي كه در آن زندگي مي كنید چیست؟ (لطفاً آدرس كامل خود را ننویس ید ) 8 . فرزند شما اغلب چه شبكه(یا شبكه هایي )را تماشا مي كند؟(لطفا فقط یک گزینه را انتخاب کنید) الف )شبكه هاي ملي ایران(مانند شبکه های1 ، 2 ، 3 ، 4 ) ب )شبكه هاي ماهواره اي تركیه ج )بكه ش هاي ماهواره اي فارسي د ) تلویزیون(شبكه ) سهند(شبكه تبریز) سایر(لطفاً نام ببرید............................................................................ :) 9 . كدام عبارت بهتر شما را توصیف مي كند ؟ الف )زبان مادري من آذري است. برگه اطالعات براي والدين ب ) زبان مادري من فارسي است و من آذري را به عنوان زبان دوم آموخته ام. ج )زبان مادري من فارسي است ولی من آذري را به عنوان زبان دوم نیآموخته ام. 192 11 . لطفاً با خط كشیدن دور اعداد زیر، مهارت خود را در صحبت كردن، درك كردن(فهمیدن) ، خواندن و نوشتن به زبان آذري درجه بندي كنید. ( 1 = ًتقریبا بدون مهارت ؛11 = مهارت كامل(تسلط در حد زبان مادري)) صحبت كردن: 1 2 3 4 5 6 7 8 9 11 درك كردن : 1 2 3 4 5 6 7 8 9 11 خواندن : 1 2 3 4 5 6 7 8 9 11 نوشتن: 1 2 3 4 5 6 7 8 9 11 11 . لطفاً با خط كشیدن دور اعداد زیر، مهارت خود را در صحبت كردن، درك كردن(فهمیدن) ، خواندن و نوشتن به زبان فارسي درجه بندي كنید. ( 1 = تقریباً بدون مهارت ؛11 = مهارت كامل(تسلط در حد زبان مادري)) صحبت كردن: 1 2 3 4 5 6 7 8 9 11 درك كردن : 1 2 3 4 5 6 7 8 9 11 خواندن : 1 2 3 4 5 6 7 8 9 11 نوشتن: 1 2 3 4 5 6 7 8 9 11 12 . با تماشاي شبكه هاي ماهواره اي تركیه، در حال فراگیري زبان تركي هستم(یاد گرفته ام:) □ درست□ غلط 13 . فرزندم با تماشاي شبكه هاي ماهواره اي تركیه، در حال فراگیري زبان تركي است(یاد گرفته است:) □ درست□ غلط 14 . فرزندم با تماشاي شبكه هاي آذري(تلویزیون سهند یا شبكه هاي ماهواره اي آذري) ، در حال فراگیري زبان آذري است(یاد گرفته است:) □ درست□ غلط 15 . پیش از اینكه فرزندم به مدرسه برود با او فارسي صحبت مي كردم. □ درست□ غلط 16 . پیش از اینكه فرزندم به مدرسه برود، او را تشویق مي كردم كه فارسي صحبت كند. □ درست□ غلط 17 . پیش بیني مي كنید زبان آذري تا چه زماني در تبریز مورد استفاده قرار بگیرد؟ □ 51 - 111 سال□ 111 - 211 سال□ بیش از211 سال□ براي همیشه چرا چنین پیش بیني دارید؟ ……………………………………………………………………………………………… ……………………………………………………………………………………………… ……………………………………………………………………………………………… ……………………………………………………………………………………………… ……………………………………………………………………………………………… ……………………………………… ................................... 11 . لطفاً با خط كشیدن دور اعداد زیر، مهارت خود را در صحبت كردن، درك كردن(فهمیدن) ، خواندن و نوشتن به زبان آذري درجه بندي كنید. برگه اطالعات براي والدين ( 1 = ًتقریبا بدون مهارت ؛11 = مهارت كامل(تسلط در حد زبان مادري)) صحبت كردن: 1 2 3 4 5 6 7 8 9 11 درك كردن : 1 2 3 4 5 6 7 8 9 11 خواندن : 1 2 3 4 5 6 7 8 9 11 نوشتن: 1 2 3 4 5 6 7 8 9 11 11 . لطفاً با خط كشیدن دور اعداد زیر، مهارت خود را در صحبت كردن، درك كردن(فهمیدن) ، خواندن و نوشتن به زبان فارسي درجه بندي كنید. ( 1 = تقریباً بدون مهارت ؛11 = مهارت كامل(تسلط در حد زبان مادري)) صحبت كردن: 1 2 3 4 5 6 7 8 9 11 درك كردن : 1 2 3 4 5 6 7 8 9 11 خواندن : 1 2 3 4 5 6 7 8 9 11 نوشتن: 1 2 3 4 5 6 7 8 9 11 12 . با تماشاي شبكه هاي ماهواره اي تركیه، در حال فراگیري زبان تركي هستم(یاد گرفته ام:) 16 . پیش از اینكه فرزندم به مدرسه برود، او را تشویق مي كردم كه فارسي صحبت كند. □ درست□ غلط 193 لطفاً هر گونه نظر یا پیشنهادي در خصوص پرسشنامه، سواالت و یا تحقیق دارید مطرح نمایید: ------------------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------ … با سپاس ... … با سپاس ... 194 Dear Parents: I am writing to ask for your help with a research project. I would like you to fill out the questionnaire below, and I would like to interview your child about some language issues. The interview will take 5-10 minutes and it will be about your child’s language behavior as well as his/her attitudes towards different TV channels, and it will be carried out before/after the English class in his/her teacher’s presence. If you are willing for your child to participate in this research please sign the form below. Note: Neither your name nor your child’s name will be used in the research. you are willing for your child to participate in this research please sign the form below. Note: Neither your name nor your child’s name will be used in the research. Thank you for your cooperation. Appendix Two: Interview Schedules Consent Form Dear Parents: Respondent’s signature consenting to the child’s involvement in the interview 195 والدین گرامی Children's (7-10 years old) interview schedule Children's (7-10 years old) interview schedule A. Language behavior رضایت نامه رضایت نامه نامه ای که پیش رو دارید جهت اعالم رضایت شما براي مصاحبه با فرزندتان طراحی شده است . این مصاحبه پنج الی ده دقیقه طول خواهد کشید و قبل یا بعد از کالس زبان فرزندتان در کانون زبان انجام خواهد شد . از فرزند شما در مورد رفتار زباني اش در منزل و نیز رفتار او در خصوص تماشاي تلویزیون سواالتي پرسیده خواهد شد . شما مي توانید در صورت تمایل رضایت خود را با امضاي این نامه اعالم نمایید. شایان ذکر است که شرکت در این تح قیق اختیاری خواهد بود و شرکت کنندگان می توانند هر لحظه که بخواهند از مشارکت در تحقیق انصراف دهند. همچنین پاسخ های شما کامالً محرمانه بوده و اسامی شما یا فرزندتان در پروژه ذکر نخواهد شد . پیشاپیش از همکاری صمیمانه شما سپاسگزاری می نمایم؛ چه، انجام این تحق یق بدون همکاری شما امکان پذیر نخواهد بود . سید هادی میرواحدی سید هادی میرواحدی نام و نام خانوادگی مصاحبه شونده(فرزند............................................................................. :) امضای پدر یا مادر : ....................................................................................................... تاریخ....................................................................................................................... : 196 Children's (7-10 years old) interview schedule A. Language behavior  How many languages can you speak?  Could you speak Farsi before you went to school? If yes, how did you learn it?  Have you attended a kindergarten?  What language do you speak with your parents? With your siblings? With your friends at school? With your teachers?  Are there any situations in which you speak Farsi with your parents/siblings/teachers?  If yes, when and why do you speak Farsi?  Is there a rule with respect to which channel you should watch in the home?  If yes, what is it? B. Media  How long do you spend watching TV every day?  Which channel do you watch most?  Do you ever watch Sahand TV? If no, why?  If yes, what is your favorite program?  If you had the choice between Azeri cartoons and Farsi cartoons, which one would you prefer to watch? Why?  Do you ever listen to the radio? If yes, which radio station(s)?  Do you ever listen to Tabriz radio? A. Language behavior  How many languages can you speak?  Do you ever listen to the radio? If yes, which radio station(s)?  Do you ever listen to Tabriz radio? 197 Main interview questions with the authorities of the kindergartens:  Is there a governmental and official policy as to which language you should use in the kindergarten? If yes, what is it?  If no, is there a policy set by the institute itself with respect to the language of the instruction? If yes, what is it?  If no, what language is used as the medium instruction then?  Why? 198
W4317459858.txt	http://fiver.ifvcns.rs/bitstream/123456789/3210/1/bitstream_9025.pdf	en		Seedborne fungi on stored onion seeds	Zbornik Matice srpske za prirodne nauke	2,022	cc-by	1,252	МАТИЦА СРПСКА MATICA SRPSKA СЕДМИ МЕЂУНАРОДНИ НАУЧНИ СКУП THE 7th INTERNATIONAL SCIENTIFIC MEETING МИКОЛОГИЈА, МИКОТОКСИКОЛОГИЈА И МИКОЗЕ MYCOLOGY, MYCOTOXICOLOGY, AND MYCOSES Књига резимеа Book of abstracts 2 – 3. ЈУН 2022. / 2 – 3 JUNE 2022 МАТИЦА СРПСКА, НОВИ САД, СРБИЈА / MATICA SRPSKA, NOVI SAD, SERBIA ГЛАВНИ ОРГАНИЗАТОР СКУПА / MAIN ORGANIZER OF THE MEETING Матица српска, Нови Сад, Србија Matica Srpska, Novi Sad, Serbia СУОРГАНИЗАТОРИ СКУПА / CO-ORGANIZERS OF THE MEETING Технолошки факултет, Универзитет у Новом Саду, Србија Faculty of Technology, University of Novi Sad, Serbia Природно-математички факултет, Универзитет у Новом Саду, Србија Faculty of Sciences, University of Novi Sad, Novi Sad, Serbia Пољопривредни факултет, Универзитет у Новом Саду, Србија Faculty of Agriculture, University of Novi Sad, Serbia Научни институт за ветеринарство Србије, Србија Scientific Veterinary Institute of Serbia, Serbia Научни институт за ветеринарство „Нови Сад”, Србија Scientific Veterinary Institute „Novi Sad”, Serbia Институт за ратарство и повртарство у Новом Саду, Србија Institute of Field and Vegetable Crops in Novi Sad, Serbia Удружење микробиолога Србије, Србија Serbian Society for Microbiology, Serbia The 7th International Scientific Meeting: Mycology, Mycotoxicology, and Mycoses II Научни одбор Седмог међународног научног скупа: Микологија, микотоксикологија и микозе Scientific Committee of the 7th International Scientific Meeting: Mycology, Mycotoxicology, and Mycoses Проф. др Милан Матавуљ, председник (СРБИЈА) Prof. dr Milan Matavulj, President (SERBIA) Проф. др Ференц Баги (СРБИЈА) Prof. dr Ferenc Bagi (SERBIA) Проф. др Александра Булајић (СРБИЈА) Prof. dr Aleksandra Bulajić (SERBIA) Проф. др Ђузепе Вентурела (ИТАЛИЈА) Prof. dr Guiseppe Venturella (ITALY) Проф. др Гордана Димић (СРБИЈА) Prof. dr Gordana Dimić (SERBIA) Др Весна Жупунски, научни сарадник (СРБИЈА) Dr Vesna Župunski, research associate (SERBIA) Др Мануела Задравец, виши научни сарадник (ХРВАТСКА) Dr Manuela Zadravec, senior research associate (CROATIA) Проф. др Алесандра Замбонели (ИТАЛИЈА) Prof. dr Alessandra Zambonelli (ITALY) Проф. др Георгиос Зервакис (ГРЧКА) Prof. dr Georgios Zervakis (GREECE) Проф. др Игор Јајић (СРБИЈА) Prof. dr Igor Jajić (SERBIA) Проф. др Бреда Јаковац Страјн (СЛОВЕНИЈА) Prof. dr Breda Jakovac Strajn (SLOVENIA) Др Елизабет Јанић Хајнал (СРБИЈА) Dr Elizabet Janić Hajnal (SERBIA) Др Марко Јауковић, научни сарадник (СРБИЈА) Dr Marko Jauković, research associate (SERBIA) Проф. др Радивоје Јевтић, научни саветник (СРБИЈА) Prof. dr Radivoje Jevtić, science advisor (SERBIA) Проф. др Митко Караделев (СЕВЕРНА МАКЕДОНИЈА) Prof. dr Mitko Karadelev (North Macedonia) Проф. др Маја Караман (СРБИЈА) Prof. dr Maja Karaman (SERBIA) The 7th International Scientific Meeting: Mycology, Mycotoxicology, and Mycoses III Академик Рудолф Кастори (СРБИЈА) Akademic Rudolf Kastori (SERBIA) Проф. др Анита Клаус (СРБИЈА) Prof. dr Anita Klaus (SERBIA) Др Александар Кнежевић, виши научни сарадник (СРБИЈА) Dr Aleksandar Knežević, senior research associate (SERBIA) Др Јована Кос, виши научни сарадник (СРБИЈА) Dr Jovana Kos, senior research associate (SERBIA) Проф. др Костас Коутсоманис (ГРЧКА) Prof. dr Kostas Koutsoumanis (GREECE) Доц. др Сунчица Koцић-Танацков (СРБИЈА) Doc. dr Sunčica Kocić-Tanackov (SERBIA) Др Весна Крњаја, научни саветник (СРБИЈА) Dr Vesna Krnjaja, science advisor (SERBIA) Проф. др Рудолф Крска (АУСТРИЈА) Prof. dr Rudolf Krska (AUSTRIA) Проф. др Сомањана Кхатуа (ИНДИЈА) Prof. dr Somanjana Khatua (INDIA) Проф. др Милица Љаљевић Грбић (СРБИЈА) Prof. dr Milica Ljaljević Grbić (SEBRIA) Проф. др Ксенија Марков (ХРВАТСКА) Prof. dr Ksenija Markov (CROATIA) Проф. др Стеван Маширевић (СРБИЈА) Prof. dr Stevan Maširević (SERBIA) Др Драгана Милошевић, виши научни сарадник (СРБИЈА) Dr Dragana Milošević, senior research associate (SERBIA) Др Армин Мешић, виши научни сарадник (ХРВАТСКА) Dr Armin Mešić, senior research associate (CROATIA) Др Антонио Морети (ИТАЛИЈА) Dr Antonio Moretii (ITALY) Проф. др Јелена Недељковић-Траиловић (СРБИЈА) Prof. dr Jelena Nedeljković-Trailović (SERBIA) Проф. др Виктор Недовић (СРБИЈА) Prof. dr Viktor Nedović (SERBIA) Проф. др Худа Нето (МАУРИЦИЈУС) Prof. dr Hudaa Neetoo (MAURITIUS) The 7th International Scientific Meeting: Mycology, Mycotoxicology, and Mycoses IV Др Ксенија Нешић, виши научни сарадник (СРБИЈА) Dr Ksenija Nešić, senior research associate (SERBIA) Проф. др Драгојло Обрадовић (Србија) Prof. dr Dragajlo Obradović (SERBIA) Др Марија Павловић (СРБИЈА) Dr Marija Pavlović (SERBIA) Проф. др Хрвоје Павловић (ХРВАТСКА) Prof. dr Hrvoje Pavlović (CROATIA) Проф. др Петер Распор (СЛОВЕНИЈА) Prof. dr Peter Raspor (SLOVENIA) Проф. др Катерина Русевска (СЕВЕРНА МАКЕДОНИЈА) Prof. dr Katerina Rusevska (NORTH MACEDONIA) Проф. др Соња Смоле-Можина (СЛОВЕНИЈА) Prof. dr Sonja Smole-Možina (SLOVENIA) Др Славица Станковић, научни саветник (СРБИЈА) Dr Slavica Stanković, science advisor (SERBIA) Др Биљана Стојановска Димзоска (СЕВЕРНА МАКЕДОНИЈА) Dr Biljana Stojanovska Dimzoska (NORTH MACEDONIA) Проф. др Љиља Торовић (СРБИЈА) Prof. dr Ljilja Torović (SERBIA) Др Војислав Тркуља (РЕПУБЛИКА СРПСКА) Dr Vojislav Trkulja (REPUBLIKA SRPSKA) Др Игор Ујчић Врховник (СЛОВЕНИЈА) Dr Igor Ujčić Vrhovnik (SLOVENIA) Проф. др Зехра Хајрулаи-Мусли (СЕВЕРНА МАКЕДОНИЈА) Prof. dr Zehra Hajrulai-Musliu (NORTH MACEDONIA) Доц. др Бојан Шаркањ (ХРВАТСКА) Doc. dr Bojan Šarkanj (CROATIA) Проф. др Данијела Сморгровичова (СЛОВАЧКА) Prof. dr Daniela Šmogrovičová (SLOVAKIA) Проф. др Ван Абд Ал Кадр Имад Ван Мохтар (МАЛЕЗИЈА) Prof. dr Wan Abd Al Qadr Imad Wan Mohtar (MALESIA) The 7th International Scientific Meeting: Mycology, Mycotoxicology, and Mycoses V Организациони одбор Седмог међународног научног скупа: Микологија, микотоксикологија и микозе Organizing Committee of the 7th International Scientific Meeting: Mycology, Mycotoxicology, and Mycoses Доц. др Сунчица Koцић-Танацков, председник (СРБИЈА) Doc. dr Sunčica Kocić-Tanackov, President (SERBIA) Др Јована Трбојевић Јоцић, секретар (СРБИЈА) Dr Jovana Trbojević Jocić, Secretary (SERBIA) Доц. др Саша Деспотовић (СРБИЈА) Doc. dr Saša Despotović (SERBIA) Проф. др Симонида Ђурић (СРБИЈА) Prof. dr Simonida Đurić (SERBIA) Др Добрила Јакић-Димић, научни саветник (СРБИЈА) Dr Dobrila Jakić-Dimić, science advisor (SERBIA) Др Сандра Јакшић, виши научни сарадник (СРБИЈА) Dr Sandra Jakšić, senior research associate (SERBIA) Проф. др Маја Караман (СРБИЈА) Prof. dr Maja Karaman (SERBIA) Проф. др Јелена Пејин (СРБИЈА) Prof. dr Јелена Пејин (SERBIA) The 7th International Scientific Meeting: Mycology, Mycotoxicology, and Mycoses VI Садржај / Content МИКОТОКСИКОЛОГИЈА / MYCOTOXICOLOGY 1‒23 МИКОЛОГИЈА / MYCOLOGY 24‒57 МИКОЗЕ / MYCOSES 58‒79 The 7th International Scientific Meeting: Mycology, Mycotoxicology, and Mycoses IX МИКОЗЕ / MYCOSES SEEDBORNE FUNGI ON STORED ONION SEEDS Slađana Medić-Pap1, Sonja Tančić-Živanov1, Dario Danojević1, Maja Ignjatov1, Aleksandra Ilić1, Jelica Gvozdanović Varga1 1 Institute of Field and Vegetable Crops, National Institute of the Republic of Serbia, Novi Sad, Serbia Corresponding author: sladjana.medicpap@ifvcns.ns.ac.rs Seed as a highly-valuable resource is preserved in collections for many years. Although the seed is kept under optimal conditions, monitoring of germination and the presence of fungi during seed preservation is of great importance. Therefore, the aim of this paper was to examine the seed health status and germination of forty-three onion accessions kept in the timespan of 15 years in the Institute of Field and Vegetable Crops collection. Germination of seed samples varied from 7-93%. The presence of fungi in the collection was determined on thirty-three tested samples. Fungi from the genera Acremonium, Alternaria, Aspergillus, Cladosporium, Epicoccum, Fusarium and Penicillium were developed. The following Fusarium species identified on the seeds were F. proliferatum, F. graminearum, F. sporotrichoides, F. solani, F. pseudograminearum and F. equiseti. Based on factor analysis, Fusarium and Penicillium were the species which affected germination, while the occurrence of Alternaria species on onion seed is connected to the year of harvest. Key words: onion, seed, fungi, germination, collection. The 7th International Scientific Meeting: Mycology, Mycotoxicology, and Mycoses 68 CIP - Каталогизација у публикацији Библиотеке Матице српске, Нови Сад 582.28(048.3) MЕЂУНАРОДНИ научни скуп "Микологија, микотоксикологија и микозе" (7 ; 2022 ; Нови Сад) Књига резимеа [Електронски извор] / Седми међународни научни скуп "Микологија, микотоксикологија и микозе", 2–3. јун 2022, Нови Сад = Book of abstracts / The 7th international scientific meeting "Mycology, mycotoxicology, and mycoses", 2–3 June 2022, Novi Sad. - Novi Sad : Matica srpska, 2022. - 1 elektronski optički disk (CD-ROM) : tekst ; 12 cm Nasl. sa naslovnog ekrana. ISBN 978-86-7946-387-6 а) Микологија - Апстракти COBISS.SR-ID 67647241 The 7th International Scientific Meeting: Mycology, Mycotoxicology, and Mycoses 80
https://openalex.org/W2094588855	https://europepmc.org/articles/pmc4029282?pdf=render	English	null	Comparisons of normal saline and lactated Ringer’s resuscitation on hemodynamics, metabolic responses, and coagulation in pigs after severe hemorrhagic shock	Scandinavian journal of trauma, resuscitation and emergency medicine	2,013	cc-by	10,262	Abstract Background: Ongoing improvements in trauma care now recommend earlier use of blood products as part of damage control resuscitation, but generally these products are not available at far forward battlefield locations. For the military, questions continue to arise regarding efficacy of normal saline (NS) vs. lactated Ringer’s (LR). Thus, this study compared the effects of LR and NS after severe hemorrhage in pigs. Methods: 20 anesthetized pigs were randomized into control (n = 6), LR (n = 7), and NS (n = 7) groups. Hemorrhage of 60% estimated total blood volume was induced in LR and NS groups by removing blood from the left femoral artery using a computer-controlled pump. Afterwards, the pigs were resuscitated with either LR at 3 times the bled volume or the volume of NS to reach the same mean arterial pressure (MAP) as in LR group. Hemodynamics were measured hourly and blood samples were taken at baseline (BL), 15 min, 3 h and 6 h after resuscitation to measure changes in coagulation using thrombelastograph®. Results: MAP was decreased by hemorrhage but returned to BL within 1 h after resuscitation with LR (119 ± 7 ml/kg) or NS (183 ± 9 ml/kg, p < 0.05). Base excess (BE) was decreased by hemorrhage; resuscitation with LR recovered BE but not with NS. Total peripheral resistance was decreased with NS and LR, with a larger drop shown in NS. Serum potassium was increased with NS, but not affected with LR. Coagulation changes were similar between LR and NS. Results: MAP was decreased by hemorrhage but returned to BL within 1 h after resuscitation with LR (119 ± 7 ml/kg) or NS (183 ± 9 ml/kg, p < 0.05). Base excess (BE) was decreased by hemorrhage; resuscitation with LR recovered BE but not with NS. Total peripheral resistance was decreased with NS and LR, with a larger drop shown in NS. Serum potassium was increased with NS, but not affected with LR. Coagulation changes were similar between LR and NS. Conclusions: NS may be inferior to LR in resuscitation due to its vasodilator effects and the risks of metabolic acidosis and hyperkalemia. Keywords: Hemorrhagic shock, Oxygen metabolism, Coagulation, Pre-hospital resuscitation and Throm survival benefit [7]. NS contains 154 mM Na+ and Cl-, with an average pH of 5.0 and osmolarity of 308 mOsm/L. ORIGINAL RESEARCH Open Access Comparisons of normal saline and lactated Ringer’s resuscitation on hemodynamics, metabolic responses, and coagulation in pigs after severe hemorrhagic shock Wenjun Z Martini, Douglas S Cortez and Michael A Dubick Martini et al. Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine 2013, 21:86 http://www.sjtrem.com/content/21/1/86 ORIGINAL RESEARCH Open Access Comparisons of normal saline and lactated Ringer’s resuscitation on hemodynamics, metabolic responses, and coagulation in pigs after severe hemorrhagic shock Wenjun Z Martini, Douglas S Cortez and Michael A Dubick Martini et al. Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine 2013, 21:86 http://www.sjtrem.com/content/21/1/86 Martini et al. Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine 2013, 21:86 http://www.sjtrem.com/content/21/1/86 Martini et al. Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine 2013, 21:86 http://www.sjtrem.com/content/21/1/86 Abstract LR solution has an average pH of 6.5, is hypo-osmolar (272 mOsm/L), and has similar electrolytes (130 mM Na+, 109 mM Cl-, 28 mM lactate, etc.) to plasma; thus, it was considered a more physiologically compatible fluid than NS. Since its initial use in 1831 for treating cholera [8,9], NS has been recommended as therapeutic intravenous (IV) fluid in various clinical situations, such as kidney transplantation, blood storage, and blood transfusion [10,11], although development of hyperchloremic acidosis is commonly observed [12-14]. LR’s acid base balance is superior to that of NS’s [15,16], but it has not been Comparisons of normal saline and lactated Ringer’s resuscitation on hemodynamics, metabolic responses, and coagulation in pigs after severe hemorrhagic shock Comparisons of normal saline and lactated Ringer’s resuscitation on hemodynamics, metabolic responses, and coagulation in pigs after severe hemorrhagic shock Wenjun Z Martini, Douglas S Cortez and Michael A Dubick Wenjun Z Martini, Douglas S Cortez and Michael A Dubick © 2013 Martini et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. * Correspondence: wenjun.z.martini.civ@mail.mil US Army Institute of Surgical Research, JBSA Ft, 3698 Chambers Pass, Sam Houston, TX 78234, USA Background During sur- gical instrumentation, anesthesia was maintained with 1% to 3% isoflurane in 30% oxygen in air using a ventila- tor and monitor (Fabius gas anesthesia system and Infinity Explorer monitoring system, Draeger Medical, Telford, PA). Tidal volume was set at 7 ml/kg with a rate of 25 breaths/minute. Ventilation was adjusted to reach an end tidal pCO2 of approximately 40 mmHg at baseline. Afterwards, no adjustments in ventilation setting were made throughout the experiment. Polyvinyl chloride catheters were inserted into the thoracic aorta via the carotid artery to measure mean arterial pressure (MAP), systolic and diastolic blood pressure, and heart rate. A Swan-Ganz thermodilution catheter was inserted in the pulmonary artery via the left jugular vein to meas- ure cardiac output and temperature. The right femoral artery was cannulated for arterial blood sampling and in- duction of bleeding. The right jugular vein was cannulated for venous blood sampling. The left femoral vein was cannulated for resuscitation. The right femoral vein was cannulated for IV anesthesia during the study. No splenec- tomy was performed in this study. approved by the Association of American Blood Bank for use with blood products in the same iv-line [11]. Thus, it is necessary and clinically relevant to comprehensively and systemically examine the resuscitative effects of these two crystalloids. Limited experimental evidence has contributed to the difficulty over the selection of resuscitation fluids. LR’s better acid base balance and survival has been shown in a rat model with massive hemorrhage [17] and an un- anesthetized swine model with rapid hemorrhage [18], suggesting the superiority of LR under severe trauma setting. In pigs with uncontrolled hemorrhage, Kiraly et al. showed that LR resuscitation was associated with hypercoagulability and less blood loss over a 2-hour post-injury period [19]. It is unclear whether the hyper- coagulability is transient or is likely to be a concern. On the other hand, a recent multicenter survey of prehospi- tal intervention at a combat zone showed that IV fluids administered in the field favored the use of NS by 73% to 17% LR [20], even though Hextend® (manufactured by BioTime, California, USA, with 6% hetastarch, a mean molecular weight of 670 kD and a degree of hydro- xyethyl group substitution of 0.75 (HES 670/0.75)) is the fluid recommended by the Committee on Tactical Com- bat Casualty Care [21]. Background Normal saline (NS) and lactated Ringer’s (LR) solution have been used as crystalloid fluids for decades [1,2], but controversies continue as to which crystalloid is best. Although damage control resuscitation was introduced recently to initiate early use of blood products for se- verely injured hypotensive trauma patients [3-6], blood products are not generally available at pre-hospital and far forward military settings. Crystalloids remain to be used as resuscitation fluids under these conditions with Page 2 of 12 America Regent, Shirley, NY, USA) and Telazol® (Tileta- mine HCl and Zolazepam HCl, 6 mg/kg, Pfizer, NY, NY, USA). Anesthesia was induced via a facemask with approximately 5% Isoflurane (Forane, Baxer, Deerfield, IL, USA) in 100% Oxygen. The pigs were then intubated with a cuffed endotracheal tube (7.5 mm, Rusch, Tele- flex Medical, Research Triangle Park, NC). During sur- gical instrumentation, anesthesia was maintained with 1% to 3% isoflurane in 30% oxygen in air using a ventila- tor and monitor (Fabius gas anesthesia system and Infinity Explorer monitoring system, Draeger Medical, Telford, PA). Tidal volume was set at 7 ml/kg with a rate of 25 breaths/minute. Ventilation was adjusted to reach an end tidal pCO2 of approximately 40 mmHg at baseline. Afterwards, no adjustments in ventilation setting were made throughout the experiment. Polyvinyl chloride catheters were inserted into the thoracic aorta via the carotid artery to measure mean arterial pressure (MAP), systolic and diastolic blood pressure, and heart rate. A Swan-Ganz thermodilution catheter was inserted in the pulmonary artery via the left jugular vein to meas- ure cardiac output and temperature. The right femoral artery was cannulated for arterial blood sampling and in- duction of bleeding. The right jugular vein was cannulated for venous blood sampling. The left femoral vein was cannulated for resuscitation. The right femoral vein was cannulated for IV anesthesia during the study. No splenec- tomy was performed in this study. America Regent, Shirley, NY, USA) and Telazol® (Tileta- mine HCl and Zolazepam HCl, 6 mg/kg, Pfizer, NY, NY, USA). Anesthesia was induced via a facemask with approximately 5% Isoflurane (Forane, Baxer, Deerfield, IL, USA) in 100% Oxygen. The pigs were then intubated with a cuffed endotracheal tube (7.5 mm, Rusch, Tele- flex Medical, Research Triangle Park, NC). Background In addition, the Israeli Defense Force has recently recommended LR as initial resuscita- tion of their combat casualties [22]. Thus, the greater use of NS by medics than the recommendation for Hextend® [20,21], recommendation for pre-hospital LR by the Israeli Defense Force [22], and the inquiry from the US Army’s Combat Developer’s office regarding NS vs. LR for pre-hospital resuscitation, all together prompted this current study. In order to assess more comprehensively the physiological and biochemical effects of LR and NS, we compared the resuscitative effects of LR and NS on hemodynamics, oxygen metabolism, and coagulation in a swine model with severe hemorrhagic shock over a 6 h post resuscitation period; the time period in which most trauma patients die from hemorrhage [23]. Upon completion of surgical procedures, anesthesia was switched to a combination of isoflurane (0.5%) and continuous IV drip of Ketacine® (Ketamine HCl, 0.25 mg/ (kgmin), Putney, Boise, ID, USA) in all pigs throughout the entire study period. After a 10-min stabilization period, blood samples were taken from the femoral artery and the right jugular vein for baseline measurements. The urine bag was emptied before urine output measurement started at baseline. Hemorrhage was then induced by bleeding from the femoral artery into sterile empty blood bags containing standard anticoagulant citrate phosphate dextrose solution. The pigs were hemorrhaged 60% of their estimated total blood volume exponentially over 60 min using a computer-controlled pump as described in our laboratory [24]. Upon the completion of hemorrhage, pigs were randomly assigned to LR or NS group. In the LR group, pigs were resuscitated with LR at 3 times the bled volume given over a 45-min period. In the NS group, resuscitation with NS was given to reach the same MAP as achieved after resuscitation in the LR group. Therefore, an NS resuscitated pig was done after a LR resuscitated pig. The LR of 3 times the bled volume was used since this ratio was conventionally recommended as initial fluid re- suscitation of hemorrhagic shock for prehospital trauma life support [25]. Pigs in the sham control group were not hemorrhaged or resuscitated. Pigs in all three groups were Martini et al. Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine 2013, 21:86 http://www.sjtrem.com/content/21/1/86 Experimental design This animal study was approved by the Institutional Ani- mal Care and Use Committee of the US Army Institute of Surgical Research and was conducted in compliance with the Animal Welfare Act and the implementing Animal Welfare Regulations and in accordance with the principles of the Guide for the Care and Use of Laboratory Animals. A total of 20 pigs (34.6 ± 1.2 kg) were randomly allocated into three experimental groups: sham control (n = 6), hemorrhage with LR resuscitation group (LR, n = 7), and hemorrhage with NS resuscitation group (NS, n = 7). After an overnight fast, the animals were pre-anesthetized with Glycopyrrolate (Glycopyrronium bromide, 0.1 mg/kg, Martini et al. Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine 2013, 21:86 http://www.sjtrem.com/content/21/1/86 Martini et al. Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine 2013, 21:86 http://www.sjtrem.com/content/21/1/86 Page 3 of 12 Oxygen delivery was calculated as cardiac output (L/min) × arterial oxygen content (ml O2/100 ml blood) × 10/ body wt (kg). Oxygen consumption (VO2) was calculated from the arterial (CaO2) and mixed venous (CvO2) oxygen content using established formulas. Percentage oxygen extraction was calculated as (CaO2 – CvO2)/CaO2. As we described previously [26], oxygen demand was calculated as plasma lactate (mmol/L from the arterial blood gas sample) + oxygen consumption (ml O2/min/kg), using the assumption as per Hannon and colleagues [27]. The oxy- gen delivery to oxygen demand ratio, an index of oxygen debt, was calculated as oxygen delivery/oxygen demand. given the same amount of anesthesia and maintenance fluid (0.04 ml/kg/min) and no significant changes in hemodynamics and coagulation were observed in the sham control pigs during the entire study period, suggesting the stability of the anesthesia regimen. The entire study was depicted in Figure 1. No heparin was used in this study. given the same amount of anesthesia and maintenance fluid (0.04 ml/kg/min) and no significant changes in hemodynamics and coagulation were observed in the sham control pigs during the entire study period, suggesting the stability of the anesthesia regimen. The entire study was depicted in Figure 1. No heparin was used in this study. All pigs were observed for 6 h after the completion of resuscitation. Blood samples were taken at baseline, after hemorrhage, 15 min, 3 h, and 6 h after resuscitation for measurements of blood gas, blood chemistry, and coagu- lation (Figure 1). Experimental design At the end of 6 h, surviving animals were euthanized with Fatal-Plus® (sodium pentobarbital, 100 mg/kg, Vortech, Dearborn, MI, USA, intravenously). Martini et al. Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine 2013, 21:86 http://www.sjtrem.com/content/21/1/86 Hemodynamic and metabolic measures Measurements of blood gas [pH, base excess, lactate, hematocrit (Hct), bicarbonate (HCO3 -), etc.] and blood chemistry (total protein, electrolytes, etc.) were deter- mined by standard clinical laboratory analysis. Hct and platelet counts were measured from citrated blood using an ABX Pentra 120 Hematology Analyzer (ABX Diagnos- tics, Inc., Irvine, CA). Plasma fibrinogen concentration, prothrombin time (PT), and activated partial prothrombin MAP and heart rate were recorded continuously during the study. Cardiac output was determined by thermodilu- tion in triplicate at baseline, at the end of hemorrhage, and then hourly throughout the remainder of the study. Stroke volume (SV) was calculated as cardiac output (ml/ min)/heart rate (beat/min). Total peripheral resistance was calculated as (MAP-CVP)(mmHg)/CO (L/min). 0 Time (h) Blood sampling 3 Study Protocol Hemorrhage (60%) 6 LR resuscitation (1:3) NS resuscitation (to reach the same MAP as in LR) Sham Control (no hemorrhage or resuscitation) - Blood sampling for blood chemistry, blood gas, and coagulation analysis Figure 1 Experimental protocol. Study Protocol Sham Control (no hemorrhage or resuscitation) Hemorrhage (60%) - Blood sampling for blood chemistry, blood gas, and coagulation analysis Figure 1 Experimental protocol - Blood sampling for blood chemistry, blood gas, and coagulation analysis Martini et al. Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine 2013, 21:86 http://www.sjtrem.com/content/21/1/86 Page 4 of 12 time (aPTT) were measured using the BCS Coagulation System (Dade Behring, Deerfield, IL). time (aPTT) were measured using the BCS Coagulation System (Dade Behring, Deerfield, IL). No significant changes in cardiac output, stroke volume, total peripheral resistance, oxygen content, or oxygen ex- traction were observed in controls during the experiment (Table 1). No significant changes in temperature were ob- served in any groups during the study. Cardiac output returned to pre-hemorrhage levels at the end of LR resus- citation (15 min). In contrast, cardiac output was increased 50% over BL by NS resuscitation and remained elevated in this group for the 6 h experimental period. Stroke volume returned to pre-hemorrhage levels with NS resuscitation (15 min), but remained below pre-hemorrhage levels for the 6 h experimental period after LR resuscitation (Table 1). Total peripheral resistance was decreased after resuscita- tion with LR or NS, with a lower resistance shown in the NS group vs. the LR group (Table 1). Hemodynamic and metabolic measures The coagulation profile was assessed in fresh whole blood with pig thromboplastin by thrombelastograph® (TEG) using a TEG 5000 Hemostasis Analyzer (Haemo- scope, Niles, IL) In TEG measurements, reaction time (R time) is the latency time for initial clot formation, K time reflects the speed to reach a certain level of clot strength, angle α reflects the rapidity of clot build-up and cross-linking, MA represents the maximum strength or stiffness of the clot, and LY60 indicates the percentage of clot lysis at 60 min after maximum clotting is achieved. Statistical analysis D Data were expressed as means ± standard error of the mean (SEM) and analyzed using SAS statistical software (Cary, NC). A two-way analysis of variance (ANOVA) with repeated measures using a Tukey adjustment was used to compare the changes over time between the groups. A one-way ANOVA with repeated measures using a Dunnett adjustment was used to compare the changes to baseline within the group. The statistically significant level was set at p < 0.05. Oxygen metabolic responses to hemorrhage and resus- citation with LR or NS were described in Table 2 and Figure 3. Arterial PO2 and PCO2 were similar among the 3 groups during the study (Table 2). Venous PO2 returned to pre-hemorrhage levels 15 min after LR or NS resuscitation, but declined during the remaining 6 h with a larger decrease observed in LR group (Table 2). There were no differences in venous PCO2 among the 3 groups during the study (Table 2). Arterial oxygen con- tent was decreased for 6 h after hemorrhage and resusci- tation with LR or NS (Table 2). Venous oxygen content was also decreased for 6 h after hemorrhage and resusci- tation with LR or NS (Table 2). Oxygen extraction was increased for 6 h after resuscitation with LR solution or NS. Oxygen delivery was reduced after resuscitation with LR, with no significant changes observed in the NS group (Figure 3A). Oxygen consumption remained un- changed in all three groups during the study (Figure 3B). Oxygen demand did not change after NS resuscitation, but doubled immediately after LR resuscitation, but returned to baseline values by 3 h (Figure 3C). Oxygen delivery to oxygen demand ratio, an index of oxygen debt, was decreased after NS resuscitation but returned to baseline value within 3 h, whereas a larger drop was observed after LR resuscitation and remained low over the course of the study (Figure 3D). Martini et al. Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine 2013, 21:86 http://www.sjtrem.com/content/21/1/86 Hemodynamics, cardiac-pulmonary function, and oxygen metabolic responses Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine 2013, 21:86 http://www.sjtrem.com/content/21/1/86 Martini et al. Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine 2013, 21:86 http://www.sjtrem.com/content/21/1/86 Page 5 of 12 mmHg BPM 0 50 100 150 200 250 Baseline Hemo Resus 1h 2h 3h 4h 5h 6h Control LR NS 0 20 40 60 80 100 Baseline Hemo Resus 1h 2h 3h 4h 5h 6h Control LR NS mL Mean Arterial Pressure Heart Rate Urine Output 0 200 400 600 800 1000 Baseline Hemo Resus 1h 2h 3h 4h 5h 6h Control LR NS * * * * * * * * * * * * * * * * * * * * * * * * * * * * Figure 2 Changes in mean arterial pressure (MAP), heart rate (HR), and urine output after hemorrhage and resuscitation with lactated Ringer’s solution (LR, 119 ± 7 ml/kg) or normal saline (NS, 183 ± 9 ml/kg) in pigs. p < 0.05 compared to the corresponding baseline values. ♣p < 0.05 compared to corresponding control values. Hemodynamics, cardiac-pulmonary function, and oxygen metabolic responses mmHg BPM 0 50 100 150 200 250 Baseline Hemo Resus 1h 2h 3h 4h 5h 6h Control LR NS 0 20 40 60 80 100 Baseline Hemo Resus 1h 2h 3h 4h 5h 6h Control LR NS Mean Arterial Pressure Heart Rate * * * * * * * * * * * * * * * * * * * * * * * mmHg 0 20 40 60 80 100 Baseline Hemo Resus 1h 2h 3h 4h 5h 6h Control LR NS Mean Arterial Pressure * * * * * * * * * * * * * * mmHg 0 20 40 60 80 100 Baseline Hemo Resus 1h 2h 3h 4h 5h 6h Control LR NS Mean Arterial Pressure * * * * * * * * * * * * * * Mean Arterial Pressure BPM 0 50 100 150 200 250 Baseline Hemo Resus 1h 2h 3h 4h 5h 6h Control LR NS Heart Rate * * * * * * * * * * mL Urine Output 0 200 400 600 800 1000 Baseline Hemo Resus 1h 2h 3h 4h 5h 6h Control LR NS * * * * * Figure 2 Changes in mean arterial pressure (MAP), heart rate (HR), and urine output after hemorrhage and resuscitation with lactated Ringer’s solution (LR, 119 ± 7 ml/kg) or normal saline (NS, 183 ± 9 ml/kg) in pigs. p < 0.05 compared to the corresponding baseline values. ♣p < 0.05 compared to corresponding control values. differences in Hct (Table 1) or total protein between LR and NS groups during the 6 h after resuscitation. Bicarbonate HCO3 - levels were decreased by hemorrhage but returned to pre-hemorrhage values by 3 h after LR resuscitation, whereas no return was observed with NS resuscitation (Figure 4). Arterial pH was decreased from a pre-hemorrhage value of 7.41 ± 0.02 to 7.35 ± 0.01 after NS resuscitation (p < 0.05) but returned to pre-hemorrhage value at the 3 h time point. No significant changes in arter- ial pH occurred in the control or LR group during the study. Base excess fell below zero after hemorrhage and returned to pre-hemorrhage levels with LR resuscitation by 3 h but remained below pre-hemorrhage levels for 6 h with NS resuscitation (Figure 4). Hemodynamics, cardiac-pulmonary function, and oxygen metabolic responses All animals survived to the end of the 6 h study. No sig- nificant changes in the hemodynamics were observed in the control group over the experimental period. A 60% hemorrhage dropped MAP from the baseline value of 79 ± 6 mm Hg to 31 ± 3 mm Hg (p < 0.05). In the LR group, resuscitation with LR at 3 times bled volume (119 ± 7 ml/kg) led to a MAP that peaked briefly near pre-hemorrhage levels, but then fell and remained at hypotensive levels around 60 mmHg (Figure 2). In the NS group, 183 ± 9 ml/kg of NS was infused to achieve MAP similar to that in the LR group (Figure 2). Heart rate was increased by hemorrhage, followed by a partial decline after resuscitation in both groups. Heart rate returned to near baseline levels in the NS group, but remained higher over baseline in the LR group (Figure 2). Urine output rose with resuscitation in both groups with a larger increase in the NS group, followed by a decline to baseline values at 2 h after resuscitation in the LR group (Figure 2). From the start of resuscitation to 15 min after the completion of resuscitation, urine output in the NS group (23 ± 5 ml/kg) doubled that in the LR group (10 ± 3 ml/kg, with 1 ± 0 ml/kg in the control group). The total urine output over the 6 h resuscitation period in the NS group (45 ± 5 ml/kg) also doubled that in the LR group (21 ± 4 ml/kg, with 10 ± 1 ml/kg in the control group, all p < 0.05 LR versus NS, and LR or NS versus control). Hemodilution, acid base balance, and electrolytes No significant changes in Hct, base excess, or lactate concentrations were observed in the control group over the entire experimental period. Significant hemodilution was observed after hemorrhage and resuscitation with LR or NS (Table 1). Total protein dropped from baseline value of 5.6 ± 0.2 g/dL to 2.7 ± 0.1 g/dL at 15 min and 3.3 ± 0.1 g/dL at 6 h after LR resuscitation and from baseline value of 5.7 ± 0.2 g/dL to 3.0 ± 0.2 g/dL at 15 min and 3.5 ± 0.2 g/dL at 6 h after NS resuscitation (all p < 0.05 vs. baseline). There were no significant Martini et al. Hemodynamics, cardiac-pulmonary function, and oxygen metabolic responses p < 0.05 compared to corresponding baseline values within the group; §p < 0.05 LR vs NS groups; and ♣p < 0.05 compared to corresponding control values. Table 1 Changes of cardiac-pulmonary functions and hemodilution after hemorrhage and resuscitation with lactated Ringer’s (LR) solution or normal saline (NS) in pigs Baseline Resuscitation 3 h 6 h Cardiac output (L/min) Control 3.9 ± 0.3 4.3 ± 0.3 4.4 ± 0.4 3.6 ± 0.2 LR 3.7 ± 0.2 3.9 ± 0.3 3.5 ± 0.2 3.3 ± 0.2 NS 3.8 ± 0.3 5.9 ± 0.5§♣ 4.7 ± 0.3§ 4.5 ± 0.2§♣ Stroke volume (ml/beat) Control 36.5 ± 3.5 36.8 ± 2.2 36.8 ± 3.7 35.5 ± 3.9 LR 41.4 ± 3.3 24.9 ± 1.6§♣ 21.4 ± 2.3§ 24.7 ± 2.1§ NS 38.2 ± 4.9 45.7 ± 4.7 38.1 ± 3.3 40.1 ± 3.3 Total Peripheral resistance (mm Hg/(L/min)) Control 20.4 ± 1.4 18.6 ± 2.0 17.3 ± 2.6 17.7 ± 1.3 LR 22.9 ± 1.0 17.7 ± 0.9* 17.9 ± 1.3* 17.4 ± 0.7* NS 23.1 ± 1.8 12.5 ± 1.4§ 12.5 ± 0.7§ 12.0 ± 0.4§ Hct (%) Control 29.0 ± 0.8% 28.9 ± 0.9% 28.0 ± 0.9% 27.6 ± 1.0% LR 30.2 ± 1.0% 13.1 ± 0.9%♣ 14.0 ± 0.7%♣ 14.7 ± 0.6%♣ NS 29.0 ± 1.0% 11.6 ± 0.8 %♣ 12.5 ± 0.6%♣ 12.2 ± 0.7%♣ Animal groups include control (C, n = 6), hemorrhage with LR resuscitation (LR, n = 6), and hemorrhage with normal saline resuscitation (NS, n = 6). Data are expressed as means ± SE. p < 0.05 compared to corresponding baseline values within the group; §p < 0.05 LR vs NS groups; and ♣p < 0.05 compared to corresponding control values. Table 1 Changes of cardiac-pulmonary functions and hemodilution after hemorrhage and resuscitation with lactated Ringer’s (LR) solution or normal saline (NS) in pigs Table 2 Changes in oxygen content and extraction after hemorrhage and resuscitation with lactated Ringer’s (LR) solution or normal saline (NS) in pigs There were no significant changes in Na+, K+, Ca++, or Cl- in the control group during the entire study period (Table 3). Na+ was increased after NS resuscitation but returned to pre-hemorrahge level within 6 h. K+ did not change initially after NS resuscitation but was elevated at 6 h afterwards (Table 3). No changes in Na+ or K+ were observed in pigs with LR resuscitation (Table 3). Hemodynamics, cardiac-pulmonary function, and oxygen metabolic responses Ca++ was similarly decreased at 15 min after resuscitation with LR or NS but returned to pre-hemorrhage levels by 6 h in both groups (Table 3). Cl- was elevated for 6 h after NS resuscitation, with no changes shown after LR resuscitation (Table 3). Hemodynamics, cardiac-pulmonary function, and oxygen metabolic responses Plasma lactate levels rose about 4.5-fold above baseline values by hemorrhage and returned to pre-hemorrhage value within 15 min after NS resuscitation and at 6 h after LR resuscitation (Figure 4). Martini et al. Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine 2013, 21:86 http://www.sjtrem.com/content/21/1/86 Martini et al. Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine 2013, 21:86 http://www.sjtrem.com/content/21/1/86 Martini et al. Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine 2013, 21:86 http://www.sjtrem.com/content/21/1/86 Page 6 of 12 Page 6 of 12 There were no significant changes in Na+, K+, Ca++, or Cl- in the control group during the entire study period (Table 3). Na+ was increased after NS resuscitation but returned to pre-hemorrahge level within 6 h. K+ did not change initially after NS resuscitation but was elevated at 6 h afterwards (Table 3). No changes in Na+ or K+ were observed in pigs with LR resuscitation (Table 3). Ca++ was similarly decreased at 15 min after resuscitation with LR or NS but returned to pre-hemorrhage levels by 6 h in both groups (Table 3). Cl- was elevated for 6 h after NS resuscitation, with no changes shown after LR resuscitation (Table 3). Coagulation Plasma fibrinogen concentration in control pigs remained unchanged during the study. Plasma fibrinogen concen- tration was decreased from a baseline value of 193 ± 12 mg/dL to 95 ± 5 mg/dL after LR resuscitation (p < 0.05) and from a baseline value of 189 ± 9 mg/dL to 101 ± 5 mg/dL after NS resuscitation (p < 0.05). Platelet count was decreased from the baseline value of 359 ± 54 103/μL to 172 ± 28 103/μL after LR resuscitation and from a base- line value of 427 ± 58 103/μL to 207 ± 29 103/μL after NS resuscitation (both p < 0.05). There were no significant differences between LR and NS groups in fibrinogen concentrations or platelet count during the 6 h after resuscitation There were no changes in TEG measurements in the control group during the study (Figure 5). R time and K times were shortened by hemorrhage but returned to near pre-hemorrhage values after resuscita- tion with LR or NS (Figure 5). Clotting speed (α-angle) was increased by hemorrhage and returned to pre- hemorrhage values after LR or NS resuscitation (Figure 5). Hemodynamics, cardiac-pulmonary function, and oxygen metabolic responses Clot strength (MA) was not changed by hemorrhage but was similarly reduced by resuscitation with LR or NS, followed by return to baseline values within 3 h after resuscitation (Figure 5). Hemodynamics, cardiac-pulmonary function, and oxygen metabolic responses No significant changes in fibrin- olysis (LY60) were observed in any group during the study (data not shown) Table 2 Changes in oxygen content and extraction after hemorrhage and resuscitation with lactated Ringer’s (LR) solution or normal saline (NS) in pigs Baseline Resuscitation 3 h 6 h Arterial O2 (mmHg) Control 475 ± 18 460 ± 15 497 ± 16 470 ± 15 LR 493 ± 15 513 ± 10 515 ± 15 499 ± 11 NS 478 ± 25 474 ± 21 490 ± 17 470 ± 16 Arterial CO2 (mmHg) Control 47 ± 4 48 ± 4 45 ± 3 45 ± 4 LR 44 ± 3 43 ± 3 45 ± 3 43 ± 2 NS 47 ± 2 43 ± 2 44 ± 2 44 ± 1 Venous O2 (mmHg) Control 55 ± 5 60 ± 4 58 ± 5 50 ± 5 LR 56 ± 2 53 ± 3 36 ± 1♣ 36 ± 1♣ NS 54 ± 2 53 ± 2 46 ± 2♣ 43 ± 1♣§ Venous CO2 (mmHg) Control 60 ± 5 57 ± 4 50 ± 3 51 ± 5 LR 53 ± 2 50 ± 2 56 ± 2 56 ± 1 NS 59 ± 5 50 ± 2 55 ± 1 55 ± 1 Arterial oxygen content (ml O2/dL blood) Control 13.8 ± 0.6 13.8 ± 0.5 13.6 ± 0.3 13.3 ± 0.7 LR 14.7 ± 0.3 7.6 ± 0.3♣ 8.5 ± 0.6♣ 7.8 ± 0.4♣ NS 13.2 ± 0.9 6.4 ± 0.4♣ 7.9 ± 0.4♣ 7.3 ± 0.4♣ Venous oxygen content (ml O2/dL blood) Control 9.9 ± 0.5 9.9 ± 0.7 9.3 ± 0.6 7.7 ± 0.9 LR 10.8 ± 0.2 4.3 ± 0.2♣ 3.5 ± 0.2♣ 3.1 ± 0.2♣ NS 8.6 ± 0.6 4.1 ± 0.3♣ 4.2 ± 0.3♣ 3.8 ± 0.3♣ Oxygen extraction (%) Control 28 ± 4 29 ± 3 32 ± 3 32 ± 3 LR 28 ± 2 43 ± 2* 57 ± 3* 60 ± 3* NS 35 ± 5 36 ± 3 47 ± 3* 49 ± 3* Animal groups include control (C, n = 6), hemorrhage with LR resuscitation (LR, n = 6), and hemorrhage with normal saline resuscitation (NS, n = 6). Data are expressed as means ± SE. Coagulation l f b There were no changes in TEG measurements in the control group during the study (Figure 5). R time and K times were shortened by hemorrhage but returned to near pre-hemorrhage values after resuscita- tion with LR or NS (Figure 5). Clotting speed (α-angle) was increased by hemorrhage and returned to pre- hemorrhage values after LR or NS resuscitation (Figure 5). Clot strength (MA) was not changed by hemorrhage but was similarly reduced by resuscitation with LR or NS, followed by return to baseline values within 3 h after resuscitation (Figure 5). No significant changes in fibrin- olysis (LY60) were observed in any group during the study (data not shown). Plasma fibrinogen concentration in control pigs remained unchanged during the study. Plasma fibrinogen concen- tration was decreased from a baseline value of 193 ± 12 mg/dL to 95 ± 5 mg/dL after LR resuscitation (p < 0.05) and from a baseline value of 189 ± 9 mg/dL to 101 ± 5 mg/dL after NS resuscitation (p < 0.05). Platelet count was decreased from the baseline value of 359 ± 54 103/μL to 172 ± 28 103/μL after LR resuscitation and from a base- line value of 427 ± 58 103/μL to 207 ± 29 103/μL after NS resuscitation (both p < 0.05). There were no significant differences between LR and NS groups in fibrinogen concentrations or platelet count during the 6 h after resuscitation. Martini et al. Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine 2013, 21:86 http://www.sjtrem.com/content/21/1/86 Page 7 of 12 0 2 4 6 8 10 12 14 16 18 Baseline Resuscitation 3h 6h Control LR NS 0 1 2 3 4 5 6 7 8 Baseline Resuscitation 3h 6h Control LR NS 0 2 4 6 8 10 12 14 16 Baseline Resuscitation 3h 6h Control LR NS 0 1 2 3 4 Baseline Resuscitation 3h 6h Control LR NS A. Oxygen delivery (ml O2/min/kg) B. Oxygen consumption (ml O2/min/kg) C. Oxygen demand (ml O2/min/kg) D. Oxygen delivery/ Oxygen demand * § * * * § * § * § * * Figure 3 Changes in oxygen delivery (A), oxygen consumption (B), oxygen demand (C), and oxygen delivery/oxygen demand (D) after hemorrhage and resuscitation with lactated Ringer’s solution (LR, 119 ± 7 ml/kg) or normal saline (NS, 183 ± 9 ml/kg) in pigs. p < 0.05 compared to the corresponding baseline values. Coagulation l f b §p < 0.05 LR group compared to NS group. ♣p < 0.05 compared to corresponding control values. A. Oxygen delivery (ml O2/min/kg) B. Oxygen consumption (ml O2/min/kg) 6h 0 Baseline Resuscitation D. Oxygen delivery/ Oxygen demand C. Oxygen demand (ml O2/min/kg) Figure 3 Changes in oxygen delivery (A), oxygen consumption (B), oxygen demand (C), and oxygen delivery/oxygen demand (D) after hemorrhage and resuscitation with lactated Ringer’s solution (LR, 119 ± 7 ml/kg) or normal saline (NS, 183 ± 9 ml/kg) in pigs. p < 0.05 compared to the corresponding baseline values. §p < 0.05 LR group compared to NS group. ♣p < 0.05 compared to corresponding control values. Plasma PT or PTT was not changed by hemorrhage. PT was similarly prolonged by resuscitation with LR (from 11.2 ± 0.2 sec at baseline to 12.1 ± 0.2 sec at 6 h) and NS (from 11.2 ± 0.3 sec at baseline to 12.3 ± 0.3 sec at 6 h, both p < 0.05). Plasma aPTT was also similarly prolonged by resuscitation with LR (from 17.1 ± 0.5 sec baseline to 20.1 ± 1.2 sec at 6 h) or NS (from 17.1 ± 0.3 sec baseline to 19.1 ± 0.5 sec at 6 h, both p < 0.05). oxygen delivery-to-oxygen demand ratio as an index of oxygen debt. This better response appears to be primarily due to vasodilation effects as suggested by the large in- crease in cardiac output compared to the LR group. Thus, in the current severe hemorrhage model, NS had better tis- sue perfusion and oxygen metabolism than LR. However, since the oxygen delivery to demand ratio was 1 or higher in the LR and NS groups at 6 hrs, both fluids seemed adequate in maintaining survival at least through 6 h. Discussion Significant separation was observed between LR and NS in acid base balance in this study, as expected. LR resuscitation returned BE and bicarbonate to pre- hemorrhage levels within 3 h, but no return of BE or bicarbonate was observed for 6 hr with NS resuscitation. Similar changes of BE and bicarbonate from LR or NS have been shown in different animal models and in patients [18,19,28]. The impact of acid base status from resuscita- tion fluids on survival has been assessed by Traverso et al. in an anaesthetized swine model with fatal hemorrhage [18]. With equal volume resuscitation from NS (pH 5.0), LR (pH 6.5, 28 mM lactate), and Plasmalyte-A (pH 7.4, 27 mM acetate), the LR group had the best survival and the Plasmalyte-A had the worst survival rate [18]. But BE Using a large-animal model with severe hemorrhage and 6 h follow-up, we performed a comprehensive assessment of the physiologic and biochemical effects of LR and NS. To achieve a similar hypotensive resuscitation level 50% more volume of normal saline was required than LR to achieve similar responses in MAP. Hemodilution, however, was not different between groups indicating that that the additional NS did not stay in the vascular, as supported by the greater urine output in the NS group. In addition, NS resulted in a lower peripheral resistance and a higher stroke volume, possibly due to its vasodilation effects. Des- pite similar oxygen extraction and oxygen consumption, NS resuscitation resulted in better oxygen delivery and Martini et al. Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine 2013, 21:86 Page 8 of 12 http://www.sjtrem.com/content/21/1/86 Martini et al. Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine 2013, 21:86 http://www.sjtrem.com/content/21/1/86 Martini et al. Discussion Since the metabolism of lactate in humans takes a few hours and involves producing H+ and HCO3 - [31], a similar time frame observed in the present study indicates simi- lar lactate metabolism in humans and pigs. In addition, current blood bank guidelines state that LR should not be mixed with blood to prevent the risk of clot forma- tion from calcium included in LR, which can diminish the anti-coagulation effect in stored blood. Thus, LR resuscitation should not be given with blood through the same iv-line and crystalloids should be avoided in patients with blood transfusion. Hemorrhage and resuscitation caused disturbances in the coagulation process. PT and aPTT were prolonged for 6 h after hemorrhage and resuscitation, suggesting a hypocoagulable states. In contrast, the TEG data suggested a hypercoagulable states. These differences between TEG and standard plasma assays were also observed in burn and trauma patients [32,33]. Clotting initiation was shortened, and clotting speed was ac- celerated by hemorrhage; but both returned to pre- hemorrhage values after resuscitation. Clot strength was compromised after resuscitation but returned within 3 h. Despite these dynamic changes in the coagulation pro- file, there were no differences between LR and NS resus- citation during the study, suggesting the equivalent effects of the two fluids on coagulation. In contrast, a faster clotting speed and better clot strength from LR resuscitation were reported by Kiraly et al. [19]. This dis- crepancy is likely due to the differences in study design. The present study used a swine model with controlled hemorrhage with 50% more volume of NS, whereas Kiraly et al. used a swine model of liver injury and an uncontrolled hemorrhage with 150% more volume of NS used. The additional hemodilution from the 150% NS might contribute to the different findings. In addition, a sham control group was included in the present study and our data showed that coagulation profiles from LR and NS resuscitation returned to pre-hemorrhage values and became similar to those of the control values. Thus, potential thrombotic risk from LR resuscitation is unlikely. patients resuscitated with LR or NS, although the patient group with NS resuscitation was more acidotic. Using a rat model with hemorrhage and simultaneous resuscitation, Healey et al. found that NS and LR had equivalent survival rates under moderate hemorrhage (36% of estimated blood volume). Discussion Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine 2013, 21:86 http://www.sjtrem.com/content/21/1/86 Page 8 of 12 BE 0 2 4 6 8 10 12 14 Baseline Hemorrhage Resuscitation 3h 6h Control LR NS 0 5 10 15 20 25 30 35 40 Baseline Hemorrhage Resuscitation 3h 6h Control LR NS Lactate HCO3- mM mM mM -6 -4 -2 0 2 4 6 8 10 12 Baseline Hemorrahge Resuscitation 3h 6h Control LR NS * * § * * § * § * * * * * * * * * § * § Figure 4 Changes in acid-base status after hemorrhage and resuscitation with lactated Ringer’s solution (LR, 119 ± 7 ml/kg) or normal saline (NS, 183 ± 9 ml/kg) in pigs. p < 0.05 compared to the corresponding baseline values. §p < 0.05 LR group compared to NS group. ♣p < 0.05 compared to corresponding control values. BE mM -6 -4 -2 0 2 4 6 8 10 12 Baseline Hemorrahge Resuscitation 3h 6h § * § * * * * mM mM Figure 4 Changes in acid-base status after hemorrhage and resuscitation with lactated Ringer’s solution (LR, 119 ± 7 ml/kg) or normal saline (NS, 183 ± 9 ml/kg) in pigs. p < 0.05 compared to the corresponding baseline values. §p < 0.05 LR group compared to NS group. ♣p < 0.05 compared to corresponding control values. Clinical trials have failed to show significant differences in outcomes of LR and NS resuscitation. In comparative trials of LR and NS performed in patients who underwent ab- dominal aortic aneurysm repair or renal transplant, Waters et al. [15] and Khajavi et al. [28] reported that there were no differences in clinical outcomes, such as duration of ven- tilation, ICU stay, or incidence of complication between and pH from LR and Plasmalyte-A resuscitation were simi- lar and higher than those from NS [18], suggesting a better acid base status was not necessarily attributable to a better survival. Nevertheless, as an index of shock, improved BE should be a goal to improve outcome and acidosis is asso- ciated with well-known detrimental effects on the cardio- vascular system and coagulation [29,30]. Page 9 of 12 Martini et al. Discussion Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine 2013, 21:86 http://www.sjtrem.com/content/21/1/86 Table 3 Changes of electrolytes after hemorrhage and resuscitation with lactated Ringer’s (LR) solution or normal saline (NS) in pigs Baseline After Hemorrhage and Resuscitation 6 h Na+ (mM) Control 138 ± 3 139 ± 6 141 ± 2 LR 140 ± 3 138 ± 3 132 ± 3 NS 141 ± 2 150 ± 3§♣ 144 ± 6 K+ (mM) Control 4.5 ± 0.2 4.7 ± 0.4 4.6 ± 0.2 LR 4.3 ± 0.2 4.3 ± 0.2 5.2 ± 0.4 NS 4.5 ± 0.2 4.5 ± 0.3 5.7 ± 0.3* Ca++ (mM) Control 9.2 ± 0.2 9.4 ± 0.3 8.9 ± 0.1 LR 9.5 ±0.4 7.7 ± 0.3♣ 8.3 ± 0.8 NS 9.4 ± 0.3 7.9 ± 0.2♣ 9.0 ± 0.3 Cl - (mM) Control 97 ± 2 109 ± 8 100 ± 1 LR 101 ± 2 102 ± 2 97 ± 3 NS 102 ± 3 121 ± 6* 123 ± 6* Animal groups include control (C, n = 6), hemorrhage with LR resuscitation (LR, n = 6), and hemorrhage with normal saline resuscitation (NS, n = 6). Data are expressed as means ± SE. p < 0.05 compared to corresponding baseline values within the group; §p < 0.05 LR vs NS groups; and ♣p < 0.05 compared to corresponding control values. Table 3 Changes of electrolytes after hemorrhage and resuscitation with lactated Ringer’s (LR) solution or normal saline (NS) in pigs net fluid inflow of 4.1 L. During the same period a total of 6.1 ± 0.1 L of NS was infused for resuscitation while urine output was 0.81 ± 0.18 L, with a net fluid inflow of 4.9 L. This 20% volume difference (4.1 L of LR vs. 4.9 L of NS) would not be able to fully explain the observed 3.5fold difference of lactate between the LR (8.7 mM) and NS (2.6 mM) group. Thus, the difference of lactate levels at 15 min after resuscitation may relate to the lactate load from LR resuscitation, at least in part. How- ever, this difference gradually disappeared within 6 h with a simultaneous increase of HCO3 - in the LR group, suggesting that an ongoing lactate metabolism in pigs and the production of bicarbonate may contribute to the better acid-base status from lactate resuscitation. Discussion TEG-K time Control LR Control LR NS Figure 5 Changes in Thrombelastograph® (TEG) measurements after hemorrhage and resuscitation with lactated Ringer’s solution (LR, 119 ± 7 ml/kg) or normal saline (NS, 183 ± 9 ml/kg) in pigs. R time: latency time for initial fibrin formation. K time: a measure of speed to reach a certain level of clot strength. Angle a: the rapidity of fibrin build up and cross-linking. MA: maximum strength of the developed clot. p < 0.05 compared to the corresponding baseline values. In order to make a valid comparison, a common physiological endpoint after resuscitation is needed to assess the effects of resuscitation. In this study, we used post-resuscitation MAP as the physiological endpoint to compare the effects of LR and NS on hemodynamics, coagulation and metabolism. We found that NS required 50% more volume to reach the same physiological end- point. If the same volume of resuscitation was used in LR and NS groups, we suspected that the blood pressure after NS resuscitation would be lower than that of LR due to its vasodilator effects. volumes infused and more hemodilution and coagula- tion impairment. Considering that restriction on resus- citation fluid has been shown improving survival in uncontrolled bleeding patients [35] and animals [36], a larger volume of NS, as compared to LR, would increase bleeding with resultant higher morbidities and mortality in trauma patients. In this study, to reach the same resuscitative physio- logical endpoint, NS required 50% more volume and was associated with a higher cardiac output and lower periph- eral resistance, as compared to LR resuscitation. These dif- ferences are possibly due to the vasodilator effects from NS. It is worth emphasizing that these effects are likely to be exaggerated under uncontrolled hemorrhage, resulting in more fluid volume, a larger hemodilution, and more se- vere impairment in coagulation. Further, an elevation of K+ was observed at 6 h post NS resuscitation, while no change of K+ was observed after LR resuscitation. The mechanism for the increase of K+ from NS is not fully known, but may be due to an extracellular shift of potassium caused by changes in blood hydrogen ion concentration, as the result A fixed volume controlled hemorrhage model was used in this study to investigate metabolic, hemodynamic and coagulation effects of hypovolemia and fluid resuscitation. Discussion But with a massive hemorrhage (218% of esti- mated blood volume), LR resuscitation resulted in better survival [17]. Thus, it appears that the separation of out- comes from LR and NS becomes apparent only under ex- treme circumstance, such as when greater than 1 blood volume of fluid is given. Lactate rose about 5 fold after 60% hemorrhage in this study and returned to pre-hemorrhage levels after resus- citation with LR or NS. The recovery rates, however, were different: NS returned lactate to pre-hemorrhage level within 15 min, but lactate did not return to near pre-hemorrhage level for at least 3 h after resuscitation. At 15 min after resuscitation, lactate levels were 8.7 ± 1.2 mM in the LR group and 2.6 ± 0.8 mM in the NS group. This difference could result from lactate load from LR resuscitation or from a larger-volume dilution from NS resuscitation. Calculation of fluid inflow and outflow suggested that the latter is unlikely. From the start of resuscitation to 15 min after the completion of resuscitation, a total of 4.6 ± 0.3 L of LR was infused and urine output was 0.46 ± 0.15 L in the LR group, with a Martini et al. Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine 2013, 21:86 Page 10 of 12 http://www.sjtrem.com/content/21/1/86 Martini et al. Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine 2013, 21:86 http://www.sjtrem.com/content/21/1/86 Page 10 of 12 0 1 2 3 4 5 6 7 Control LR NS 0 1 1 2 2 3 3 4 Control LR NS 50 60 70 80 Control LR NS 50 60 70 80 Control LR NS TEG-K time TEG-R time TEG-MA TEG-Angle (α) min min mm Degree * * * * * * * * Figure 5 Changes in Thrombelastograph® (TEG) measurements after hemorrhage and resuscitation with lactated Ringer’s solution (LR, 119 ± 7 ml/kg) or normal saline (NS, 183 ± 9 ml/kg) in pigs. R time: latency time for initial fibrin formation. K time: a measure of speed to reach a certain level of clot strength. Angle a: the rapidity of fibrin build up and cross-linking. MA: maximum strength of the developed clot. *p < 0.05 compared to the corresponding baseline values. Acknowledgments h h 20. Lairet JR, Bebarta VS, Burns CJ, Lairet KF, Rasmussen TE, Renz EM, King BT, Fernandez W, Gerhardt R, Butler F, et al: Prehospital interventions performed in a combat zone: a prospective multicenter study of 1,003 combat wounded. J Trauma Acute Care Surg 2012, 73(2 Suppl 1):S38–S42. The authors appreciate the support received from the Veterinary Service Support Branch and Laboratory Support Branch at the U.S. Army Institute of Surgical Research. The authors appreciate the support received from the Veterinary Service Support Branch and Laboratory Support Branch at the U.S. Army Institute of Surgical Research. 21. Butler FK: Tactical combat casualty care: update 2009. J Trauma 2010, 69(Suppl 1):S10–S13. This study was supported by the U.S. Army Medical Research and Materiel Command. The opinions or assertions contained herein are the private views of the author and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense. 22. Lipsky AM, Ganor O, Abramovich A, Katzenell U, Glassberg E: Walking between the drops: Israeli defense forces’ fluid resuscitation protocol. J Emerg Med 2013, 44(4):790–795. Received: 22 August 2013 Accepted: 5 December 2013 Published: 11 December 2013 23. Holcomb JBSP: Optimal use of blood in trauma patients. Biologicals 2010, 38(1):6. 24. Burns JW, Baer LA, Darlington DN, Dubick MA, Wade CE: Screening of potential small volume resuscitation products using a severe hemorrhage sedated swine model. Int J Burns Trauma 2012, 2(1):59–67. Discussion This model mimics situations where trauma patients bleed an amount of blood before hemorrhage is con- trolled [34]. However, it is limited in reflecting coagula- tion changes under a clinical scenario of continued bleeding (or rebleeding). It is likely the on-going bleed- ing from uncontrolled hemorrhage will exaggerate the impact of vasodilation effects from NS, with higher Page 11 of 12 of hyperchloremic acidosis from NS resuscitation [37]. Consistent with our current results, clinically significant hyperkalemia from NS administration has been reported in patients during renal transplantation [16,28]. Thus, NS is associated with vasodilator effects and the risks of meta- bolic acidosis and hyperkalemia. Currently, military first responders have NS, LR and Hextend available [20]. How- ever, the results from the current study and our recent study [38] suggest that all of these fluids have limitations in managing hemodynamics, metabolic responses, and co- agulation. The optimum resuscitation fluid awaits further development. of hemostatic resuscitation: a multi-institutional analysis. J Trauma Acute Care Surg 2013, 75(1):76–82. Carlino W: Damage control resuscitation from major haemorrhage in polytrauma. Eur J Orthop Surg Traumatol 2013 [Epub ahead of print] Dutton RP: Haemostatic resuscitation. Br J Anaesth 2012, 109(Suppl 1 7. Spoerke N, Michalek J, Schreiber M, Brasel KJ, Vercruysse G, MacLeod J, Dutton RP, Duchesne JC, McSwain NE, Muskat P, et al: Crystalloid resuscitation improves survival in trauma patients receiving low ratios of fresh frozen plasma to packed red blood cells. J Trauma 2011, 71(2 Suppl 3):S380–S383. 8. Foex BA: How the cholera epidemic of 1831 resulted in a new technique for fluid resuscitation. Emerg Med J 2003, 20(4):316–318. 9. Awad S, Allison SP, Lobo DN: The history of 0.9% saline. Clin Nutr 2008, 27(2):179–188. 10. Belani KG, Palahniuk RJ: Kidney transplantation. Int Anesthesiol Clin 1991, 29(3):17–39. 11. Banks AAoB: Accreditation Requirements Manual. 5th edition. Arlington: American Association of Blood Banks; 1994. Authors’ contributions 19. Kiraly LN, Differding JA, Enomoto TM, Sawai RS, Muller PJ, Diggs B, Tieu BH, Englehart MS, Underwood S, Wiesberg TT, et al: Resuscitation with normal saline (NS) vs. lactated ringers (LR) modulates hypercoagulability and leads to increased blood loss in an uncontrolled hemorrhagic shock swine model. J Trauma 2006, 61(1):57–64. WM designed and performed the study, analyzed the data with assistance from statistician Mr John Jones, and wrote the manuscript. DC performed the study. MD designed study and edited the manuscript. All authors read and approved the final manuscript. Competing interests The authors declare that they have no competing interests. Competing interests The authors declare that they have no competing interests. 18. Traverso LW, Hollenbach SJ, Bolin RB, Langford MJ, DeGuzman LR: Fluid resuscitation after an otherwise fatal hemorrhage: II. Colloid solutions. J Trauma 1986, 26(2):176–182. Conclusions 12. Wilkes NJ, Woolf R, Mutch M, Mallett SV, Peachey T, Stephens R, Mythen MG: The effects of balanced versus saline-based hetastarch and crystalloid solutions on acid-base and electrolyte status and gastric mucosal perfusion in elderly surgical patients. Anesth Analg 2001, 93(4):811–816. We compared resuscitative effects of NS and LR in a swine model with 60% controlled hemorrhage. Although LR and NS had equivalent effects on hemodynamics and oxygen metabolism, NS required a larger resuscitation volume and was associated with poor acid base status and elevated serum potassium in this model. Thus, NS may be inferior to LR in resuscitation due to its vasodilator effects and the risks of metabolic acidosis and hyperkalemia. 13. Williams EL, Hildebrand KL, McCormick SA, Bedel MJ: The effect of intravenous lactated Ringer’s solution versus 0.9% sodium chloride solution on serum osmolality in human volunteers. Anesth Analg 1999, 88(5):999–1003. 14. Scheingraber S, Rehm M, Sehmisch C, Finsterer U: Rapid saline infusion produces hyperchloremic acidosis in patients undergoing gynecologic surgery. Anesthesiology 1999, 90(5):1265–1270. 15. Waters JH, Gottlieb A, Schoenwald P, Popovich MJ, Sprung J, Nelson DR: Normal saline versus lactated Ringer’s solution for intraoperative fluid management in patients undergoing abdominal aortic aneurysm repair: an outcome study. Anesth Analg 2001, 93(4):817–822. Abbreviations BE B BE, Base excess; BL, Baseline; IV, Intravenous; LR, Lactated Ringer’s; MAP, Mean arterial pressure; NS, Normal saline; PT, Prothrombin time; aPTT, Activated partial prothrombin time; TEG, Thrombelastography®; TEG measurements, ; R time, Latency time for initial fibrin formation; K time, a measure of speed to reach a certain level of clot strength; Angle α, the rapidity of fibrin build up and cross-linking; MA, Maximum strength of the clot; LY60, Percentage of clot lysis at 60 min after MA is achieved. 16. 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Hahn RG: Fluid therapy in uncontrolled hemorrhage–what experimental models have taught us. Acta Anaesthesiol Scand 2013, 57(1):16–28. 37. Halperin ML, Kamel KS: Potassium. Lancet 1998, 352(9122):135–140. 38. Martini WZ, Dubick MA, Blackbourne LH: Comparisons of lactated Ringer’s and Hextend resuscitation on hemodynamics and coagulation following femur injury and severe hemorrhage in pigs. References J Trauma Acute Care Surg 2013, 74(3):732–740. 38. Martini WZ, Dubick MA, Blackbourne LH: Comparisons of lactated Ringer’s and Hextend resuscitation on hemodynamics and coagulation following femur injury and severe hemorrhage in pigs. J Trauma Acute Care Surg 2013, 74(3):732–740. doi:10.1186/1757-7241-21-86 Cite this article as: Martini et al.: Comparisons of normal saline and lactated Ringer’s resuscitation on hemodynamics, metabolic responses, and coagulation in pigs after severe hemorrhagic shock. Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine 2013 21:86. doi:10.1186/1757-7241-21-86 Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission
https://openalex.org/W2225925591	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0146491&type=printable	English	null	Model Selection in Historical Research Using Approximate Bayesian Computation	PloS one	2,016	cc-by	8,239	RESEARCH ARTICLE Editor: John P. Hart, New York State Museum, UNITED STATES Editor: John P. Hart, New York State Museum, UNITED STATES Received: May 25, 2015 Accepted: December 17, 2015 Published: January 5, 2016 Xavier Rubio-Campillo* Computer Applications in Science & Engineering department, Barcelona Supercomputing Centre, Barcelona, Spain Computer Applications in Science & Engineering department, Barcelona Supercomputing Centre, Barcelona, Spain * xavier.rubio@bsc.es * xavier.rubio@bsc.es * xavier.rubio@bsc.es a1111 Case Study This work examines an alternate approach to this evaluation based on a Bayesian-inspired model selection method. The validity of the classical Lanchester’s laws of combat is exam- ined against a dataset comprising over a thousand battles spanning 300 years. Four varia- tions of the basic equations are discussed, including the three most common formulations (linear, squared, and logarithmic) and a new variant introducing fatigue. Approximate Bayesian Computation is then used to infer both parameter values and model selection via Bayes Factors. Copyright: © 2016 Xavier Rubio-Campillo. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: Dataset is distributed under the terms of a Creative Commons Attribution- ShareAlike 4.0 International License and accessible at https://github.com/xrubio/lanchester Formal Models and History Computational models are increasingly being used to study historical dynamics. This new trend, which could be named Model-Based History, makes use of recently published data- sets and innovative quantitative methods to improve our understanding of past societies based on their written sources. The extensive use of formal models allows historians to re- evaluate hypotheses formulated decades ago and still subject to debate due to the lack of an adequate quantitative framework. The initiative has the potential to transform the disci- pline if it solves the challenges posed by the study of historical dynamics. These difficulties are based on the complexities of modelling social interaction, and the methodological issues raised by the evaluation of formal models against data with low sample size, high variance and strong fragmentation. OPEN ACCESS Citation: Rubio-Campillo X (2016) Model Selection in Historical Research Using Approximate Bayesian Computation. PLoS ONE 11(1): e0146491. doi:10.1371/journal.pone.0146491 Introduction The discipline of History presents its ideas as descriptive models expressed in natural language. Historians use the flexibility of this communication system to explain the complexity and diversity of human societies though their written records. The approach is different than the majority of scientific disciplines, which formulate their theories in formal languages such as mathematics. Formal languages are not as flexible as natural languages, but they are much bet- ter defining concepts and relations without ambiguities [1]. Hypotheses defined in formal lan- guage can then be falsified against empirical evidence, and quantitative methods can then be applied to compare predictions generated by a theory to observed patterns. As a consequence, an old theory can be replace by a new one when it has superior explanatory power. This evaluation of ideas does not happen in History. Quantitative methods cannot be used to falsify descriptive models or perform cross-temporal and cross-spatial comparison. These inabilities are central to the current methodological debates of the discipline [2–6]. There is a clear desire to identify both the common trajectories and the observed differences between case studies with diverse spatiotemporal coordinates. However, it is unclear how this could be achieved using the common methods of the discipline. A possible approach to tackle this challenge is to shift the discipline from descriptive to for- mal models [7]. This innovation could allow historians to know under what extent a working hypothesis explains a historical dynamic by quantifying the distance between the predictions of a model and the patterns observed in the evidence. This new approach has clear benefits, but it is not an easy task as it requires a) formal models, b) quantified datasets and c) methods to compare both components. These debates are intrinsically linked with the increasing number of available databases and historical research using formal models [8–10]. The rise of what we could define as Model- Based History is changing the way researchers study historical trajectories [2]. To date, this new approach to the past has been focused on three main topics: trade networks [11], sociocul- tural evolution [7] and warfare [12–14]). The increase in the number of works is diversifying the topics examined by Model-Based History, and now it includes fields such as knowledge exchange [15] or the evolution of religion [16]. Quantitative comparison between models and observations is one of the advantages of this new approach. Impact Funding: Funding for this work was provided by the SimulPast Consolider Ingenio project (CSD2010- 00034) of the former Ministry for Science and Innovation of the Spanish Government and the European Research Council Advanced Grant EPNet (340828). Results indicate decisive evidence favouring the new fatigue model. The interpretation of both parameter estimations and model selection provides new insights into the factors guid- ing the evolution of warfare. At a methodological level, the case study shows how model selection methods can be used to guide historical research through the comparison between existing hypotheses and empirical evidence. Competing Interests: The author has declared that no competing interests exist. 1 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0146491 January 5, 2016 ONE \| DOI:10.1371/journal.pone.0146491 January 5, 20 Model Selection in Historical Research Using ABC Model Selection in Historical Research Using ABC increasing popularity due to the current debates on the use of statistics analysis for scientific research [19–23]. It is worth mentioning that neither method seems better than the other one, and the choice will depend on the aim of the research: Null Hypothesis Significance Testing aims to know if the observed process could be explained without the working hypothesis, while model selection aims to choose which hypothesis is better at matching evidence. The model selection approach provides a set of new methods to evaluate models. Most of them quantify the loss of information from each model to the evidence using information crite- ria [24]. Two of the most widely used methods are Akaike Information Criterion and Bayesian Information Criterion. Both of them fit the different models to the observed patterns using maximum likelihood methods, and then they calculate an index of information loss (i.e. low values indicate better models). A different solution is to use the Bayesian statistical framework. It is based on the idea that the knowledge of a given system with uncertainty can be gradually updated through new evi- dence. The process is achieved by computing the probability that a given hypothesis is correct, considering both existing knowledge and new data. The main advantage of this approach is that it seems better fitted to evaluate competing models under high levels of uncertainty and equifinality [25]. Despite its interest, scientific research did not start using the Bayesian frame- work until recent years, even if it was formulated 200 years ago [26]. The delay on the adoption was mainly caused by the mathematical complexities of applying Bayesian statistics to non- trivial problems. The development of new computational methods such as Markov Chain Monte-Carlo and Approximate Bayesian Computation (ABC) has mainly solved this limita- tion, thus explaining the current success of Bayesian inference. Historians constantly deal with competing explanations of uncertain datasets, so it seems that Bayesian model selection can be useful to the discipline. This potential can also be inferred from the fact that other historical disciplines such as biology and archaeology are part of this Bayesian renaissance. Biology is particularly active in using these methods in fields such as population genetics and ecology [27, 28]. Archaeology traditionally limited Bayesian inference to C14 dates [29], but model selection techniques are becoming popular beyond this applica- tion [30–33]. Materials and Methods Case study: the evolution of combat These examples suggest that Bayesian model selection can be applied to History, considering the similarities between the three disciplines. First, all these fields study temporal trajectories using data with high levels of uncertainty. Second, the analysis of these datasets implies that they need to evaluate the plausibility of multiple competing hypotheses. Finally, all of them want to identify patterns generated as an aggregate of individual behaviour. As a con- sequence, it seems clear that Bayesian model selection would have significant utility for historians. This paper presents the use of Bayesian inference to perform model selection in historical research. The utility of a Bayesian-inspired computational method known as Approximate Bayesian Computation is discussed. The use of ABC is then illustrated with a classical example of formal model used in History: the classical Lanchester’s laws of warfare. Next section pres- ents the case study, the model selection framework and the competing models. Third section shows the results of the method, both in terms of model selection and parameter estimation. The text then interprets these results and concludes with an evaluation of the approach in the context of Model-Based History. Introduction The most common statistical framework to perform this evaluation is Null Hypothesis Significance Testing. First, the problem to solve is defined as a clear research ques- tion and a working hypothesis H1. This hypothesis is a possible answer which could be falsified by existing evidence. The explanation provided by H1 will then compete against a null hypoth- esis H0. H0 is an alternative that does not take into account H1. H1 is translated into a formal model, usually a computer simulation of the dynamics encapsulated in the hypothesis. Model- Based History often prefers bottom-up techniques such as Agent-Based Models [17] or com- plex network analysis [18]. Classical equation-based models are also applied, but these innova- tive approaches seem better suited to the type of social processes examined by the discipline. The created model defines the system at a small-scale level (e.g. individual or groups), and it evolves through the interaction between these entities. The emergence of distinctive large-scale patterns generated by this set of interactions is then compared to empirical data. If the proba- bility of getting the observed patterns without H1 is less than a given confidence interval (i.e. the p-value) we can reject H0, thus accepting H1. Null Hypothesis Significance Testing is useful to prove that our model has higher predictive power than a random process. However, it is not designed to compare multiple potentially valid explanations. Model selection is a different approach designed to quantify by how much a model is better at explaining evidence than alternate models. Model selection is having 2 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0146491 January 5, 2016 Model Selection in Historical Research Using ABC officers on managing armies and fighting the enemy. These practices had a major impulse dur- ing Second World War with the creation of Operations Research. This new research field focused on developing formal models able to help commanders on decision-making [34]. The introduction of the first computers expedited the use of these quantitative methods during the Cold War, establishing them as a standard procedure for training and planning. In contrast, History is only now incorporating some of these techniques to the study of past conflicts [35]. Boardgames, mathematical models and computer simulations are proving their utility in the task of studying warfare understood as an unfortunate part of human culture [36]. The theoretical model formulated by F.W. Lanchester in 1916 is one of the most popular mathematical formulations used in the field [37]. Lanchester aimed to design the laws predict- ing the casualties of two enemy forces engaged in land battle. He proposed a system of coupled differential equations where casualties were dependent on two factors: a) force size and b) fighting value. The first factor takes into account the importance of sheer numbers on the out- come of military conflict, while the second factor encapsulates qualitative differences between individual fighting skills (e.g. morale, training, technology, etc.). Two models were initially pro- posed: the linear law and the square law. The linear law aimed to capture the dynamics of ancient battles, where the supremacy of hand-to-hand combat meant that each soldier could only attack an opponent at a given moment. The equations defining the rate of casualties in a battle between armies Blue and Red are defined in Eq 1: dB dt ¼ rBR dR dt ¼ bRB ð1Þ ð1Þ with B, R as the size of the forces and r, b as their ﬁghting value. The rate of casualties is propor- tional to both sizes, so even highly disproportionate odds would cause similar casualties to both opponents. The square law models warfare after the introduction of gunpowder-based weapons. This technological innovation increased the range, thus allowing each soldier to attack multiple ene- mies. Case study: the evolution of combat Warfare is probably the first human activity ever explored with formal models. Their use began in early XIXth century in the form of boardgames such as kriegsspiel. They were used to train 3 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0146491 January 5, 2016 The squared law models the casualties of a force as the enemy’s force size multiplied by the fighting value of its individuals, as seen in Eq 2: dB dt ¼ rR dR dt ¼ bB ð2Þ ð2Þ The Lanchester’s laws generated a large amount of interest during the Cold War [38–42]. h d b d h l d f h l d l d d h f l g g g The debate was centred on the actual predictive power of the laws, and it included the formula- tion of alternate proposals such as the popular logarithmic model. It suggested that the casual- ties suffered by a force are not dependant on the enemy’s size, but on its own size as defined in Eq 3 [40]: dB dt ¼ rB dB dt ¼ bR ð3Þ ð3Þ Several works discussed the validity of the laws [42, 43]. Other contributions extended the original framework introducing concepts such as spatial structure or system dynamics [44, 45]. The utility of the model was also expanded beyond its initial purpose, and has been successfully applied to study competition dynamics in ecology [46–48], evolutionary biology [49] or eco- nomics [50]. Model selection were also applied to compare the plausibility of these models against histor- ical evidence. The most extensive effort was made by Charles D. Allen’s in his monograph [51]. The author tested the validity of different models to explain a dataset of 1080 land battles from the middle of XVIIth century to the beginnings of the XXth century. The analysis suggested 4 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0146491 January 5, 2016 Model Selection in Historical Research Using ABC that the logarithmic model has higher explanatory power than the two classical models. How- ever, the coarse-grained results assumed that this power remained constant during the whole period, thus not examining the validity of the models for the different phases of warfare. Simi- lar works used Bayesian inference to evaluate the Lanchester’s laws in specific scenarios. They included biological case studies [48], daily casualties during Inchon-Seoul campaign in 1950 [52] or attrition during the battle of the Ardennes in 1944 [53, 54]. All these results suggests that the Lanchester’s laws are useful to understand if casualties are more influenced by quantitative or qualitative factors. Some authors suggested that the models should introduce dynamic parameters such as variable fighting values or fatigue [44]. However, as some of these works highlights, a pure Bayesian framework could hardly cope with the mathematical difficulties added by this new complexity. The dataset. The dataset used in this study is based on Allen’s list of battles, originally compiled in a previous work [55]. The introduction of weapons with longer ranges over 300 years should be reflected in a gradual increase in the validity of the squared model over the lin- ear model. In order to test this idea the span has been divided in four periods, based on prior opinions of decisive transitions in the evolution of warfare [56]: 1. Pike and Musket (1620–1701). The first period was characterised by deep formations of sol- diers (i.e. tercios and regiments) armed with muskets and pikes. 1. Pike and Musket (1620–1701). The first period was characterised by deep formations of sol- diers (i.e. tercios and regiments) armed with muskets and pikes. 2. Linear warfare (1702–1792). The War of the Spanish Succession (1702–1714) saw a shift in battle tactics and technological innovations. Armies were deployed in thin formations exclu- sively armed with muskets, while pikes were substituted by bayonets. 3. Napoleonic Wars (1793–1860). The French Revolution forced another major transition in warfare, which was mainly adopted during the Napoleonic wars. The new concept of citizen armies allowed the states to increase the size of their forces up to the limits imposed by pre- industrial logistics. 4. American Civil War (1861–1905). The impact of industry development became explicit on the battlefield during the American Civil War. The size of armies and the lethality of their weapons steadily increased until fully industrialised armies were deployed in the Russo-Jap- anese War. This conflict was the prelude of what would be seen during the two world wars. Exploratory Data Analysis has been used to identify structural patterns in the dataset. A time series of the number of battles can be seen in Fig 1, while size and casualty ratios are depicted in Fig 2. These visualisations shows how the dataset has relative small sample size and high variance. These are common properties seen in historical data. The figures suggest that the number of battles remained constant during the 300 years with the exception of the Napo- leonic Wars. At the same time, the gradual increase on average army size seems linked to a decrease on casualty ratios. PLOS ONE \| DOI:10.1371/journal.pone.0146491 January 5, 2016 The model selection framework Standard Bayesian inference updates a set of prior beliefs considering new evidence and a given likelihood function. Prior beliefs aggregate the existing knowledge of a given topic, and the degree of credibility of this knowledge. These beliefs are translated into parameters of the model. The possible values for each parameter receive an initial probability following a specific statistical distribution. The likelihood function is used to compute the probabilities of any given result considering the value of the input parameters. The updated knowledge (i.e. the the 5 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0146491 January 5, 2016 simplicity and lack of assumptions makes it perfect for illustrative purposes. It is defined as follows: simplicity and lack of assumptions makes it perfect for illustrative purposes. It is defined as follows: 1. Initialise parameters sampling the prior distributions 2. Run the model and compute the distance to evidence 3. If distance is within the closest runs below a tolerance level τ keep values of parameters; oth- erwise discard them. This algorithm is executed a large number of runs, and the set of kept parameter values is used as the posterior distribution. Model Selection in Historical Research Using ABC Fig 1. Number of battles by decade. The three identified transitions correlate with periods of intensive warfare. doi:10.1371/journal.pone.0146491.g001 Fig 1. Number of battles by decade. The three identified transitions correlate with periods of intensive warfare. Fig 1. Number of battles by decade. The three identified transitions correlate with periods of intensive warfare. doi:10.1371/journal.pone.0146491.g001 doi:10.1371/journal.pone.0146491.g001 posterior distribution) is then computed following Bayes’ rule: posterior distribution) is then computed following Bayes’ rule: PðyjDÞ ¼ PðDjyÞ PðyÞ PðDÞ PðyjDÞ ¼ PðDjyÞ PðyÞ PðDÞ PðDÞ being θ the considered value and D the observed data. This can be translated as (following [57]): being θ the considered value and D the observed data. This can be translated as (following [57]): posterior ¼ likelihood prior evidence posterior ¼ likelihood prior evidence A barrier to the adoption of Bayesian inference is the difficulty to derive likelihood functions when the examined model is not a standard statistical distribution. This constraint limits the use of the framework for computer simulations encapsulating complex dynamics such as the ones explored in Model-Based History. A major breakthrough to this issue is the recent devel- opment of ABC [58, 59]. ABC comprises a family of computationally-intensive algorithms able to approximate pos- terior distributions without using likelihood functions. These methods identify the regions of the prior space producing the closest results to the evidence. This capability of extending the Bayesian framework to any computer simulation has exponentially increased the popularity of ABC during the last decade, including the other historical disciplines: biology [60–63], and archaeology [31, 33, 64, 65]). The analysis performed in this work implements the simplest ABC method: the rejection algorithm [66]. It is not the most efficient ABC method (see [67, 68] for alternatives), but its 6 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0146491 January 5, 2016 PLOS ONE \| DOI:10.1371/journal.pone.0146491 January 5, 2016 Model Selection in Historical Research Using ABC Fig 2. Size and casualty ratio by battle. The total number of soldiers involved in each battle is defined in the Y axis while the size of each point shows the casualty ratio of the battle. Fig 2. Size and casualty ratio by battle. The total number of soldiers involved in each battle is defined in the Y axis while the size of each point shows the casualty ratio of the battle. The total number of soldiers involved in each battle is defined in the Y axis while the size of each point shows the doi:10.1371/journal.pone.0146491.g002 doi:10.1371/journal.pone.0146491.g002 Definition of competing models We will evaluate the plausibility of four different variations of the Lanchester equations: the two original laws (linear and squared), the popular logarithmic variation and a new model add- ing fatigue effects. For convenience the models have been here transformed to difference equa- tions as seen in Eqs 4, 5 and 6: Linear: Btþ1 ¼ Bt rBtRt Rtþ1 ¼ Rt bRtBt ð4Þ ð4Þ 7 / 18 7 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0146491 January 5, 2016 Model Selection in Historical Research Using ABC Squared: Squared: Btþ1 ¼ Bt rRt Rtþ1 ¼ Rt bBt ð5Þ ð5Þ Logarithmic: Btþ1 ¼ Bt rBt Rtþ1 ¼ Rt bRt ð6Þ ð6Þ The fourth model adds fatigue to the logarithmic model. This factor is modelled as a gradual decrease in the efficiency of the armies as defined in Eq 7): Fatigue: Btþ1 ¼ Bt rBt logðe þ tÞ Rtþ1 ¼ Rt bRt logðe þ tÞ ð7Þ ð7Þ Fighting value b is scaled to the maximum number of casualties that B can inflict to R in a time step. In order to avoid disparate values b is defined following Eq 8 for the linear model and Eq 9 for the other three. Linear law: Linear law: b ¼ 100 Bt¼0 Rt¼0 ð8Þ ð8Þ Other models: b ¼ 100 maxðBt¼0; Rt¼0Þ ð9Þ ð9Þ The enemy’s fighting value r is then defined as b multiplied by an odds ratio P. In this way the individual value of a Red soldier is expressed as a ratio of Blue’s value (e.g. P = 2 would mean that each Red soldier is as lethal as two Blue soldiers). Distinctive dynamics for each model are observed in Fig 3. All models are initialised as a battle where an army is being opposed by a smaller force with higher fighting value. In the lin- ear model the forces have similar casualty rates, while size has a bigger impact in the squared model. The logarithmic model increases the weight of fighting value over size as the smaller force finishes with more soldiers. The fatigue model generates similar casualties than the loga- rithmic model, but they are distributed over a longer period of time. Experiment Design Previous authors suggested that the deterministic nature of the original laws was too rigid to perform a proper comparison with long-term observations. Using a fixed P for a large number of battles would ignore any slight variation on the fighting value odds from one engagement to the next one. The issue has been solved introducing stochasticity in P, which is sampled every battle from a gamma distribution with shape κ and scale θ. For convenience the input parame- ters are expressed as mean μ and standard deviation σ, which are then used to compute k ¼ m s 2 and y ¼ s2 m . The outcome of each battle is generated using as parameters the sampled P 8 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0146491 January 5, 2016 Model Selection in Historical Research Using ABC and initial army sizes Bt = 0, Rt = 0 set to historical values. The chosen Lanchester variant as deﬁned in Eqs 4–7 is then iterated until one of the forces has suffered as many casualties as recorded in the historical data. The entire workﬂow is depicted in Fig 4. The rejection algorithm calculates a distance between the results of a single run and obse ti A p p l pp h i th p i f t ti ti ti th t Fig 3. Time series of attrition. Casualties of two forces as computed by the four competing models with P = 1.5, Bt = 0 = 15000 and Rt = 0 = 10000. doi:10.1371/journal.pone.0146491.g003 Fig 3. Time series of attrition. Casualties of two forces as computed by the four competing models with P = 1.5, Bt = 0 = 15000 and Rt = 0 = 10000. doi:10 1371/journal pone 0146491 g003 Fig 3. Time series of attrition. Casualties of two forces as computed by the four competing models with P = 1.5, Bt = 0 = 15000 and Rt = 0 = 10000. doi:10.1371/journal.pone.0146491.g003 doi:10.1371/journal.pone.0146491.g003 doi:10.1371/journal.pone.0146491.g003 and initial army sizes Bt = 0, Rt = 0 set to historical values. The chosen Lanchester variant as deﬁned in Eqs 4–7 is then iterated until one of the forces has suffered as many casualties as recorded in the historical data. The entire workﬂow is depicted in Fig 4. The rejection algorithm calculates a distance between the results of a single run and observa- tions. Model selection The fatigue model is decisively selected for all periods when using the lowest τ = 0.0005 (see Fig 5 left). The two original models (linear and squared) are not present in this set comprising the best 500 runs, while the logarithmic is only present for the XVIIth century. Larger tolerance levels increase the relevance of the linear and logarithmic models, while the squared model is never selected. The estimation of distances in Fig 6 shows that the plausibility of the models is not constant over the different periods. The four models followed the same trend, as their ranks remain con- stant over the different phases. In addition, all of them performed much worse for the battles of the third period (i.e. Napoleonic wars). Model Selection in Historical Research Using ABC being discussed [69]. This experiment avoids the debate by directly comparing the set of casu- alties for each battle and side. The distance between a simulation run and evidence is the abso- lute difference between simulated and historical casualties divided by historical casualties, thus normalising the weight of all battles regardless their total size. This comparison is performed identifying both in the evidence and simulation the Red army R as the side with lower casualty ratio in each battle. Uninformed prior beliefs were used for the two parameters (μ and σ). The limits of their uniform distributions were defined as Uð0; 5Þ, based on Allen’s results. Each competing model was ran 1 million times for each period. Sensitivity to tolerance levels was accounted by storing posterior distributions for different thresholds (τ = 0.05, τ = 0.005 and τ = 0.0005). The model selection method is based on Bayes Factors. They quantify the relative likelihood of different competing models against the evidence expressed as an odds ratio [70]. This ratio was quantified with the common method of introducing a third parameter m as a model index variable [59]. It was used within a hierarchical model where m identified which of the four vari- ants of the Lanchester’s laws was used during the run. Bayes Factors are then computed as the posterior distribution of m within the tolerance level τ. Experiment Design A popular approach is the comparison of summary statistics aggregating the outcome of a run against the evidence. However, this solution has theoretical issues which are currently The rejection algorithm calculates a distance between the results of a single run and observa- tions. A popular approach is the comparison of summary statistics aggregating the outcome of a run against the evidence. However, this solution has theoretical issues which are currently 9 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0146491 January 5, 2016 Model Selection in Historical Research Using ABC xample for a experiment using 1000 runs and tolerance τ = 0.01. Left side illustrates the rejection algorithm f the Lanchester model. ework. Example for a experiment using 1000 runs and tolerance τ = 0.01. Left side illustrates mulation of the Lanchester model. ig 4. Flowchart for the ABC framework. Example for a experiment using 1000 runs and tolerance τ = 0.01. L while the green panel details the simulation of the Lanchester model. 4. Flowchart for the ABC framework. Example for a experiment using 1000 runs and tolerance τ = 0.01. Left side illustrates the rejection algorithm le the green panel details the simulation of the Lanchester model. 10 1371/journal pone 0146491 g004 Fig 4. Flowchart for the ABC framework. Example for a experiment using 1000 runs and tolerance τ = 0.01. Left s while the green panel details the simulation of the Lanchester model. Fig 4. Flowchart for the ABC framework. Example for a experiment using 1000 runs and tolerance τ = 0.01. Left side illustrates the rejection algorithm while the green panel details the simulation of the Lanchester model. doi:10.1371/journal.pone.0146491.g004 doi:10.1371/journal.pone.0146491.g004 PLOS ONE \| DOI:10.1371/journal.pone.0146491 January 5, 2016 10 / 18 Model Selection in Historical Research Using ABC Fig 6. Distance from fatigue model to evidence with τ = 0.0005. Absolute distances (Y axis) of the best 500 runs ordered by rank (X axis, being 1 the best one), model (colour) and historical period (left to right). doi:10.1371/journal.pone.0146491.g006 Fig 6. Distance from fatigue model to evidence with τ = 0.0005. Absolute distances (Y axis) of the best 500 runs ordered by rank (X axis, being 1 the best one), model (colour) and historical period (left to right). doi:10.1371/journal.pone.0146491.g006 The parameter μ exhibits a dynamic of gradual decrease over the three centuries. Three main blocks can be observed: the oldest period (1620–1701) has the largest mean value (2.4), while the following 150 years (second and third period) have smaller means (around 1.9) and the latest period has the lowest peak (1.6). The σ distribution is similar for all periods except for the oldest one. The combination of the two posterior distributions as seen in Fig 9 illustrates the interaction between μ and σ. The dispersion of the posterior distribution for the first period is much larger than the rest of the examined periods. In addition all results follow a distinctive pattern: the largest values of σ are only selected if the μ value is also large. Parameter estimation The posterior distribution for parameters μ and σ is now examined for the fatigue model at τ = 0.0005. Fig 7 and Fig 8 show that both parameters follow unimodal distributions for all periods. The complete set of posterior distributions can be observed in SI 1, where similar pat- terns are observed for the other models (see S1 Fig for parameter μ and S2 Fig for parameter σ). Fig 5. Model selection for different tolerance levels. Proportion of the models used in the best runs for the four historical periods and three τ values (corresponding to the selection of left: 500, centre: 5000 and right: 50000 best runs). doi:10 1371/journal pone 0146491 g005 Fig 5. Model selection for different tolerance levels. Proportion of the models used in the best runs for the four historical periods and three τ values (corresponding to the selection of left: 500, centre: 5000 and right: 50000 best runs). doi:10.1371/journal.pone.0146491.g005 PLOS ONE \| DOI:10.1371/journal.pone.0146491 January 5, 2016 11 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0146491 January 5, 2016 Discussion These results confirm that the original Lanchester’s laws (i.e. linear and squared) are a poor match to historical evidence. The outcome is similar to other studies, which highlighted the better match of the logarithmic model [51]. Beyond this replication of past results, the use of the ABC framework provides new insights to the discussion. The decisive advantage of the fatigue model shows that this formulation is better supported by historical evidence than the rest of the models. The extreme psychological and physical stress conditions in the battlefield caused a gradual decrease on the efficiency of the armies. The better fit of the fourth model would suggest that this process had an impact in the final outcome. The performance of the logarithmic model is similar to the fatigue model, even though it shows slightly lower match to evidence. The explanatory power of the two classical models is much lower, as they are consistently below the best runs for any tolerance level. The credibility of the models is not constant over the entire time span. The best matches are the oldest and more recent periods, while the third period (1793-1860) is revealed as more unpredictable. The period was dominated by the French Revolutionary Wars and the Napole- onic Wars, where traditional European tactics were transformed at a scale not previously seen. 12 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0146491 January 5, 2016 Model Selection in Historical Research Using ABC Fig 7. Posterior distribution of μ. Results for the fatigue model and τ = 0.0005. doi:10.1371/journal.pone.0146491.g007 Fig 7. Posterior distribution of μ. Results for the fatigue model and τ = 0.0005. doi:10.1371/journal.pone.0146491.g007 This outcome would suggest that the generalist approach undertaken by the Lanchester’s laws is not suited to study transition periods with higher rates of change. Posterior distributions for parameters μ and σ suggest a gradual decrease of the relevance of individual fighting value. In particular, P values calculated for XVIIth century battles are larger and more diverse than the rest of the dataset. This result suggests that the non-professional armies of this era produced a much wider set of results under similar conditions, as the fighting value of the soldiers was much relevant than their numbers. The gradual standardisation of tac- tics and training would give more relevance to the size because individual fighting value was equalised between all armies. PLOS ONE \| DOI:10.1371/journal.pone.0146491 January 5, 2016 Model Selection in Historical Research Using ABC The study of different tolerance levels also provides a cautionary tale on the use of ABC. As its name indicates it approximates the posterior distributions, and the method needs additional parameters such as the tolerance level τ. It means that τ also needs to be explored, as any other parameter. Results of the case study are a good example of the need of this exploration, as Bayes Factors for τ = 0.05 are radically different than the other two values. Any study using ABC should acknowledge this issue and integrate this discussion in the experiment design. Computational models are becoming a relevant quantitative tool for historical research. This new approach allows historians to evaluate the plausibility of competing hypotheses beyond what has been discussed in natural language. It is clear that History presents a unique set of issues and challenges to formal modelling, often related to the uncertainty of the datasets collected by the researchers. In this context, the integration of model selection methods such as ABC with new datasets and computer models can provide solutions to some of the current debates of the discipline. Supporting Information S1 Fig. Complete parameter estimation for parameter μ. Parameter μ posterior distribu- tion for the four models and historical periods. Results obtained from the four initial experi- ments with τ = 0.0005. (TIFF) S2 Fig. Complete parameter estimation for parameter σ. Parameter σ posterior distribution for the four models and historical periods. Results obtained from the four initial experiments with τ = 0.0005. S1 File. Dataset and Source code. Dataset. Dataset is distributed under the terms of a Creative Commons Attribution-ShareAlike 4.0 International License. The models and the ABC rejec- tion algorithm were implemented in Python programming language. Source code of the model is licensed under a GNU General Public License. Last versions for both data and source code can be downloaded from https://github.com/xrubio/lanchester. (ZIP) Author Contributions Conceived and designed the experiments: XRC. Performed the experiments: XRC. Analyzed the data: XRC. Contributed reagents/materials/analysis tools: XRC. Wrote the paper: XRC. Acknowledgments We would like to thank Mark Madsen and two anonymous reviewers for their comments on previous versions of the manuscript. We would also like to thank Enrico R. Crema for his con- tributions in many discussions regarding the method, Victor Pascual for his suggestions on data visualization, Maria Yubero for comments on the text and Francesc Xavier HernÃ ndez Cardona on the evolution of warfare. Discussion The variability of fighting value P within the same period is basi- cally constant after XVIIth century. Mean values are similar for the second and third period, while showing a significant decrease after 1861. This would suggest that the evolution of war- fare, now dominated by mass-production, would give even more relevance to sheer numbers while differences between individuals would then become a minor factor. Beyond the examined scenario, the case study illustrates how Model-Based History could benefit from a Bayesian-inspired framework. The use of a meta-model to compute Bayes Fac- tors allows the researcher to compare hypotheses while generating credible posterior PLOS ONE \| DOI:10.1371/journal.pone.0146491 January 5, 2016 13 / 18 Model Selection in Historical Research Using ABC Fig 8. Posterior distribution of σ. Results for the fatigue model and τ = 0.0005. doi:10.1371/journal.pone.0146491.g008 Model Selection in Historical Research Using ABC Fig 8. Posterior distribution of σ. Results for the fatigue model and τ = 0.0005. doi:10.1371/journal.pone.0146491.g008 Fig 8. Posterior distribution of σ. Results for the fatigue model and τ = 0.0005. doi:10.1371/journal.pone.0146491.g008 doi:10.1371/journal.pone.0146491.g008 doi:10.1371/journal.pone.0146491.g008 distributions. It also shows how the original framework can be easily extended to test new hypotheses, as seen in the fatigue model. It is worth mentioning that Bayes Factors already take into account parsimony because complex models with larger number of parameters will gener- ate wider posterior distributions. As a result, models with more parameters will be more times below the tolerance threshold, thus promoting simpler models. distributions. It also shows how the original framework can be easily extended to test new hypotheses, as seen in the fatigue model. It is worth mentioning that Bayes Factors already take into account parsimony because complex models with larger number of parameters will gener- ate wider posterior distributions. As a result, models with more parameters will be more times below the tolerance threshold, thus promoting simpler models. Fig 9. Relation between μ and σ values with τ = 0.0005. Large σ values are only selected when μ value is also large. doi:10 1371/journal pone 0146491 g009 Fig 9. Relation between μ and σ values with τ = 0.0005. Large σ values are only selected when μ value is also large. doi:10.1371/journal.pone.0146491.g009 Fig 9. Relation between μ and σ values with τ = 0.0005. Large σ values are only selected when μ value is also large. doi:10.1371/journal.pone.0146491.g009 PLOS ONE \| DOI:10.1371/journal.pone.0146491 January 5, 2016 14 / 18 References 1. Epstein JM. Why model? Journal of Artificial Societies and Social Simulation. 2008; 11(4):12. 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https://openalex.org/W3201411987	https://www.researchsquare.com/article/rs-29313/latest.pdf	English	null	Willingness to perform induced abortion and associated factors among graduating Midwifery, Medical, Nursing, and Public health officer students of University of Gondar, Northwest Ethiopia: Institution based Cross sectional study	Research Square (Research Square)	2,020	cc-by	4,085	Willingness to Perform Induced Abortion and Associated Factors Among Graduating Midwifery, Medical, Nursing, and Public Health Officer Students of University of Gondar, Northwest Ethiopia: Institution based Cross sectional study Mihretu Molla Enyew (  mihretumolla143@gmail.com ) Research Posted Date: May 20th, 2020 Abstract Background: In developing countries, abortion is often unsafe and a signiﬁcant cause of maternal morbidity and mortality accounting for about 8% (4.7%–13.2%) of maternal mortality worldwide. Internationally, safe abortion services are recognized as reducing maternal mortality, and liberalized abortion laws are associated with reduced mortality resulting from unsafe abortion procedures. However health care providers have moral, social and gender-based reservations that affects their willingness towards providing induced abortion services. The purpose of this study was to assess willingness to perform induced abortion and associated factors among graduating Midwifery, Medical, Nursing, and Public health officer students of University of Gondar. Methods: Institution based cross sectional study was conducted from March 29 to May 30, 2019. All graduating students available during data collection period were considered as study population. Stratified simple random sampling technique was used to select 424 study participants. Pre tested, semi- structured, self-administered questionnaire was used to collect data. Data analysis was done using SPSS version 20. Ethical clearance was obtained from School of midwifery under the delegation of institutional review board of university of Gondar. Results: 290 students out of 424 students were willing to perform induced abortion for indications supported by Ethiopian abortion law, making a proportion of 68.4% (95%Cl: 64.2, 72.9). Sex (Being male (AOR = 4.89, 95%CI: 3.02, 7.89)), religion (being orthodox than protestant (AOR = 10.41, 95%CI: 3.02, 21.57)), being Muslim than protestant (AOR = 5.73, 95%CI: 1.37, 15.92)) and having once or less a week religious attendance (AOR=2.00, 95% CI: 1.20, 3.34) were factors associated with willingness towards performing induced abortion. Conclusions: According to this study willingness of students towards providing induced abortion services was good. However female students, protestant followers and those students with more than once a week religious attendance should be encouraged in abortion care provisions. DOI: https://doi.org/10.21203/rs.3.rs-29313/v1 DOI: https://doi.org/10.21203/rs.3.rs-29313/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at BMC Pregnancy and Childbirth on November 10th, 2020. See the published version at https://doi.org/10.1186/s12884-020-03382-0. Page 1/15 Page 1/15 Plain English Summary Even though Ethiopia has made significant progress in expanding comprehensive abortion care, prevention of maternal and morbidity and mortality from unsafe abortion has not yet been achieved. One of the main reasons why unsafe abortion is still practiced is the unwillingness of some health care providers to provide induced abortion/safe termination services due to various religious, cultural and biological factors. Therefore, Assessing Graduating students’ willingness and associated factors towards induced abortion provision is an important indication of their intention and capacity to provide induced abortion services during their future careers. Relevant data form graduating students were collected by using semi-structured, self-administered questionnaire. Page 2/15 290 graduating students out of 424 students were willing to perform induced abortion for indications supported by Ethiopian abortion law. Students’ sex, religion and religious practices where identified as associated factors with their willingness to provide induced abortion care services. 290 graduating students out of 424 students were willing to perform induced abortion for indications supported by Ethiopian abortion law. Students’ sex, religion and religious practices where identified as associated factors with their willingness to provide induced abortion care services. In conclusion, willingness of students towards providing induced abortion services was good when compared with other findings. However female students, protestant followers and those students with more than once a week religious attendance should be encouraged in induced abortion care provisions. As a limitation this study lacked triangulation. I.e. the reasons for graduating students who were not willing to perform induced abortion was not explored qualitatively and Students’ willingness to perform induced abortion was not supported by action research. Background Over 25 million unsafe abortions occurred every year worldwide, the majority (97%), occurred in developing countries, attributing 4.7% – 13.2% of maternal deaths annually (1, 2). Induced abortion (safe termination) services are recognized as key interventions in reducing maternal mortality and morbidity associated with unsafe abortion procedures (3, 4). However, health care providers in Sub-Saharan Africa (SSA) have moral, social and gender based reservations that affect their willingness towards performing induced abortion (5). As a result, complication of unsafe abortion is among the leading cause of maternal morbidities and mortalities in the region (6). The government of Ethiopia revised and liberalized the abortion law in 2005 to increase the accessibility of safe termination services by taking the amount of Unsafe abortion and its health detrimental consequences on maternal health (7). However, a major reduction of morbidity and mortality from the complication of unsafe abortion has not yet been achieved (8). The shortage of health care providers who can provide comprehensive abortion care (CAC) is still critical and this is again exacerbated by the unwillingness of some health care providers to provide induced abortion services due to various religious, cultural and biological factors (9, 10). Graduating students’ willingness towards induced abortion provision is an important influence on their intention and capacity to provide induced abortion services during their future careers (11). Even though there is no concrete evidence in Ethiopia, studies done in Asia and Africa revealed a significant level of unwillingness towards performing induced abortion by graduating students in their future careers. For instance, medical students involved in a study done in Maharastra (India) described fear due to social norms and illegality of the procedure to provide abortion in their future practice(12). Another study done in South Africa (SA) revealed only 23% of students were willing to perform induced abortions once they are qualified (13). Thus, this study will have a fundamental role in pinpointing recommendations to develop positive attitude among graduating students towards providing safe abortion care services thereby reducing maternal morbidity and mortality. Study setting University of Gondar, College of medicine and health sciences located in Gondar city, Northwest Ethiopia. The University of Gondar is one of the oldest and most well-established higher education institutions in the country. There were a total of 805 graduating Midwifery, Medicine, Nursing and public health officer (PHO) students in the College of Medicine and Health Sciences for 2018/19 academic year. Page 3/15 Page 3/15 Page 3/15 Methods Study design and period Institution-based cross-sectional study was conducted from March 29 to May 30, 2019. Characteristics of participants Proportionally selected graduating midwifery, medical, nursing, and PHO of 2018/19 academic year were included in the study. Students who were not available due to social or medical reasons during the data collection period were excluded. Variables of the study Dependent variable willingness of graduating students (willing, not willing) Independent variables Stratified random sampling technique was used to select study participants (Fig. 1) Variables of the study Sampling Sample size was calculated using single population proportion sample size calculation formula. Assuming willingness of students to be 50%, Zα/2 value of 1.96 and marginal error of 5%, Sample size was calculated as follows: n= (Zα/2)2pq/w2 n = (1.96)2(.5) (.5)/ (0.05)2 n = 385 n= (Zα/2)2pq/w2 n = (1.96)2(.5) (.5)/ (0.05)2 n = 385 Adding 10% non-response rate Adding 10% non-response rate Stratified random sampling technique was used to select study participants (Fig. 1) Liberalized components of induced/safe abortion services Nationally supported indications to terminate pregnancy such as pregnancy following rape, pregnancy following incest, minority pregnancy, pregnancy endangering the woman’s life and pregnancy complicated by gross fetal defects (22). Data collection tools and procedures Data were collected by self-administered interviews using a semi-structured and pre-tested questionnaire. Six Bsc midwives and two Msc midwives were assigned to collect data and to supervise the data collection process respectively. Data quality control The quality of data was assured by proper designing and pre-testing of the questionnaires on 5% of study participants at Debretabor University and by giving training for the data collectors and supervisors before the actual data collection. Every day after data collection, questionnaires were reviewed and checked for consistency and completeness by the supervisors. Data clean up and cross-checking was done before analysis. Induced abortion deliberate termination of pregnancy before viability for reasons of nationally liberalized indications. Data processing and analysis All the questionnaires were checked for completeness, manually and coded, then entered into Epi info 7 and exported to SPSS version 20 software package for further analysis. Descriptive analysis results were presented in the form of table, figure, and text using frequencies and summary statistics such as mean, standard deviation and percentage. Bivariate logistic regression analysis was used to determine the association of each independent variable with the outcome variable and multivariable logistic regression analysis was employed to adjust the influence of various independent variables (confounding effects) on the outcome variable. Odds ratio (OR) with 95% confidence interval (CI) was used to see the association between independent variables and the dependent variable. Willingness of graduating students status and level of willingness of students in performing induced abortion following graduation for indications supported by Ethiopian abortion law. Independent variables Age, field of study, marital status, religion, ethnicity, sex, family place of residence, frequency of religious attendance, exposure to sexual practice, exposure to abortion care servicesfi Page 4/15 Results Page 5/15 Page 5/15 Socio-Demographic Characteristics A total of 424 graduating students were included in the study, making a 100% response rate. The age of the study participants was between 20 and 30 years with mean (± SD) age 23.15 ± 1.77 years. (Table 1). Table 1 Socio demographic characteristics of study participants of attitudes and associated factors on induced abortion among graduating midwifery, medical, nursing and health officer students of university of Gondar, northwest y Ethiopia, 2018. n = 424 y Ethiopia, 2018. n = 424 Variable Frequency Percent (%) Age 20–24 372 87.7 25–29 42 9.9 30–35 10 2.4 Sex Male 225 53.1 Female 199 46.9 Field of study Midwifery 140 33 Medicine 100 23.6 Nursing 158 37.3 Public health officer 26 6.1 Family place of residence Urban 149 35.1 Rural 275 64.9 Marital status Single 392 92.5 Married 32 7.5 Page 6/15 (continued) Variable Frequency Percent (%) Religion Orthodox 339 80 Muslim 64 15 Protestant 21 5 Ethnicity Amhara 245 57.8 Kemant 47 11.1 Oromo 56 13.2 Tigray 23 5.4 Sidama 47 11.1 Others* 6 1.4 Religious service attendance More than once a week 279 65.8 Once a week or less 145 34.2 Exposure to sexual practice Yes 124 29.4 No 300 70.6 Exposure to abortion care services Yes 377 88. 9 No 47 11.1 s of students towards performing induce udy participants, 290 of them were willing to perform induced abortion making a proportion of 68.4% (95%Cl: 64.2, 72.9). About 70.7% of mid udents, 72.8% of nursing students and 57.7% of PHO were willing to pr Of the total 424 study participants, 290 of them were willing to perform induced abortion for indications supported by law, making a proportion of 68.4% (95%Cl: 64.2, 72.9). About 70.7% of midwifery students, 61% of medical students, 72.8% of nursing students and 57.7% of PHO were willing to provide induced abortion services. Of the total 424 study participants, 290 of them were willing to perform induced abortion for indications supported by law, making a proportion of 68.4% (95%Cl: 64.2, 72.9). About 70.7% of midwifery students, 61% of medical students, 72.8% of nursing students and 57.7% of PHO were willing to provide induced abortion services. Of the total 424 study participants, 290 of them were willing to perform induced abortion for indications supported by law, making a proportion of 68.4% (95%Cl: 64.2, 72.9). About 70.7% of midwifery students, 61% of medical students, 72.8% of nursing students and 57.7% of PHO were willing to provide induced abortion services. Page 7/15 Page 7/15 Factors Associated with willingness of graduating students towards performing induced abortion In bivariate logistic regression analysis, willingness towards performing induced abortion was associated with male sex, single marital status, urban family place of residence, orthodox and Muslim religions and frequent religious attendance. Among variables found to be significantly associated with willingness towards doing induced abortion using bivariate logistic regression analysis, male sex (AOR = 4.89, 95%CI: 3.02, 7.89), being orthodox than protestant (AOR = 10.41, 95%CI: 3.02, 21.57) being Muslim than protestant (AOR = 5.73, 95%CI: 1.37, 15.92) and less than once a week religious attendance (AOR = 2.00, 95% CI: 1.20, 3.34) were also found to be significantly associated in multivariate logistic regression analysis (Table 2). Page 8/15 Table 2 Bivariate and multivariate logistic regression analysis of Factors Associated with willingness towards performing induced abortion among graduating midwifery, medical, nursing and health officer students of university of Gondar, northwest Ethiopia, 2018. n = 424 Variable Willing to do abortion COR (95% CI) AOR (95% CI) P value Yes No Sex Frq Frq Male 189 36 5.09 (3.24, 8.01) 4.89 (3.02, 7.89) .000 Female 101 98 Reference Family residence Urban 116 33 2.04 (1.29, 3.23) 1.52(0.91, 2.54) .108 Rural 174 101 Reference Marital status Single 281 111 6.47 (2.90,14.42) 1.59 (0.58, 4.32) .369 Married 9 23 Reference Religion Orthodox 249 90 16.60(4.78,57.69) 10.41 (3.02, 21.57) .000 Muslim 38 26 8.77(2.34, 32.83) 5.73 (1.37, 15.92) .017 Protestant 3 18 Reference Religious attendance ≤ once a week 116 29 2.41 (1.50, 3.88) 2.00 (1.20, 3.34) .008 >once a week 174 105 Reference Di i Table 2 Discussion This study assessed willingness of graduating midwifery, medical, nursing and PHO students towards providing induced abortion services and found 290 students out of 424 students willing to perform induced abortion for indications supported by law, making a proportion of 68.4% (95%Cl: 64.2, 72.9). This finding was in line with findings in California-66.21% (14) and Poland-70% (15). The result was high when compared with a study done in Argentina-3.34% (16). This was mainly due to lack of accurate information about legal frameworks of abortion care provision by the Argentinian side. The finding was also high when compared with another study done in Canada (17) where fewer than 30% of students planned to provide any type of abortion. The possible explanation might be Lack of perceived social support for providing CAC and lack of interest to specialize in health care fields containing abortion Page 9/15 by Canadian students. The result was also high when compared with a study done Turkey-50.1% (18) and Iran-43.7% (19) respectively. This difference might be due to religious preferences as these two countries are being governed by strict Islamic doctrines. The magnitude was again high when compared with the finding Ireland-58.8%(20). This might be due to the difference in the year of study of students as the later were not graduating students. by Canadian students. The result was also high when compared with a study done Turkey-50.1% (18) and Iran-43.7% (19) respectively. This difference might be due to religious preferences as these two countries are being governed by strict Islamic doctrines. The magnitude was again high when compared with the finding Ireland-58.8%(20). This might be due to the difference in the year of study of students as the later were not graduating students. Male students were about five times (AOR = 4.89, 95%CI: 3.02, 7.89) more likely to provide induced abortion services than their female counterparts. This finding is contrary to the findings of previous studies done in Iran (21). The possible explanation might be long-lasting male dominance in every aspects of participation in our country when compared with Iran. Concerning students’ religion, being orthodox were about ten times (AOR = 10.41, 95%CI: 3.02, 21.57) more likely to show willingness to induced abortion services than being protestant. Being Muslim was also about six times (AOR = 5.73, 95%CI: 1.37, 15.92) more likely to show willingness to induced abortion services than being protestant. Discussion This finding is in line with the findings of the studies done SSA (5), SA (13), Iran (19) and Ethiopia (9). This might be tailored to the fact that religious restrictions of newly introduced Protestantism might have been practiced better than those of relatively older doctrines. Those students who had religious attendance less than once a week irrespective of the type of religion were two times (AOR = 2.00, 95% CI: 1.20, 3.34) more likely to show willingness to induced abortion services than those who had more than once a week religious attendance. This finding was consistent with the finding of the study done in Iran and Chile (19,(22)). The possible explanation might be the more students attend religious activities the more they become conservative in providing induced abortion services as they might directly link it with doing sin and vice versa. Conclusions According to this study willingness of graduating students towards performing induced abortion was good when compared with other findings. However female students, protestant followers and those students with more than once a week religious attendance should be encouraged in induced abortion care provisions. List Of Abbreviations Declarations Declarations Ethics approval and consent to participate Ethical approval was obtained from department of midwifery under the delegation of Institutional Review Board (IRB) of University of Gondar. Written informed consent was obtained from each study subject before data collection process. During data collection process the data collectors informed each study participant about the purpose and anticipated benefits of the research project and the study participants were also informed on their full right to refuse, withdraw or completely reject part or all of their part in the study. They were also informed that all data obtained from them would be kept confidential by using codes instead of any personal identifiers. List Of Abbreviations Page 10/15 Page 10/15 AOR Adjusted Odds Ratio CAC Comprehensive Abortion Care CI Confidence Interval COR Crude Odds Ratio EMwA Ethiopian Midwives Association Frq Frequency IRB Institutional Review Board Km Kilo Meters Nf Final Sample size PHO Public Health Officer SA South Africa SPSS Statistical Package for Social Sciences SSA Sub- Saharan Africa ST Safe Termination UNICEF United Nations International Children's Emergency Fund WHO World Health Organization Funding The funding body (Ethiopian Midwives association in collaboration with Amplify change) had a role in the coverage of the financial costs related to the design and data collection of this study. However, the funding body had no role in the analysis, interpretation of data and in writing of the manuscript. Author’s contributions wrote the proposal, supported and supervised data collection, analyzed the data and drafted the paper and prepare the final manuscript. Competing interests "The author declare that he has no competing interests" Others* Gurage, Somali, Hadiya, Wolayta, Kembata Gurage, Somali, Hadiya, Wolayta, Kembata Acknowledgments I would like to thank the Ethiopian midwives Association for providing such a chance and Amplify change for its genuine financial support. I would also like to thank college of medicine and health science register workers who provide me all the necessary information concerning my study area. I would also like to thank college of medicine and health science register workers who provide me all the necessary information concerning my study area. I would also very delighted to thank study participants, data collectors, and supervisors. would also very delighted to thank study participants, data collectors, and s Consent for publication p Not applicable Availability of data and material Not applicable Availability of data and material Page 11/15 Page 11/15 Page 11/15 All data generated or analysed during this study are included in this published article [and its supplementary information files]. References 1. Ganatra B, Gerdts C, Rossier C, Johnson BR Jr, Tunçalp Ö, Assifi A, et al. Global, regional, and subregional classification of abortions by safety, 2010–14: estimates from a Bayesian hierarchical model. The Lancet. 2017;390(10110):2372–81. 2. Say L, Chou D, Gemmill A, Tuncalp O, Moller AB, Daniels J, et al. Global causes of maternal death: a WHO systematic analysis. The Lancet Global health. 2014;2(6):e323-33. Page 12/15 Page 12/15 Page 12/15 3. Ganatra B, Faundes AJBP, Obstetrics RC, Gynaecology. Role of birth spacing, family planning services, safe abortion services and post-abortion care in reducing maternal mortality. 2016;36:145– 55. 4. Lim LM, Singh KJBP, Obstetrics RC, Gynaecology. Termination of pregnancy unsafe abortion. 2014;28(6):859–69. 5. Loi UR, Gemzell-Danielsson K, Faxelid E, Klingberg-Allvin M. Health care providers’ perceptions of and attitudes towards induced abortions in sub-Saharan Africa and Southeast Asia: a systematic literature review of qualitative and quantitative data. BMC Public Health. 2015;15(1):139. 6. Gebremedhin M, Semahegn A, Usmael T, Tesfaye GJSr. Unsafe abortion and associated factors among reproductive aged women in Sub-Saharan Africa: a protocol for a systematic review and meta-analysis. 2018;7(1):130. 7. Wada TJMLR. Abortion law in Ethiopia: a comparative perspective. 2008;2(1):1–32. 8. Gebrehiwot Y, Fetters T, Gebreselassie H, Moore A, Hailemariam M, Dibaba Y, et al. Changes in morbidity and abortion care in Ethiopia after legal reform: National results from 2008 and 2014. 2016;42(3):121. 9. Holcombe SJ, Berhe A, Cherie AJSifp. Personal beliefs and professional responsibilities: Ethiopian midwives' attitudes toward providing abortion services after legal reform. 2015;46(1):73–95. 10. Sintayehu Y, Hordofa B, Shiferaw KJJWsHC. Health Care Providers’ Perception and Associated Factors towards Safe Abortion in Selected Health Facilities in Adama. Ethiopia. 2018;7(428):2167- 0420.1000428. 11. Teffo ME, Rispel, LCJGha. ‘I am all alone’: factors influencing the provision of termination of pregnancy services in two South African provinces. Global health action. 2017;10(1):1347369. 12. Sjöström S, Essén B, Gemzell-Danielsson K, Klingberg-Allvin M. Medical students are afraid to include abortion in their future practices: in-depth interviews in Maharastra, India. BMC medical education. 2016;16(1):8. 13. Wheeler SB, Zullig LL, Reeve BB, Buga GA, Morroni C. Attitudes and intentions regarding abortion provision among medical school students in South Africa. International perspectives on sexual and reproductive health. 2012:154–63. 13. Wheeler SB, Zullig LL, Reeve BB, Buga GA, Morroni C. Attitudes and intentions regarding abortion provision among medical school students in South Africa. International perspectives on sexual and reproductive health. 2012:154–63. 14. References Rosenheck RE, Feigal C, Brown N, Barcellos T, Bender N. Attitudes on Abortion: A Cross-Sectional Analysis of Medical Students in Their Preclinical Years [3F]. Obstetrics Gynecology. 2016;127:51S. 14. Rosenheck RE, Feigal C, Brown N, Barcellos T, Bender N. Attitudes on Abortion: A Cross-Sectional Analysis of Medical Students in Their Preclinical Years [3F]. Obstetrics Gynecology. 2016;127:51S. 15. Michalik A, Zdun-Ryżewska A, Pięta B, Basiński K, Kiełbasińska J, Mazurkiewicz B, et al. Multicenter study on midwifery students’ attitudes towards abortion and it’s place in their future practice– comparison of respondents at early and late stages of the university education. Nurse education in practice. 2019. 16. Ramos S, Romero M, Michel AR. Health care providers’ opinions on abortion: a study for the implementation of the legal abortion public policy in the Province of Santa Fe, Argentina. Reproductive health. 2014;11(1):72. Page 13/15 Page 13/15 17. Myran DT, Carew CL, Tang J, Whyte H, Fisher WA. Medical students’ intentions to seek abortion training and to provide abortion services in future practice. Journal of Obstetrics Gynaecology Canada. 2015;37(3):236–44. 18. Kesen NF, Polat G, Daşbaş S. Opinions on abortion among a group of university students in Turkey. International Journal of Humanities Social Science. 2015;7(1):89–95. 18. Kesen NF, Polat G, Daşbaş S. Opinions on abortion among a group of university students in Turkey. International Journal of Humanities Social Science. 2015;7(1):89–95. 19. Inaloo R, Tghipour N, Razeghi A, Rahmanian E. Attitudes of students of Jahrom University of medical sciences towards abortion: a descriptive, cross-sectional study. Journal of Fundamental Applied Sciences. 2018;10(6S):80–91. 20. Fitzgerald JM, Krause KE, Yermak D, Dunne S, Hannigan A, Cullen W, et al. The first survey of attitudes of medical students in Ireland towards termination of pregnancy. Journal of medical. 2014;40(10):710–3. 21. Yanikkerem E, Üstgörül S, Karakus A, Baydar O, Esmeray N, Ertem G. Turkish nursing students' attitudes towards voluntary induced abortion. JPMA The Journal of the Pakistan Medical Association. 2018;68(3):410–6. 22. Biggs MA, Casas L, Ramm A, Baba CF, Correa SV, Grossman DJBo. Future health providers’ willingness to provide abortion services following decriminalisation of abortion in Chile: a cross- sectional survey. 2019;9(10). Figures Figures Figures Page 14/15 Figure 1 Figure 1 Page 14/15 Schematic presentation of enrolment of study participants of willingness to perform induced abortion and associated factors among graduating midwifery, medical, nursing and PHO students of university of Gondar, 2019. N=424 Figure 2 Proportion of willing students to perform induced abortion among graduating midwifery, medical, nursing and PHO students of university of Gondar, 2019. N=424 Figure 2 Proportion of willing students to perform induced abortion among graduating midwifery, medical, nursing and PHO students of university of Gondar, 2019. N=424 Proportion of willing students to perform induced abortion among graduating midwifery, medical, nursing and PHO students of university of Gondar, 2019. N=424 Page 15/15
https://openalex.org/W2969876676	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0220652&type=printable	English	null	The barn owls’ Minimum Audible Angle	PloS one	2,019	cc-by	10,830	Abstract Malmierca, Universidad de Salamanca, SPAIN Received: April 16, 2019 Accepted: July 19, 2019 Published: August 23, 2019 Editor: Manuel S. Malmierca, Universidad de Salamanca, SPAIN Editor: Manuel S. Malmierca, Universidad de Salamanca, SPAIN Received: April 16, 2019 Accepted: July 19, 2019 Published: August 23, 2019 Copyright: © 2019 Krumm et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Bianca Krumm1,2, Georg M. Klump1, Christine Ko¨ppl2, Ulrike LangemannID1* Bianca Krumm1,2, Georg M. Klump1, Christine Ko¨ppl2, Ulrike LangemannID1* 1 Cluster of Excellence “Hearing4all”, Division for Animal Physiology and Behaviour, School of Medicine and Health Sciences, Department of Neuroscience, Carl von Ossietzky University of Oldenburg, Oldenburg, Germany, 2 Cluster of Excellence “Hearing4all”, Division for Cochlea and auditory brainstem physiology, School of Medicine and Health Sciences, Department of Neuroscience, Carl von Ossietzky University of Oldenburg, Oldenburg, Germany * ulrike.langemann@uni-oldenburg.de * ulrike.langemann@uni-oldenburg.de RESEARCH ARTICLE The barn owls’ Minimum Audible Angle RESEARCH ARTICLE a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Abstract Interaural time differences (ITD) and interaural level differences (ILD) are physical cues that enable the auditory system to pinpoint the position of a sound source in space. This ability is crucial for animal communication and predator-prey interactions. The barn owl has evolved an exceptional sense of hearing and shows abilities of sound localisation that outperform most other species. So far, behavioural studies in the barn owl often used reflexive responses to investigate aspects of sound localisation. Furthermore, they predominately probed the higher frequencies of the owl’s hearing range (> 3 kHz). In the present study we used a Go/NoGo paradigm to measure the barn owl’s behavioural sound localisation acuity (expressed as the Minimum Audible Angle, MAA) as a function of stimulus type (narrow- band noise centred at 500, 1000, 2000, 4000 and 8000 Hz, and broad-band noise) and sound source position. We found significant effects of both stimulus type and sound source position on the barn owls’ MAA. The MAA improved with increasing stimulus frequency, from 14˚ at 500 Hz to 6˚ at 8000 Hz. The smallest MAA of 4˚ was found for broadband noise stimuli. Comparing different sound source positions revealed smaller MAAs for frontal com- pared to lateral stimulus presentation, irrespective of stimulus type. These results are con- sistent with both the known variations in physical ITDs and variation in the width of neural ITD tuning curves with azimuth and frequency. Physical and neural characteristics combine to result in better spatial acuity for frontal compared to lateral sounds and reduced localisa- tion acuity at lower frequencies. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Krumm B, Klump GM, Ko¨ppl C, Langemann U (2019) The barn owls’ Minimum Audible Angle. PLoS ONE 14(8): e0220652. https:// doi.org/10.1371/journal.pone.0220652 Editor: Manuel S. Malmierca, Universidad de Salamanca, SPAIN Received: April 16, 2019 Accepted: July 19, 2019 Published: August 23, 2019 Copyright: © 2019 Krumm et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. OPEN ACCESS Citation: Krumm B, Klump GM, Ko¨ppl C, Langemann U (2019) The barn owls’ Minimum Audible Angle. PLoS ONE 14(8): e0220652. https:// doi.org/10.1371/journal.pone.0220652 OPEN ACCESS Citation: Krumm B, Klump GM, Ko¨ppl C, Langemann U (2019) The barn owls’ Minimum Audible Angle. PLoS ONE 14(8): e0220652. https:// doi.org/10.1371/journal.pone.0220652 Editor: Manuel S. * ulrike.langemann@uni-oldenburg.de Data Availability Statement: All relevant data are within the manuscript. Data Availability Statement: All relevant data are within the manuscript. Barn owls predominately hunt at night and rely on their auditory system to locate potential prey [1,2]. In situations like harsh winters or in the breeding season barn owls depend on their ability to strike prey for day-to-day survival [3]. In order to save energy, high localisation acu- ity is advantageous for hunting. Catching prey with high precision implies that the barn owl is capable of pinpointing the target with high precision [4–6]. In the present study we aimed to determine the barn owl’s behavioural sound localisation acuity in the laboratory. Funding: This study was funded by the Deutsche Forschungsgemeinschaft (TRR 31, EXC 1077) to GMK and CK. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Sound localisation in vertebrates largely relies on two binaural cues: interaural time (ITD) and interaural level difference (ILD). In mammals these cues are used for sound localisation in Competing interests: The authors have declared that no competing interests exist. 1 / 19 PLOS ONE \| https://doi.org/10.1371/journal.pone.0220652 August 23, 2019 Barn owl MAA azimuth in a complementary fashion: ITDs are relied on to localize low-frequency sounds and ILDs to localize high-frequency sounds (referred to as duplex theory, see [7]). This means that binaural cues are analysed separately in low- and high-frequency processes [8,9]. Due to the structure of their fleshy pinnae, mammals are, in addition, able to use monaural spectral cues for sound localisation in elevation [9]. Similar to mammals, most birds use both ITD and ILD for sound localisation in azimuth. Barn owls, however, rely primarily on ITDs for sound locali- sation in azimuth, while ILDs play a minor role here [2,10–13]. Instead, the asymmetrical facial ruff of the barn owl generates ILDs that provide a cue for sound localisation in elevation [2,10,14]. Barn owls possess space-specific neurones that are tuned to specific sound source positions in azimuth and elevation. These neurones are located in the external part of the inferior colli- culus (ICx) and represent a physiological map of auditory space [15]. Knudsen and his col- leagues were the first to observe that space-specific neurones representing frontal space have more confined receptive fields, both in azimuth and elevation, than neurons tuned to locations in the periphery [15,16]. Data Availability Statement: All relevant data are within the manuscript. The azimuthal spatial tuning of neurones in the barn owl IC is deter- mined mainly by the interaural time difference (ITD), corresponding to the large variation of this acoustic cue with azimuth [11]. As a general pattern, the width of the neurones’ ITD tun- ing curves increases with increasing azimuth and with decreasing frequency [17]. In other words, the ITD tuning is sharper for high- frequency sounds compared to low-frequency sounds and for azimuthal source positions in frontal space compared to lateral space. These observations predict that localisation acuity for sounds in the frontal space is superior com- pared to sounds presented in the periphery and that localisation acuity is also better for high- frequency compared to low-frequency sound sources. These assumptions are supported by behavioural studies in humans (Homo sapiens) [18], cats (Felis silvestris catus) [19] and Euro- pean starlings (Sturnus vulgaris) [20]. Behavioural studies measuring the localisation of static sounds are classified either as abso- lute or relative localisation tasks [5,21]. An absolute sound localisation task requires the subject to identify the absolute position of a single sound source. Such a task measures localisation accuracy as well as localisation precision, corresponding to constant and random errors in sound localisation, respectively [4,5]. Absolute localisation tasks often evaluate motor actions like head or gaze orientation towards the perceived sound source position [10,22–25], and the localisation performance is often referred to as the minimum resolvable angle (MRA) [6]. In contrast, in a relative localisation task the position of one sound source is located with refer- ence to another sound source. The thresholds are expressed as the minimum audible angle (MAA), a measure of localisation acuity [4,5,20]. A behavioural paradigm for deriving the MAA thus measures how well the position of a test stimulus can be discriminated from the position of a reference stimulus [18]. So far, behavioural studies measuring the barn owl’s localisation abilities have employed one of the following paradigms: pupillary dilation response (PDR) [26,27], head-turning behaviour [10,22] or target-approaching behaviour [1,28]. These studies used either reflexive responses, or trained the owls to fly to the target loudspeaker. Furthermore, they measured the localisation ability mainly in frontal space and predominately for the higher frequencies of the owls’ hearing range, i.e. above 3 kHz. In the present study, we determined the azimuthal MAA of barn owls using a Go/NoGo paradigm. PLOS ONE \| https://doi.org/10.1371/journal.pone.0220652 August 23, 2019 Subjects The subjects were 3 European barn owls (Tyto alba) aged between 2 and 24 years. Two of the birds hatched in 2015 at the University of Oldenburg, the third bird hatched in 1993 at the Technical University Munich. All birds were hand reared from the age of around 12 days. The owls were kept in individual indoor aviaries each and were transferred from the aviary to the experimental chamber on the fist. The food reward during the experiment and supplementary food consisted of pieces of one-day chickens (Gallus gallus). The owls’ weight was monitored daily and the motivation of the animals was controlled by maintaining a weight of about 10 to 15% below their free feeding weight. The care and treatment of the birds were approved by the Landesamt fu¨r Verbraucherschutz und Lebensmittelsicherheit (LAVES), Lower Saxony, Ger- many (permit numbers: Az 33.9-42502-04-11/0647, Az 33.19-42502-04-16/2339). Experimental set-up Experiments were carried out in a sound-attenuating echo-reduced chamber (IAC type 1203-A; outside dimensions: 2.8 m x 2.7 m x 2.5 m; internal dimensions: 2.2 m x 2.1 m x 2.0 m.), lined with sound-absorbing acoustic foam (PLANO 50/0 covered with WAFFLE 65/125 Seyboth & Co., cut-off frequency 500 Hz, α > 0.99; total attenuation 48 dB at 500 Hz, > 57 dB for frequencies 1 kHz). There was no cage, so the owls could move freely in the chamber. In the middle of the chamber was a pedestal to position the owl. Two perches were mounted on the pedestal, a waiting perch and a target perch. A stainless steel ring with a diameter of 1.8 m was wall-mounted 1.2 m above the floor. The ring supported 30 loudspeakers (Vifa XT25T G30-04, ASE) that were positioned at the height of the owl’s head. The loudspeakers were arranged in a semicircle with an azimuthal distance of 6˚ between two adjacent loudspeakers as viewed from the position of the head of the owl sitting on a waiting perch. To monitor the owl’s behaviour and location, the two perches were equipped with infrared light barriers. A custom-built automatic feeder was mounted in front of the target perch. The feeder provided up to 24 rewards for correct behavioural responses. A webcam (QuickCam Pro 9000, Logitech) was used to observe the owls during the experiments. A Linux-operated workstation controlled the experimental protocol by operating an enhanced real-time processor (RP2, Tucker-Davis Technologies). An external 32-channel soundcard (Hammerfall DSP Multiface II, RME) gen- erated all acoustic stimuli. The soundcard’s output was fed to four amplifiers (RMB-1048, Rotel) driving the 30 loudspeakers. Based on measured loudspeaker impulse responses, the sound pressure level and frequency responses of the 30 loudspeakers were equalized individu- ally with a 128th order minimum phase FIR filter for each loudspeaker. Data Availability Statement: All relevant data are within the manuscript. We investigated the effects of stimulus type and the spatial position of the reference stimulus on the barn owls’ sound localisation acuity. We were able to compare the barn owls’ localisation acuity at high and low frequencies. Furthermore, we were able to compare frontal and lateral localisation acuity. 2 / 19 PLOS ONE \| https://doi.org/10.1371/journal.pone.0220652 August 23, 2019 Barn owl MAA Barn owl MAA Procedures Go/NoGo task. The owls were trained in a Go/NoGo paradigm to report a change in stimulus position by leaving the waiting perch. The birds had learned to generally orient their head towards 0˚ in azimuth. A trial was started when the owl was sitting on the waiting perch and interrupting the light barrier. In each trial, reference stimuli were repeatedly presented within a random time interval of between 8 to 20 s ("waiting time"). Stimulus repetition rate was 1.3 s. When the waiting time had elapsed and the owl was still oriented towards 0˚ in azi- muth, the next stimulus was presented from either another speaker location (test stimulus) or from the same speaker location (catch stimulus) as the reference stimulus. If the owl perceived the change in stimulus position it would fly to the target perch to obtain its food reward. A response within a report interval of 5.5 s after the beginning of the test stimulus was scored as a “Hit”. Each “Hit” was rewarded with a piece of one-day chicken. In contrast, no response within the report interval was scored as a ‘‘Miss” and initiated the beginning of the next trial. In “catch trials” with no change of stimulus location the owl should remain on the waiting perch during the report interval. If the owl, however, would take off within the report interval in a catch trial, this was scored as a “false alarm” (FA). The false-alarm rate reflects a subject’s strategy and provides information about its decision criterion. If the owl would take off before the random time interval had elapsed (reference stimuli still running), this behav- iour was rated as an “early alarm” (EA) and triggered a 10 s time out after which the waiting time restarted. Since test and catch stimuli are rare events, i.e., constitute only about 2% of the broadcast signals, the probability of random hits and false alarm is very low. Thresholds were obtained by the method of constant stimuli [33], using a set of stimuli with a specific distribution of angular separations of the sound sources. These stimuli (test and catch stimuli) were presented in blocks of trials in random order. The number of blocks was different for the two paradigms (see below). In trials with test stimuli, the range of angular sep- aration between reference and test was adjusted according to stimulus type and to the individ- ual owl. Test signals Stimulus types comprised narrowband noise signals (termed NB noise, bandwidth 40Hz) with centre frequencies of 500, 1000, 2000, 4000 and 8000 Hz and broadband noise signals (termed BB noise, frequency range 500 to 8000 Hz). For NB noise signal with a bandwidth of 40 Hz the transients and envelope fluctuations are slow [29]. Since all noise signals had the same bandwidths and did not provide distinct envelope cues, these do not provide useful ITDs for binaural comparison. Furthermore it has been shown that in the barn owl onset ITDs are unimportant in comparison to ingoing ITDs [30]. Signals had an effective duration of 100 ms (with 10 ms Hanning ramps) and were presented at a level of 40 dB SPL, which is well above the owls’ auditory threshold [31]. In order to rule out potential discrimination cues based PLOS ONE \| https://doi.org/10.1371/journal.pone.0220652 August 23, 2019 3 / 19 Barn owl MAA either on loudness differences or on differences between single loudspeaker characteristics, a level roving of ±3 dB was implemented. Stimuli were presented either from a single loudspeaker (true loudspeaker position), or from two adjacent loudspeakers simultaneously playing identical stimuli and thus simulating a sound source position between the two loudspeakers, using "summing localisation". This procedure was necessary in order to improve the spatial resolution of our setup. Due to their physical dimension the loudspeakers could not be moved any closer to each other. Previous studies had shown that this is a valid approach. Keller and Takahashi [32] have shown that an owl turns its gaze towards the space between the two loudspeakers when two speakers are activated simultaneously indicat- ing that the owl perceived a single stimulus, located between two loudspeakers, i.e. the owl expe- rienced summing localisation. Furthermore, Feinkohl and Klump [20] have shown that a starlings (Sturnus vulgaris) perception does not differ if stimuli are presented either from a "true" loud speaker position or from a position "simulated" by using summing localisation. PLOS ONE \| https://doi.org/10.1371/journal.pone.0220652 August 23, 2019 Procedures Overall the angular separation varied between ±1.5˚ and ±27˚. A negative sign denotes positions to the left of the reference whereas positive sign denotes positions to the right. Within one experimental session only one stimulus type (i.e. NB noise of a given centre frequency or BB noise) was tested. The direction of the change in speaker location, left or right from the reference position was randomized between trials. To minimize training effects, the sequence of stimulus types was randomized for each individual. Single reference and multiple reference paradigms. We determined the minimum audi- ble angle (MAA) with two different paradigms. In the single reference paradigm, the MAA 4 / 19 PLOS ONE \| https://doi.org/10.1371/journal.pone.0220652 August 23, 2019 Barn owl MAA was measured in frontal space (three owls, six stimulus types: NB noise centred at 500, 1000, 2000, 4000, 8000 Hz, BB noise). Here, the reference position was fixed at 0˚ in azimuth. Each session consisted of 42 trials divided into 6 blocks of 7 trials each. Each block consisted of 2 catch trials and a set of 5 test trials with different angular separations. In the multiple reference paradigm, the MAA was determined both in frontal and lateral space (two owls, four stimulus types: NB noise centred at 500, 2000, 8000 Hz, BB noise). The reference position varied between -45˚, 0˚, and +45˚ within one session. The reference position in each trial was selected in a pseudo-random order. Each session consisted of 42 trials divided into 2 blocks of 21 trials each. Each block consisted of 6 catch trials and a set of 15 test trials with five different angular separations for each of the three reference positions. Results We determined the barn owl’s relative localisation acuity, i.e., the minimum audible angle. The owls’ MAA was strongly affected by the factors stimulus type and reference position. There was no significant difference between the individual owls. Derivation of the minimum audible angle To derive the owls’ discrimination performance between reference and test stimuli, we employed the sensitivity measure d’ [34,35]. In the single reference paradigm four valid ses- sions were required to obtain the MAA. In the multiple reference paradigm ten valid sessions were necessary. To obtain a psychometric function we first looked at the relationship between the propor- tion of correct responses and the angular separation between test and reference stimuli. Then we calculated the sensitivity for discriminating, separately for each angular separation. The thresholds defining the barn owls’ MAA were derived by fitting a cumulative normal distribu- tion to the data points constituting the psychometric function and by estimating the angular separation at which a d’ of 1.0 was reached (see S1 Fig for an example). In the single reference paradigm, a session was considered valid if (1) the owl achieved at least 12 Hits, (2) the false alarm rate was 20%, (3) the d’ value of the smallest angular separa- tion was below 1.0, (4) the d’ value exceeded 1.8 for one of the larger angular separations. Since it became apparent that the first two validity criteria were already good predictors for a stimu- lus driven response, the number of validity criteria were reduced for the second paradigm. Thus in the multiple reference paradigm, experimental sessions were included in the analysis if (1) the owl achieved at least 12 Hits and (2) the false alarm rate was 20%. Data analysis We performed a general linear mixed model (GLMM) analysis of variance to test for the effects of stimulus type and reference position. The dependent variable was the MAA. Statistical anal- yses were performed using SPSS 24.0 (IBM SPSS Statistics). A p value of 0.05 was the crite- rion for a significant effect. Since there was no significant difference between the frontal MAAs obtained with either paradigm, the data of the single reference and the multiple refer- ence paradigms were pooled in the analysis. Effect of stimulus type on localisation acuity The barn owls’ individual psychometric functions observed in the single reference paradigm are depicted in Fig 1. These functions show that the sensitivity varied between a d’ of about 0 and 3. Furthermore, the steepness of the psychometric functions increased with increasing test 5 / 19 PLOS ONE \| https://doi.org/10.1371/journal.pone.0220652 August 23, 2019 Barn owl MAA frequency. Individual MAA values that were obtained in either paradigm are listed in Tables 1 and 2. Generally the owls’ MAA decreased with increasing centre frequency. In the single ref- erence paradigm, the largest MAA of 13.7˚ ± 3.2˚ (mean ± s.d.) was obtained at 500 Hz and MAA decreased to 8.4˚ ± 1.4˚ at 8000 Hz (Table 1). With BB noise, the MAA further decreased to 4.9˚ ± 0.3˚. The owls’ localisation acuity differed significantly between stimulus types (F = 39.942, p < 0.001). Pairwise comparison (Bonferroni corrected) revealed that localisation acuity for all stimulus types was significantly different from that for the 500 Hz condition (p < 0.001). In addition, acuity obtained with BB noise was significantly different to that for the 1000 Hz, 2000 Hz and 8000 Hz conditions (p = 0.014, p < 0.001, and p = 0.007, respec- tively). Furthermore, localisation acuity for the 4000 Hz condition was significantly different from that for the 1000 Hz and the 2000 Hz conditions (p = 0.047 and p < 0.001, respectively). All other comparisons yielded no significant results. Effect of reference position on localisation acuity This corresponds to a larger MAA for lateral compared to frontal stimulus presentation. Irrespective of the stimulus type, the “frontal MAA” was significantly smaller compared to the “lateral MAA” (see statisti- cal results below). For any of the three reference positions, MAA values increased with decreasing centre frequency, similar to the data obtained with frontal stimulus presentation only (Fig 1). Again, the largest MAA values were obtained at 500 Hz (Table 2). “Lateral MAAs” were on average 19.9˚ at 500 Hz, 13.3˚ at 2000 Hz, 11.5˚ at 8000 Hz, and 8.2˚ for BB noise. “Frontal MAAs” obtained in the multiple reference paradigm were on average 14.5˚, 10.2˚, 6.6˚, 3.4˚, at 500 Hz, 2000 Hz, 8000 Hz and BB noise, respectively (Table 2). The barn owls’ localisation acuity differed significantly between reference positions (F = 26.829, 0 001) P i i i (B f i t d) l d i ifi t diff Effect of reference position on localisation acuity In the multiple reference paradigm, reference stimuli were presented from three azimuthal positions (-45˚, 0˚, +45˚). The psychometric functions of two barn owls, obtained in the multi- ple reference paradigm, are shown in Fig 2. The psychometric functions of the lateral reference positions were both shifted to the right relative to the frontal reference. This corresponds to a larger MAA for lateral compared to frontal stimulus presentation. Irrespective of the stimulus type, the “frontal MAA” was significantly smaller compared to the “lateral MAA” (see statisti- cal results below). For any of the three reference positions, MAA values increased with decreasing centre frequency, similar to the data obtained with frontal stimulus presentation only (Fig 1). Again, the largest MAA values were obtained at 500 Hz (Table 2). “Lateral MAAs” were on average 19.9˚ at 500 Hz, 13.3˚ at 2000 Hz, 11.5˚ at 8000 Hz, and 8.2˚ for BB noise. “Frontal MAAs” obtained in the multiple reference paradigm were on average 14.5˚, 10.2˚, 6.6˚, 3.4˚, at 500 Hz, 2000 Hz, 8000 Hz and BB noise, respectively (Table 2). The barn owls’ localisation acuity differed significantly between reference positions (F = 26.829, p < 0.001). Pairwise comparisons (Bonferroni corrected) revealed a significant difference between the frontal and each of the lateral reference positions (p < 0.001), but no significant difference between the two lateral reference positions. For the lateral positions we tested whether the direction of the change in stimulus position had a significant effect on the barn owls’ behaviour, i.e., we checked whether the behavioural response was biased towards "inward" or "outward" shift of location (relative to the reference position). We did not find such an effect (chi-square tests, using Hit and Miss responses). Moreover, there was no inter- action between stimulus type and reference position indicating that stimulus types and refer- ence position had an additive effect. In other words, the change of MAA with centre frequency was similar at all reference positions and the MAA was generally smaller in frontal space com- pared to lateral space. In the multiple reference paradigm, reference stimuli were presented from three azimuthal positions (-45˚, 0˚, +45˚). The psychometric functions of two barn owls, obtained in the multi- ple reference paradigm, are shown in Fig 2. The psychometric functions of the lateral reference positions were both shifted to the right relative to the frontal reference. https://doi.org/10.1371/journal.pone.0220652.g001 Stimulus type and localisation acuity Under natural conditions, barn owls will hear prey signals that are more likely broadband than narrowband in frequency, for example, rustling leaves stirred up by a mouse rushing to cover [1]. Broadband signals will stimulate a considerable proportion of the barn owl’s sensory epi- thelium in the inner ear and thus provide multiple localisation cues in a larger frequency range than narrowband signals. Hence sound localisation acuity for broadband signals is expected to be superior to localisation acuity for narrowband signals. The results from our study confirm that the barn owls’ localisation acuity is indeed best with BB noise. The MAA for BB noise sig- nals presented within frontal space was about 4˚. This remarkable acuity is in accordance with previous studies [10,22,27] investigating the sound localisation ability of the barn owl. Similar to our study, the behavioural studies referred to in Table 3 used either broadband and narrow- band stimuli or tones. Typically, the behavioural localisation performance deteriorates with decreasing centre frequencies. This was also true for the barn owls’ MAA values obtained in our study (Fig 1, Table 1, and Fig 3A). A study by Cazettes et al. [17] indicates that the width of ITD tuning curves of space-specific neurones in the external part of the inferior colliculus (ICx) becomes narrower with increasing frequency (Fig 4A in [17]). Since neural ITD responses are based on measurements of interaural phase difference (IPD), this is a direct con- sequence of the IPD sensitivity of their input neurones that are narrowly frequency selective responses are based on measurements of interaural phase difference (IPD), this is a direct con- sequence of the IPD sensitivity of their input neurones that are narrowly frequency selective [37]. In particular, the period length of a stimulus becomes shorter as the frequency increases [37], which also requires a higher temporal precision of the neurons [38]. Table 2. MAA values in degree of two barn owls (Ugle and Sova) as a function of reference position and stimulus type. Data were obtained in the multiple reference paradigm. Discussion In this study, we investigated the barn owls’ MAA for different stimulus types and different spatial locations. The different stimulus types covered nearly the entire hearing range of the barn owl [31,36]. In particular, we strove to also cover the low-frequency range (< 3 kHz) which had only been sampled sparsely in previous studies [10], allowing for a valid comparison of localisation acuity at high and low frequencies. Furthermore, using a conditioned beha- vioural response, different reference positions could be tested, enabling for the first time the comparison between frontal and lateral localisation acuity. 6 / 19 PLOS ONE \| https://doi.org/10.1371/journal.pone.0220652 August 23, 2019 Barn owl MAA Fig 1. Psychometric functions for all six stimulus types and three owls obtained with a single reference position at 0˚ in azimuth. The barn owls sensitivity (d’) is plotted as a function of angular separation between reference and test stimuli. The different colours represent the different owls. https://doi.org/10.1371/journal.pone.0220652.g001 Fig 1. Psychometric functions for all six stimulus types and three owls obtained with a single reference position at 0˚ in azimuth. The barn owls sensitivity (d’) is plotted as a function of angular separation between reference and test stimuli. The different colours represent the different owls. https://doi.org/10.1371/journal.pone.0220652.g001 7 / 19 PLOS ONE \| https://doi.org/10.1371/journal.pone.0220652 August 23, 2019 Barn owl MAA Table 1. MAA values in degree, mean values and s.d. of three barn owls (Ugle, Sova, and Weiss) as a function of stimulus type. Data were obtained in the single reference paradigm. stimulus type Ugle Sova Weiss mean s.d. 500 Hz 15.0 16.0 10.0 13.7 3.2 1000 Hz 12.3 12.9 7.7 11.0 2.8 2000 Hz 9.8 13.1 12.3 11.7 1.7 4000 Hz 7.3 6.6 6.3 6.7 0.5 8000 Hz 7.0 9.8 8.5 8.4 1.4 BB noise 4.9 4.6 5.2 4.9 0.3 https://doi.org/10.1371/journal.pone.0220652.t001 Table 1. MAA values in degree, mean values and s.d. of three barn owls (Ugle, Sova, and Weiss) as a function of stimulus type. Data were obtained in the single reference paradigm. https://doi.org/10.1371/journal.pone.0220652.t001 Stimulus type and localisation acuity reference position stimulus type Ugle Sova - 45˚ 500 Hz 20.3 19.7 2000 Hz 13.8 12.9 8000 Hz 10.9 11.7 BB noise 8.8 7.2 0˚ 500 Hz 14.1 14.9 2000 Hz 10.2 10.1 8000 Hz 5.5 7.7 BB noise 4.1 2.7 + 45˚ 500 Hz 20.0 19.7 2000 Hz 13.5 13.0 8000 Hz 10.7 12.5 BB noise 10.2 6.6 htt //d i /10 1371/j l 0220652 t002 able 2. MAA values in degree of two barn owls (Ugle and Sova) as a function of reference position and stimulus pe. Data were obtained in the multiple reference paradigm. 8 / 19 PLOS ONE \| https://doi.org/10.1371/journal.pone.0220652 August 23, 2019 PLOS ONE \| https://doi.org/10.1371/journal.pone.0220652 August 23, 2019 8 Barn owl MAA PLOS ONE \| https://doi.org/10.1371/journal.pone.0220652 August 23, 2019 9 / 19 Barn owl MAA Fig 2. Psychometric functions for four stimulus types and two owls obtained in the multiple reference paradigm (reference stimuli were presented from -45˚, 0˚, and +45˚). The barn owls sensitivity (d’) is plotted as a function of angular separation between reference and test stimuli. The different colours represent the different reference positions. https://doi.org/10.1371/journal.pone.0220652.g002 https://doi.org/10.1371/journal.pone.0220652.g002 Although the behavioural sensitivity of barn owls extends to frequencies as low as about 200 Hz [36], and even lower best-frequency responses have been observed neurally [39], no previous behavioural sound localisation study has used stimuli with an centre frequency lower than 1000 Hz (Table 3). In our study, we determined the barn owl’s localisation acuity down to a centre frequency of 500 Hz. The barn owl’s MAA was about 14˚ at 500 Hz, a value that is nearly twice as large as the MAA of 8˚ obtained at 8000 Hz. In contrast, estimates of human sound localisation acuity at 500 and 8000 Hz revealed an MAA of about 1˚ and 3.5˚, respec- tively [18] (Fig 3A). At first glance, human subjects thus appear to outperform barn owls. Humans, however, benefit from a much larger head that provides larger ITD and ILD cues. Generally, MAA values obtained in humans are smallest in the low-frequency range which plays a major role in communication [18,40]. A more appropriate comparison to owls with respect to head size is the cat, a predatory mammal with good low-frequency hearing and a slightly larger head than the barn owl (cat about 60 mm, barn owl about 44 mm) [41–43]. Stimulus type and localisation acuity Mar- tin and Webster [44] showed that the cat’s MAA for BB noise was about 4˚. The cat’s MAA val- ues for pure tones generally increased from 0.5 to 32 kHz [44]. This is in contrast to the barn owl, were MAA values generally decrease with increasing frequency. One explanation for that is, that the cats’ auditory system shows phase locking (the temporal synchrony to the wave- form) only up about 4 kHz [45]. Thus ongoing ITDs can only be neurally represented and used by the cat at frequencies below 4 kHz. At higher frequencies, ILDs become more effective [44,45]. Barn owls are most sensitive at frequencies between about 4 and 8 kHz [36], rely mainly on high-frequency cues for hunting [2] and show their smallest MAA at 8 kHz. Since the barn Table 3. Barn owl sound localisation performance for different stimulus types measured in selective studies. Study N procedure threshold criterion Stimuli localisation ability [˚] Knudsen and Konishi 1979 1 Search coil technique head turning accuracy (localisation error) 2 kHz 75 ms 3 kHz 75 ms 4 kHz 75 ms 6 kHz 75 ms 8 kHz 75 ms 9 kHz 75 ms 10 kHz 75 ms BB noise 75 ms 12 11 10 11 12 11 20 5 Knudsen et al. 1979 1 Search coil technique head turning accuracy (localisation error) BB noise 75 ms 3 Bala and Takahashi 2000 3 MAA, Pupillary dilation response (PDR) D > 0.8 4.5 kHz 100 ms BB noise 100 ms 5 3 Bala et al. 2003 3 MAA, Pupillary dilation response (PDR) D > 0.8 BB noise 100 ms 3 Present study 3 MAA, Go/NoGo d’ 1.0 (equivalent to 0.76 p(c)) 0.5 kHz 100 ms 1 kHz 100 ms 2 kHz 100 ms 4 kHz 100 ms 8 kHz 100 ms BB noise 100 ms 14 (± 3.5) 11 (± 3.1) 11 (± 1.7) 7 (± 0.7) 7 (± 1.4) 4 (± 1.3) PLOS ONE \| https://doi.org/10.1371/journal.pone.0220652 August 23, 2019 PLOS ONE \| https://doi.org/10.1371/journal.pone.0220652 August 23, 2019 10 / 19 10 / 19 Barn owl MAA Fig 3. Minimum Audible Angle (MAA) as a function of stimulus type in different species. Stimulus type and localisation acuity PLOS ONE \| https://doi.org/10.1371/journal.pone.0220652 August 23, 2019 11 / 19 Barn owl MAA owl’s auditory system shows phase locking up to 10 kHz, barn owls are able to use ITDs at high frequencies [38,46]. Furthermore high frequencies are over-represented on the barn owl’s basilar papilla: more than half of its length represents the frequency range between 4 and 8 kHz [47]. Based on all these findings, head size alone clearly is an unsuitable predictor for sound localisation acuity (see also [48]). Instead, ecological constraints like catching prey or sensitivity for communication signals might put sound localisation abilities and the underlying neural mechanisms under more or less selective pressure in different species [39]. owl’s auditory system shows phase locking up to 10 kHz, barn owls are able to use ITDs at high frequencies [38,46]. Furthermore high frequencies are over-represented on the barn owl’s basilar papilla: more than half of its length represents the frequency range between 4 and 8 kHz [47]. Based on all these findings, head size alone clearly is an unsuitable predictor for sound localisation acuity (see also [48]). Instead, ecological constraints like catching prey or sensitivity for communication signals might put sound localisation abilities and the underlying neural mechanisms under more or less selective pressure in different species [39]. Previous behavioural studies in the barn owl have also used different modes of stimulus presentation. Stimuli were either presented in a closed-loop condition (allowing for head turns during the presentation of the sound) [10,22,28] or in an open-loop condition (stimuli termi- nate before the subject can initiate head turns in response to the sound) [10,22]. Bala and col- leagues [26,27] presented their stimuli also under an open-loop condition, however, instead of orienting responses they measured pupillary dilation responses with the head fixated. It has been previously shown that stimulus duration affects localisation performance [49]. Stimulus duration can either support open-loop or closed-loop behavioural responses [21]. Closed-loop experiments with long stimulus durations allow the subjects to correct the initial trajectory of the behavioural responses based on the sensory feedback [21]. Therefore, long stimulus dura- tions (closed-loop condition) can yield better sound localisation thresholds (e.g. lower MAA or MRA values) than short stimulus durations [20,50]. Stimulus type and localisation acuity In the starling it has been shown that the mean MAA for BB noise improved by 11.2˚ when stimulus duration was increased from 100 ms to 1000 ms, whereas the mean MAA for a 2 kHz tone improved by 3.2˚ when presented with a long stimulus durations of 1000 ms [20]. Since closed-loop experiments do not provide much information about the mechanism of sound localisation, it is difficult to draw valid con- clusions on the subjects’ localisation acuity. Thus, data obtained under closed-loop conditions are not directly comparable to data obtained under open-loop conditions. Table 3 therefore refers only to behavioural studies that presented stimuli under open-loop conditions. Stimulus type and localisation acuity Data from different sources: (A) owl (blue dot, present study, N = 3), owl (red dot, Bala and Takahashi 2000, N = 5), starling (green dot, green circle, Feinkohl and Klump 2013, N = 4), cat (orange squares, Martin and Webster 1987, N = 5), human (black dot, Mills 1958, N = 3); (B) owl (blue square, red circle, green square, present study, N = 2), cat (orange dot, orange circle, Heffner and Heffner 1988, N = 4), human (black dot, black circle, Heffner and Heffner 1988, N = 4), human (black squares, Mills 1958, N = 3). The reference position in (A) was at 0˚ in azimuth and in (B) different reference positions were used. htt //d i /10 1371/j l 0220652 003 Fig 3. Minimum Audible Angle (MAA) as a function of stimulus type in different species. Data from different sources: (A) owl (blue dot, present study, N = 3), owl (red dot, Bala and Takahashi 2000, N = 5), starling (green dot, green circle, Feinkohl and Klump 2013, N = 4), cat (orange squares, Martin and Webster 1987, N = 5), human (black dot, Mills 1958, N = 3); (B) owl (blue square, red circle, green square, present study, N = 2), cat (orange dot, orange circle, Heffner and Heffner 1988, N = 4), human (black dot, black circle, Heffner and Heffner 1988, N = 4), human (black squares, Mills 1958, N = 3). The reference position in (A) was at 0˚ in azimuth and in (B) different reference positions were used. Fig 3. Minimum Audible Angle (MAA) as a function of stimulus type in different species. Data from different sources: (A) owl (blue dot, present study, N = 3), owl (red dot, Bala and Takahashi 2000, N = 5), starling (green dot, green circle, Feinkohl and Klump 2013, N = 4), cat (orange squares, Martin and Webster 1987, N = 5), human (black dot, Mills 1958, N = 3); (B) owl (blue square, red circle, green square, present study, N = 2), cat (orange dot, orange circle, Heffner and Heffner 1988, N = 4), human (black dot, black circle, Heffner and Heffner 1988, N = 4), human (black squares, Mills 1958, N = 3). The reference position in (A) was at 0˚ in azimuth and in (B) different reference positions were used. PLOS ONE \| https://doi.org/10.1371/journal.pone.0220652 August 23, 2019 Comparison of localisation acuity with physical cues The interaural connections are assumed to amplify sound localisation cues, like the ITD, especially in the low frequency [51,53]. According to that, HRTFs do not reflect what the barn owl actually perceives, especially at low frequencies. To reevaluate the effect of the internally coupled ears, Kettler and colleagues [54] measured eardrum vibrations in the barn owl, by using laser Doppler vibrometry. It was shown that low-frequency directionality increased with decreasing frequency in a narrow frequency band from 1.5 and 3.5 kHz [54]. Even though an effect of the internally coupled ears on directionality was apparent below 1.5 kHz, the authors stated that their eardrum vibration measurements were only reliable in a fre- quency range between 1.5 and 6.3 kHz [54]. More data on the directionality of the peripheral auditory system responses at low frequen- cies and on the contribution of the interaural connections are provided by a study from Cal- ford and Piddington [55]. They measured cochlear microphonics (CM) in the grass owl (Tyto longimembris), a Tyto species with a head size similar to that of the barn owl (T. longimembris 42.5 mm [55]; T.alba about 45 mm [42]). The results of Calford and Piddington [55] demon- strated that the overall ITD range available to the owl is clearly frequency dependent. The ITD range at frequencies above 1 kHz was similar to an ITD range estimated mainly by the path length around the head (approx. ±150 – ±220 μs) and as shown in HRTFs (S2 Fig). In contrast, at frequencies below 1 kHz, the ITD range available to the owl was much larger, up to ±400 and ±550 μs [55]. This supports the hypothesis that the internal coupling of the middle ears has a greater role at lower frequencies whereas at higher frequencies, these interaural connec- tions appear to be functionally irrelevant [55,56] (S2 Fig). At higher frequencies, the ITD range perceived by the owl should thus approximate its acoustic range of ITDs (measured as ±250 to ±300 μs [52], S2 Fig). From the CM-derived relation between sound source azimuth and ITD in the grass owl [55] we estimated the change in ITD per degree in azimuth using the slope of a regression line (panel B of S2 Fig). Comparison of localisation acuity with physical cues Azimuthal sound localisation acuity in the barn owl depends on the ability to process differ- ences in the arrival times of sounds at the two ears (ITDs). Sensitivity to ITDs depends on tem- poral coherence of the timing of the action potentials and the waveform (i.e., phase locking). The temporal information of a stimulus is encoded by phase locking of auditory-nerve fibre responses [51]. Avian auditory-nerve fibres typically phase lock up to 3–4 kHz [38]. The barn owl, however, extends phase locking up to frequencies of about 10 kHz [38,46]. The quality of phase locking is commonly expressed as vector strength. It is known that vector strength decreases with increasing frequency [38]. In the case of the barn owl, the vector strength of phase locking decreases from 0.9 at 0.4–0.5 kHz to 0.2 at 9 kHz [38]. Head-related transfer functions (HRTFs) are direction-specific acoustic filters that repre- sent all relevant cues that might be used by the auditory system for locating sound sources in space. It is possible to derive single spatial cues from HRTFs. We used HRTFs of barn owls [52] to convert our MAA values (depicted in Tables 1 and 2) into the corresponding ITDs. We estimated the change in ITD per degree in azimuth using the slope of a regression line (panel B of S2 Fig). The slope of the ITD representing frontal space, within ±10˚ in azimuth, was about 5 μs/degree for 0.5 kHz and only about 2.5 to 4 μs/degree for frequencies of 1 kHz and above. Using these slopes we derived “minimum audible ITDs” of about 71 μs, 44 μs, 44 μs, 27 μs, and 19 μs, for 500, 1000, 2000, 4000 and 8000 Hz, respectively. The slope of the ITD rep- resenting between ±35˚ and ±55˚ lateral azimuth was about 3.5 μs/degree for 0.5 kHz and about 1.4 to 4 μs/degree for frequencies of 1 kHz and above. Using these slopes, we derived PLOS ONE \| https://doi.org/10.1371/journal.pone.0220652 August 23, 2019 12 / 19 Barn owl MAA minimum audible ITDs of about 70 μs, 33 μs, and 46 μs, for 500, 2000, and 8000 Hz, respec- tively. Note that since the frequencies of HRTF and behavioural did not correspond exactly, we matched 3000 and 7000 Hz from HRTF data to MAAs at 4000 and 8000 Hz, respectively. HRTF recordings, however, do not consider the effect of the internally coupled middle ears of birds [53]. Comparison of localisation acuity with physical cues The slope of the ITD representing frontal space, within ±10˚ in azimuth, was about 11 μs/degree for 0.5 kHz but only about 2 to 5 μs/degree for frequencies of 1 kHz and above. The steeper slopes for frequencies below 1 kHz clearly reflect the contribution of the internally coupled middle ears, i.e., ITDs perceived by the owl are enhanced (which should improve localisation acuity at low frequencies). For the barn owls’ MAAs, these slopes predict the following equivalent minimum audible ITDs: the frontal MAA values for 500, 1000, 2000, 4000 and 8000 Hz correspond to an ITD of about 155 μs, 50 μs, 55 μs, 20 μs and 15 μs, respec- tively. The slope of the ITD representation between ±35˚ and ±55˚ lateral azimuth was about 4 μs/degree for 0.5 kHz and about 2 μs/degree for frequencies of 1 kHz and above. The lateral MAA values for 500, 2000 and 8000 Hz thus correspond to minimum audible ITDs of about 80 μs, 30 μs and 23 μs, respectively. These numbers for minimum audible ITDs are clearly larger for frequencies below 1 kHz than those for frequencies above 1 kHz. In addition, for 2000 Hz and 8000Hz, frontal and lateral minimum audible ITDs are similar while for 500 Hz, frontal minimum audible ITDs are considerably larger than lateral ones. Note that the above conversion of behavioural MAA to minimum audible ITD normalises out all known variations of the physical ITD cue, whether they arise from differential diffrac- tion in frontal vs. lateral space, or from the internally coupled middle ears across frequencies. In other words, if the neural processing of ITD were invariant across frequencies, the mini- mum audible ITD should not vary across frequency. However, it clearly does. This indicates that the underlying neural computation of ITDs is, firstly, frequency dependent (more PLOS ONE \| https://doi.org/10.1371/journal.pone.0220652 August 23, 2019 13 / 19 Barn owl MAA accurate at high than at low frequencies) and secondly, for a frequency of 500 Hz, is more accurate for lateral sound source positions than for frontal ones. The fact that the neural com- putation of ITDs is less accurate at lower frequencies might be explained by the temporal dis- persion in phase locking. Even though the quality (vector strength) of phase locking decreases with increasing frequency, the temporal dispersion (temporal jitter) is reduced because the period of the sound signal decreases with increasing frequency [37,57,58]. Localisation acuity as a function of sound source position In the present study, the barn owls’ localisation acuity was best when the reference stimuli were presented from 0˚ in azimuth and the test stimuli were also presented within frontal space. Irrespective of stimulus type, MAA values deteriorated when the reference stimulus was shifted into lateral space (Fig 3B). This is in accordance with a previous study in the barn owl using head-turning responses as a measure of localisation accuracy in an absolute localisation task [22]. Here the localisation error for a target sound presented within ± 10˚ in azimuth was about 2˚ (frontal space, open-loop condition, BB noise). The localisation error increased to about 6˚ and 9˚ for target sounds presented at ± 50˚ and ± 70˚, respectively [22]. When mea- suring azimuthal head-turning behaviour barn owls generally tended to underestimate the position of a noise burst presented laterally, i.e. their localisation accuracy decreased [10,59]. This implies a bias towards frontal sound source positions. The barn owl’s higher sound locali- sation acuity in frontal space not only holds for stationary sounds. They are also more sensitive in distinguishing leftward and rightward moving auditory stimuli presented in frontal space compared to lateral space [60]. Reduced localisation acuity for lateral sounds was also observed for behavioural responses of cats and humans (Fig 3B). Mills [18] showed for human subjects that localisa- tion acuity of tones was better in front compared to the periphery. The smallest MAA values for humans were about 1˚ at frequencies between 250 and 1000 Hz for a reference position of 0˚ in azimuth. The MAA increased when reference positions were shifted into lateral space. For frequencies below 1000 Hz, the MAA values were about 2.5 and 7˚ for reference positions of 45˚ and 75˚, respectively [18]. At higher frequencies, MAA values were much larger (see [18]. Heffner and Heffner [19] obtained similar results in humans and cats with broadband noise. For humans they reported thresholds of 1.3˚, 2.8˚, 4.4˚ and 9.7˚ for sound sources centred at 0˚, 30˚, 60˚, and 90˚, respectively [19]. In cats the thresholds for broad- band noise signals increased from 4.8˚ to 7.5˚, 6.8˚, and 9˚ for 0˚, 30˚, 60˚ and 90˚, respec- tively [19]. In an absolute localisation task Nodal and colleagues [24] found that the ferret’s (Mustela putorius furo) localisation performance also decreased when test signals were pre- sented from lateral positions [24]. Comparison of localisation acuity with physical cues This results in an enhanced temporal precision at higher frequencies compared to low frequencies [58]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0220652 August 23, 2019 Localisation acuity as a function of sound source position All of these behavioural studies show best localisation performance in frontal space, irrespective of head sizes and ecological constraints. Since azi- muthal sound localisation in mammals depends both on ITDs and ILDs, localisation acuity might not only be affected by ITDs. ILDs have been shown to become non-monotonic with increasing azimuth and thus ILDs possibly effect the localisation performance (e.g. [61]). In contrast to mammals, barn owls exploit ITD and ILD cues differently. CM recordings as well as HRTF recordings in the barn owl have shown that ITDs vary almost exclusively with azimuth, while ILDs vary less in azimuth than in elevation and are thus predicted to play only a minor role in azimuthal sound localisation [11,14,62]. Indeed, head orienting responses to dichotic stimuli delivered via headphones confirmed that ILDs vary only slightly with azimuth [12]. From these results we conclude that the barn owls’ localisation acuity probably was mainly affected by ITD cues and less by ILD cues. PLOS ONE \| https://doi.org/10.1371/journal.pone.0220652 August 23, 2019 14 / 19 Barn owl MAA Barn owl MAA In general, neurophysiological studies in the barn owl midbrain also suggested that localisa- tion acuity should be better in frontal space compared to the periphery. Knudsen and col- leagues found that the majority of space-specific neurones were tuned to the frontal space (about ± 30˚ in azimuth) and had more confined receptive fields in both azimuth and elevation than neurones tuned to lateral space [15,16]. Thus, the frontal space is clearly overrepresented in the barn owls’ neural map of auditory space [15,16]. In addition, Cazettes and colleagues [17] conclude that the ITD tuning width of the space-specific neurones in the ICx varied as a function of azimuthal location [17]. Fischer and Peña [63] applied a model to explain the barn owls’ better performance at frontal positions, i.e., superior localisation performance in frontal space than in lateral space. By using a Bayesian estimator, they were able to explain the sound localisation performance. Our behavioural observations on the MAA and the corresponding minimum audible ITDs for different frequencies (see above) are in agreement with these neurophysiological results. Interestingly, there also is a match between physiology and behaviour of the barn owl when relating the minimum audible ITDs derived from the MAA and frequency-specific variation in spatial tuning of ICx neurones. Localisation acuity as a function of sound source position Cazettes and colleagues [17] observed that high-frequency neurones in the ICx had more narrow ITD tuning curves than low-frequency neurones. Fur- thermore, neurons tuned to low frequencies preferentially responded to larger ITDs (corre- sponding to lateral sound source positions) whereas neurones tuned to higher frequencies preferentially responded to smaller ITDs, i.e., frontal sound source positions [64]. This matched the physical cue reliability of interaural phase differences for natural stimuli filtered by a typical owl’s HRTF: low frequencies provide a more reliable cue for stimuli emanating in lateral space and vice versa, i.e. better reliability of high-frequency components for stimuli emanating in frontal space [17,64]. Finally, Cazettes et al. [65] showed that these biases are conveyed, via convergent projections, to pre-motor neurones driving the owl’s spontaneous head-turning behaviour. Mean firing of the pre-motor neurone population predicted the owl’s general behavioural bias for greater localization accuracy in frontal vs. lateral space and also its greater bias when responding to high-frequency as compared to low-frequency band-pass noises [65]. For the owl’s behaviour in the present experiments, this predicts that the minimum audible ITDs corresponding to frontal and lateral MAA values are frequency-dependent. As shown above, the derived values for the minimum audible ITDs corresponding to frontal and lateral MAAs are consistent with these predictions. As expected based on the ITD tuning of ICx neu- rones at different frequencies, minimum audible ITDs were smaller at high frequencies than at low frequencies. Furthermore, whereas for high frequencies the minimum audible ITD was larger for lateral than for frontal positions, at the lowest frequency of 500 Hz the minimum audible ITD was the same for lateral and frontal positions. This is in agreement with the obser- vation of a greater bias towards frontal space in the accuracy of spontaneous head turns to high-frequency band-pass noises when compared to those elicited by low-frequency band-pass noises [65]. Supporting information S1 Fig. Description of fitting procedure for estimating the minimum audible angle (MAA). The data points representing response rate in relation to the angular separation of the sound sources were fitted by a cumulative normal distribution, applying the Generalized Reduced Gradient nonlinear method for minimizing the total RMS error of the differences between the fitted values and the measured data points using the "Solver Add-in" in the Micro- soft Excel version 2010. Adjusted parameters of this fitted psychometric function (blue line) 15 / 19 15 / 19 PLOS ONE \| https://doi.org/10.1371/journal.pone.0220652 August 23, 2019 Barn owl MAA were the lapsing rate (i.e., upper limit), the false-alarm rate (i.e., lower limit), the inflection point and the slope of the function at the inflection point. Based on the false-alarm rate, the hit rate that represented a sensitivity d’ of 1.0 for detecting the change in sound source location was calculated. For a false-alarm rate of 0.071 and a threshold d’ of 1.0 the hit rate at threshold is 0.319. Based on this threshold hit rate (green arrow) the threshold angular separation (black arrow, i.e., the MAA) of 4.9˚ is determined from the fitted psychometric function. (TIF) S2 Fig. ITD as a function of frequency and azimuth. (A) Dependence of ITD was calculated from cochlear microphonics (CM; closed symbols and solid lines) and head-related transfer functions (HRTF; open symbols and dashed lines). Data are estimated from Moiseff 1989 [11] (CM Barn owl, N = 1), from Calford and Piddington 1988 [55] (CM Grass owl, N = 1), and from Hausmann et al. 2010 [52] (HRTF Barn owl, N = 1). HRTF data represent the passive acoustic case, and CM data represent the internally coupled ears case. (B) From the data depicted in (A) we calculated the slopes of the ITD representation. Slopes representing frontal space within ±10˚ in azimuth are represented by closed symbols and solid lines, whereas slopes representing lateral space are represented by open symbols and dashed lines. 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https://openalex.org/W4252281733	https://geusbulletin.org/index.php/raekke5/article/download/7019/12889	Danish	null	Nordvestsjællands Geologi	Deleted Journal	1,943	cc-by	61,001	Danmarks Geologiske Undersøgelse. g V. Ræ kke. Nr. 6. Danmarks Geologiske Undersøgelse. V. Ræ kke. Nr. 6. Nord vestsjællands Geologi Ved V. Milthers Med 2 Kort og en Tabel Kø ben havn I Kommission hos C. A. Reitzels Forlag 1943 Danmarks Geologiske Undersøgelse. V. Ræ kke. Nr. 6. Trykt hos Nielsen & Lydiche (Axel Simmelkiær), Kobenhavn. Indhold. Indledning Side 5 Aflejringer ældre end Istiden . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Danium (Xyere Kridt) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Paleocæn . . . . . . . . . . .. . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Eocæn . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . 13 Istidsdannelserne og yngr e Jordlag . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 Inter glaciale Jordlag . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 Glaciale Aflejringer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 Moræneaflejringer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 Moræneler . . . . . . . . . . . . Indhold. . . . . . . . . 26 Morænesand og Morænegrus ....... . . . . . . . Lagdelte Glacialaflejringer ....... . .. .. .. . . Diluvialsand og -grus Stenfrit Diluvialler .. Stenene i Istidslagene ..... .. ........ . . . Landoverfladens Udformning ved Istidens Slutning Egnene mod Sydvest og Syd .. .. . . . .. . Røsnæs-Asnæs med Bagland (Røsnæs. - Kaastrup- Rugtved. - Asnæs- Ugerløse. - Svallerup-Svebølle. - Indlands- 32 34 34 42 56 61 62 isens Tilførselsretninger til Røsnæs og Asnæs) . . . . . . . . . . 62 Egnen Vest og Syd for Tissø . . . . . . . . . . . . . . . . . . . 73 Omraadet Syd for Aamose mellem Tissø og Stenlille. . 7-1 Omraadet mellem Haraldsted og Tølløse . . . . . . . . . . . . . . . . . . . . . . 80 Aamosedalen og dens Afløb ved Istidens Slutning . . . . . . . . . . . . . . 84 De centrale og nordlige Egne . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86 Morænefladen med de h atformige Bakker S f. Lammefjord.. .. 86 Højlandet mellem Skarresø og Sejerø Bugt med vestligere Forland . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 Bakkeomraadet Nord for Aamose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96 Omraadet mellem Ugerløse og Holbæk Fjord, samt Tuse Næs 97 De ·bueformige Højdedrag i Odsherred mellem Dragsholm og Højby .... .. .. .. .. .. . . . . ........ ......... .. . . .. .. . . . . . . ... 102 Odsherreds nordligste Del . . . . . . . . . . . . . . . . Nord vestsjællands Geologi Ved V. Milthers Med 2 Kort og en Tabel Indhold. . . . . . . . . . . . . . . . . . . . . . . 113 Sejerø .... ... . . .. ... . . . . .. . . . ......... .. . . ...... .. .. .. . .... . . . . ... 115 Side Senglaciale Aflejringer ................................................ 117 Hedesletter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117 Fossilførende, senglaciale Lag . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118 Postglaciale Aflejringer . . ... ... .. . . . . ...... ... ....... ........... . ...... 129 Ferskvandsaflejringer . .... . . . .... . . .. . . ...... . . . . ........ . . . ....... 130 Mosekalk og Kildekalk .... . .. ........ . ... .. . .. .................. 131 Tørv og Tørvemoser . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137 Saltvandsallej ringer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154 Havindskæringerne og deres Fauna . . . . . . . . . . . . . . . . . . . . . . . . . . . 154 Strandafl ejringerne og deres Højde .............. .. .... ..... . .. 163 Flyvesand . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Indhold. . . . . . . . . . . . . . . 173 Henvisninger og No ter .. . . . . .. ........ ..... . .... . . .... ...... ... . .. . . ... . .. 176 Stedfortegnelse .................. ... . .. . .. . . .. ... .. . . ... ............. 181 Indledning. N ordvestsjælland er den mest uregelmæssige Del af Sjæl- land, hvad Form og Overfladeforhold angaar. De lange Odder, der alle peger mod Vest og Nordvest, de fremtræ- dende, lange, buede Højdedrag og naturgivne Kysters tun- gede Forløb med de mange Bugter og dybe Indskæringer giver tilsammen et Landskabspræg, som gør Nordvestsjæl- land fuldstændig enestaaende, ikke blot inden for Sjælland, men inden for hele det danske Landskab. N Som noget helt for sig ligger de fremtrædende Odder og Næs. De har allerede i den danske Geologis Barndom været Genstand for vaagen, geologisk Interesse. I 1810 nævner HENRICH STEFFENS i sine »Geognostisch-geologische Auf- satze«, at man »ved Kalundborg skal have fundet Spor af Sandsten, ja, endog af Granit«. Det laa for ham nær at samstille Røsnæs med Kullen i Skaane, eftersom han gik ud fra, at Sydvestsveriges Grundfjæld strakte sig vidt mod Vest, udgjorde Bunden af Kattegat og traadte i Dagen i Hirtsholmene ved Frederikshavn. I sin første Danmarks Geologi i 1835 nævner FORCHHAM- MER vel ikke udtrykkelig Røsnæs, men paa det medfølgende Kort ligger Næsset i den Linie, fra Riigen, over Møns Klint til Mors, langs hvilken han mente, at der havde fundet Hævninger og Forstyrrelser Sted, hvorved Undergrundens Lag var skudt op til Jordoverfladen. Da han nogle Aar senere, i 1840, besøgte Røsnæs og her saa de forstyrrede, opskudte Lag af Plastisk Ler, var det naturligt, at han deri fandt en Bekræftelse af de Forestillinger, han havde om Danmarks geologiske Opbygning. 6 Aflejringer ældre end Istiden. D er er ingen Steder i Nordvestsjælland, hvor Under- laget for Istidens Aflejringer naar op til Jordover- fladen. Dets Beskaffenhed og Højdebeliggenheden af dets Overflade kendes kun fra Boringer. Da det er i ret be- grænset Omfang, at der er udført saa dybe Boringer, at den prækvartære Undergrund er naaet, kan Grænserne for de forskellige Jordlags Udstrækning kun bestemmes i store Træk. Det er - saa vidt det kendes - tre Formationer, som i hvert sit Omraade udgør det direkte Underlag for de kvartære Lag, nemlig D 1) Danium, yngste Etage af Kridtformationen. 2) Paleocæn, ældste Etage af Tertiærformationen. 3) Eocæn, næstældste Etage af Tertiærformationen. 6 Da FORCHHAMMER i 1858 paany udgav en Skildring af Danmarks Undergrund, fremhævede han stærkt sin For- modning om, at de tre lange Næs ud fra Sjællands Nordvest- kyst svarede til de tre >~Forbjerge« ved Sjællands Østkyst, nemlig Sjællands Odde til Amager, Sejerø til Stevns Klint og Røsnæs til Møn. Han siger, at Røsnæs »hører til Møens- kjædens Fortsættelse og viser den samme Forstyrrelse og de samme lodrette Lag, som Kridtet i Møens hvide Klint.« I Modsætning til saadanne Formodninger vides det nu, at Nordvestsjællands ydre Form ikke staar i nogen Aar- sagsforbindelse med Højdebeliggenheden af de Jordlag i Omraadets dybere Undergrund, som er ældre end Istiden. Disse Jordlag ligger overalt i betydelig Dybde under Hav- fladen og viser ingen Relation til den nuværende Overflades Højdeforhold. Mellem Fremkomsten af disse dybtliggende Jordlag og Fremkomsten af de Jordlag, der kommer til Syne i Over- fladen, ligger Tidsrum af lang Varighed, 1lemlig hele den mellemste og yngre Del af Tertiærtiden samt den ældste og den mellemste Del af Istiden. Det er derfor kun Sporene fra den sidste Del af Istiden og de efterfølgende Tidsrum, der træder umiddelbart frem for vore Øjne. De rummer dog en Ud-vjkling, der ikke blot har givet Landskabet dets nuværende, karakteristiske Træk, men ogsaa i den for- holdsvis korte Tid efter Istidens Ophør har medført ind- gribende Ændringer i Omraadets geografiske Fremtræden og ydre Form. Danium (Nyere Kridt). Daniet udgør den yngste af Kridtformationens Aflejrin- ger. Det er den, der udgør den prækvartære Undergrund i den nordøstligste Del af Nordvestsjælland, og den overlej- res mod Sydvest af Tertiærformationens ældste Afdeling, Paleocæn. Den omtrentlige Grænse imellem dem kan drages fra Orø over Tuse Næs, Lammefjordsdæmningen, Øst om Vig og Vest om Ellingegaard til Kattegatkysten imellem Lumsaas og Overby. Inden for det Omraade, hvor Danskekalken ligger direkte dækket af Istidens Aflejringer, synes dens Overflade at ligge i nogenlunde ens Højde, nemlig imellem 25 og 45 m under Havfladen. Dette fremgaar af det ringe Antal Boringer, der foreligger fra Omraadet: 8 Lumsaas Mejeri, 500 m Ø for Byen . . . . . . . . . . . . . . . . 7 40,5 m Svagbørnskoloni, Nykøbing Nordstrand, N for Skæreby Bakke . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + 42,5 - Rørvig Vandværk . . .. . ....... . .......... . .... . . . . + 37,0 - Nykøbing Vandværk, nær ved Jærnbanestationen .... + 44,5 - Odsherreds Destruktionsanstalt, 1 km VSV for Nykø- bing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + 30,0 - Ellingegaard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + 29,5 - Sidingefjord, henimod Udløbet . . . . . . . . . . . . . . . . . . . . 7 25,0 - I Omraaderne længere mod Syd og Sydvest, hvor Kalk- lagene er overlejrede af Lag fra den efterfølgende, tertiære Lagserie, ligger Danskekalkens Overflade gennemgaaende langt lavere og i betydelig mere vekslende Højde. Danium (Nyere Kridt). Direkte kendes Beliggenheden kun ved 3 Steder, nemlig Holbæk (+ 102,5 m), Herrestrup Andelsmejeri (+ 67,5 m), samt Od- dens Andelsmejeri, omtrent midt imellem Yderby og Overby, hvor Grænsen imellem Daniet og det overliggende dog ikke ligger dybere end + 42 m, d. v. s. paa det nærmeste samme Højde som ved Lumsaas Mejeri. Men indirekte fremgaar Kalkoverfladens dybere Beliggenhed - og rimeligvis stærkt varierende Højde - af, at de overliggende paleocæne og eocæne Lag stedvis har betydelig Tykkelse og gaar ned til stor Dybde (under Havfladens Niveau), uden at deres Un- derkant er naaet. Der kan derom henvises til de efterføl- gende to Lister over Lagdybder og Lagtykkelser. Efter hvad der foreligger fra Boringer ned i Daniels Kalklag, er det væsentligst Bryozokalk der forekommer i denne Del af Sjælland, ligesom Tilfældet er i Nordøstsjæl- land. Hvor stor den samlede Lagtykkelse af Daniet er, vides ikke, da man ingen Steder i Omraadet er naaet ned til dets Underlag, Skrivekridtet. Dette Lag træder kun i den syd- ligste Del af Sjælland frem som direkte Underlag for Kvar- tæret, bortset fra en lille Plet ved Køge Bugt. Som løsrevne Flager i Istidslagene er der dog enkelte Steder truffet Skrivekridt i denne Del af Sjælland. Dette gælder særlig den sydøstligste Del af det her om- handlede Omraade; det er fra gammel Tid kendt, at der i Allindelille Fredskov og Kastrup Skov findes Lag af Skrive- kridt. Det er de nordligste af et stort Antal Forekomster 9 af »brokket Kridt«, spredt over den sydlige Del af Sjælland. Det maa paaregnes, at Indlandsisen har medbragt disse Kridtflager fra Østersøegne, der slutter sig nær til Omraadet omkring Møn. Særlig paafaldende er Forekomsten af en Flage af Skrivekridt fra Bunden af Lammefjord NØ f. Gis- linge. Flagen havde kun en Tykkelse af ca. 1 m. Hvorfra dens Tilførsel til Lammefjord kan være sket, fra Østersøen eller Kattegat, er ganske usikkert. af »brokket Kridt«, spredt over den sydlige Del af Sjælland. Det maa paaregnes, at Indlandsisen har medbragt disse Kridtflager fra Østersøegne, der slutter sig nær til Omraadet omkring Møn. Særlig paafaldende er Forekomsten af en Flage af Skrivekridt fra Bunden af Lammefjord NØ f. Gis- linge. Flagen havde kun en Tykkelse af ca. 1 m. Hvorfra dens Tilførsel til Lammefjord kan være sket, fra Østersøen eller Kattegat, er ganske usikkert. 15, 7 m Lys, graa, uhærdnet Kertemindemergel, 67 ,3 °/o CaCO 3 • 14, 7 - Lidt grovere Grønsand, omkring 64 °/o CaCO 3 ; en- kelte mørkere, mere lerede Korn 36 °/o CaCO3 • 11,0 - Fint, graat Grønsand, 74,7-84 °/o CaCO 3 • 2,2 111 Hvidgraa Kalksten, lig Lellinge Grønsandskalk, 92,5 °/o CaCO 3 • 2,5 - Hvidgraat Kalksand, 56,8 0/o CaCO; Flint, der an- tyder, at Daniet er naaet. Med Undtagelse af det øverste Lag er hele Serien rig paa Glaukonit, der anses som Tegn paa, at Havvandet, hvori Udfældningen har fundet Sted, har været iltholdigt. Ved Stestrup Mejeri 5 km S f. Tølløse ved en Højde af ca. 56 111 er der ved Boring truffet Paleocæn i en Dybde fra 9·1,0--153 111, uden at Underlaget naaedes. Prøver fra 96 111 til 127 111 viser en Lagrække, der veksler mellem kalk- rig Kertemindemergel og kalkfattig Grønsandsten og med vekslende Indhold af Glaukonit. Efterfølgende Liste giver en Oversigt over, ved hvilken Højde i Forhold til Havfladen man ved Boring har truffet Grænsen mellem Paleocænet og de overliggende Istidslag. De bageste Tal angiver Tykkelsen af de gennemborede pal- eocæne Lag, og med et + er betegnet, at deres Underkant ikke er naaet. Oddens Andelsmejeri, midt imellem Y derby og Over by . . . . . . . . . . . . . . . . . . . . . . . . . . . . + 34 m; 8 m Vig Vandværk .......... . ....... ... ..... + 24 15 + Herrestrup Andelsmejeri ....... ......... + 27,5 - ; 40 Statsungdomslejr ved Avdebo ............ 7 42,5 - ; 23 - + Holbæk Vandværk,• Boring 1 km V for Hav- nen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + 54,5 - ; 46 Høng Vandværk ........................ 7 18,5 7,5 - + Ruds-Vedby Vandværk .................. 7 36,5 24,5 - + Nordruplund . . . . . . . . . . . . . . . . . . . . . . . . . . . . + 51 44 + Store Merløse Mejeri . . . . . . . . . . . . . . . . . . . . + 31 63 + Stestrup Mejeri . . . . . . . . . . . . . . . . . . . . . . . . 7 35 62 + Mejeriet Skovvang, 700 m N for Skee. . . . . . Paleocæn. Sydvest for Grænselinien for Daniet i Odsherred og paa Tuse Næs er det Tertiærets nederste Etage, Paleocænet, der udgør Underlaget for Istidsdannelserne. Dette er Tilfældet i hele den sydøstlige Del af Omraadet. Vestgrænsen gaar fra Sydøstkysten af Sejerø Bugt nær forbi Asnæs, Øst om Vognserup og Vest om Ruds-Vedby til Store-Bælts Kyst Syd for Mullerup. Ved denne Grænse overlejres det af Plastisk Ler. Paleocænets Underlag er naaet ved Boringer ved Hol- bæk og Herrestrup samt ved to Boringer ved Oddens Mejeri, midt imellem Overby og Yderby. Overdækket af Plastisk Ler er Paleocænet truffet ved Sejerby Mejeri, ved Hallebygaard Nord for Tissø og ved Gørlev Vandværk og Mullerup Teglværk. Blandt en lang Serie af Boringer, som er udført ved Hol- bæk langs Kysten af Holbæk Fjord, er man ved to Borin- ger Vest for Byen naaet ned i paleocæn Kertemindemergel som Istidslagenes Underlag (Fig. 7). Ved den vestligste Bo- ring i hele Serien naaedes dette Lag i Dybden + 51,5 m; ved den anden Boring, i Strandparken, 150 m Vest for Vand- værket, strakte Paleocænet sig fra + 54,5 m til + 102,6 m, hvor det hvilede paa Flintlag fra Danium. Den samlede Lagtykkelse var saaledes her 46 m. Lagfølgen var : 15, 7 m Lys, graa, uhærdnet Kertemindemergel, 67 ,3 °/o CaCO 3 • 11,0 - Fint, graat Grønsand, 74,7-84 °/o CaCO 3 • 14, 7 - Lidt grovere Grønsand, omkring 64 °/o CaCO 3 ; en- kelte mørkere, mere lerede Korn 36 °/o CaCO3 • 1 10 2,2 111 Hvidgraa Kalksten, lig Lellinge Grønsandskalk, 92,5 °/o CaCO 3 • 7 52 15 + Det er saaledes kun fra faa Steder det vides, ved hvilken Højde Paleocænets Underkant træffes. Dets Overflade synes ikke at rumme store Højdeforskelle; rimeligvis skyldes Overfladens nuværende Beliggenhed i væsentlig Grad den Bortskæring og Udjævning, som er sket i Istiden, og staar næppe i noget paaviseligt Forhold til de Kræfter, der har 11 frembragt og udformet selve de paleocæne Lag. Disse har sandsynligvis i en Del af Tertiærtiden ligget dækkede af eocæne og yngre Aflejringer, som er blevet fjærnede ved en senere foregaaet Denudation. frembragt og udformet selve de paleocæne Lag. Disse har sandsynligvis i en Del af Tertiærtiden ligget dækkede af eocæne og yngre Aflejringer, som er blevet fjærnede ved en senere foregaaet Denudation. Til et endnu dybere Niveau end nogen af de nævnte Bo- ringer, nemlig til + 120 m, er en Boring naaet ved Gislinge Vandværk og er dog ikke i denne Dybde naaet ned til Is- tidsaflejringernes Underlag. Indblandede Stykker af paleo- cænt Ler i de nedre Lag i Borehullet antyder, at der i de nærmere Omgivelser findes paleocæne Lag i mindre Dybde, end der her er boret til. Ved et andet Sted, nemlig Sofieholm, Vest for Tølløse, hvor der ved + 28 m er truffet 5 m sort, sandet Ler, og derunder 2 m »Flint, gulagtigt Ler« og 2,5 m » Kalkskifer«, er det maaske ikke udelukket, at det drejer sig om ter- tiære Lag. Der er inden for det Omraade, hvor Paleocænet udgør Undergrunden, en særlig Lejlighed til at lære et af dets vigtigste Jordlag at kende, nemlig Kerte minde m er- g e 1 en. Det er, hvor denne forekommer som løsrevne Fla- ger i Istidslagene i Egnene Syd for Regstrup Station, Sydvest for Holbæk. Kortet Fig. 1 viser Udbredelsen af et stort Antal af saadanne Forekomster. Den største er den, der paa Kor- tet er betegnet med en sort Rektangel, Nordøst for Hanerup ved Regstrup. De udfyldte Cirkler betegner Steder, hvor der ved Forekomsternes Kortlægning i 1930 var friske Profiler. I Forekomsten ved Hanerup er der en ca. 200 X 100 m stor Grav, som dog ikke nær omfatter hele den Flage, der ligger her. Dens Tykkelse naar op til 20-----25 m eller mere. Paa visse Steder ligger den paleocæne Mergel tæt under Overfladen og er paa andre dækket af Moræneler. 2,2 111 Hvidgraa Kalksten, lig Lellinge Grønsandskalk, 92,5 °/o CaCO 3 • I ufor- vitret Tilstand har den en svagt blaalig Farve; der ved For- vitring bliver mere lys gullig, og ved Tørring bliver Merge- len næsten hvid. Den er meget fed, kun ca. 6 0/o har Korn- størrelse over 0,02 mm; den indeholder omkring 70 0/o kul- sur Kalk, og blandt de større Korn udgør Glaukonit kun en yderst ringe Del, medens Svovlkis er en væsentlig Be- standdel. Det angives derved, at Aflejringen udgør en Dynd- Fig. 1. Kort over Forekomster med paleocæn Mergel. Se Teksten Side 11. Efter S1Gu110 HANSEN. (D. G. U. IV. Hk. Bel. 2. Nr. 7). . Kort over Forekomster med paleocæn Mergel. Se Teksten Side 11. Efter S1Gu Mergel. Se Teksten Side 11. Efter S1Gu110 HANSEN. (D. G. U. IV. Hk. Bel. 2. Nr. 7). 13 facies, afsat i stærkt iltfattigt Vand i nogen Lighed med Forholdene i Sortehavet. Det er kun en ringe Mængde Skal- ler af Muslinger, der hidtil er fundet i Kertemindemergelen ved Hanerup; de er fundet i nogle konkretionsagtige Lag og i daarlig bevaret, stærkt knust Form. De fundne Arter er: Dentalium rugiferum v. K., Nucula sp., Lucina sp., Mo- cliola sp., Natica sp. og Cyliclrna? sp. Der er endnu en Forekomst med omlejrede paleocæne Bjærgarter, der maa nævnes, nemlig: Klintebjærg i det nord- ligste Odsherred. I de mægtige Gruslag her forekommer der et stort Antal Blokke af Grønsandskalk med en rigelig Mængde Fossiler, fra en Fauna der har nogen Lighed med Faunaen i det paleocæne Grønsand ved København og Lel- linge. Hvorfra Blokkene stammer er imidlertid ganske usik- kert. Klintebjærg ligger inden for den Del af Nordvestsjæl- land, hvor den prækvartære Undergrund udgøres af Da- niels Kalklag. Tilførslen af Materiale hertil maa være fore- gaaet fra Retninger imellem Nordøst og Sydøst. Foruden Lagene af Grønsandsten og Grønsandskalk fra Nedre Paleocæn og kalkrig Kertemindemergel fra Mellem Paleocæn indeholder den paleocæne Etage øverst en Afde- ling med kalkfrit Ler (Øvre Paleocæn), som efter al Sand- synlighed ogsaa vil vise sig at være repræsenteret i den nordvestsjællandske Undergrund, men den er endnu ikke direkte paavist. Eocæn. Denne Etages Jordlag udgør den prækvartære Undergrund i den vestligste Del af Nordvestsjælland fra Sejerø Bugt ved Høve Strand til Store-Bælts Kyst Syd for Mullerup. Det mest almindelige af Eocænets Jordlag er det Plastiske Ler, der ogsaa i Nordvestsjælland fremtræder som vigtige Dag- forekomster, nemlig ved Sydkysten af Røsnæs. Ved adskil- lige af Borestederne er man ved Boringen naaet igennem det Plastiske Ler ned i dets paleocæne Underlag af Kerteminde- ler eller Grønsandslag. Ved Sejerby Mejeri er der boret ned i graat Kertemindeler, og ved Faarevejle er Paleocænet rime- ligvis ogsaa naaet. Sandsten med Glaukonit er truffet nederst 14 i den særdeles dybe Boring ved Hallebygaard, og det er rimeligvis lignende Lag af »Sandsten«, der er naaet ved Mullerup Teglværk. Ved Gørlev Vandværk er en· Boring ned til + 121 m ført langt ned i Kertemindeler. i den særdeles dybe Boring ved Hallebygaard, og det er rimeligvis lignende Lag af »Sandsten«, der er naaet ved Mullerup Teglværk. Ved Gørlev Vandværk er en· Boring ned til + 121 m ført langt ned i Kertemindeler. Nedenstaaende Liste viser Beliggenheden af det Plastiske Lers Overkant og Tykkelse af de gennemborede Lag af Eo- cæn ( og Paleocæn) : Sejerby Mejeri . . . . . . . . . . . . . . . . . . . . . . . . . + 66 m; Asnæs Vandværk . . ... . ................ + 74 Faarevejle Vandværk ................... + 25,5 Kalundborg Vandværk . .... ....... . . . .. . + 49 Kalundborg Radio, Gisseløre . . . . . . . . . . . . + 70 Avnsøgaard ....................... . ..... + 28 Vesterbygaard ............. . ........ . . . . + 38 Hallebygaard . . . . . . . . . . . . . . . . . . . . . . . . . . + 89 Kirke-Helsinge Vandværk . . . . . . . . . . . . . . + 35,5 Mullerup Teglvæ rk . . . . . . . . . . . . . . . . . . . . Fig. 2. Forekomster af Plastisk Ler (de sortfarvede Partier) langs Sydkystcn af Røsnæs. 1 : 80.000. Efter Geodætisk Instituts Kort. Eocæn. + 60 38 m + 26 + 89,5 + 0,5 + 4 + 29,5 + 41,5 + 103,5 + 15,5 - + 45,5 - + Listen viser, at Lagene liggev ved væsentlig lavere Niveau end længere mod Nordøst, og ligeledes ligger den prægla- ciale Overflade med mere vekslende Højder end i de øst- ligere Egne. Til den nederste Afdeling af Eocænet hører Lagene af vul- kansk Aske, der paa Mors og andre Steder, i Veksellejring med Lag af Diatomekisel, optræder som den bekendte Moler- formation. Det har vist sig ved adskillige dybe Boringer, hvorfra der er foretaget Undersøgelse, at der over Moler- formationen følger rødfarvet Plastisk Ler, der paa de fleste Steder er kalkholdigt. Det Plastiske Lers Farve kan iøvrigt variere stærkt mellem brun, graa, rød og grøn. Ligesaa kan der inden for hinanden nærliggende Lag af Plastisk Ler være en stadig gentagen Veksling imellem kalkfrit og kalk- holdigt Ler. Kendskaben til det Plastiske Ler i det nordvestlige Sjæl- land stammer overvejende fra Forekomsterne langs S y d- k y s ten a f R ø s næ s, hvor det dog ikke er faststaaende Lag der findes, men derimod Flager i Istidslagene. Der er paa spredte Steder truffet Lag af Plastisk Ler paa Stræk- ningen fra Fyrpynten til Kysthospitalet. Almindeligst er ·~ . I . -_,,,.-----------· -·------ / , \ \ Fig. 2. Forekomster af Plastisk Ler (de sortfarvede Partier) langs Sydkystcn af Røsnæs. 1 : 80.000. Efter Geodætisk Instituts Kort. ,_ 0 ,_.. 01 16 KELD MILTHERS fol. 1943. Fig. 3. Skred i Plastisk Ler med lodretstaaende Lag. Kysten SV f. Kongstrup. KELD MILTHERS fol. 1943. Fig. 3. Skred i Plastisk Ler med lodretstaaende Lag. Kysten SV f. Kongstrup. Forekomsterne fra Røsnæsgaard til Hellesklint SØ f. Snoge- kær Gaard, i Omegnen af Ulstrup, samt i Egnen fra 1 ½ km Vest for Kongstrup til Syd for Nostrup. I sin rene Tilstand er det Plastiske Ler usædvanlig finkornet. En Prøve har ved Slæmning vist følgende Korn- størrelser: 0,02 - 0,006 mm . ... . . . . ..... .. . . .. . 0,006- 0,002 ................... . 0,002- 0,0006 - ...... .... .. .. ... . . . < 0,0006 .. . ......... .... .. . . Eocæn. 1,0 0/o 6,8 - 2,6 - 89,6 - 17 I tør Tilstand er Stykkerne af Leret benhaarde; i stærkt vandfyldt Tilstand bliver det overordentlig blødt og glider let ud, naar det udsættes for Tryk af overliggende Jordlag. Hvor det fo rekommer ved Kysterne og Udglidningen ikke mød~r Modstand, opstaar derfor Skredterrasser, der især kan frembringe særdeles afvekslende Kystformer, hvor det li. ELO M ILT II E HS fo t. 1943. Fig. 4. Grav i Plastisk Ler, dækkel af lagdelt Sand. Syd for Kongstrup. li. ELO M ILT II E HS fo t. 1943. Fig. 4. Grav i Plastisk Ler, dækkel af lagdelt Sand. Syd for Kongstrup. Plastiske Ler grænser op til Istidslag, som kan modstaa Trykket. Saa længe der er Lag af Plastisk Ler, der k an give Anledning til yderligere Skred, efterfulgt af Havets Bort- fjærnelse af det udflydte Ler, er Kystens Udseende derfor udsat for stadige Ændringer. Som F ølge af dets overordent- lige F inkornethed har det Plastiske Ler ikke kunnet finde nogen praktisk Anvendelse førend i den allernyeste Tid, da det udgør Grundlaget for Fremstillingen af et porefyldt Byg- ningsma teriale, Betonklinker. 2 2 vulkanske Askelag. l{ysten af Røsnæs SV for Snogekær Efter S. A. ANDERSEN. (D. G. U. Il. R. Nr. 59). s. 19. vulkanske Askelag. l{ysten af Røsnæs SV for Snogekær Efter S. A. ANDERSEN. (D. G. U. Il. R. Nr. 59). s. 19. 1 19 d imod Kalundborg Fjord, neden for et Hus Sydvest for Snogekær Gaard er der et 30 n1 langt og 10 m højt Profil med bueformigt opskudte Lag, der har vist sig at indeholde »mo- lerlignende Lag«. Ved en Undersøgelse i 1935 er der h eri maalt en Serie paa 50- iO v u 1 kanske Aske 1 ag, der svarer til den nedre Del af den positive Lagrække paa Mors. En Bjærgart af lignende Art er i sin Tid fundet Sydvest for Ulstrup, hvor der har været Plastisk Ler fremme ved Ky- sten. I de allernærmeste Aar omkring 1940 er der forelaget ret omfattende Boringer for Undersøgelse af det Plastiske Lers Udstrækning og Beliggenhed for det forannævnte praktiske Formaal. Der er derved paa Strækningen fra Sydvest for Ulstrup til Øst for Sognegrænsen mellem Kongstrup og Nostrup flere Steder fundet Forekomster af nogle Hektars Udstrækning og med Laglykkelse paa indtil 25 m og der- over. Eocæn. Lagene omkring Kongstrup har overalt vist sig at være kalkfri ; derimod h ar det i et Omraade Sydvest for Ulstrup vist sig, at det rødfarvede Plastiske Ler var k alk- holdigt, n-iedens det grønlige og graa Ler ikke indeholdt kulsur Kalk. Der ligger maaske heri en An tydning af, at disse Lag ved Ulstrup h ører til i den nedre Del af den eocæne Etage sammen med nærliggende vulkanske Askelag. Lagene af Plastisk Ler i Omraadet omkring Kongstrup indeholder derimod blandt Farverne ogsaa rødfarvet Ler, uden at der her til denne Farve er knyttet noget Kalkind- hold som i Lagene ved Ulstrup. Det ligger i hvert Fald deri som en Muligh ed, at det Plastiske Ler i Kongstrup Omraadet h ører til en yngre Afdeling af Etagen end de vulkanske Askelag, d. v. s. til Mellem Eocæn og ikke til Nedre Eocæn . Bortset fra Farveforskellighederne er det Plastiske Ler særdeles ensartet; af Fossiler kan der findes en Del Forami- niferer. Jævnlig kan der træffes Gipskrystaller, ligesom der undertiden findes udskilt Mangankonkretiioner af stærkt varierende Form. Paa forskellige Steder er der ogsaa et Bortset fra Farveforskellighederne er det Plastiske Ler særdeles ensartet; af Fossiler kan der findes en Del Forami- niferer. Jævnlig kan der træffes Gipskrystaller, ligesom der undertiden findes udskilt Mangankonkretiioner af stærkt varierende Form. Paa forskellige Steder er der ogsaa et gult Mineral Natrojarosit (Guljærnerts) i smalle Spalter i det Plastiske Ler. Istidsdannelserne og yngre Jordlag. D en Udvikling, der har fulgt Istidens Indtræden og se- nere Tidsrum, har for Nordvestsjælland som for hele Danmark været præget af en stærk og stadig Skiften. I Løbet af de omfattende Tidsrum, som Istiden rumrn.ede, har kolde Afsnit med Indlandsis over Landene 3- 4 Gange veks- let rn.ed varme Klimaforhold, under hvilke der har værel lige saa gode og bedre Levebetingelser for Planter og Dyr som i Danmarks nuværende Land- og Havomraader. D e I det sidste kolde Tidsafsnit, da isfrit Land dukkede frem af Isdækket i Jylland, var Nordvestsjælland Genstand for, at Isstrømme skiftevis skød sig frem fra forskellige Side, og Landskabet fik derved formet vigtige Træk af sit karakteri- stiske Præg. - I den Tid der er forløbet efter Istiden, h ar Landet undergaaet Hævninger og Sænkninger i Forhold til Havfladen, som fra Tid til anden har ændret Kystliniens Beliggenhed og Landets Omrids i overordentlig Grad. Tykkelsen af Istidslagene er højst forskellig fra Egn til Egn. Hvis de underliggende Lags Overflade laa i ens Højde, vilde de kvartære Lags samlede Mægtighed være udtrykt ved Jordoverfladens Højdeforhold. Dette er dog ingenlunde Tilfældet, og der er desuden ingen som. helst Aarsagsforbin- delse mellem de to Overfladers Højdebeliggenhed. Mægtig- heden fremtræder næsten udelukkende som et Resultat af Indlandsisens Virksomhed i Istiden. Interglaciale Jordlag. I Istidens sidste varme Tidsrum, sidste Interglacialtid, naaede der i to fra hinanden adskilte Afsnit Havarme ind 21 over Dele af Danmark (se Fig. 6). I det første Tidsafsnit strakte der sig fra Vest, tværs over Slesvig og Holsten og de sydligste Dele af Danmark langt ind i Østersøens Dal en Hav- over Dele af Danmark (se Fig. 6). I det første Tidsafsnit strakte der sig fra Vest, tværs over Slesvig og Holsten og de sydligste Dele af Danmark langt ind i Østersøens Dal en Hav- MARINE AFLEJRINGER fra '<-'"-.,,_...,._'---:_-_-_-_~~--_-_-_-_-:___--::. SIDSTE INTERGLACIALTID =: Skærumhede-Seriens AOejringer 1111 Eem-Aflejringer 0 Aflejringer paa primært Leje L::!. Aflejringer paa sekundært Leje 10 l Holbæk 2_ Nordruplund 3_ Slrandegaa.rds Dyrehave 4. Høve 5. SkambæksMølle 6. Høng 7. K,benhavnsf'rihavn 8. Nebbegaard 9. Hven IO. StubberupHave Il. Dornbusch ~ ~ ~ -rr ffT TT TT Fig. 6. Efter H. ØouM (D. G. U. IV. H. Bd. 2. Nr. 10). MARINE AFLEJRINGER fra '< SIDSTE INTERGLACIALTID Fig. 6. Efter H. ØouM (D. G. U. IV. H. Bd. 2. Nr. 10). bugt - Eemhavet - med et rigt Indhold af varmeelskende Mollusker. Denne stærkt tempererede Havperiode afløstes af en Landhævning m ed et Tilløb til en Overskridning af Ind- landsis. Med et nyindtrædende mildere Klima fulgte igen en Landsænkning, der m edførte, at der fra Nord, over Vend- bugt - Eemhavet - med et rigt Indhold af varmeelskende Mollusker. Denne stærkt tempererede Havperiode afløstes af en Landhævning m ed et Tilløb til en Overskridning af Ind- landsis. Med et nyindtrædende mildere Klima fulgte igen en Landsænkning, der m edførte, at der fra Nord, over Vend- 22 syssel og Kattegat, kunde strække sig en Havbugt over Sjæl- land og videre ind i Østersøens Dal. Medens Eemhavet havde Betingelser for a t rumme en varmeelskende Dyrever- den, kunde den ny Havarm - Skærumhedehavet - ikke faa et lignende Dyresamfund fra de Dele af Verdenshavet, som det stod i Forbindelse med. syssel og Kattegat, kunde strække sig en Havbugt over Sjæl- land og videre ind i Østersøens Dal. Medens Eemhavet havde Betingelser for a t rumme en varmeelskende Dyrever- den, kunde den ny Havarm - Skærumhedehavet - ikke faa et lignende Dyresamfund fra de Dele af Verdenshavet, som det stod i Forbindelse med. Fra den første af disse to Havindskæringer kendes der ikke paa Sjælland Forekomster paa primært Leje. Interglaciale Jordlag. I Nord- vestsjælland er der dog to Steder, hvor der er fundet Jordlag med Skalrester, der kan stamme fra dette Havs Molluskind- hold. Det ene er H ø n g, hvor der i 1905 ved en Boring for Vandværket, i en Dybde af 56111 (ved + 20m), fandtes Grus med delvis rullede Skaller og Skalrester. Indholdet af Skaller fremgaar af Listen Side 25, Faunaen ligner meget Eemhavets Fauna og kan m ed Sikkerhed siges at stamme derfra, ved at Indlandsisen og Smeltevand fra Isen har ført det skalholdige Materiale frem til Vestsjælland fra Hav- omraaderne Syd for Store-Bælt. Foruden denne Boring h ar to senere udførte Boringer ved Høng Vandværk gennemtrængt skalførende Lag, som her laa ved Højder imellem ca. + 9 m og ca. + 2 m . Skallerne (og de ved Borearbejdet itubrudte Stykker ) er ualmindeligt velbevarede, saa at det kan anses for aldeles sikkert, at de fra første Færd er aflejret (ligger paa primært Leje) i det Sand, som Boringen er gaael n ed igennem. Derimod er selve det gennemborede Sandlag rimeligvis en Flage, som Ind- landsisen i sidste Afsnit af Istiden har revet løs fra det op- rindelige Lejested og ført til Høng fra sydligere Egne. Fau- naen har vist sig meget rig baade paa Arter og Individer, saa at der blandt Skaller fra en eneste Boring er fundet mange flere Arter, end der er fundet i de velbekendte Lag af samme Alder i Ristinge Klint paa Langeland. Flere af Ar- terne optræder i usædvanlig store og tykskallede Eksempla- rer, og Faunaen tyder paa stor Saltholdighed i det Hav, hvori Dyrene har levet. De for Eemhavet karakteristiske Arter er næsten alle fundet i Boreprøverne. (Om Indholdet af Arter h envises til Noterne Side 177 ). +20 r 0 7 +20 .51 .76 .71 .13 .JO .9 .!9 .17 .50 .12 .8 .7 .6 .2 .20 .18 .41 .7 I) .5 Arkiv Nr. 198. 0 0 ,10 f- 0 0 0 I +T. Q D . 0 0 0 0 0 0 0 0 I- ~-. - 0 - - 0 -- 0 - - 0 - 0 - - - 0 - ~ - 0 . 0 . 0 Q 0 0 .. 0 () D . Interglaciale Jordlag. 0 0 0 0 0 D 0 Q C, Q ::~:· 0 -70 f- 0 0 !/ ~ 0 ~1 i no 0 0 0 0 0 • ::.;. 0 0 . ?~ 0 0 0 C) 0 Q 0 ~ • 0 0 0 c20 f- m 0 o, 7 +20 . 0 •:• ;·\ 0 0 +30 f- ■ ::-: D :•· .. l dO I\.? :~: D i:,, +4o 1 ! r +40 - lnterglaoall mannl Ler +50 1 ~ 13:.J 01/uv/a!sand o_g-Grus [j 7 "50 M Moræneler +60 r § Pa!eocæn §I -\ +60 Meter -1 § 2700Me/er Fig. 7. Boreprofiler i og Vest for Holbæk. Se Teksten Side 9 og 24. E·ter H1LMA11 ØouM. (D. G. U. IV. R. Bd. 2. Nr. 10). +T. Fig. 7. Boreprofiler i og Vest for Holbæk. Se Teksten Side 9 og 24. E·ter H1LMA11 ØouM. (D. G. U. IV. R. Bd. 2. Nr. 10). 2 24 Det andet Findested er ved Røsnæs Sydkyst, imellem Skambæks Mølle og Kongstrup. Der er her i sin Tid fundet en skalførende Lerart, muligvis en Flage i Morænen, men om de nærmere Lejringsforhold vides intet. De fundne Skal- ler stemmer nærmest overens med Eemhavets; de for Eem- havet mest karakteristiske Former har dog ikke været re- præsenteret i den udtagne Lerprøve; desuden er den formo- dede Flage snarest kommet nordfra til Sydkysten af Røsnæs, og maa da antages at være omlejret to Gange. Medens det af Eemhavets Aflejringer kun er omlejrede Dele der forekommer, er der derimod paa et Par Steder i Nordvestsjælland truffet Lag fra Skærumhedehavet, lig- gende paa samme Sted hvor de oprindelig blev aflejret, nemlig ved Holbæk og ved Nordruplund. g g p Ved Hol b æ k er der paa en lang Strækning fra Ar- bejdsanstalten i Øst til 2 km Vest for Havnen, truffet marint, fossilførende Ler ved en Række Boringer. Planen Fig. 7 viser Boringernes indbyrdes Placering; det største Antal er sat ned langs Stranden vestpaa fra Byen. Lerets regelmæssige Op- træden viser med tilstrækkelig Tydelighed, at det ligger paa primært Leje. Den største Højde, Leret naar op til, er ca. + 24 m; den største fundne Mægtighed er ca. 18 m. Leret er meget fedt, i løs Tilstand lysegraat med Smaapletter af mørkere Farve som Følge af organisk Stof. Lagdelingen an- tydes stedvis af lysere, mere sandede Striber. Liste over de fundne fossile Mollusker. u .,: ., C C: :... "C ., "' ::l " ., ·;:: .,: "i5. 6 .a ., l\l .,: ::l ~ 6 VJ .a l\l :... ;:::l::: s "" 6..!l .a "C "' "" ... ., " .,- 0 .... > ~ æ·;:: ., "' ,.:.c! 0 "' .,: ., ... 0:: V:"' 0:: z 0:: I- (/)[/) Arktiske Arter: I li I I As/arte sp. . ... . ... . . . ... I X X X Bela Trevelyana ... .. .. . . . ? I I X Cylichna scalpta .. . . ....... X I I X Leda permzla ...... . ... .. X X ? X Mya lruncala . . .. . ...... . X X X X Nuculu tenuis .... . . . .. . . . X X X X Porllandia arclica ..... . . . . X X Saxicava arclica . .. ... . . . . X X X Tellina calcarea . ..... . ... X X ? X Boreale (nordlige) Arter: Cardium f ascialum . . . . . . . . ? I ? I X Cyprina islandica . .... .. . X X ? X Hydrobia ulvae .. ... .... . . X X I I Mylilus edulis . .. .. . . ... . . X X X Venus gallina .. . .. . ... .. . X I X X I Lusitanske (sydlige) Arter: I Bittium reticulatum . . .. .... X X I X Cardium echinatum . . . . . . . X X X - edule .. . .. . ... . X X X - exiguum ........ X X I Corbula gibba . . . . . . . . . . . . X X X Lucina divaricata .. . . . .. . X X I Mactra subtruncata . .... . X X Nassa I I pygmaea ... . ... .. .. . X X Nassa reticulata . . . . ... . .. X X I I X Nalica intermedia .. ... . . . X X X Nuculu cfr. nucleus . . .. . . X X I ? Nucula sulcata . . . . . . . . . . . . X X X ? Oslrea edulis . ......... .. . X X X Scalar•ia communis .. . . . . . . Interglaciale Jordlag. I de sammen- hængende Boreprøver laa Molluskskallerne samlede, men knuste. Foruden et betydeligt Antal Mollusker er der ogsaa i Leret fundet en Art af Smaakrebs, Balanus crenatus, samt 0 to liter (Øres ten) af den højarktiske Gadus saida (Polar- torsk). Det Dyresamfund, som Lagene indeholder Rester af, svarer nøje til Skærumhedehavets Indhold af Mollusker. Det andet Sted i Nordvestsjælland, hvor der er fundet Lerlag med en tilsvarende Fauna, er N o r d r up lun d, 11 km Nordøst for Slagelse. Laget laa her i en Dybde fra 42 til 59 m under Overfladen. Skallerne laa i knust Til- stand, men med Brudstykkerne samlede. Foruden de i Li- sten Side 25 anførte Mollusker fandtes der i Leret mange 25 Liste over de fundne fossile Mollusker Liste over de fundne fossile Mollusker. X X Scrobicularia piperata ... . X X X Tapes senescens . .. . .. . . .. X X Turbonilla lactea ..... .. . X X I Turritella terebrn .. . . .. . .. X X I X 26 ubestemmelige Fragmenter af Muslinger, samt mange For- aminiferer. Et tredje Sted, hvor der er fundet Ler med Skaller af samme Art, er 700 m NNV for Landsbyen Høve i Ods- herred, hvor det i 189'4 var til Syne i en gammel Mergel- grav. Det stenfri, meget fede Ler dækkedes af 1,6 m Mo- ræneler og havde en Mægtighed af mindst 3 m. Det inde- holdt især talrige Eksemplarer af Muslingen Tellina calca- rea, der alle var knuste, men ikke sjældent laa med Brud- stykkerne samlede. Lagets Højde, 18 m over Havet, viser, at det ligger paa sekundært Leje, men den regelmæssige Lag- deling og Skallernes Beskaffenhed antyder, at Lerflagen kun har været Genstand for en kortvarig Transport. Foruden Bløddyrskallerne fandtes mange Foraminiferer og Ostra- coder (Muslingekrebs). Medens de marine Skaller paa de tre nævnte Steder ligger i de Lerlag, hvor de er aflejret fra. første Færd, kendes saa- danne Skalrester ogsaa under Forhold, hvor de af Indlands- isen er revet ud fra deres oprindelige Leje, og Skallernes søndersplittede Dele er blevet blandet med Morænemate- riale. Paa denne Maade er der 1 km Sydvest for Tengsle- mark i det nordlige Odsherred fundet Skalfragmenter ind- lejret i Moræneler. Dette var lodret bænket, med skalførende Lag skiftende med skalfri Morænelerslag af mørkere Farve end de skalførende. Moræneler. Det stenholdige Ler, Moræneleret, er den Istidsjordart, som mest umiddelbart viser Beskaffenheden af det Materiale, Indlandsisen medførte og efterlod sig, da den smeltede bort. De Isdækker, som afsatte Moræneleret i denne Del af Sjæl- land, er kommet hertil fra vidt adskilte Dele af Skandinavien, hvilket fremgaar af de hjemstedsbestemmelige Sten, de saa- kaldte »Ledeblokke«, som Istidslagene indeholder. De Ret- 27 ninger, ad hvilke Indlandsisen til forskellig Tid er kommet, har kunnet være Nord- Syd, Øst- Vest, ja endog Syd- Nord. Da Isen som Følge heraf har bevæget sig hen over et Under- lag af vidt forskellige Bjærgarter, hvorfra der er indblandet Brudstykker i Isdækkets Morænemateriale, er Moræneleret derved blevet et sandt Bjærgartsmuseum, med store og smaa Best::i,nddele liggende fuldkommen regelløst indæltede imel- lem hinanden. Fra de skandinavisk e Fjælde er h entet Gra- niter , Gnejser og mange andre krys tallinske Bjærgarter; fra de omliggende Havomraader er medført Sandsten og Skifre, graa og røde Kalksten og andre lagdelte Bjærgarter, der- iblandt ogsaa Flint og Kalk fra Kridtformationens Lag i Sjællands nærmeste Omgivelser. ninger, ad hvilke Indlandsisen til forskellig Tid er kommet, har kunnet være Nord- Syd, Øst- Vest, ja endog Syd- Nord. Da Isen som Følge heraf har bevæget sig hen over et Under- lag af vidt forskellige Bjærgarter, hvorfra der er indblandet Brudstykker i Isdækkets Morænemateriale, er Moræneleret derved blevet et sandt Bjærgartsmuseum, med store og smaa Best::i,nddele liggende fuldkommen regelløst indæltede imel- lem hinanden. Fra de skandinavisk e Fjælde er h entet Gra- niter , Gnejser og mange andre krys tallinske Bjærgarter; fra de omliggende Havomraader er medført Sandsten og Skifre, graa og røde Kalksten og andre lagdelte Bjærgarter, der- iblandt ogsaa Flint og Kalk fra Kridtformationens Lag i Sjællands nærmeste Omgivelser. I nogen Dybde under Jordoverfladen er Moræneleret af blaagraa Farve og indeholder sædvanligvis større eller min- dre Mængder af kulsur Kalk, saa at det kan bruges som Mergel. Det er den Farve og Beskaffenhed, som det op- rindeligt aflejrede Materiale havde. Den gule til gulbrune Farve i Lerets øverste Del skyldes senere k emiske Om- dannelser, idet Ilt fra Atmosfæren og Kulsyre der af ned- sivende Vand er ført ned igennem Jordlagene, har iltet J ernforbindelserne og virket opløsende paa den kulsure Kalk. Derved er Lerets gule Farve fremkommet, og den op- løste kulsure Kalk er vasket bort, saa at Lerets øverste Lag k an være aldeles kalkfri i Modsætning til de dybere lig- gende Lag. Moræneler. Som Følge af at Transportvejene for Indlandsisen var for- skellige, og Adgangen til at optage Kalk fra Isens Underlag var meget uens, blev Morænelerets Kalkindhold paa nogle Steder stor men paa andre Steder kun lille. Særlig stor er Kalkm.ængden i visse Egne mod Sydøst. Der optræder h er paa nogle Steder Flager af rent, brokket Skrivekridt, i hvis Kridtmasse der ikke er blevet indblandet andre Dele af Ind- landsisens Morænemateriale. I Tilslutning hertil forekommer der paa mange Steder Syd for en Linie: Døjringe- Flinte- rup- Kyringe- Sk ee- Allindelille saakaldt »Hvid.ler«, hvor- ved forstaas særdeles kalkholdigt Moræneler. Ved Indblan- ding af indtil 65 °/o Kridt og derover i Isens øvrige Materiale 28 er Farven blevet ganske lys, hvad det stedlige Navn »Hvid- ler« antyder. I andre Dele af Nordvestsjælland optræder der ligeledes paa enkelte Steder Moræneler med højt Indhold af kulsur Kalk, som· Følge af at der har været Adgang for Isen til at optage kulsur Kalk fra det Underlag, som den har passeret paa sin Vej til Aflejringsstedet. Bortset fra Stederne og Egnene med de særlig kalkrige Morænelag ligger Indholdet af kulsur Kalk i Nordvestsjæl- lands Moræneler hovedsagelig mellem 10 °/o og 30 0/o. En Række Undersøgelser af Morænemergel her viser en yder- ligere Forskellighed fra Egn til Egn, som nok har nogen praktisk Interesse, men hvor det dog mere er geologiske Grunde, der giver Anledning til at hæfte sig ved dem, idet de sammen med andre Forhold peger hen paa, fra hvilke forskellige Sider Morænematerialet er ført til denne Del af Sjælland. Det fremgaar nemlig af disse Undersøgelser, at Kalkindholdet i Odsherred og i Egnene videre mod Sydvest langs Sejerø Bugt, til og med Røsnæs, hos den overvejende Del af Prøverne ligger under 20 0/o. I de sydøstligere Egne, inde i Landet, ligger Indholdet gennemgaaende lidt højere, men det er især i Landskabet, der grænser op til Store Bælt, Syd for Kalundborg Fjord, at der er en Tilvækst at spore. Her viser den største Del af Morænemergelen et Indhold af over 20 0/o kulsur Kalle Det Dække af Indlandsis, som har aflejret sit Morænemateriale her, har altsaa haft Lejlighed til paa sin Transportvej at opsamle et kendeligt større Kalk- indhold end det Isdække, der naaede frem til Egnene om- kring Sejerø Bugt. Senere hen skal det i Sammenhæng med en anden Side af Nordvestsjællands geologiske Forhold vises, hvorledes denne Forskellighed i Kalkindholdet kan være hidført. Moræneler. Det er allerede tidligere nævnt, at Moræneleret bestaar af en Sammenblanding af yderst uensartede Bestanddele. Dette gælder Materialets Ari:, og det gælder i samme Grad Bestand- delenes Kornstørrelse. Moræneleret kan indeholde det fineste Lerslam, Sand, og fra Smaasten op til Stenblokke paa mange Kubikmeters Størrelse. Paa de fleste Steder optræder Leret umiddelbart i denne inderlig sammenæltede, usorterede 29 Form, saaledes som det ligger i Jorden, og som det kan ses i nøgne Strandklinter, Lergrave og Mergelgrave eller andre kunstige Nedskæringer. De kantstødte Morænesten kan ligge paa Fladen eller staa paa Højkant, saa at der ikke er nogen Art af Orientering at øjne. Der er dog ogsaa Steder, hvor der er en Lagordning, en Bænkning til Stede i Leret. Ved den geologiske Korllægning i Nordvestsjælland er der flere Steder set en saadan Lag- ordning, endog med Overgang til tydelig Lagdeling. I Al- mindelighed er Lagstillingen ikke vandret, men hældende under alle mulige Vinkler. Leret er sandsynligvis oprindelig aflejret 111ed vandret Lagstilling, og Lerbænkene har senere ved Isens Tryk faaet den hældende Stilling. Lagene kan være saa tynde, at Bænkningen gaar over til en virkelig Skifrig- hed, saa at de enkelte Morænelag let kan skilles fra hin- anden uden at være adskilte ved Sandlag. Paa flere Steder er der tillige iagttaget en mere tydelig Lagdeling i Moræne- leret med vandret Lagstilling og hist og her med smaa mel- lemlejrede Lag af Sand og Grus. En udpræget Lokalitet af denne Art var Risbakke ved Sydspidsen af Nakkeland Syd for Rørvig. Der er her set et ca. 300 m langt Snit i Kyst- klinten med en 1,6 m h øj Væg af Moræneler, dækket af 2,2 m Strandgrus. I Moræneleret var der i hele Profilets Længde en meget Lydelig Lagordning med regelmæssige, horisonlale Lag. I en nærliggende Lergrav noget højere oppe viste Moræneleret ligeledes en udpræget Lagdeling. - Lig- nende Forhold har været til Syne i en Lergrav midt imellem Veddinge og Høve og andre Steder. Den vandrette Lagdeling i det gule, forvitrede Moræneler markeredes ved Veddinge yderligere ved tynde Lag af Sand og Grus. Et Profil med næsten lodret staaende Bænkning er set 1 km SV for Tengslemark i den nordlige Del af Odsherred. Moræneleret her udmærkede sig ved at indeholde Brud- stykker af Molluskskaller. Moræneler. Skalførende Lag vekslede med mørkere, skalfri Lag af Moræneler, saa at det heraf maa fremgaa, at Lagserien er bleven rejst paa Kant, efter at den oprindelig var aflejret med horisontal Lagstilling. 30 Hvor Udfældningen af Indlandsisens Morænemateriale, Moræneleret, har kunnet foregaa under rolige Forhold og uden at der har været Smeltevand i Virksomhed, er der Grund til at antage, at den aflejrede Bundmoræne er afsat Lag for Lag nogenlunde svarende til den nysnævnte ho- risontale Bænkning. Hvor Underlaget for denne Bund- moræne var plan, har Moræneleret kunnet aflejres ovenpaa som et Dække med jævn Overflade, hvad de bekendte Mo- ræneflader fra Østsjælland, Nordfyn og Lolland giver et ud- præget Udtryk for. Saadanne Moræneflader forekommer og- saa adskillige Steder i Nordvestsjælland, omend ikke i en saa ubrudl Form og af en saadan Udstrækning som i de nævnte Egne. I Nordvestsjælland findes der navnlig Syd for Lamme- fjord en saadan Moræneflade, hvis Præg af Flade dog i Egnen omkring Kundby og Sydvest derfor afbrydes af nogle Bakker, hvis Indre bestaar af Gruslag, men som er helt eller næsten helt overdækket af Moræneler, saa at kun den øverste Top rager op igennem Lerdækket. Paa lignende Maade som her i Kundby Egnen er Moræne- leret i store Dele af det nordvestsjællandske Landskab ud- bredt som et sammenhængende Dække over Jordlag af for- skellig Art. Disse Jordlag er ofte Sand og Grus eller ud- slæmmede Lag af stenfrit Ler, men det kan lige saa vel være tidligere aflejrede Lag af Moræneler. Det er blot i dette Tilfælde ikke altid saa umiddelbart let at se, hvor Grænsen er mellem det yngste Morænelersdække og dets Underlag af ældre Moræneler. Mange Steder er man dog i Stand til at finde Forskellene frem. De kan vise sig i Form af forskellig Kornstørrelse, ved Forskel i Stenindhold eller paa anden Maade. Dette er noget, der særlig kan være Lejlighed til at iagttage i Kystklinter af tilstrækkelig Højde, saaledes som de findes paa flere Steder for Eks. ved Lumsaas eller ved Sydsiden af Røsnæs. I høje Kystklinter og dybe Udgravninger af forskellig Art er der ogsaa Lejlighed til at se, at Moræneleret kan optræde paa anden Maade end som et Dække, nemlig som Indlag i Istidsaflejringerne, Side om Side med Jordlag af anden Art. I Kystklinten langs Sydsiden af Røsnæs er der særdeles god 31 Lejlighed til a t se Moræneler i denne Form. Moræneler. og ikke blot væltede Gruslag, saaledes som det er Tilfældet ved de saakaldte »hatformige Bakker« i Egnen ved Kundby, som senere skal omtales. Saaledes som det geologiske Kort viser det, bestaar Over- fladelagene i Nordvestsjæliand overvejende af Moræneler. Disse Overfladelag repræsenterer det Morænedække, som Indlandsisen efterlod ved sin Bortsmeltning fra Landet ved Slutningen af Istidens sidste kolde Afsnit, den sidste Gla- cialtid. Dets førnævnte Forskelligheder fra Egn til Egn hæn- ger som nævnt sammen med, at det er ført til Aflejrings- stederne fra forskellig Side, dels fra Øst og dels fra Syd. Disse Forhold vil senere blive nærmere omtalt i Forbindelse med de enkelte Egnsbeskrivelser og Forløbet af Israndens Opholdslinier i det Tidsrum, da Bortsmeltningen fandt Sted. Moræneler. Saadanne Ind- lag optræder h er sammen med Sand og Grus paa de mange Steder, hvor der træder Plastisk Ler frem i Klinterne og har foraarsaget Skred, hvorved Moræneleret er blevet blottet. Ved Skredterrassernes Sider, ud mod Kysten, ses Moræne- leret mange Steder som »Næser« af betydelig Højde rage op bag Forstranden og skille imellem to af de ved Skred op- staaede Fordybninger. Andre Steder staar det som Søjler, paa alle Sider omgivet af Istidsaflejringer af Sand eller Grus. Saadanne stejltstaaende Morænelersnæser , som ikke h ar del- taget i det Plastiske Lers Udskriden, forekommer f. Eks. langs Hellesklint SV for Ulstrup og ved Kongstrup Klinter (Fig. 19), hvor især Timeglasklint kan nævnes. Disse Mo- rænelerspartier og andre Istidslag tillige med de tilgræn- sende Lag af Plastisk Ler er tilsammentagne a t betragte som Flager af et vældigt Omfang, der af Indlandsisen er revet løs fra deres oprindelige Plads og fly ttet til den nu- værende. Af den ringe indbyrdes Sammenblanding af de for- skelligartede Lag kan m an slutte, at Indlandsisen kun har transporteret de omfattende Flager et kort S Lykke Vej, f. Eks. fra et Sted i Sejerø Bugt. Paa lignende Maade k an man flere andre Steder træffe omflyttede Lerlag af Moræneler. Hvor saadanne sammen- blandede Lag optræder, giver det sig jævnlig Udtryk i Over- fladeformerne ved a t udgøre Underlaget for et uregelmæssigt kuperet Landskab, h vis Overfladelag bestaar af Moræn eler, der som et Dække er aflejret oven paa det ujævne Underlag. Man træffe1· ogsaa stejltstaaende Lag af Moræneler og Grus- lag gaaende næsten h elt til Overfladen. Nævnes k an saale- des den sydligste af en Række Bakketoppe, »Ulvebjærge« SV for Svenstrup (Vest for Saltbæk Vig). I Bakken - der nu (1941) er gennemgravet paa tværs - h ar der som en 10'-- 20 m bred Revle imellem. indtil 5 n1 h øje Vægge af Moræne- ler staaet stejlt oppressede Lag af smaastenet Grus og finere lagdelt Materiale. Gruslagenes Strygningsretning varierer mellem SV-NØ og NV- SØ og er saaledes yderst uregel- mæssig; sammen med de opragende Partier af Moræneler tyder dette paa, at det h er drejer sig om virkelig omflyttede 32 og ikke blot væltede Gruslag, saaledes som det er Tilfældet ved de saakaldte »hatformige Bakker« i Egnen ved Kundby, som senere skal omtales. Morænesand og Morænegrus. Disse to Former af Moræneaflejringer er der ikk~ ved den oprindelige Kortlægning lagt Vægt paa at betegne paa det geologiske Kort, men vil være at finde under Betegnelserne lagdelt Sand og Grus. Den overvejende sandede Form er dog at betragte som yderst sjælden; derimod optræder Moræne- grus paa ikke saa faa Steder, men næsten altid under sær- lige Forhold, nemlig i saadanne Strøg, hvor Isranden en Tid har gjort Holdt under Tilbagerykningen, og hvor derfor en særlig stor Rest grove Dele af udvasket Morænemateriale er blevet levnet tilbage efter Isens endelige Bortsmeltning. Mo- rænegruset kan næppe noget Steds anses for at være pri- mært tilført Materiale, men er overalt en Udvaskningsrest, selvom det har den fuldstændig ikkelagdelte Beskaffenhed, som er Moræneaflejringernes Særpræg. I nogle Tilfælde ses Morænegruset at hvile paa Moræne- ler, men paa de fleste Steder underlejres det af lagdelt Grus. I begge Tilfælde naar Laget af Morænegrus sjældent en Tyk- kelse af mere end ganske faa Meter. Paa adskillige Stræk- ninger, hvor Morænegrusets Optræden er nær knyttet til Opholdslinier for Isranden, ledsages disse Strøg af en Rig- dom paa store Sten, der oprindelig har været jordfaste eller 33 33 KELD MILTIIEllS fol. 1943. Fig. 8. Fredet Stenbestrøning. Ubberup Højskole. Se Teksten Side 34. KELD MILTIIEllS fol. 1943. Fig. 8. Fredet Stenbestrøning. Ubberup Højskole. Se Teksten Side 34. i,ELu .,, 11 .T11E11s ro 1. rn43. Fig. 9. Fredet Stenbestrøning. Ubberup Højskole. I Baggrunden tilbøjre ses Bøgebjærg. Se Teksten Side 34. 3 i,ELu .,, 11 .T11E11s ro 1. rn43. Fig. 9. Fredet Stenbestrøning. Ubberup Højskole. I Baggrunden tilbøjre ses Bøgebjærg. Se Teksten Side 34. i,ELu .,, 11 .T11E11s ro 1. rn43. Fig. 9. Fredet Stenbestrøning. Ubberup Højskole. I Baggrunden tilbøjre ses Bøgebjærg. Se Teksten Side 34. 3 34 har henligget løst paa Markoverfladen, men som ved Op- dyrkningen er samlet sammen i Stengærder eller i store Stendynger. Saadanne store Stenbestrøninger paa Markerne, som i ældre Tid har været meget almindelige i visse Egne, findes der endnu enkelte Spor af. Nævnes kan saaledes en særlig smuk, fredet Stenbestrøning ved Ubberup Højskole. En forlængst fjærnet, pragtfuld Stenbestrøning fandtes i sin Tid ved A vnsøgaard, hvor der har været en udstrakt Aflej- ring af Morænegrus, og hvor der endnu i 1941 i et 100 m langt Profil kunde ses et indtil 6 m tykt Lag af Moræne- grus. Store, jordfaste Sten er bl. a. Morænesand og Morænegrus. set paa en Del af Viel- sted Aas NV for Gyrstinge Sø og paa Keldbjærg ved Ubbe- rup. - Den nære Forbindelse mellem Morænegrus og lag- delt Grus med store Sten bliver der ogsaa Lejlighed til at omtale senere. Diluvialsand og -grus. Et Blik paa det geologiske Overfladekort viser umiddel- bart, at der træder Istidssand og -grus frem til Jordover- fladen paa mange og ofte temmelig store Strækninger i Nordvestsjælland. Paa nogle Steder optræder Lagene af Sand og Grus som jævne Flader og i nær Tilslutning til Flader med stenfrit Ler. Eller de ligger i Veksellejring med Lag af stenfrit Ler. I begge Tilfælde giver Lagenes Optræden umid- delbart Udtryk for, hvorledes deres Opstaaen er gaaet for sig, nemlig i et afdæmmet Bassin eller som en Deltaaflej- ring under skiftende Strømforhold. Som Eksempler kan nævnes Omraaderne ved Tysinge Syd for Tølløse, mellem Bromme og Aamose, og et lille Sandparti ved Svallerup Nordvest for Tissø. Forholdene her nærmer sig de Tilfælde, hvor Sandet er afsat som Hedesletter, Sandflader, der er formet af Smeltevandet foran Indlandsisens Rand (ekstra- marginalt), idet Aflejringens Terrænform er bevaret ganske uforandret fra sin Opstaaen. Andre Steder danner Sandet »Fladbakker«, hvis Over- flade er ganske plane, men som ligger hævet op over de nærmeste Omgivelser, saa at man deraf kan slutte, at de ved Lagenes Opstaaen har ligget som et isoleret Delta, omgivet af et senere bortsmellet Isdække. Eksempler herpaa har man enkelte Steder Nord og Nordøst for Kalundborg og i Egnene Nord og Syd for Stenlille. I Tilslutning hertil kommer saa de Omraader med Sand og Grus, hvor Landskabsformen er ganske uregelmæssig med en stadig Vekslen mellem Bakker og Dale, og hvor den nuværende Terrænform ikke kan have været den, der fandtes ved de øverste Jordlags Opstaaen. Der maa dengang have været en Isophobning til Stede, som har givet en helt anden synlig Overflade end Nutidens, idet navnlig de nu- værende Sænkninger maa have været fyldt med jorddækket og senere bortsmeltet Is. Landskaber, som nu er ganske ujævne og af stærkt vekslende Form, kan ved disse sandede og grusede Overfladelags Opstaaen have været temmelig plane. Af Omraader, hvor vi kan tænke os, at saadanne For- hold har raadet, kan nævnes Bakkeegnene omkring Jyde- rup Stationsby, Sandomraaderne langs Sydkysten af Røsnæs og de bakkede Egne med Sand og Grus i Odsherred. Der k an i alle de forskellige Tilfælde være stor Forskel paa Kornstørrelsen af det udskyllede Sand- og Grusmate- riale, som Smeltevandsfloderne har efterladt sig under deres Løb. Det kan variere fra fint, ensartet Sand, igennem Sand- lag med Smaasten - eller en stadig Skiften mellem Sand- lag og Gruslag - til groft Grus med Stenblokke af stor Størrelse. Diluvialsand og -grus. Der kan ikke gives nogen almindeligt gældende Regel for Forholdel mellem Landskab og det afsatte Mate- riales Grovhed. Oftest vil dog nok stenfrit Sand være knyt- tet til plane Sandflader (Eks.: Omraaderne ved Tysinge og Svallerup), hvor Strømhastigheden kun har været ringe. Af- lejringen af særlig groft Materiale med Sten op Lil 112- 1 m i Tværmaal er fortrinsvis knyttet til Steder nær Smelte- vandsflodernes Udspring, og hvor en stillestaaende Isrand i nogen Tid har haft sin Plads. Blandt Eksemplerne herpaa kan nævnes Egnene ved Avnsøgaard, Ubberup Øst for Ka- lundborg, Bakker NØ for Grevinge og Bakkerækkerne mel- lem Højby og Svinninge i Odsherred. Man ser paa mange 36 Steder ved Gruslag af denne Art, at der i nogle Meters Dybde er godt lagdelt Materiale, vekslende mellem mere sandede og mere stenrige Lag; men opadtil aftager Lagdelin- gen samtidig med, at Stenmængden tager til, saa at der i de øverste Meter kun findes Morænegrus med sammenpak- kede Sten uden Antydning af Lagdeling. KELD MILTHERS fol. 1943. Fig. 10. Lagdelt Grus dækket af Morænegrus. Keldbjærg SV f. Ubberup. Se Teksten Side 35 og 69. KELD MILTHERS fol. 1943. Fig. 10. Lagdelt Grus dækket af Morænegrus. Keldbjærg SV f. Ubberup. Se Teksten Side 35 og 69. I Almindelighed ligger Lagene af det lagdelte .Sand og Grus nogenlunde horisontalt uden at være undergaaet væ- sentlige Forstyrrelser. Dette er let forstaaeligt, hvor velud- formede Lag gaar nær til Jordoverfladen, uden at der er Tegn paa, at de har været overskredet af noget Isdække; men ogsaa hvor Sandlagene ligger dækkede af Moræneler og saaledes har været isdækkede, kan de ligge uforstyrrede. Ofte kan der forekomme Spring og Smaaforskydninger som Følge af Sammensynkning inden for Lagene. Men i mange Tilfælde har Lagene været Genstand for gennemgribende 37 Forstyrrelser; de ligger med hældende eller foldede Lag, eller de ligger skudt ind som Revler i Moræneler. En saadan Lagstilling kan navnlig træffes i Bakker, der kan være frem- kommet ved Istryk. Ved den geologiske Kortlægning af Nordvestsjælland er saadanne forstyrrede Lag truffet flere Steder i den Række af Bakker, der strækker sig fra Melby, Syd for Kalundborg, i sydøstlig Retning hen imod Udby, og ligeledes i Bakkeomraaderne Nord for Aamose. Nævnes l(ELU ~ II LT II EHS fu t. 1V42. Fig. 11. Stejllstillede Sandlag. Hyrdehøj Syd for Kundby. l(ELU ~ II LT II EHS fu t. 1V42. Fig. 11. Stejllstillede Sandlag. Hyrdehøj Syd for Kundby. !{ELO MILTIIEHS fol. 1942. KELD MlLTH EHS fol. 1942. Fig. 12. Slejllslillede Sandlag i hatformig Bakke. Fig. 13. Stejltstillede Sandlag i hatformig Bakke. Enkeltpartier i samme Profil som Fig. 11 viser. Hyrdehøj Syd for Kundby. Diluvialsand og -grus. kan ogsåa den lidet omfattende, men forholdsvis h øje Bakke i Ulvebjærge Sydvest for Saltbæk. Langs Sydkysten af Røs- næs ses forstyrrede, oppressede Lag af Sand jævnlig Side om Side med de oppressede Lag, der her forekommer af Plastisk Ler, og ligeledes Side om Side med opragende Par- tier af Moræneler, der altsaa maa have været Genstand for Omlejring sammen med de andre Jordlag. kan ogsåa den lidet omfattende, men forholdsvis h øje Bakke i Ulvebjærge Sydvest for Saltbæk. Langs Sydkysten af Røs- næs ses forstyrrede, oppressede Lag af Sand jævnlig Side om Side med de oppressede Lag, der her forekommer af Plastisk Ler, og ligeledes Side om Side med opragende Par- tier af Moræneler, der altsaa maa have været Genstand for Omlejring sammen med de andre Jordlag. En særlig Gruppe af oprejste Lag af Diluvialaflejringer knytter sig til den Art af Bakker, der paa Grund af Form og Placering har faaet Betegnelsen: »hatformige Bakker «. De er alle mere eller mindre ellipsoideformede, og hvor de KELD MlLTH EHS fol. 1942. Fig. 13. Stejltstillede Sandlag i hatformig Bakke. Fig. 11 viser. Hyrdehøj Syd for Kundby. !{ELO MILTIIEHS fol. 1942. Fig. 12. Slejllslillede Sandlag i hatformig Bakke. Enkeltpartier i samme Profil !{ELO MILTIIEHS fol. 1942. KELD MlLTH EHS fol. 1942. Fig. 12. Slejllslillede Sandlag i hatformig Bakke. Fig. 13. Stejltstillede Sandlag i hatformig Bakke. Enkeltpartier i samme Profil som Fig. 11 viser. Hyrdehøj Syd for Kundby. <:..; (X) !{ELO MILTIIEHS fol. 1942. Fig. 12. Slejllslillede Sandlag i hatformig Bakke. Enkeltpartier i samme Profil !{ELO MILTIIEHS fol. 1942. KELD MlLTH EHS fol. 1942. 12. Slejllslillede Sandlag i hatformig Bakke. Fig. 13. Stejltstillede Sandlag i hatformig Bakke. Enkeltpartier i samme Profil som Fig. 11 viser. Hyrdehøj Syd for Kundby. 39 optræder paa iøvrigt fladt Terræn, har de ofte det hatformige Udseende, som har givet Anledning til Navnet. De findes især spredt over Morænefladen Syd for den inddæmmede Lammefjord og Svinninge Vejle, som igen mod Syd grænser op til det store Højdedrag omkring Skamstrup og Holm- strup, og mod Vest begrænses af det kuperede Højdedrag Vest for Hjembæk. Bakkernes Indre bestaar overalt af lag- delt Materiale, overvejende Sand med Gruslag i meget veks- lende Mængdeforhold. Undertiden forekommer der under- ordnede Lag af stenfrit Ler. Ofte træffer man en ganske usædvanlig livlig Vekslen mellem finere og grovere Lag af ganske ringe Tykkelse. Lagene er overalt bragt i en mere eller mindre opretstaaende Stilling. Diluvialsand og -grus. I Almindelighed er Lagenes Strygningsretning inden for hver enkelt Bakke nogenlunde ens, men undertiden kan La- gene være forskudt og bøjede, saa at de fremtræder som foldede og siksak.formede Linier igennem Profilet. Dette fremtraadte især tydeligt ved Kortlægningen i 1894 i et Profil i Lundebjærg 2 km SV f. Kundby, som Fig. 14 viser en Tegning fra. Lagene havde en Tykkelse af 1- 10 cm; de var konformt siksakbøjede, saa at de i Profilet fremtraadte som utallige, uregelmæssigt formede Savtakker, hvoraf kun de tydeligste og mest fremtrædende er vist paa Figuren. For- skydningens Størrelse beløb sig ingen Steder til mere end 20 cm. Som Figuren viser, var Lagene øverst undergaaet en Bøj- ning, saa at de laa omtrent vandret, med Lagenderne ven- dende mod Nordøst, i samme Retning som Bakkeoverfla- dens Hældning paa vedkommende Sted. Denne Bøjning maa antages at hidrøre fra en Slæbning, der enten kan tænkes at være foregaaet samtidig med, at Lagene blev rejst paa Højkant eller ved senere Bevægelser i det dækkende Islag i Bakkens Hældningsretning. De hatformige Bakkers kantstillede Lag er altid dækket af en Morænerest, der kan være af forskellig Mægtighed og Beskaffenhed. I Almindelighed bestaar Overfladelaget i Bak- kernes højeste Parti af Grus og Sand og er rimeligvis op- 40 staaet ved Isens Udtværing af Lagerie i Bakkernes Indre, saa at Lagdelingen er forsvundet. Paa enkelte Steder er Bakkerne dækket af Moræneler. staaet ved Isens Udtværing af Lagerie i Bakkernes Indre, saa at Lagdelingen er forsvundet. Paa enkelte Steder er Bakkerne dækket af Moræneler. I Lundebjærg var der i 1894 et 70 m langt Profil med lodret slillede Lag vinkelret paa Profilet. Forsaavidt de hele SV 1 :i 1, Fig. 14. Profil i Lundebjærg SV f. Kundby. Efter D. G. U. I. R. Nr. 8. 1 :i 1, Fig. 14. Profil i Lundebjærg SV f. Kundby. Efter D. G. U. I. R. Nr. 8. Bakken igennem staar paa samme Maade, vil Lagenes sam- lede Mægtighed her være mindst 200 m, nemlig det samme som Bakkens Udstrækning vinkelret paa Strygningen. Et lignende Resultat kommer man til for de nærliggende Bak- ker Gedebjærg og Kirkebjærgs Vedkommende. Det samme Maal finder man ved Tornved Bjærg, der ligger sydligere. Diluvialsand og -grus. Dalen er helt og holdent udformet i Tilslutning til Dannelsen af Aasbakkerne ved Vielsted og Gravningen af Gyrstinge Søs Dal, og den munder ud i de bredere Dalfor- mer Nord for Stenlille. Ud imod disse brede Dale har ogsaa Udmundingsstedet været for to andre korte Aase; de ligger paa hver sin Side af Sandlyng Aa Dal, den ene Vest og Nordvest for Stenlille By, den anden, sydligere, Sydøst for Tjørntved. I den syd- ligste Del af Stenlille Aas har der igennem en lang Aar- række været store Grusgrave med regelmæssigt, vandret lejrede Lag; deriblandt er en betydelig Mængde fint Sand, og nordligere i Bakken skal Lagfølgen i Følge stedligt Ud- sagn være Grus og derunder stenfrit Ler hvilende paa Sand. Syd for Ruds-Vedby strækker der sig fra Syd mod Nord en lang, lav Sandbanke af Aaskarakter. Den har sit højeste Punkt længst mod Syd i Tindingebanke. Ligesom ved Aas- bakkerne ved Stenlille har Vandtilstrømningen fundet Sted fra Syd. Ved Aagaard, Vest for Tissø, er der en lille Aas, Lange- bjærg, der med sin Retning Sydvest-Nordøst viser, fra hvil- ken Side Isen sidst er kommet hertil, nemlig fra Sydvest. Ved Trønninge, Sydvest for Holbæk Fjord, er der en Sandbakke, som her har en ret udpræget Aasform, der taber sig ganske i Grusomraadet ved Butterup og videre mod Syd- øst langs Kal veaa. Diluvialsand og -grus. Da Terrænet, bortset fra de opragende hatformige Bakker, er en jævn Moræneflade, er der kun ringe Sandsynlighed for, at Indlandsisen skulde have brudt saa mægtige Lag op 41 fra sit Underlag og atter aflejret dem med nogenlunde pa- rallel og indbyrdes lidet forstyrret Lagstilling, saaledes som det her er sket. Derimod er der al Grund til at antage, at de kanlstillede, mægtige Lag oprindelig er afsat i Spalter i Isdækket; de har derved kunnet opnaa den meget anselige Mægtighed, samtidig med at Lagenes horisontale Udstræk- ning kun var forholdsvis ubetydelig. Ved Isdækkets Bort- smeltning maatte de saaledes aflejrede Sand- og Gruslag næsten nødvendigvis komme til at hælde eller blive lodret stillede, ganske som Tilfældet er med Lagene i de hatformige Bakker. Paa nogle Steder fremtræder Bakkerne med Sand og Grus i Form af lange Enkeltbakker eller Bakkerækker, der er kendt under Navnet Aase. I Almindelighed betegner disse Bakkeformer Beliggenheden af Smeltevandsfloder, der enten har haft deres Løb under Isdækket eller paa anden Maade i snæver Forbindelse med Isen. Deres Længderetning viser Bevægelsesretningerne for Isdækket i Tidsrummet kort for- inden dets Bortsmeltning. De mest udprægede Repræsentanter for disse »subgla- ciale« Bakker og den dertil knyttede »Tunneldal« udgøres af »Aasen« Nord for Haraldsted Langsø, »Aasbanke« og sammenhørende Bakker ved Vielsted samt Haraldsted Sø og Gyrstinge Sø. Den fremtrædende Haraldsted Aas stræk- ker sig nogle Kilometer fra Øst mod Vest parallelt med Langsø. Profiler i Aasen viser flere Steder, at der under dens Opstaaen er afsat Lag af Moræneler ind mellem Aasens Gruslag. Tunneldalen Haraldsted Sø- Gyrstinge Sø viser det sub- glaciale Flodløbs Fortsættelse mod Vest, og der er langs Dalens Sider enkelte smaa Aasbakker, der afspejler Flo- dens Grusaflejringer. Ved Vielsted, Nordvest for Gyrstinge Sø, er der en Række smukt aasformede Bakker, der umid- delbart ved Byen skifter Retning fra Syd- Nord til Øst- Vest, og fortsætter videre i nordvestlig Retning frem til en veludformet Erosionsdal Nordøst for Ostrup. I Aasbak- kerne ved Vielsted By er der Grave med Grusmateriale af meget forskellig Størrelse, men stedvis stærkt sammenkittet, 42 delvis med Stenpakning og med talrige, kubikfod-store Sten liggende som Lag i finere Grus. Paa den nordvestligere Strækning er Aasfonnen ikke saa veludviklet, men der fin- des en Rigdom paa Sten og mange jordfaste Stenblokke. Langs Grusbæltet her strækker der sig et Dalstrøg, der er stærkt præget af subglacial Erosion og derved adskiller sig afgørende fra det tilgrænsende, smaakuperede Moræneland- skab. Stenfrit Diluvialler. I Modsætning til det stenholdige Ler, Moræneleret, der er udbredt som Dække over udstrakte Dele af Landskabet, er det kun som spredte, mindre Partier, at der forekommer stenfrit Ler; dette er ligesom lagdelt Sand og Grus opstaaet 43 ved, at Smeltevand h ar udslæmmet og sorteret Dele af Ind- landsisens Indhold af Morænemateriale. Det udslæmmede Materiale blev af Smeltevandsfloderne afsat efter Kornstør- relse og efter som Flodernes Strømstyrke var i Stand til a t transportere Materialet. Det grove, først Grus og derefter Sand, kunde efterlades paa Flodlejets Bund; det fine Lerslam førtes med, indtil der naaedes et Bassin, hvor det kunde bundfældes i stillestaaende Vand. Saadanne Bassiner har kunnet findes paa isbar Mark, m en Forholdene viser, at de lige saa ofte har kunnet være til Stede paa en isdækket Mark.overflade, hvor Bassinet har været helt eller delvis begrænset af stilleliggende, »død« Is. I Bassiner af denne Art er en stor Del opstaaet af de stenfri Leraflej- ringer, som forekommer i Nordvestsjælland, og som skal omtales i det følgende. Nogle af de stenfri Leraflejringer, som oprindelig er afsat i aabne Bassiner, er senere blevet dækkede af andre Istidslag, Sand- og Gruslag eller Moræne- ler, saa at kun mindre Dele af Lagserien nu træder frem ved Jordoverfladen. ved, at Smeltevand h ar udslæmmet og sorteret Dele af Ind- landsisens Indhold af Morænemateriale. Det udslæmmede Materiale blev af Smeltevandsfloderne afsat efter Kornstør- relse og efter som Flodernes Strømstyrke var i Stand til a t transportere Materialet. Det grove, først Grus og derefter Sand, kunde efterlades paa Flodlejets Bund; det fine Lerslam førtes med, indtil der naaedes et Bassin, hvor det kunde bundfældes i stillestaaende Vand. Saadanne Bassiner har kunnet findes paa isbar Mark, m en Forholdene viser, at de lige saa ofte har kunnet være til Stede paa en isdækket Mark.overflade, hvor Bassinet har været helt eller delvis begrænset af stilleliggende, »død« Is. I Bassiner af denne Art er en stor Del opstaaet af de stenfri Leraflej- ringer, som forekommer i Nordvestsjælland, og som skal omtales i det følgende. Nogle af de stenfri Leraflejringer, som oprindelig er afsat i aabne Bassiner, er senere blevet dækkede af andre Istidslag, Sand- og Gruslag eller Moræne- ler, saa at kun mindre Dele af Lagserien nu træder frem ved Jordoverfladen. Af Forekomster m ed stenfrit Istidsler, der ligger i nuti- digt lukkede Bassiner eller Lavninger, er der adskillige. Stenfrit Diluvialler. I saadanne Sænkninger er der aflejret stenfrit Ler ved det nedlagte Teglværk ved Asnæs i Odsherred, ved et nu ned- lagt Teglværk mellem Jyderup Station og Jyderup Kirkeby, ved Tølløse Teglværk og ved Ruds-Vedby Teglværk, hvor der ogsaa forekommer senglaciale, fossilførende Lag. Lige- saa i et Leromraade Læt Sydøst for Nyrød Gaard Øst for Stemnagle, hvorfra der er taget Ler til Vanløse Teglværk. Af Forekomster m ed stenfrit Istidsler, der ligger i nuti- digt lukkede Bassiner eller Lavninger, er der adskillige. I saadanne Sænkninger er der aflejret stenfrit Ler ved det nedlagte Teglværk ved Asnæs i Odsherred, ved et nu ned- lagt Teglværk mellem Jyderup Station og Jyderup Kirkeby, ved Tølløse Teglværk og ved Ruds-Vedby Teglværk, hvor der ogsaa forekommer senglaciale, fossilførende Lag. Lige- saa i et Leromraade Læt Sydøst for Nyrød Gaard Øst for Stemnagle, hvorfra der er taget Ler til Vanløse Teglværk. I flade Bassiner, som i mindre Grad har været begrænset af Is, er der mange Steder aflejret stenfrit Ler. Ved Gørlev Teglværk grænser en saadan Flade op til Engene langs Hel- singe Aa. Mellem Svallerup og Udby er der et stort Om- raade af lignende Art. Det bliver nærmere omtalt senere. Nord og Nordvest for Munke-Bjærgby er der et stort, fladt Omraade med stenfrit Ler. Det naar kun en Højde af 2- 3 m over den tilstødende Engflade Nordøst for Munke-Bjærgby og er baade mod Nord og mod Syd begrænset af højere Terræn . Ind mod Munke-Bjærgby, ved Teglværket, begræn- I flade Bassiner, som i mindre Grad har været begrænset af Is, er der mange Steder aflejret stenfrit Ler. Ved Gørlev Teglværk grænser en saadan Flade op til Engene langs Hel- singe Aa. Mellem Svallerup og Udby er der et stort Om- raade af lignende Art. Det bliver nærmere omtalt senere. Nord og Nordvest for Munke-Bjærgby er der et stort, fladt Omraade med stenfrit Ler. Det naar kun en Højde af 2- 3 m over den tilstødende Engflade Nordøst for Munke-Bjærgby og er baade mod Nord og mod Syd begrænset af højere Terræn . Ind mod Munke-Bjærgby, ved Teglværket, begræn- 44 ses det af et Bakkedrag med en Horisont af stenfrit Ler dækket af Sand. Ved Orebo Teglværk SØ for Niløse er der et Leromraade, der mod Nord og Vest maa have være begrænset af Is, hvorimod der ved de andre Sider er højere Terræn. Stenfrit Diluvialler. Under nært tilsvarende Forhold ligger et Leromraade Nord for Assentorp, NV for Stenmagle. Læn- gere mod Øst, i Egnen ved Vanløse, i et særdeles fladt Sand- omraade er der flere Partier af stenfrit Ler, grænsende op til Mosedrag, som Lerhorisonten paa sine Steder gaar ind under. Paa lignende Maade som her optræder der store Lerarealer Nord og Vest for det udstrakte Moseomraade, Tysinge, Syd for Tølløse. Landskabet her er overordentlig fladt, af Karakter som et Deltaomraade med Vandtilstrøm- ning fra Østsiden ; den nuværende Mosesænkning maa have ligget fyldt af Is op i Højde med den omgivende Flade. En dybt nedskaaren, Øst- Vest gaaende Dalfure Nord for Kirke-Eskilstrup er et sidste Spor efter Vandtilførslen fra Øst. Afløbet fra det flade Deltabassin har været hæmmet af omkringliggende Dødis, gennem hvilket Tysinge Aa har banet sig et slingrende Løb frem imod Halleby Aa. Ved disse og andre lignende Forekomster med stenfrit Istidsler, hvor Leromraaderne optræder med ganske plan Overflade og kun i ringe Udstrækning har været begrænset af Dødis eller af stærkt isfyldte Jordlag, kan man af Over- fladens plane Form med temmelig stor Sikkerhed drage den Slutning, at Bunden af de Søer, hvori Udfældningen fandt Sted, bestod af isfri Mark. Disse Forekomster staar derfor, ligesom dem i nutidige Bassiner, tæt ved Overgan- gen til »senglaciale« Aflejringer, hvad Aflejringstiden an- gaar. De har dog ganske samme Præg som de stenfri Ler- lag, hvis Overfladeform viser, at de er opstaaet under mere glaciale Forhold, og som nu skal omtales. Det drejer sig om et stort Antal Steder, hvor der fore- kommer lagdelt, stenfrit Ler i kuplede eller flade Bakker, » P 1 a t e a u b a k k e r«, som helt eller næsten til alle Sider rager højere op end det omgivende Landskab. Da Lagenes Beskaffenhed viser, at Leret oprindelig maa være slæmmet ud i lukkede Bassiner, er det indlysende, at Aflejringen er 45 foregaaet i Issøer, d. v. s. Søer paa Isoverfladen, og helt om.givet af stillestaaende, »død« Is. Disse Forekomster op- træder især i Omraadet mellem Røsnæs og Tissø samt i Egnen mellem Knabstrup og Aamose; de er fortrinsvis knyttede til Strækninger, der har ligget i snæver Forbin- delse med en stillestaaende Isrands Opholdslinie. De er af en saadan Interesse, at de maa gives en nærmere Omtale. yttede t St æ ge , de a gget s æve o b delse med en stillestaaende Isrands Opholdslinie. Stenfrit Diluvialler. De er af en saadan Interesse, at de maa gives en nærmere Omtale. I det n o r d ø s t 1 i g e B æ 1 t e a f R ø s n æ s ligger der to saadanne Lerforekomster, begge helt og holdent om- givne af Moræneler. Den ene ligger Sydøst for Landsbyen Aagerup. Det er et 800 X 400 m stort Omraade med nyre- formig Omkreds. Det naar en Højde af 28 m, flere Meter højere end Omgivelserne med Undtagelse af den runde Morænebakke, Ravnshøj, der halvt omkredset af det sten- fri Ler rager op til 37 Meters Højde ved Sydsiden af Om- raadet. - Det andet Lerparti ligger noget længere mod Sydøst, mellem Langedams Gaard og Nostrup Gaard. Det har en Længde af 1 ½ km og en største Bredde af 400 m. Det naar overalt en større Højde end de tilgrænsende Om- givelser, men Omraadets største Højde, 34 m, ligger længst mod Sydsydøst, ved Nostrup Gaard, medens det ved den modsatte Ende, ved Langedams Gaard, kun naar ca. 15 m. Ved den nordøstlige Side har Kallerup Teglværk en Ler- grav. Leret naar her en Tykkelse af ca. 4 m. Boringer paa Langedams· Gaards Mark i 1939 viste en Maksimaltykkelse af 6,5 m. Den aftagende Højde fra SSØ mod NNV antyder, at Søens Bund har bestaaet af Is, saaledes at den gradvis er sunket, eftersom Isunderlaget smeltede bort. I Sammen- hæng dermed staar muligvis det, at Lerbakkens Overflade paa en Strækning er noget ujævn og ikke af Kuppelform saaledes som Lerbakken ved Aagerup. Terrænets Form og andre Forhold viser, at man her har et Israndsstrøg med Forløb NV-SØ. - Til et lidt senere Stadium af Israndens Tilbagerykning mod Nordøst hører nogle »Plateaubakker« Øst for Kallerup og omkring Vollerup. Det er Bakker med plan Overflade i Modsætning til det omliggende, lavere og ujævne Landskab, men Bakkerne indeholder kun Sand og Grus; deres største Højde er 24 m. 46 I nær Tilslulning til samme Israndsstrøg som de nævnte Plateaubakker ligger en Lerbakke af samme Art ved Ubberup, Øst for Kalundborg. Den er formet som en noget afrundet Trekant med ca. 400 m lange Sidelinier. Dens højeste Punkt, 31 m, naar 10 m op over det omgi- vende, temmelig udjævnede Landskab, hvis Fladepræg af- brydes af mange fremtrædende Bakker. Stenfrit Diluvialler. Aflejringen af det stenfri, tydeligt lagdelte L_er har en Tykkelse af 4- 5 m og hviler paa Moræneler. I en Lagrække paa 271 cm har der kunnet maales 7 Aarsvarv (se Side 55), uden at Lerels Bund var naaet. Enkelte af »Varvene« indeholdt et stort Antal Finlag, saakaldte »Døgnvarv«. I Omraadet længere mod Sydøst, Egnen Syd for V æ r s 1 e v, findes der flere fremtrædende, stenfri Ler- aflejringer, nogle meget omfattende, andre ganske smaa. Den, der landskabelig træder stærkest frem, er Lerbjærg 1 ½ km Øst for Forsinge Station. Med sin største Højde, 57 111, naar den 20-30 m op over det omgivende Landskab. Den kuplede Bakkes Omkreds er pæreformig; den maaler 1 km i Længden og 700 m som største Bredde, og det stenfri, fede Ler naar næsten overalt helt ud til Bakkens Side- skraaninger. En mere sjælden Fremtræden viser Forekomsten af Pla- teauler mellem Klovby og Værslev Gaard. Hele Leromraadet her omfatter omtrent 1 km 2 • Paa en Strækning vestpaa fra Klovby optræder det med næsten horisontal Overflade, der længst mod Vest ligger 15 m højere end det vestligere Mo- ræneland. Mod Sydøst gaar det stenfri Lerareal uden Ter- rængrænse jævnt over i Morænelersterræn. - I modsat Retning, mod Nordvest, paa en Strækning af 1 km har den stenfri Leroverflade et jævnt Fald paa 15 m fra Fladen ved Klovby til Værslev Gaard, hvor den uden Højdeforskel gaar over i det tilgrænsende Morænelandskab ved samme Højde som Plateaubakkens Fod Vest for Klovby. Hvorledes denne skraanende Leroverflade er opstaaet, er det vanske- ligt at forstaa, da den jævne Overflade gør det mindre sand- synligt, at den er fremkommet ved Eftersynkning efter et bortsmeltet Underlag af Is. ~"' •J . , .•. ' ~.1--,,:L,'--,/-0/'r"~-. / : ~ , _____ -_-_ ... --~-. ·-:-:-~-~.~~-~~~-:r·::.f' ~,,· ff -:, te/fJk_ ; );y11;~;.\ ,' y.·~ '•<!fv"--7'.\,;; ~- ,__, l :-, :: -. ~ ,:,J ~-- -- -- - / } ; I' ~ Fig. 15. Bakk er og Flader med Plateau-Ler. Maaleslok 1 : 40.000. Se Teksten Side 46. Autoriseret Gengivelse efter l\Iaalebordsblad c 6 Værslev, 1895. ~ -.J ~-- -- -- - / } ; I' ~ Fig. 15. Bakk er og Flader med Plateau-Ler. Maaleslok 1 : 40.000. Se Teksten Side 46. Stenfrit Diluvialler. Autoriseret Gengivelse efter l\Iaalebordsblad c 6 Værsle 48 Mellem Udby og Svallerup og nordpaa fra denne sidste By optræder der stenfrit Ler i et ret stort Omraade. Men medens Leromraadet mellem Klovby og Værslev Gaard væ- sentlig er formet, dels som en næsten horisontal Del og dels som en Skraaflade, bestaar Omraadet Nordøst for Svallerup af to sammenstødende Dele, hvis Fællesgrænse er en ca. 5 m høj, brat Skraaning. Afdelingen oven for Skraaningen strækker sig fra Syd til Nord fra Katholm og Drengager Huse over Yppenbjærg Huse til Dysselodden Øst for Uger- løse. Dens Terrænhøjde varierer m ellem 32 og 36 m. Den lavere Lerflade strækker sig fra Udby næsten til Svallerup og videre mod NNV næsten til Gaarden Nygaard Sydøst for Ugerløse. Dens Højde naar fra 30 m ved Skraaningens Fod til 20 m længst mod Sydøst. Det kan med næsten fuld Sikkerhed antages, at Aflejrin- gen af dette sammenhørende, men todelte Leromraade er foregaaet i to Sæt, først den højere og sidst den lavere Afde- ling, efter alt at dømme foran et mod Sydvest og Sydøst vigende Isdække. Mod Nordøst gaar den højtliggende Afde- ling terrænmæssig fuldstændig i et med det tilgrænsende Morænelerlandskab, bortset fra dettes højere opragende Bakketoppe. Det underliggende Moræneler dukker ogsaa paa nogle faa Steder op igennem det stenfri Lerdække ved Skraaningen ned til den lavere Afdeling. En Mergelgrav 200 m SØ for Nygaard, der i Tiden omkring Aarhundred- skiftet stod med friske Vægge, vist~, at der her var et mindst 3 m mægtigt Lag af lagdelt, stenfrit Ler. - Ved Overgangen fra den højere til den lavere Afdeling maa der være sket en Sænkning af Vandstanden paa ca. 5 m , maaske i Forbindelse med Bortsmeltning af Isdækket Sydvest og Syd for den højtliggende Sø. At Søens Is-Begrænsning er veget tilbage, dels mod Syd- vest og dels mod Sydøst, fremgaar tydeligt af Forholdene. Lerfladen mellem Udby og Svallerup falder svagt mod SØ og grænser til den Side op til lavere liggende Moræneler og Engdrag. Søens Begrænsning har altsaa her været et Is- dække. Lersletten Nord for Svallerup er mod Sydvest be- grænset af fint Sand, der uden Terrængrænse udvider Fla- 49 Fig. 16. Plateau-Lerbakker i smaakuperet Morænelandskab. Se Teksten Side 50. Autoriser et Gengivelse efter Maalebordsblad M 3223 Mørkøv, 1937. 1 : 40.000. Fig. 16. Plateau-Lerbakker i smaakuperet Morænelandskab. Se Teksten Side 50. Autoriser et Gengivelse efter Maalebordsblad M 3223 Mørkøv, 1937. Stenfrit Diluvialler. Den har nær Udløbet, ved »Huldige« Sydvest for Kongsdal, en Dybde af henved 10 m og har skaaret sig videre tilbage mod Nord, saa at den som en 800 m lang Dal gennemskærer hele Lerplateauet fra Nord til Syd. Denne Art af Slugter, der er et Særkende for omfattende Issø-Lerbakker i andre Egne af Danmark, har tjent som Aftapningsrender fra ·Søerne ved Bortsmeltningen af Isen ved Søranden. det store Omfang, flere Afløbsrender, af hvilke den ene fremtræder som en særdeles udpræget Slugt. Den har nær Udløbet, ved »Huldige« Sydvest for Kongsdal, en Dybde af henved 10 m og har skaaret sig videre tilbage mod Nord, saa at den som en 800 m lang Dal gennemskærer hele Lerplateauet fra Nord til Syd. Denne Art af Slugter, der er et Særkende for omfattende Issø-Lerbakker i andre Egne af Danmark, har tjent som Aftapningsrender fra ·Søerne ved Bortsmeltningen af Isen ved Søranden. Umiddelbart ved Knabstrup Teglværk eir der omfattende og dybe Grave i stenfrit Istidsler. Da Leret h er særlig har geologisk Interesse ved sit Indhold af Plante- og Dyre- levninger, bliver det omtalt senere i Afsnittet om fossilfø- rende, sen glaciale Aflejringer. Forud for at den indtil 16 m dybe Bassinudfyldning fandt Sted, er en lidt østligere, 15 m højere Plateaubakke bleven overklædt med Issøler. Dette udgør dog kun et indtil 1 m tykt Overtræk over Moræneler. Selve Bakken her maa have været udformet med sin fulde Højde, inden Udslæmningen af stenfrit Ler i den nydannede Issø fandt Sted; Omraadet ved Teglværket maa da paa samme Tid have været fyldt med et 30 m tykt Lag Is for at naa samme Højde. Ved Undløse Teglværk lidt S y dø s t f o r U n d 1 ø s e By er der et lille Omraade med stenfrit Ler, der ikke træ- der stærkt frem i Landskabet. Lerlaget naar en Tykkelse af mindst 4½ m og er muligvis oprindelig opstaaet som en isdæmmet Bassinudfyldning. Det er dækket af et tyndt Lag sandet og stenet Muld, og paa et enkelt Sted har Dæk.laget nederst indeholdt et decimetertykt Tørvelag af senglacial Alder (Allerødlag). Det stenfri Ler hviler paa lagdelt, sten- frit Issøsand. Det er selv smukt lagdelt og »varvigt« (se Fig. 17). I det højereliggende Omraade ved Skjoldenæsholm er der enkelte fremtrædende Bakker af den Form, som Forekom- sterne af Plateauler sædvanligvis er knyttet til. Stenfrit Diluvialler. 1 : 40.000. 50 den mod Sydvest. Ved Landevejen mellem Svallerup og Ugerløse er der en kilometerbred Sænkning, hvorigennem en Del af den forhenværende Søbund har Afløb direkte til Store-Bælt, mens den i Issøens Tid har været tillukket af Isbarrieren. Den endelige Tømning af den isdæmmede Sø har dog først kunnet ske, dengang Sænkningen mellem Svallerup og Udby blev frigjort for Isdækket, og der blev aabnet Afløb mod Sydøst til Halleby Aa umiddelbart Vest for Tissø. Det kunde paa Forhaand ligge nær at anse den lavest lig- gende af de to Lerhorisonter for at være opstaaet ved Ud- slæmning fra den højere liggende. Af en saadan Udslæm- ning er der dog kun svage Spor. Lermaterialet tyder heller ikke i den Retning. 700 m Nordøst for Svallerup er der aabnet en Lergrav, hvorfra der føres Ler til Gørlev Tegl- værk. Der ses her 2½ m normalt lagdelt Diluvialler med 1- 2 cm tykke Lag, hvilende paa Moræneler. I det store Bakkeomraade Nord for Store Aamose er der paa Strækningen m e 11 em K n ab s trup og Kong s- d al en stor Gruppe flade Bakker, omgivne af et Terræn med mere toppede Bakker og smaakuperet Landskab, be- staaende af Moræneler og Sandaflejringer. De fleste af de flade Bakker indeholder stenfrit Dih~vialler; de hører med blandt de højeste Bakker i deres nærmeste Omegn, og deres Fremkomst kan kun forklares ved, at de oprindelig var Bunden af. Søer, der laa omgivne af et højere liggende Isdække, fra hvis Overflade Leret slæmmedes ud i de op- staaede Fordybninger. Ved Smeltningen af den omgivende Is kom de udslæmmede Leromraader efterhaanden til at fremtræde som de Plateaulerbakker, vi nu ser, og som i denne Egn rager indtil 35 m op over deres nærmeste Om- givelser (Fig. 16}. Ligesom disse Bakker af Plateauler landskabeligt i høj Grad præger Egnen ved deres fremtrædende Højde, gør de det ogsaa ved deres Udstrækning. Mens enkelte af dem kun naar et Omfang af 15- 20 ha, er andre betydelig større, og en enkelt, umiddelbart Vest for Kongsdal, omfatter ~ver 1 km2• Denne sidste Lerbakke rummer, i Sammenhæng med 51 det store Omfang, flere Afløbsrender, af hvilke den ene fremtræder som en særdeles udpræget Slugt. Stenfrit Diluvialler. Foruden den Bakke, hvis højeste Punkt er Gyldenløves Høj, er der den 118 m høje Højbjærg, der ligger noget vestligere og S te- st ru p Bjærg, 4 km Vest for Gyldenløves Høj. Kun paa 52 denne sidste er der dog et Dække af Plateauler, og det kun paa Bakkens sydøstlige Halvdel. Lerfladen h er naar en Højde af 93 m; den er ved Afløbsfurer skilt fra Bjærgets nordvestlige, kuplede Del, der naar en Højde af 102 m. Det er vist den højestliggende Forekomst af Plateauler paa Sjælland. denne sidste er der dog et Dække af Plateauler, og det kun paa Bakkens sydøstlige Halvdel. Lerfladen h er naar en Højde af 93 m; den er ved Afløbsfurer skilt fra Bjærgets nordvestlige, kuplede Del, der naar en Højde af 102 m. Det er vist den højestliggende Forekomst af Plateauler paa Sjælland. I det stærkt bakkede Højdeomraade mellem Hjembæk og Bregninge er der en enkelt højt opragende, fladt kuplet Bakke, der indeholder stenfrit Diluvialler, nemlig den 84 m høje Linde b j ær g, Sydvest for Favrbo. I dens Nord- side er der en stor Teglværksgrav m ed l0L-12 rn. høje Vægge. Øverst er Leret fuldstændig slenfrit men indeholder nederst spredte Sten. Igennem hele Højden mangler Leret fuldstændig Lagdeling. Med sin Overflade danner den om- fattende Bakke en stærk Modsætning til det tilgrænsende, rigt kuperede Landskab med regelløst liggende, toppede Bakker og mellemliggende, uregelmæssige Sænkninger. Paa Indersiden af den mægtige Bakkebue Vejrhøj- Høve, der omkranser Lammefjord mod Vest, er der flere Partier med Diluvialler, hvori der har været eller er Profil. Ved et nu nedlagt Teglværk ved Ris, Nord for Faarevejle, var der i 1894 Profil i stenfrit Ler med smaa Sandlag. Paa et Sted var den ca. 4 m høje, vestlige Lervæg smukt gennemsat af Spring. I Retningen Ø-V havde Lagene ganske svagt, vestligt Fald; i en Væg med denne Retning fandtes der og- saa Spring i Lagrækken. Asnæs Teglværk, der nu ligger ved Landevejen midt imellem Faarevejle og Høve, har sin Lergrav S yd ø s t f o r V e d d i n g e, i et Leromraade der har en Længde- udstrækning paa 1 ½ km, og hvis hvælvede Overflade særlig har Hældning mod Sydøst. I den lange Teglværksgrav ses et indtil lQL-12 m højt Snit i stenfrit Ler uden kendelig Lagdeling. Stenfrit Diluvialler. Tidligere har Asnæs Teglværk ligget kort Nord for Asnæs By, hvor der fandtes et Leromraade af ganske ringe Ud- stræknin1:, men med en Lertykkelse paa mere end 9 m. Leret var udpræget lagdelt og vekslede mellem sandrige og fede Ler horisonter. 5 53 Et Omraade med stenfrit Ler ved Lyng huse, Nordøst for Grevinge, bliver, som Følge af sin Beliggenhed i For- hold til Bakkebuerne i Odsherred, omtalt senere (Side 107 ). hold til Bakkebuerne i Odsherred, omtalt senere (Side 107 ). Medens det stenfri Istidsler paa alle de hidtil nævnte Ste- der ligger uforstyrret og har naaet helt eller saa nær til Overfladen, at det deraf fremgaar, at det ikke har været overskredet af Indlandsis, er der andre Steder, hvor saa- danne Lerlag Jigger dækkede af Sand og Grus og kan være undergaaet betydelige Forstyrrelser. Under saadanne For- hold træffer man stenfrit Ler flere Steder i Egnen mellem Munke-Bjærgby og Aamose. - Ved Munke-Bjærgby og Rude- Eskilstrup træder der stenfrit Ler frem til Jordoverfladen i Skraaningen af de fremtrædende Bakker, som de to Byer ligger paa. Paa begge Steder hviler Leret paa Sandlag og er dækket af Sand og Grus. Lerlagene naar en Tykkelse af over 5 m og strækker sig som en Lerhorisont ind i eller igennem Bakkerne; nederst i Skraaningerne er det dæk- kende Sand og Grus tyndest eller mangler h elt, men maa i Bakkernes højeste Partier have mange Meters Mægtighed. - I Grusomraadet videre mod Syd, Øst for Bromme, op- træder der mange Steder Lerlag af mindre Tykkelse, dækkede af Grus og Sand eller vekslende med Sandlag. I Alminde- lighed ligger Lagene uforstyrrede; dog er der i en Bakke SV f. Munke-Bjærgby iagttaget bøjede og foldede Lag af Sand og stenfrit Ler, dækkede af Diluvialsand. Under lignende Forhold optræder der ogsaa stenfrit Ler i Sandomraaderne ved Aamose. Tæt Nord for Sandlynggaard er der i et Profil set stenfrit Ler af ukendt Mægtighed, dæk- ket af 4 m Diluvialsand. Leret var tydeligt lagdelt og havde næsten lodret Lagstilling. I de vestligere Sandomraader Syd for Aamose, hen imod Skellingsted Bro, stikker der flere Steder i Skrænterne ud imod Moserne stenfrit Ler frem, dækket af Diluvialsand. Paa denne Maade ligger det ved Bodal, hvor det i sin Tid har været anvendt til Teglværks- brug. Dette er ligeledes Tilfældet Nordvest for Verup, hvor der har været to Teglværker. Stenfrit Diluvialler. I den nordlige Grav var Leret dækket af 2- 3 m Sand. I den sydlige Grav gik Leret helt til Overfladen; i et 4 m højt Profil saas, at det øverst 54 var svagt lagdelt, nederst indeholdt det smaa, sammenkrøl- lede Sandlag. I det øverste, gule Ler fandtes mange Kalk- konkretioner, »Marlekor«, som voldte Vanskeligheder for Brændingen. Nord for Aamose optræder der stenfrit Ler under ganske tilsvarende Forhold som i Sandomraaderne ved Aamosens Sydside. V ed Øgaarde er der saaledes i et Profil set Ler under 3 m Grus; paa en Strækning gaar det ogsaa her til Overfladen. Det er ved Omtalen af de forskellige Forekomster , hvor der har været Snit i de stenfri Lerlag, nævnt, om Leret var lagdelt eller ikke. Nogle af Stederne mangler det fuldstæn- dig Lagdeling, medens det almindeligste er, at det indehol- der Lagdeling. Efter Undersøgelse af det stenfri Ler paa en talrig Mængde Steder i Danmark anses det for godtgjort, at de to Former afspejler forskellige Trin i Leraflejringernes Afsætning i Issøerne. Det ikke lagdelte, homogene Ler tænkes da aflejret under stærkt glacialt prægede Forhold, med en kraftig lssmeltning omkring Søen og rigelig Bundfældning af Materiale, uden at der har kunnet finde nogen Sortering og Lagordning Sted. I Modsætning hertil skulde Fremkom- sten af Leraflejringens Afsætning i lagordnet Form staa i Forbindelse med et mere fremskredet Stadium i en Issøs Udvikling med friere Vandoverflade og mindre Slam- mængde. I en Aflejring som den ved Favrbo Teglværk, hvor Lag- deling fuldstændig mangler, kan da lssøens Levetid anses for at have været saa kort, at en lagordnet Lerafsætning ikke har naaet at komme i Stand. Det synes ikke udelukket, at dette kan staa i Forbindelse med, at Lerbakken, Linde- bjærg, ligger højt, omgivet af et stærkt kuperet Dødisland- skab, hvis Isdække Søbunden da har haft Mulighed for at hæve sig op over allerede saa tidlig, at Søtilstanden kun blev kortvarig og en langsommere Slamtilførsel ikke naaede at indtræde, inden Issøen ophørte at eksistere. De fleste af de omtalte Issøaflejringer med stenfrit Ler indeholder en tydelig Lagdeling. Hertil er der i nogle Til fælde knyttet en ensartet gentagen Lagordning med »Varv«. der afspejler den skiftevise Afsætning i Løbet af et Aar 55 (Sommerlag og Vinterlag). Et godt Eksempel paa Udform- ningen findes i Lergraven ved Undløse Teglværk, hvorfra Fig. 17 viser et Billede, den nederste Del af en samlet Serie paa henved 30 Aarslag. Stenfrit Diluvialler. Lagdelingen med Udformningen som (Sommerlag og Vinterlag). Et godt Eksempel paa Udform- ningen findes i Lergraven ved Undløse Teglværk, hvorfra Fig. 17 viser et Billede, den nederste Del af en samlet Serie paa henved 30 Aarslag. Lagdelingen med Udformningen som Fig. 17. Varv i det stenfri Ler ved Undløse Teglværk. Efter S1GURD HANSEN (D. G. U. II. R. Nr. 63). Fig. 17. Varv i det stenfri Ler ved Undløse Teglværk. Efter S1GURD HANSEN (D. G. U. II. R. Nr. 63). Varv begynder ved Leraflejringens Bund og afsluttes paa et enkelt Sted af et Allerødlag. Forholdet her staar da i skarp Modsætning til Aflejringen ved Favrbo Teglværk med den fuldstændige Mangel paa Lagdeling. Til Forskel fra det højt opragende Lindebjærg ligger Leraflejringen ved Undløse Teglværk lavt i et temmelig jævnt Landskab. Den umiddelbare Overlejring af de ægte senglaciale Alle- 56 rødlag viser ogsaa, at de varvformede Lerlag var afsat i den nærmest mulige Forbindelse med de rent glaciale Is- forholds Afslutning. Tilsvarende Forhold er ogsaa til Stede ved andre af Forekomsterne med stenfrit Ler, f. Eks. Ruds- Vedby Teglværk og andre Steder. rødlag viser ogsaa, at de varvformede Lerlag var afsat i den nærmest mulige Forbindelse med de rent glaciale Is- forholds Afslutning. Tilsvarende Forhold er ogsaa til Stede ved andre af Forekomsterne med stenfrit Ler, f. Eks. Ruds- Vedby Teglværk og andre Steder. Der har været gjort Forsøg paa ved Hjælp af »Varvdia- grammer« fra de Lokaliteter, hvor der har kunnet paavises Varv, at finde de paagældende Aflejringers indbyrdes Aldersfølge. En nærmere, indgaaende Undersøgelse har imidlertid vist, at de forskellige danske Lokaliteter gen- nemgaaende har for faa Varv og ligger i for stor Afstand fra hinanden til, at en Sammenstilling til Brug for en saadan relativ Aldersberegning har kunnet skaffes til Veje. Stenene i Istidslagene. Det er paa adskillige Steder i det foregaaende omtalt, at der i Moræneaflejringerne optræder Indlag af Jordarter, som maa være hentet fra Sjællands nærmeste Omegn. Det drejer sig om Lag af Skrivekridt, paleocæn Mergel, Plastisk Ler, interglaciale Lerlag samt omlejrede Lag af glacialt Grus og Moræneler. Saadanne Indlag, som ikke er blevet fuldstændig indæltet i eller sammenæltet med Moræneleret, kan være af højst forskellig Dimension, fra Flager af mæg- tigt Omfang til en Størrelse som store Stenblokke. At en fuldstændig Opblanding ikke har naaet at finde Sted maa skyldes, at Transporten af disse løse Jordlag ikke har drejet sig om ret store Afstande, da det kun er haarde Genstande, der har kunnet taale langvejs Transport i Isen uden at gaa i et med dennes øvrige Morænemateriale. Stenene, som forekommer i Istidslagene eller er udskyllet af dem, er netop saadanne Indlag, som har kunnet taale en langvejs Transport uden at blive fuldstændig smuldret. Op- rindelig har de hørt til i det Morænemateriale, der i sin usorterede Form optræder som Moræneler. Derfra er de af Vandstrømme fra Smeltningen af Isen skyllet ud og afsat som Gruslag. Moræneleret, Markoverfladen og de glaciale 57 eller senglaciale Gruslag er saaledes de oprindeligste Steder, hvor vi nu kan finde de af Indlandsisen hidførte Sten. Derfra kan de senere være udskyllet og samlet i Strandvolde eller foran Kystklinter. eller senglaciale Gruslag er saaledes de oprindeligste Steder, hvor vi nu kan finde de af Indlandsisen hidførte Sten. Derfra kan de senere være udskyllet og samlet i Strandvolde eller foran Kystklinter. Nogle af Stenene er af imponerende Størrelse. Dyre- s t e n, der ligger nærved Hovedlandevejen 1 km Nord for Aarby ved Kalundborg, maaler 6,GO m i Længden, 5,15 m l{ELD MILTHEHS fol. 1943. Fig. 18. Dyresten, ved Landevejen 1 km N f. Aarby. l{ELD MILTHEHS fol. 1943. Fig. 18. Dyresten, ved Landevejen 1 km N f. Aarby. som største Bredde og 1,6 m i synlig Højde. Dens Omfang er 18½ m . I anden Forbindelse (Side 97 ) nævnes en 60- 70 m 3 stor Sten i Skoven Bredevang Nordvest for Bro- mølle. Den har et Omfang af 17 m. Tæt Øst for Avnsøgaard ligger en 2 X 3 m stor Sten, delvis begravet i Jorden, saa dens Højde ikke kendes. Omraadet her har oprindelig været uhyre rigt paa store, jordfaste Sten. Stenene i Istidslagene. De her nævnte store Sten hører alle til i saadanne Strøg, hvor man ad anden Vej har Grunde til at formode, at en stagnerende Isrand har haft sin Plads. Til saadanne Strøg er der jævnlig knyttet en 58 tæt Bestrøning af store Sten. I denne Forbindelse kan ogsaa nævnes de mange store Stendysser paa Røsnæs paa Bakke- kammene fra Raklev og vestpaa. tæt Bestrøning af store Sten. I denne Forbindelse kan ogsaa nævnes de mange store Stendysser paa Røsnæs paa Bakke- kammene fra Raklev og vestpaa. Af de talrige større Sten, som oprindelig kan have ligget henstrøet paa Markoverfladen, er nu den allerstørste Del samlet i Stengærder. De udgøres overvejende af Graniter og Gnejser samt andre eruptive Bjærgarter og krystallinske Skifre. Den Slags Sten maa for den største Dels Vedkom- mende stamme fra den sydlige Halvdel af Sverige. Stenene af Sandsten og forskellige Slags Skifer stammer dels fra det sydlige Sverige, dels fra Østersøegne, fra hvis vestlige Del ogsaa Markstenene af Flint maa være ført til denne Del af Sjælland. Naar derimod de med Flinten sammenhørende Kalklag fra Kridtformationen og ligesaa Kalksten fra ældre Lag fra Østersøens Bund ikke findes som Marksten, skyldes det, at de ligesom Kalken i de øverste Jordlag er blevet fjærnet ved Hensmuldring, Opløsning og Udvaskning i Løbet af de Aartusinder, der er gaaet siden Istiden. I Istidslagene eller som Strandsten forekommer derimod mange Arter af Kalksten og andre Slags Blokke af lagdelte Bjærgarter. Der er fundet Scolithussandsten fra Egnene om- kring Kalmarsund, Antraconitskifer, Ortoceratitkalk og Graptolitskifer fra Egne mellem Øland, Sydøst-Skaane og Bornholm, forskellige Slags graa og røde Silurkalksten fra Østersøegne mellem Gotland og Øsel samt endelig Ræt-Lias- og Jurasandsten, som rimeligvis ogsaa stammer fra det bal- tiske Omraade. Fra den sydvestlige Del af Østersøen kan Indlandsisen have hidført Blokke af Grønsandsten, Skrive- kridt og dets sorte Flint. Sten af prikket Flint er fra det østlige Skaane ført af Isen til Nordvestsjælland. Fra Østersøens vestlige Del eller fra Sjællands egen Undergrund kan endelig Sten af Saltholmskalk, Limsten og paleocæn Grønsandsten være kommet. En særlig stor Mængde af saa- danne Sten forekommer som tidligere nævnt i Gruslagene i Klintebjærg i Odsherred. Af Indeslutninger, der stammer fra Istiden selv, maa næv- nes Elefantlevninger. I en Grusgrav i Nærheden af Munke- Bjærgby er fundet en ret anselig Stødtand. Stenene i Istidslagene. Ved Udtagelsen 59 af Gruslaget har Tanden desværre været Genstand for haard- hændet Medfart »under Anvendelse af Træskospark og Spadehug«, saa at der kun foreligger Brudstykker af Tan- den. Da der fra interglaciale Jordlag i Jylland foreligger Vidnesbyrd om, at der i sidste Interglacialtid har levet Ele- fanter i Danmark, er det maaske ikke udelukket, at den molesterede Tand har hidrørt fra en ægte, dansk Elefant. Hvis dette ikke har været Tilfældet, kan det med temmelig stor Sikkerhed siges, at det er fra fjærnern Egne mod Syd- øst, Indlandsisen har ført Tanden til Munke-Bjærgby. Dette antydes af, at for næsten alle de Elefantlevninger, der er fundet i Danmark, har Fundstederne ligget i de Egne af Landet, hvis Istidslag er af sydøstlig Oprindelse. Det samme er i udpræget Grad Tilfældet med de paagældende Gruslag ved Munke-Bjærgby, hvad deres øvrige Indhold af Sten viser. Hovedmængden af de krystallinske Sten maa som nævnt stamme fra den sydlige Del af Sverige, men de fleste af de Bjærgarter, som de tilhører, har ikke et saadant Præg, at deres oprindelige Hjemsted kan bestemmes inden for snævre Grænser. Anderledes er det med nogle Porfyrer og porfyriske Bjærgarter og nogle enkelte andre Bjærgarter, der har et særlig karakteristisk Præg, og hvis Hjemsted er snævert be- grænset. Nogle Bjærgarter af denne Art, som der kendes Sten af fra Danmark, forekommer i Norge, andre i Sverige og atter andre paa Ålandsøerne og i den tilgrænsende Del af Østersøen. Som Følge deraf kan da Stenene af disse Bjærgarter være vejledende ved Udregningen af, fra hvilke Retninger Indlandsisen har ført sit Materiale frem. Disse Sten betegnes derfor som Lede b 1 ok k e. I Nordvest- sjælland forekommer der Sten fra alle disse Hjemstedsom- raader. Fra Egnen omkring Oslo stammer Rhombeporfyr, Larvikit og beslægtede Bjærgarter samt Rhombeporfyr- konglomerat. Fra Dalarne i Sverige er kommet Bredvad- porfyr, Grønklittporfyrit og en hel Række beslægtede Bjærg- arter og Varieteter; Påskallavikporfyr fra Egnen ved Kal- marsund, Kinnediabas fra Vestergotland og Basalt fra Skaane. Fra Ålandsøerne stammer Kvartsporfyr, Rapakivi, 60 Granit og andre Rapakivibjærgarter, og endelig er Brun Østersøkvartsporfyr og Rød Østersøkvartsporfyr hentet fra Bunden af Østersøen Syd for Ålandsøerne. Granit og andre Rapakivibjærgarter, og endelig er Brun Østersøkvartsporfyr og Rød Østersøkvartsporfyr hentet fra Bunden af Østersøen Syd for Ålandsøerne. Ledeblokkene fra disse vidtspredte Hjemstedsomraader, Norge, Sverige og Baltikum, optræder ikke jævnt fordelt over Nordvestsjælland. Stenene i Istidslagene. Fordelingen opviser tværtimod store Forskelligheder, der er nøje knyttet til karakteristiske Træk i det fligede Landskabs Omrids og sammensatte Udform- ning. Som Hjælpemiddel til at faa Rede paa Fordelingen har man benyttet Optælling af Ledeblokkene i Grusgrave, blandt Strandsten og som Marksten Lil Brug for statistisk Behandling af det indsamlede Materiale. De øverste Jordlag i Nordvestsjælland er blevet afsat af Indlandsis, der er kommet til Omraadet fra Retningerr imellem Øst og Syd. Et vigtigt Skel mellem to Hoved- retninger for de aflejrende Isstrømme strækker sig fra Kalundborg Fjord i Retning mod Store Aamose og vi- dere mod Sydøst. I Egnene Syd for dette Skel er den alt- overvejende Mængde af Ledeblokkene af rent baltisk Her- komst, hvad der stemmer godt med andre geologiske Tegn paa, at det sidste Isdække til disse Egne er kommet fra Sydsydøst, langs Store-Bælt. I Omraadet Nord for Skel- bæltet: Kalundborg Fjord- Aamosen er et saadant Overtal af rent baltiske Ledeblokke ikke til Stede. Medens der Syd for Skellet forekommer flere Sten af de to Slags Østersø- porfyrer end af de to nævnte Stenarter fra Dalarne, er det modsatte Tilfældet Nord for Skellet. Til denne sidste Del af Nordvestsjælland er de afsluttende Isstrømme kommet fra Retninger imellem Sydøst og Øst. Paa Sejerø og ved Nord- kysten af Røsnæs viser Stenindholdet tydeligt, at Moræne- materialet er ført dertil fra Sydøst. Senere er Retningen ændret til Øst- Vest. Dette fremgaar af Stenindholdet i Odsherred; her er den relative Mængde af Østersøblokke yderligere formindsket, og desuden er Antallet af Ledeblokke dalet til et rent Minimum i Forhold til den øvrige Sten- mængde. Dette kan naturligt forklares ved, at den Isstrøm, som sidst er naaet hertil, har faaet sit Stenindhold fta den sydvestlige Del af Sverige, hvorfra ingen af Ledeblokkene 61 stammer, og at Isen kun i yderst ringe Grad har faaet ind- blandet Materiale fra den sydligste Del af Sverige og den baltiske Dal. stammer, og at Isen kun i yderst ringe Grad har faaet ind- blandet Materiale fra den sydligste Del af Sverige og den baltiske Dal. Forinden denne Tilførsel af Materiale fra Sydøst og Øst havde fundet Sted, var der fra Nord og Nordøst tilført den nordlige Del af Nordvestsjælland Ledeblokke af Rhombe- porfyr og andre fra Norge, samt Kinnediabas fra Vestergot- land. Stenene i Istidslagene. Langs Sydkysten af Røsnæs optræder disse Blokke i betydeligt Antal som Strandsten, der er skyllet ud af de Istidslag, som her ligger opskudt Side om Side med Lag af Plastisk Ler. Paa Sjællands Odde og især paa den nord- ligere liggende Ø, Hesselø, findes der et endnu m ere anse- ligt Antal af disse Ledeblokke og h er skyllet ud af selve det øvre Morænedække. Rimeligvis er Tilførslen af disse Ledeblokke fra Nord og Nordøst foregaaet i det første Af- snit af sidste Glaciallid, samtidig med at Nordjylland dæk - kedes af Is fra Nord, og forinden de mægtige skandinaviske Ismasser Øst og Syd om Sverige naaede frem til denne Del af Danmark. Dog er den Mulighed ikke udelukket, at de var ført h ertil allerede i den foregaaende Glacialtid, hvis sidste skandinaviske Isstrøm kom Lil Danmark fra Nord. (En Liste, som viser den relative Mængdefordeling af visse Ledeblokke paa Røsnæs, Asnæs og Sejerø, findes Side 71 ). Landoverfladens Udformning ved Istidens Slutning. Saaledes som det nordvestsjællandske Landomraade ligger formet fra Naturens Haand, er det den mest sammensatte og fligede Del af Sjælland; og af Nordvestsjællands snævrere Omraader vil Odsherred rimeligvis for en uvilkaarlig Be- tragtning tage sig ud som den m est splittede Del med sine dybe Fjordindskæringer, og adskilt som det er fra den øv- rige Landsdel af den omfattende Lammefjord. Af den efter- følgende Fremstilling vil dog fremgaa, at den sydlige Del af Odsherred oprindelsesmæssigt n etop ved Lammefjord som Centralomraade er snævert sammenknyttet med de Bakkelandskaber længere mod Syd, som den tilsyneladende 62 ingen Samhørighed har med. Det vil da af Fremstillingen vise sig, at der nok forefindes vigtige geologiske Skille- linier i Nordvestsjælland, men at de ligger langt sydligere end Lammefjord, nemlig ved Store Aamose, med Fortsæt- telse imod Nordvest, i Retning dels mod Saltbæk Vig og dels mod Kalundborg Fjord. ingen Samhørighed har med. Det vil da af Fremstillingen vise sig, at der nok forefindes vigtige geologiske Skille- linier i Nordvestsjælland, men at de ligger langt sydligere end Lammefjord, nemlig ved Store Aamose, med Fortsæt- telse imod Nordvest, i Retning dels mod Saltbæk Vig og dels mod Kalundborg Fjord. For at fremme Forstaaelsen af, hvorledes den Udvikling er gaaet for sig, som har ført til Fremkomsten af Nordvest- sjællands ejendommeligt formede Landskab, vil det være formaalstjenligst at opdele det i mindre Omraader og omtale hvert enkelts landskabelige og geologiske Særpræg for sig. De kan naturligt deles i to Hovedgrupper, nemlig: 1) Eg- nene mod Sydvest og Syd og 2) de centrale og nordlige Egne. Røsnæs-Asnæs med Bagland. Skiftende med disse uregel- mæssige Skredterasser staar stejle, høje Skrænter, paa sine Steder som smalle »Næser« med Terrasser paa begge Sider, andre Steder med større Udstrækning, men ogsaa i disse Til- fælde mere varierende end Nordkystens, fordi Jordindholdet kan veksle mellem Moræneler, fine Sandlag og storstenede Gruslag, og fordi Sandlagene kan være stillede paa Kant, Side om Side med Lagene af Plastisk Ler. Hele det brogede Virvar af Jordlag og Former hænger sammen med, at Skræn- terne i det Plastiske Ler og de ledsagende Lag frembyder et Snit igennem et sammenrodet Kompleks af Jordlag af uens Konsistens, som Indlandsisen har blandet imellem hinanden og stillet paa Kant med det opblødte og smidige Plastiske Ler som Smørelse. Naar saa tilmed Havet har kunnet skære sig ind og ophæve den Modstand mod Udglidning, som er til Stede, hvor dette Lagkompleks ligger inde i Landet, da er Muligheden for Kystens landskabelige Formrigdom op- staaet. Som Følge af Storm og Strøm har Havet kunnet fjærne de udgledne Jordmasser, saa at nye Skred kunde fremkomme og Variationer har kunnet vedblive at skifte, Kysten ved Nordsiden af Røsnæs er enten ganske flad eller har indtil ca. 10 m høje Skrænter ud imod en smal Forstrand. Skrænterne indeholder overvejende Moræneler. - Sydkysten har et fra Nordkysten vidt forskelligt Præg. Den har mange Steder Kystskrænter, der naar 20'--40 m i Højde og varierer stærkt saavel i Udseende som i Jordbe- skaffenhed. Der kan være en livlig Skiften mellem Mo- ræneler, Sand og Grus samt tertiært, Plastisk Ler. Om dette sidste er til Stede, giver det sig til Kende ved, at der ud mod Kysten optræder uregelmæssige Terrasser neden for de højere Skrænter. De er fremkommet ved Jordskred som Følge af Glidninger i det Plastiske Ler, der ikke kan bære de overliggende Jordlags Tryk, naar det bliver stærkt vand- fyldt og derved uhyre blødt. Skiftende med disse uregel- mæssige Skredterasser staar stejle, høje Skrænter, paa sine Steder som smalle »Næser« med Terrasser paa begge Sider, andre Steder med større Udstrækning, men ogsaa i disse Til- fælde mere varierende end Nordkystens, fordi Jordindholdet kan veksle mellem Moræneler, fine Sandlag og storstenede Gruslag, og fordi Sandlagene kan være stillede paa Kant, Side om Side med Lagene af Plastisk Ler. Røsnæs-Asnæs med Bagland. Omraadet Røsnæs-Asnæs er baade landskabeligt og geo- logisk en af de allermest ejendommelige Dele af Sjælland. Kender man dem kun fra et almindeligt Landkort, uden Angivelser af Højder og Jordbundsforhold, kan man ikke ane de to Halvøers Forskellighed. Denne træder dog hurtigt frem, hvad enten det er fra Søsiden eller fra Landsiden, man lærer Omraadet at kende. Modsætningen mellem Asnæs' Ensartethed og Røsnæs' variationsrige Landskab og Jord- bund er meget stor. Røsnæs. Landskabeligt er Røsnæs en fremtrædende Odde, med baade anselige Højder og stærkt skiftende Over- fladeformer. De største Højder - op til 64 m - findes i den sydlige Del, langs Landevejen fra Raklev til Ulstrup og videre mod Vest. Her er tillige de største indbyrdes Højdeforskelle, 20 m og derover mellem Bakketoppe og Dal- gruber. Dertil kommer saa den store, frie Udsigt til begge Sider: mod Syd den nære Kalundborg Fjord med Asnæs i Baggrunden, og mod Nord den lavereliggende, jævnt bølgede Del af Røsnæs ud imod Sejerø Bugt. Mange af Højderyg- gens Bakker er af langstrakt Form i samme Retning som hele Systemet. Bakkekammen krones desuden af et stort Fig. 19. Kysten Sydvest for Kongstrup efter Skred i Plastisk Ler ved Siden af frer:nstaacnde Morænelersnæse. 1898. Se Teksten Side 64. ~ ~ ~ ~ Fig. 19. Kysten Sydvest for Kongstrup efter Skred i Plastisk Ler ved Siden af frer:nstaacnde Morænelersnæse. 1898. Se Teksten Side 64. 64 Antal smukke Stendysser, der giver Landskabet yderligere Relief. Paa nogle Steder, mest i Egnen ved Kongstrup, kryd- ses Bakkekæden af brede, dybe Erosionsdale ned imod Ka- lundborg Fjord. Enkelte af dem synes helt at overskære Bakkedragets højeste Kam. g j Kysten ved Nordsiden af Røsnæs er enten ganske flad eller har indtil ca. 10 m høje Skrænter ud imod en smal Forstrand. Skrænterne indeholder overvejende Moræneler. - Sydkysten har et fra Nordkysten vidt forskelligt Præg. Den har mange Steder Kystskrænter, der naar 20'--40 m i Højde og varierer stærkt saavel i Udseende som i Jordbe- skaffenhed. Der kan være en livlig Skiften mellem Mo- ræneler, Sand og Grus samt tertiært, Plastisk Ler. Om dette sidste er til Stede, giver det sig til Kende ved, at der ud mod Kysten optræder uregelmæssige Terrasser neden for de højere Skrænter. De er fremkommet ved Jordskred som Følge af Glidninger i det Plastiske Ler, der ikke kan bære de overliggende Jordlags Tryk, naar det bliver stærkt vand- fyldt og derved uhyre blødt. Røsnæs-Asnæs med Bagland. Hele det brogede Virvar af Jordlag og Former hænger sammen med, at Skræn- terne i det Plastiske Ler og de ledsagende Lag frembyder et Snit igennem et sammenrodet Kompleks af Jordlag af uens Konsistens, som Indlandsisen har blandet imellem hinanden og stillet paa Kant med det opblødte og smidige Plastiske Ler som Smørelse. Naar saa tilmed Havet har kunnet skære sig ind og ophæve den Modstand mod Udglidning, som er til Stede, hvor dette Lagkompleks ligger inde i Landet, da er Muligheden for Kystens landskabelige Formrigdom op- staaet. Som Følge af Storm og Strøm har Havet kunnet fjærne de udgledne Jordmasser, saa at nye Skred kunde fremkomme og Variationer har kunnet vedblive at skifte, 65 KELD M1LTIIEHS fol. 1943. Fig. 20. Sænkningsbæller med Sideskrænter, efter usynlig, underjordisk Udskridning af Plastisk Ler. Nær Kysten SV f. Kongstrup. KELD M1LTIIEHS fol. 1943. Fig. 20. Sænkningsbæller med Sideskrænter, efter usynlig, underjordisk Udskridning af Plastisk Ler. Nær Kysten SV f. Kongstrup. i(ELD Mll.THEl!S fol. 1943. Fig. 21. Sænkningsbælte og Sideskrænt; jvfr. Fig. 20. 5 i(ELD Mll.THEl!S fol. 1943. Fig. 21. Sænkningsbælte og Sideskrænt; jvfr. Fig. 20. 5 5 KELD MILTHERS fol. 194~. Fig. 22. Randmorænebakker (Musbjærg, Kovsen og ~'lonshøjc) l\ord for J\'yrnp. Set fra Sydøst, i Bakkernes Længderetning. $e Teksten Side 67. 0, 0, KELD MILTHERS fol. 194~. dmorænebakker (Musbjærg, Kovsen og ~'lonshøjc) l\ord for J\'yrnp. Set fra Sydøst, i Bakkernes Længderetning. $e Teksten Side 67. KELD MILTHERS fol. 194~. Fig. 22. Randmorænebakker (Musbjærg, Kovsen og ~'lonshøjc) l\ord for J\'yrnp. Set fra Sydøst, i Bakkernes Længderetning. $e Teksten Side 67. 67 indtil mere stabile Forhold stedvis og tidvis naaede at ind- Lræde. indtil mere stabile Forhold stedvis og tidvis naaede at ind- Lræde. Den tredje Ting, som. vedrører Røsnæs' særlige Præg, er Overfladejordlagenes Fordeling. Kortet vise1· den. I Forbin- delse med det, der er nævnt i det foregaaende om Højder, Landskabsformer og Kysterne, fremhæver denne Fordeling yderligere Forskellen mellem Syddelen og Norddelen af Røs- næs. Den første er den højtliggende, stærkest kuperede og rigest varierede Del. Som Kortet viser, er det den sandede, magre Del, samtidig med at det er den Del, der giver Røsnæs sit sjældne landskabelige Præg. Højdedragets Sandomraader indeholder i overvejende Grad temmelig fint Sand, liggende i uforstyrrede Lag. Saa- ledes ses det ogsaa i Kystprofiler, over de forstyrrede Sand- lag. Sandet er jævnlig dækket af mere stenede Lag af ringe Tykkelse, men som stedvis indeholder Stenblokke af im- ponerende Størrelse. Røsnæs-Asnæs med Bagland. De er at opfatte som en Rest efter et stærkt udvasket, maaske tyndt Morænedække. At der har været et tyndt Isdække til Stede kan ogsaa fremgaa af, at der mellem Ulstrup og Røsmcsgaard pletvis findes smaa Partier af Moræneler, hvilende paa Sandet. Det samme er Tilfældet i andre Dele af Røsnæs. - Den Del af Halvøen, der vender mod Nord, bestaar overvejende af Moræneler. I Omraadets sydligste Del er Landskabet orienteret overens- stemmende med hele Næssets Længdeudstrækning; enkelte Bakker i det orienterede Grusunderlag mellem Nyrup og Nostrup (Fig. 22) stikker op igennem Morænelersbeklæd- ningen og viser, at det er dette Underlag, som Orienteringen hører sammen med. Det nordlige Omraade rummer ogsaa paa nogle Steder Dækker af stenfrit Ler og Gruslag, som tidligere er omtalt (Side 45 og 35). K a a s trup - Ru g tv e d. Det stærkt orienterede, sam- menhængende Bakkelandskab igennem den sydlige og cen- trale Del af Røsnæs kan ikke følges længere mod Øst end til Møllebakken ved Kalundborg og Bakkerne Nord derfor. Lidt længere mod Nordøst og Øst optræder derimod et anderledes formet Bakkeomraade, udstrakt i Retningen NV- SØ, ganske overensstemmende med Bakkeretningen 68 Raklev-Kongstrup. Ved det østlige Omraade drejer det sig imidlertid ikke om et fuldt sammenhængendeBakkedrag,men derimod om en Række eller Kæde af Enkeltbakker. Mange af Bakkerne i Centralomraadet: Kaastrup-Værslev Skovbak- ker bestaar af Grus og rager 20- 30 m op over det omgi- vende, smaakuperede Morænelandskab. Fra den højeste af KELD MILTHEHS fol. 1943. Fig. 23. Række af Morænebakker (Keldbjærg og Tømmerup Banke) Vesl for Ubberup. Set fra Sydsydøsl. KELD MILTHEHS fol. 1943. Fig. 23. Række af Morænebakker (Keldbjærg og Tømmerup Banke) Vesl for Ubberup. Set fra Sydsydøsl. Bakkerne, Bøgebjærg (48 m) ved Ubberup Valgmenig- hedskirke, falder disse Højder mod Nordvest til 31 m ved Lerchenfeld og 24 m ved Grusfladen Stensbjærg ved Kalle- rup. Efter Retningen at dømme kan Bakkestrøget se ud til oprindelsesmæssigt at hænge sammen med denne Grusflade; de faldende Højder mod Nordvest kunde da antyde, at Grus- bakkerne er opstaaet i Spalter i Isdækket parallelt med Is- randen og med Afløb i Retningen SØ-NV. De kantstillede Lag i Ulvebjærge (Side 31) gaar maaske ind i Linien. Mod 69 Sydøst naar Højden af Bakkestrøgets højeste Toppe op Lil 4-0--4 7 m h ele Vejen indtil Rugtved. - Et Særpræg for h ele denne Bakkekæde med tilgrænsende Landskab er den uhyre Rigdom paa store Sten. Røsnæs-Asnæs med Bagland. I Sydøst- siden af Keldbjærg, Sydvest for Ubberup By, er der i en 50- 60 m lang Grusgrav set følgende Profil: øverst 4 m Morænegrus med Sten op til 2/3 m i Diameter; derunder 6- 8 m temmelig fint, regelmæssigt lagdelt Grus (Fig. 10). Bakkeryggen Vest for Grusgraven er en udpræget Moræne- vold med mange store, jordfaste Stenblokke. - En Lev- ning af den oprindelige Belægning med store Sten findes paa Ubberup Højskoles Mark umiddelbart Øst for Bøge- bjærg. Billederne Fig. 8 og 9 viser Partier af den fredede Stenbelægning. A s n æ s - U g e r 1 ø s e. Medens der paa Røsnæs er be- tydelige Strækninger, der ligger højere end 50 m , er det paa Asnæs kun faa og smaa Pletter, der naar en Højde af 20 m over Havet. Landskabet kan dog ikke kaldes en Flade; der er endog Partier, der nærmest maa betegnes som smaakupe- rede. Mod Øst findes der enkelte, smaa toppede Bakker og vestligere findes enkelte grubeform ede Fordybninger, men Tørvemoser forekommer kun yderst sparsomt. Jordsmonnet er overvejende Moræneler, paa en Strækning dækket af Sandlag, men de lave Kystklinter bestaar af Moræneler. Den yderste Del af Asnæs, Dyrehaven, Vest for Vesterskov, hen- ligger i fuldt oprindelig Tilstand, med en Mængde store Blokke paa Markoverfladen. I den nære Omegn af Lerchenborg lager et Landskab fat med et andet Overfladepræg end det paa Asnæs: større Bakkeformer, adskilte ved jævne, brede Dale. I den største Del af et 2 km bredt Bælte Syd for Melby-Aarby ligger de fladt kuplede, lave Bakker regelløst spredt; kun længst mod Nord, ved Melby, optræder der Grusbakker i Rækkeform. Sydøst for Aarby, hen imod Ugerløse, tiltager Højdeforskel- len mellem Bakker og Dale, og der optræder Nord og Øst for Ugerløse Rækker af Bakker med høje Sandprofiler. Kor- tet viser Udstrækningen. 70 S v a 11 er up - S ve b ø 11 e. Imellem Ugerløse og Udby og videre mod Nordøst træffer man en bakket Landskabsform, som afviger stærkt fra de nærmeste Omgivelser mod Nord og mod Syd. I det Omraade, der strækker sig omtrent fra Svallerup til Rugtved, har Landskabet baade en særlig Overfladeform og en særlig Jordbeskaffenhed .. Jordlagenes Art fremgaar af Kortet. Det mest paafaldende er Diluvial- leret, der som et Betræk dækker en Del af Omraadets flade Bakker. Røsnæs-Asnæs med Bagland. Disse Bakker udfylder saa stor en Del af det paa- gældende Omraade, at de fuldstændig præger Landskabet. De fleste af dem naar en Højde omkring 45 m, medens deres Fod gennemgaaende ligger ved en Højde af 25- 30 m; den eneste der naar væsentlig højere (57 m), er Lerbjærg Øst for Forsinge. Den Del af Bakkerne, der er beklædt med stenfrit Ler, har gennemgaaende en jævnere Overflade end den øvrige Del, uden at dette giver sig Udslag i Højderne af Bakkernes Overflade og ved deres Fod. Diluviallerets Optræden her er omtalt tidligere (Side 46); hvilken Forbi~- delse der kan være mellem Bakkernes Opstaaen og Lerets Placering paa dem, henstaar fuldstændig i det uvisse. Omraadet med disse Bakkeformationer overskæres fra Nord til Syd af Vandskellet m.ellem Afløbene til Kalundborg Fjord og til Tissø. Disse Afløb har deres Udspring Øst for Værslev By. Skønt Passet mellem dem her er ganske lavt og smalt, og skønt Faldet er mange Gange større imod Vest end mod Syd, er det dog mod Tissø, Afvandingen herfra finder Sted. Dette, sammen med Terrænformerne, lader ane, at Landskabet Vest for Fladbakke-Omraadet, - inden for Trekanten Værslev By- Frankerup- Kærby - har ligget dækket af Dødis i nogen Tid efter, at der igennem Dalen mod Sy<;l forbi Forsinge var bleven aabne.t Passage imellem Fladbakkerne for de sidste Rester af Smeltevand. Ved om- trent samme Tid har den tidligere (Side 48) omtalte, isdæm- mt;dE; Sø mellem Svallerup og Udby k,unnet tømmes for Vand, idet · der ved Smeltning af Isspærringen aabnedes Afløb for Vandet mod Syd til Dalen forbi Flinterup. Øst for Linien Rugtved- Kelleklinte optræder Fladbak- kerne ikke. Landskabet er derimod uroligt, rummer adskil- 71 lige Grusbakker og højtliggende Partier med en Mængde store Sten. Dette er især Tilfældet mellem Saltofte og Sve- bøllegavn. I den store Bakkeknude, der ved Saltofte Ud- sigtsbakke naar en Højde af 81 m og rummer en særdeles omfattende Udsigt, er der fra Syd mod Nord, ned imod Svebølle Station, to Erosionsdale; den vestlige, som er den største, gennemskærer helt Bakkeknuden og viser, hvor- ledes den er udskaaret af Smeltevand fra et Isdække lig- gende paa Sydøstsiden af Bakken. Fortsættelsen af dette Isdækkes Randomraade kan følges videre mod Nord igen- nem Stenrand Plantage og Øst om Bregninge, hvad der senere bliver omtalt (Side 91 ). lige Grusbakker og højtliggende Partier med en Mængde store Sten. Dette er især Tilfældet mellem Saltofte og Sve- bøllegavn. Røsnæs-Asnæs med Bagland. I den store Bakkeknude, der ved Saltofte Ud- sigtsbakke naar en Højde af 81 m og rummer en særdeles omfattende Udsigt, er der fra Syd mod Nord, ned imod Svebølle Station, to Erosionsdale; den vestlige, som er den største, gennemskærer helt Bakkeknuden og viser, hvor- ledes den er udskaaret af Smeltevand fra et Isdække lig- gende paa Sydøstsiden af Bakken. Fortsættelsen af dette Isdækkes Randomraade kan følges videre mod Nord igen- nem Stenrand Plantage og Øst om Bregninge, hvad der senere bliver omtalt (Side 91 ). Indlandsisens Tilførselsretninger til Rø s næ s o g A s n æ s. I Sammenhæng med den særegne Opbygning af Røsnæs og Uligheden mellem Røsnæs og Asnæs staar ogsaa, at deres Indhold af Stenmateriale viser store Forskelligheder. Til Belysning heraf fremsættes ne- denstaaende Liste over det relative Mængdeforhold mellem visse karakteristiske » Ledeblokke«, der stammer fra de i Listen navngivne Omraader. Foruden Sten fra Røsnæs og Asnæs er der i Oversigten ogsaa medtaget Sten fra Sejerø og Reersø, som bliver omtalt senere. Antal Porfyrsten fra optalte I I Østersøens Sten Egnene Dalarne Egne ved Bund ved Oslo i Sverige Kalmar s: f. Ålands- øerne °lo °lo °lo °lo Sejerø, Kysterne . 293 1 44 13 42 Røsnæs, Nordkyst 319 1 55 18 26 - , Sydkyst. 302 13 50 9 28 Asnæs, - 75:1 11 89 Reersø, Vestkyst . 89 1 46 1 52 Forskellen mellem Stenindholdet paa Røsnæs og Asnæs er stærkt iøjnefaldende. Paa Asnæs er der et saa stærkt baltisk præget Indhold som næsten tænkeligt. Den stærkeste Modsætning hertil viser Røsnæs' Sydkyst, hvor der overalt 72 kan træffes et betydeligt Antal norske Sten, et Forhold som jævnligt forekommer, hvor det ikke alene er det allerøverste Istidslag, hvorom det drejer sig, men ogsaa dybere liggende Lag. Men Forskellen mellem Asnæs og Nordkysten af Røs- næs er ligeledes stor. Dette giver sig Udslag i Nordkystens store Antal af Dalablokke og Porfyrer fra Kalmaregnen i det sydøstlige Småland. Stenindholdet viser tydelig nok, at det er ikke tilført fra Syd, saaledes som Tilfældet maa være med det paa Asnæs. Derimod viser det et tydeligt Slægtskab med Materialet paa Sejerø, ikke mindst ved sin store Mængde af Påskallavikporfyr fra Småland. Sammenholdt med Landskabsformen viser Røsnæs sig derved at være opstaaet langs Sydvestsiden af en stor Istunge, der fra Sydøst har bevæget sig fremad i samme Retning som Sejerøs Længde- retning. Røsnæs-Asnæs med Bagland. Det samlede System af Enkeltbakker paa Røsnæs gaar da ind som Led i det store Bakkedrag, der som Dele af to flade Buer strækker sig langs Sydkysten fra Fyrspidsen til Kalundborg. - Hermed stemmer det overens, at der ved et Besøg paa Røsnæs i 1929, paa en Bakkeryg 7 50 m Sydøst for Nostrup Gaard, saas en stor, jordfast Blok, der var is- skuret fra Nordøst mod Sydvest. Stenen er senere fjærnet fra Stedet. Som tidligere nævnt er der i Sandomraadet langs Røs- næs Sydkyst mange Afløbsrender eller Erosionsdale ned imod Kalundborg Fjord. De henligger næsten alle i fuld- stændig tør, »fossil« Tilstand. Imellem Røsnæsgaard og Ul- strup har de fleste et stærkt slingrende Forløb og er smalle i Forhold til Længden. I Egnen ved Kongstrup er Dalene korte, men brede og bærer i høj Grad Præg af at være formede foran en stagnerende Isrand. - Det er saaledes en omfattende Gruppe af Forhold, som viser, at Røsnæs er opstaaet foran et Isdække fra Nord. Modsat hertil er Asnæs afsat foran et Isdække, der er kommet til Stedet fra Syd. Som allerede nævnt, fremgaar dette af Næssets Indhold af Ledeblokke, der er u_sædvanlig stærkt baltisk præget. I Tilslutning hertil kan anføres det tidligere (Side 48) omtalte Forhold, at Forekomsten af Dilu- vialler mellem Svallerup og Udby er opstaaet i en Issø, der 73 mod Syd var begrænset af Is. At der fra Syd har strakt sig en Istunge op igennem Store-Bælt og over den vestlige Del af Sjælland fremgaar af flere Forhold, som bliver om- Lalt i det følgende. mod Syd var begrænset af Is. At der fra Syd har strakt sig en Istunge op igennem Store-Bælt og over den vestlige Del af Sjælland fremgaar af flere Forhold, som bliver om- Lalt i det følgende. De Israndstadier, der har været Ophavet til Udformningen af det her beskrevne Landskab, er anført paa det medføl- gende Højdekort. Røsnæs Linien og Lerchenfeld-Rugtved Li- nien er hidført fra Nord, mens Lerchenborg-Udby-Svebølle Linien er sat op fra Syd. Ved Saltofte er den første blevet overskaaret af den sidste, som herfra er gaaet i nordlig Retning. I hvilket Omfang de to Israndstadier har været betinget af Isfremstød er usikkert; de er i hvert Fald ikke kommet i Stand ved en simpel Tilbagesmeltning af Isdæk- ket. Røsnæs-Asnæs med Bagland. Dette kommer mest uvilkaarligt til Udtryk ved selve Røsnæs, hvor de opskudte Lag og de talrige paralleltlø- bende Bakker er Udtryk for den stærke Sammenskydning der har fundet Sted. I Forbindelse hermed kan ogsaa næv- nes Røsnæs Liniens buede Form, der snarere tyder paa Fremstød end Tilbagerykning. At der imidlertid imellem de to Linier Øst for Tømmerup-Rørby har henligget Dødis, som har betinget Vandafløbet forbi Forsinge til Tissø, er tidligere nævnt. - Aldersforholdet mellem de to Linier fremgaar af, at det er Asnæs Linien, som har overskaaret Røsnæs Linien. Hvor langt den dermed sammenhørende Isinvasion har bredt sig ind over østligere Egne maa lades ubesvaret. Men det er ikke usandsynligt, at Lille Aamoses Dal, Nord for Tissø, i sit oprindelige Anlæg er en Tunnel- dal, der er udskaaret af en Syd- Nord gaaende Flod under Isen under dens Udbredelse fra Syd og Sydvest ind over disse Omraader. Egnen Vest og Syd for Tissø. Landskabet langs Store-Bælt Syd for Asnæs 10---12 km ind i Landet er hovedsagelig en lidt ujævn Moræneflade, som er ret ensartet ,udformet bortset fra dets Kystind- skæringer og nogle langstrakte Moselavninger. Til Omraa- dets vigtigste Karakter fra Istiden hører den lille SV- NØ rettede Aas ved Aagaard, som viser Isbevægelsens Retning 74 i dens Slutningsafsnit. Indholdet af Ledeblokke ved Bjærge Klint har en Sammensætning ganske svarende Lil den ved Asnæs. Dette Forh9ld har derimod ændret sig lidt sydligere, nemlig ved Reersø, saaledes som Listen Side 71 viser·. Østersøblokkenes uhyre Overtal er her svundet ind til en mere normal Procent.mængde. Fra Stenselskabet ved Nord- kysten af Røsnæs adskiller Reersøstenene sig dog ved det ringe Antal af Smålandsporfyrer. Afvigelsen fra Stensel- skabet ved Asnæs og Bjærge Klint viser altsaa, at der til Reersø er ført Is med et andet Stenmateriale end det, den forudgaaende Is havde medført. Naar man i Forbindelse der- med ser, at der fra Reersø langs Kysten mod Sydøst strækker sig et temmelig bakket Landskab, falder det naturligt at anse dette Bakkestrøg for en Israndslinie, nemlig Grænsen for Udbredelsen af det Isfremstød, som medførte det æn- drede Stenindhold. Omraadet Syd for Aamose mellem Tissø og Stenlille. Omraadet Syd for Aamose mellem Tissø og Stenlille. Landskabet mellem Tissø og Stenlille er varierende baade hvad Højder, Overfladeformer og geologisk Beskaffenhed angaar. Dets vestligste Parti mellem Tissø og Reerslev er et kompakt Højdedrag, der ved Kløveshøj naar Højden! 100 m. Det rager dermed ikke blot højt op i Forhold til Tissøs lave Vandflade og den nordligere Lille Aamose, men naar ogsaa en henved 50 m større Højde end det østligere Landskab. Som Kortet viser, bestaar dette Højdedrag næsten udeluk- kende af Moræneler. Ved Slutningen af Istiden har det utvivlsomt haft Isdækket liggende ved Vestsiden, men hvorledes den langstrakte Kuppel iøvrigt har faaet sin fremtrædende Højde er ganske uvist. Maaske kan den skyldes en tidligere Isinvasion fra Nordøst i Tilknytning til den før omtalte Røsnæs Linie. Det lavere liggende Landskab mod Sydøst, henimod Sten- lille og Munke-Bjærgby, er temmelig ujævnt i dets 1~ordlige Del, men rummer mod Sydøst store, flade Strækninger. Ujævn.hederne skyldes dels smaatoppet Landskab, dels fremtrædende ovale Bakker, hvoraf den største er Sobjærg (ved Byen af samme Navn), der med en Højde af 65 m Fig. 2~ . Morænelersbakkeslrøg Syd for Aamose. Autoriseret Gengivelse eflcr Maalebordsbladenc e 5 Ruds Vedby 1896, og I 5 Niløse. 1896. I : 40.000. -~ -._) ~· Fig. 2~ . Morænelersbakkeslrøg Syd for Aamose. 76 rager mere end 30 m op over Omgivelserne. Den mest paa- faldende Bakkeform danner dog den Kæde af Bakker, der strækker sig som et retliniet Bælte i Retning VNV-ØSØ fra Sønderød forbi Niløse til Skuerup, d. v. s. med en Længdeudstrækning af 12 km. rager mere end 30 m op over Omgivelserne. Den mest paa- faldende Bakkeform danner dog den Kæde af Bakker, der strækker sig som et retliniet Bælte i Retning VNV-ØSØ fra Sønderød forbi Niløse til Skuerup, d. v. s. med en Længdeudstrækning af 12 km. Mest udpræget fremtræder Formen ved Kongsted, Vest for Niløse (hvorfra Kortet Fig. 24 viser et Udsnit). Saavidt Iagttagelserne rækker, bestaar Bakkerne af Moræneler und- tagen Nord for Sønderød og ved Skuerup. Formen va- rierer fra cirkelrund til aflang, og der er indtil tre paral- lele Rækker. Mod Øst tabe~· Rækkeformen sig. - Hvor- ledes disse Bakkerækker er opstaaet, er temmelig usikkert. Bakkerækker med en lignende Anordning som disse ligger det nær at opfatte som »drumlins«, d. v. s. Morænebakker, hvis Længderetning falder sammen med Bevægelsesretnin- gen af det Isdække, fra hvilket de stammer (saaledes er disse Bakker tydet i den geologiske Kortbladsbeskrivelse til de nordvestsjællandske Kort, 1900). Omraadet Syd for Aamose mellem Tissø og Stenlille. Der er dog ogsaa den nærliggende Mulighed, at de afspejler Retningen af en Is- randstilling, og at deres Længderetning dermed staar vin- kelret paa Isens Bevægelsesretning. Der er nogen Grund til at antage, at denne sidste Mulighed svarer til Virkeligheden. Det ligger saaledes nær at se Bakkekædens Placering tæt langs Morænelerets Nordgrænse ved Kongsted og Niløsc som et Tegn paa, at Bakkerne er en Israndsdannelse. Dette synes ogsaa bedst at svare til, hvad andre Forhold peger i Retning af vedrørende Israndstadiernes Orientering in- den for Omraadet. Umiddelbart Nord for den nysomtalte Bakkekæde og vi- dere mod Øst til noget forbi Stenmagle er der langs Syd- randen af Store Aamose et udstrakt Omraade med lagdelte Smeltevandsaflejringer og uden Moræneler. I Almindelighed er det kun Sand eller Grus der forekommer, men enkelte Ste- der optræder der ogsaa stenfrit Ler, enten afsat oven paa Sandlagene eller liggende som en Lerhorisont dækket af Sand eller Grus. En Del af Omraadet Øst for Sandlyng Aa er landskabeligt særlig karakteriseret ved, at der paa en svagt ujævn Sandflade ligger en Del temmelig regelmæs- Fig. 25. Landskab NV og N for Slenlille. Autoriseret Gengivelse eller Maalebordsbladenc f 5 Niløsc 1896 og g 5 Stenmagle 1895. 1 : 40.000. -..J -..J Fig. 25. Landskab NV og N for Slenlille. Autoriseret Gengivelse eller Maalebordsbladenc f 5 Niløsc 1896 og g 5 S Fig. 25. Landskab NV og N for Slenlille. Autoriseret Gengivelse eller Maalebordsbladenc f 5 Niløsc 1896 og g 5 St 78 sigt runde Bakker, der rager indtil 15 m op over Omgivel- serne. De naar en Højde af 4,2- 48 m, Bakkesiderne er ret stejle ; de smaa Bakker er kuppelformige, de mere omfat- tende er gennemgaaende temmelig flade. Saaledes som .de er formede, maa man antage, at de ikke betegner Erosions- rester af et sammenhængende Plateau, men at derimod deres Form og Omrids er den oprindelige. De kan anses for at være afsat i Fordybninger i et Isdække af omtrent samme Tykkelse som Bakkernes Højde over Omgivelserne. Først efter Isdækkets Bortsmeltning kunde Sand- og Grus- lagene i de tilstødende, lavere liggende Omraader afsættes. I Sandomraadet Vest for Sandlyng Aa er der ikke en saa udpræget Forskel mellem Bakker og lavere Landskab som i det østligere Omraade. Omraadet Syd for Aamose mellem Tissø og Stenlille. Naar man lægger Højderne til Grund, ser det nærmest ud, som om det Sæt i Aflejringens Forløb, der i det østlige Omraade kun repræsenteres af Bakkerne, er det normale i det vestlige, medens den lavere liggende Sandhorisont her er stærkt underordnet. Højderne af Bakketoppene er nogenlunde jævnt faldende fra Øst (ved Stemnagle) mod Vest (ved Verup) med et Fald paa 1 : 500, nemlig fra 54 til 39 m. Der er saaledes højdemæssigt intet til Hinder for, at Bakkerne i Østpartiet er sammenhørende med den overvejende Del af Overfladelagene i det vestlige Sandomraade. Det er da ikke usandsynligt, at denne øvre Etage i Aflejringen skyldes et Øst-Vest gaaende glacialt Flodsystem, medens der i Aamose Dalen og tilstødende Dale endnu henlaa Lag af begravede Isrester, som ud- fyldte Sænkningerne. Med den ovennævnte Faldgradient (1 : 500) er det endog muligt, at Smeltevandsfloden har haft Afløb ad Afløbsdalen mod Nordvest fra Store Aamose, hvor det nuværende Paspunkt ligger ved en Højde af 25 m. Fra Stenlille strækker der sig mange Kilometer sydpaa et Sand- og Grusomraade, der mellem Bromme og Døj- ringe naar en Bredde af 5 km; derimod er Forbindelsen med det nordlige Parti kun ganske smal og bestaar ved Stenlille og Tjørntved af de to korte, tidligere nævnte Aase med den mellemliggende, smalle Dal langs Sandlyng Aa. Omraadet mod Syd har foruden at være karakteriseret ved 79 Jordbeskaffenheden ogsaa landskabeligt sit særegne Præg. Hertil m edvirker i udpræget Grad de Dalsystemer, som det rummer. Først er her at nævne det Dalsystem, der stræk- ker sig fra Nord mod Syd tæt forbi Munke-Bjærgby og Brorn.me. Saaledes som den Dal fremtræder, der indeslutter Sørækken: Eskilstrup Sø, Munke-Bjærgby Sø og videre mod Syd: Lillesø, Maglesø og Vedsø, og som yderligere er fortsat af en smal Dal mod Sydvest forbi Store Frederikslund til Skovsø - maa den betragtes som en Tunneldal, frembragt af en Flod strømmende fra Syd mod Nord under Isdækket. Søernes Vandflade ligger indtil 20-25 m lavere end de om- liggende Sandbakker. - En østligere, paralleltløbende gla- cial Overfladeflod har fra Syd, forbi Pedersborg ved Sorø, banet sig Vej frem til Døjringe og har herfra strømmet vi- dere mod Nord og formet Dalen, som nu rummer Sandlyng Aa. Vandskellet mellem denne Aa og Tilløbet til Tudeaa ligger nu i Dalen, 2 km Nord for Døjringe, ved en Højde af 32 m . Omraadet Syd for Aamose mellem Tissø og Stenlille. Ind i eller igennem Sandbakkerne ved Munke- Bjærgby, Rude-Eskilstrup, Saltofte og andre Dele af Om- raadet strækker der sig en Horisont af stenfrit Ler . Den viser, at der i Løbet af Lagkompleksets Aflejringstid er sket indgribende Ændringer i Aflejringsbetingelserne. Medens Lagene af Sand og Grus viser frie Gennemløbsmuligheder for strømmende Vand, har der i det Tidsrum, da de mægtige Lerlag afsattes, hersket Søtilstand. Sandsynligvis har denne Tilstand efterfulgt den Tid, da den subglaciale Flod udfor- mede Tunneldalen med Sørækken. Den rolige Tilstand er atter efterfulgt af en stærk Vandgennemstrømning, hvor Omraadets øvre Lag af Sand og Grus og ligeledes Aasene ved Tjørntved og Stenlille er afsat. Den sidste Fase i den glaciale Udvikling er Udformningen af de markante Ero- sionsdale i Omraadet mellem Bromme og Døjringe og der- fra mod Nord i Retning mod Aamose. - Sammen med disse sidste Faser hører ogsaa Aflejringen af stenfrit Ler i det store Omraade mellem Tersløse og Tunneldalen ved Eskilstrup Sø. Omtrent samtidig hermed er det rimeligvis, at den lave Etage i Sandomraadet Øst for Sandlyng Aa og de tilsluttede Dale Nord for Stenlille er opstaaet. 80 Omraadet mellem Haraldsted og Tølløse. Længst mod Sydøst i den her omhandlede Del af Sjæl- land er der Landskabsforhold, som man ikke træffer i det øvrige Nordvestsjælland. Alene Øst og Sydøst for Aamose er der tre hver for sig karakteristiske Landskabsformer. Det specielle Særkende for den sydligste Del er Sørækken Gyr- stinge Sø- Haraldsted Sø. Man har her Vestenden af den Tunneldal, hvis Spor strækker sig fra Stenlille i Vest til Østenden af Køge Aas tæt ved Køge, og som har en samlet Længde af 40 km. Søerne er de dybtskaarne Spor efter den Smeltevandsflod, som her har banet sig Vej, men som ogsaa har afsat Spor i Form af Aase. Nord for Haraldsted Sø ligger »Aasen«, og fra Gyrstinge Sø mod Nordvest strækker sig Vielsted Aas. Flodløbet har haft sin videre Fortsættelse mod Nordvest og Vest ad en Dal med Udmunding Nord for Stenlille tæt ved Siden af Stenlille Aas, der, som før nævnt, har faaet sin Vandtilførsel fra Syd. Fra Stenlille har det fælles Afløb fortsat sig igennem Store Aamose og videre mod Nordvest til Saltbæk Vig, som det senere skal omtales. I Allindelille Fredskov og andre Steder Nord for Harald- sted Sø træder der Flager af brokket Skrivekridt frem ved Jordoverfladen. Af saadanne løsrevne Partier af Skrivekridt, der er meget almindelige i den sydøstlige Del af Sjælland, hvortil de er ført fra nærliggende Dele af Østersøen, har vi her de yderste kendte Forekomster. - Længere mod Nord, navnlig Øst for en Linie: Stestrup- Udstrup-Allinde- lille Old, er Landskabet usædvanlig smaakuperet med yderst uregelmæssige Former. Landskabsformen tyder paa, at der her i længere Tid har henligget et Dække af død Is, hvorfra den regelløst formede Overflade har faaet sit Præg. At dette har været Tilfældet fremgaar ogsaa af, at der paa det højt opragende Stestrup Bjærg har været en Issø, hvis Bundflade ligger indtil 20-30 m højere end det omgivende Moræne- landskab; Bjærgets Top naar yderligere en halv Snes Meter højere (102 111). Langs med Halleby Aa, fra dens Udspring fra Skee Mose, forbi Skee og Skee-Taastrup hen imod Merløse og Taastrup, strækker der sig et temmelig jævnt Morænelandskab. Dets C, Fig. 26. Morænelandskab ved Balleby Aa's øvre Løb. Autoriseret Gengivelse efter Maalebordsbladene h 5 Bonderup 1896, i 5 Skjoldenæ9nolm 1897, h 4 Alindcmagle 1895 og i 4 Haraldsted 1895. 1 : 40.000. 00 .... Fig. 26. Morænelandskab ved Balleby Aa's øvre Løb. Omraadet mellem Haraldsted og Tølløse. Autoriseret Gengivelse efter Maalebordsbladene h 5 Bonderup 1896, i 5 Skjoldenæ9nolm 1897, h 4 Alindcmagle 1895 og i 4 Haraldsted 1895. 1 : 40.000. Fig. 26. Morænelandskab ved Balleby Aa's øvre Løb. Autoriseret Gengivelse efter Maalebordsbladene h 5 Bonderup 1896, i 5 Skjoldenæ9nolm 1897, h 4 Alindcmagle 1895 og i 4 Haraldsted 1895. 1 : 40.000. 82 Overfladeform er paafaldende forskellig fra det nysnævnte, stærkt kuperede Omraade og det bakkede Landskab mod Syd. Det ligger nær at anse dette Landskabsbæltes jævnere Terrænform som Tegn paa, at Isafsmeltningen her er fore- gaaet i et hurtigere Tempo end i de ujævnere Naboomraa- der. Selvom Grænserne imellem dem ikke kan betragtes som Israndslinier, · efterladte efter en tilbagevigende Isrand fra selve Indlandsisen, træder de dog i Stedet for saadanne Linier, ved at de markerer Forløbet af Isdækkets Bortsmelt- ning fra Omraadet. Det relativt flade Morænelandskab afbrydes mod Vest ved en Linie: Merløse--Bonderup af en Moselavning. Samtidig med al denne og den tilgrænsende, nysnævnte Moræneflade endnu har henligget dækket af et ringe Dødislag, har der været isfrit Land umiddelbart Vest derfor. Aflejringerne af Sand og stenfrit Ler ved Vanløse viser dette, og disse Sand- fladers Finkornethed viser, at det er svagt rindende Vand- masser, der har afsat dem. De staar derved i Modsætning til Omraadet mellem Stenmagle og Sandlyng Aa, hvor det tillige af Højdeforholdene fremgaar, at det er opstaaet under andre Forhold og paa et tidligere Tidspunkt, saa- ledes som det allerede er omtalt (Side 78- 79). Den tidsmæssige Rækkefølge af Grus- og Sandaflejringer- nes Opstaaen i hele Omraadet har da rimeligvis været: Den tidsmæssige Rækkefølge af Grus- og Sandaflejringer- nes Opstaaen i hele Omraadet har da rimeligvis været: 1) Først Bakkerne Stenmagle- Sandlyng Aa og Sandpar- tiet Vest for Sandlyng Aa; derefter 2) Fladerne Stemnagle-Sandlyng Aa, samt Omraaderne S f. Stenlille; og sidst 3) Sandomraadet ved Vanløse, samt Afløbet fra Vielsted Aas. Isen, som fra første Færd har strakt sig som et temmelig sammenhængende Dække til - og ud over - Aamosen, er under dette Aflejringsforløb smeltet bort indtil en Linie fra Stenlille forbi Merløse til Allinde- lille. Højdeforholdene i Forbindelse med Morænelerets Udbre- delse i Omraadet imellem Stenmagle, Undløse og Bonderup viser, at der ogsaa her har henligget et Dødislag, dengang Fladerne med Sand og stenfrit Ler ved Vanløse afsattes. Fig. 27. ~forænclandskab og Flade Nord for St. Merløsc. Se Teksten Side 84. Autoriseret Gengivelse citer Maalebordsblad h 5 Bonderup 1896. Omraadet mellem Haraldsted og Tølløse. 1 : 40.000. CO '-" CO '-" CO '-" Fig. 27. ~forænclandskab og Flade Nord for St. Merløsc. Se Teksten Side 84. Autoriseret Gengivelse citer Maalebordsblad h 5 Bonderup 1896. 1 : 40.000. 84 En lang, voldformig Bakkerække, der strækker sig fra Bonderup nordpaa i Retning mod Nyrup kan opfattes som en Fremstødsmoræne fra en Isrand, der rimeligvis har haft sin Fortsættelse mod Sydøst og her falder sammen med den førnævnte Terrængrænse, Nord for Skee. Efter at det bagved liggende Isdække ogsaa var gaaet over i Dødisform, er der i Omraadet omkring Tysinge Syd for Tølløse opstaaet et isfrit Omraade, hvor Smeltevandet har kunnet afsætte plane Sandflader tillige med Lag af stenfrit Ler. Afløbet derfra er foregaaet ad en slingrende Rende igennem et Dødisdække frem til Halleby Aaens Dal umiddelbart Vest for Moræne- bakken ved Bonderup. En lang, voldformig Bakkerække, der strækker sig fra Bonderup nordpaa i Retning mod Nyrup kan opfattes som en Fremstødsmoræne fra en Isrand, der rimeligvis har haft sin Fortsættelse mod Sydøst og her falder sammen med den førnævnte Terrængrænse, Nord for Skee. Efter at det bagved liggende Isdække ogsaa var gaaet over i Dødisform, er der i Omraadet omkring Tysinge Syd for Tølløse opstaaet et isfrit Omraade, hvor Smeltevandet har kunnet afsætte plane Sandflader tillige med Lag af stenfrit Ler. Afløbet derfra er foregaaet ad en slingrende Rende igennem et Dødisdække frem til Halleby Aaens Dal umiddelbart Vest for Moræne- bakken ved Bonderup. Langs en Linie fra Udstrup forbi Baarup, Stestrup og Grønnebjærg Gaard og videre mod Nordøst gaar der en Grænse mellem det førnævnte, stærkt kuperede Moræne- landskab mod Sydøst og det jævnere Terræn mod Nord- vest. Selvom det har været Dødis, der paa begge Sider af Grænsen har dækket Landskabet, kan denne Terrængrænse dog med ret stor Sikkerhed anses for at vise, i hvilken Retning Bortsmeltningen af Isdækket er foregaaet. Som Stadfæstelse deraf tjener, at denne Linies nordøstlige Fort- sættelse krydser den øvre Del af Elverdams Aaens Dal; denne Dal har oven for Skæringsstedet et subglacialt Præg i Sammenligning med den mere udtalte ekstramarginalt prægede Dalform neden for. Aamosedalen og dens Afløb ved Istidens Slutning. Aamosedalen og dens Afløb ved Istidens Slutning. Den Dal, som nu rummer Store Aamose, havde ved Is- tidens Slutning ikke blot Tilløb af Vand fra de samme Omraader som i Nutiden, men tillige længere borte fra, alt- sammen ad Dalen ved Stenlille. Fra Syd førtes der Vand- masser hertil ad Smeltevandsdale fra Sorø Egnen og lige- ledes fra et betydeligt Omraade Vest for Bromme, som nu afvandes mod Vest igennem Tudeaa. Desuden har der fun- det Vandtilstrømning Sted fra Sydøst fra Tunneldalen igen- nem Gyrstinge Sø. 8 85 Afløbet fra Aamose har fra første Færd fundet Sted ved Bromølle ved Aamoses Nordvestspids, igennem en Dal mel- lem Holmstrup og Kattrup. Den nuværende Dalbunds høje- ste Punkt ligger ved· 25 m . Hvor meget højere Dalbunden har ligget ved Afløbets Indtræden, er det næppe muligt at afgøre, selv om Grusterrassen ved I~ajemose og Højden af Terrassehakket maaske kan give Bidrag til Afgørelsen. Aadalen er begrænset af et Morænelerslandskab, der fra begge Sider hælder ned mod Dalfuren, uden at der er kendelige Spor af Floderosion. Efter Ombøjningen af Halleby Aa Nordvest for Rangle- mølle til Retningen Øst- Vest træffer man i Skoven Møller- vænge en glacial Erosionsskrænt ved 20 m med en neden- for liggende Sandterrasse. Nord for Grydemølle, 1½ km vestligere, er der igen et Terrassestykke, med Terrassehak- ket liggende _ved 15 m. Tæt ved dette Sted drejer Halleby Aas Retning nu fra Øst- Vest til Nord- Syd ved en Højde af 6--7 m. Men den Flod, som ved Istidens Slutning for- mede de nysnævnte Erosionsmærker og Terrasser, fortsatte derimod sit Løb videre mod Nordvest igennem en dybt ned- skaaren, yderst markeret Dal, som Jærnbanen overskærer 700 m Nordøst for Avnsøgaard. Denne Dal, der fortsætter sin Vej videre forbi Bregninge til Saltbæk Vig, udgør nu Lejet for Bregninge Aa. Ved Jærnbanens Skæring med Dalen ligger Udspringet af Aaen, ved en Højde af lOL-11 m; Dalen har her en Bredde af 200 m. Aarsagen til, at Afløbet fra Aamosedalen i Tilslutning til Isdækkets Bortsmeltning er gaaet for sig i Retning mod Saltbæk Vig og ikke som nu gennem Tissø, maa ligge i, at der endnu her henlaa Is, som tvang Smeltevandsfloden til at følge denne Bane. Rimeligvis har Isdækket strakt sig helt frem til Flodløbet. I den Retning taler det, at der ved den snævre Overgang fra Smeltevandsflodlejet til Lille Aa- moses Dal kun findes yderst svage Spor af Erosion. Aamosedalen og dens Afløb ved Istidens Slutning. En lavtliggende Bræmme af Sand og Grus langs visse Dele af Tissø antyder dog, at Afløbet til Store-Bælt fra denne Sø og Lille Aamoses Dal stadig i nogen Tid har været spærret af 86 et Isdække. Denne Bræmme ligger ved en Højde af indtil 10 m og kan dermed naturligt anses for at være opstaaet langs Bredden af den isdæmmede Tissø, mens denne endnu - ligesom Store Aamose - havde Afløb mod Sejerø Bugt. Morænefladen med de hatformige Bakker S f. Lammefjord. 89 de fire førstnævnte viser Kurverne altsaa Bevægelsesretnin- gen NØ- SV; ved Drusebjærg Retningen Ø- V. Disse Ret- ninger falder godt i Traad med, hvad man ad andre Veje kan vide om Isbevægelsen, der h erfra er gaaet dels mod Syd og dels mod Vest i Retning mod Israndstillinger Nord for Aamose og i Nærheden af Bregninge. de fire førstnævnte viser Kurverne altsaa Bevægelsesretnin- gen NØ- SV; ved Drusebjærg Retningen Ø- V. Disse Ret- ninger falder godt i Traad med, hvad man ad andre Veje kan vide om Isbevægelsen, der h erfra er gaaet dels mod Syd og dels mod Vest i Retning mod Israndstillinger Nord for Aamose og i Nærheden af Bregninge. Morænefladen med de hatformige Bakker S f. Lammefjord. Morænefladen med de hatformige Bakker S f. Lammefjord. Landskabet Syd for Lammefjord fra Holbæk Fjord til Arnakkegaard Vest for Svinninge fremtræder som en umiddelbar Fortsættelse af Lammefjords Flade med Højder stigende op til ca. 20 m. Mod Syd strækker Fladepræget sig omtrent til Jærnbanen Holbæk- Jyderup. Den største Del af Fladen indeholder Moræneler, der dog paa en stor Stræk- ning ligger saa lavt, at den dækkes af omfattende Moser og Enge. Længst mod Syd og Sydvest er der Sandaflejringer. Den temmelig plane Flade afbrydes paa nogle Steder af isolerede Bakker, der dog i et enkelt Omraade, nemlig Egnen ved Kundby, optræder i en saadan Mængde, at de ganske forstyrrer Fladepræget'. Som Følge af Bakkernes betydelige Højde i Forhold til deres ringe horisontale Ud- strækning har de faaet Navnet »hatformige Bakker«. Denne deres ydre Fremtræden hænger nøje sammen med Bakkernes indre Opbygning af kantstillede Grus- og Sandlag, hvad der er nærmere omtalt Side 37- 41. Saaledes som det der er nævnt, maa det antages, at Lagene oprindelig er afsat i Spalter i Isdækket og derefter stillet paa Højkant ved at vælte. Senere har Isen bevæget sig hen over de opstaaede Forhøjninger. Bevægelsens Retning kan ved enkelte af Bakkerne direkte aflæses ved Hjælp af Højdekurverne i Bakkernes nærmeste Omegn. Paa Kortet Fig. 28, der viser et Udsnit fra Kundby Egnen, ser man, at Højdekurverne fra Bakkerne . Lundebjærg, Hyrdehøj, 'Kfrkebjærg og \en sydligere Bakke er trukket ud i Retning mod Sydvest. Ved Drusebjærg Nord for Kundby er Kurverne trukket ud i Ret- ning Ø-V. Disse skraanende Bakkerygge kan naturligt op- fattes som drumlinsagtige Slæb, hvis Moræneler ved Isens Bevægelse hen over Bakkerne er afsat i Læ af disse. Ved 87 Fig. 28. :\fo rænefladc med h atformige Bakkc1·. Autoriseret Gengivelse efter i\faalebordsblad f 7 Kundby 1896. 1 : 40.000. Fig. 28. :\fo rænefladc med h atformige Bakkc1·. Autoriseret Gengivelse efter i\faalebordsblad f 7 Kundby 1896. 1 : 40.000. 88 KELD MJJ, THEHS fol. 1942. Fig. 29. Tornved Bjærg, hatformig Bakke, imellem Jyderup og Mørkøv, scl fra Øst. KELD MJJ, THEHS fol. 1942. Fig. 29. Tornved Bjærg, hatformig Bakke, imellem Jyderup og Mørkøv, scl fra Øst. KELD MILTliERS fot. 1942. Fig. 30. Stejltstillede Sandlag. Kirkebjærg, Nordøst for Stigs-Bjærgby. KELD MILTliERS fot. 1942. Fig. 30. Stejltstillede Sandlag. Kirkebjærg, Nordøst for Stigs-Bjærgby. Fig. 30. Stejltstillede Sandlag. Kirkebjærg, Nordøst for Stigs-Bjærgby. Højlandet mellem Skarresø og Sejerø Bugt med vestligere Forland. Det Landskab, som her er nævnt under eet, er af yderst forskelligt landskabeligt Præg, men den geologiske Udform- ning har haft et saadant Forløb, at det bedre kan omtales i Sammenhæng end hver for sig. Den udprægede, land- skabelige Grænselinie, som adskiller det bakkede Moræne- landskab fra det lavere liggende Grusterræn, skiller ikke imellem to samtidigt opstaaede Landskaber, men er Skellet mellem Ornraader, der væsentlig har faaet tilført sit Ma- teriale fra forskellig Side og er udformet til hver sin Tid. Det højtliggende Morænelandskabs Materiale er tilført fra Øst, medens Grusterrænet er opstaaet ved Istilførsel fra anden Side, saaledes som det vil fremgaa af det følgende. Den føromtalte Moræneflade med de hatformige Bakker har sin Vestgrænse ved en Linie fra Sydspidsen af Svin- ninge Vejle over Agersvold til Jyderup Station. Straks Vest for denne Grænse stiger Terrænet fra Højder paa H}--15 m op til meget større Højder og bliver tillige stærkt kuperet. De største Højder, noget over 80 m, naas Syd for Favrbo samt Sydvest for Bjærgsted, hvor Landet har sit særlige Præg. Det bakkede Højland har sin Nordgrænse ved en Linie Særslev- Føllenslev- Algestrup. Fra Eskebjærg h ar det dog en Fortsættelse mod Nordvest, og det kan - med lavere Højder og mindre kuperet Overflade - følges helt til Nordvestgrænsen af Alleshave. Det mest særprægede ved dette Højland er dog dets Vest- grænse, saaledes som den er formet paa Strækningen fra Eskebjærg, forbi Daverup og derfra videre Vest om Bjærg- sted. Medens nemlig Grænserne over for det fladere Land- skab mod Nord og mod Øst ganske savner faste Konturer, 90 er Grænsen over for Lavlandet mod Vest usædvanlig fast formet. Paa den 7 km lange Strækning fra Eskebjærg til et Stykke SØ for Daverup har den et ganske retliniet For- løb. Den sydligste Del af Grænselinien - omkring Dave- rup - er formet som Rækker af Grusbakker, rn.ed indtil fire Rækker bag hinanden. Saaledes som de ligger, begræn- sede af Højlandets Morænelandskab mod Nordøst og af en jævnt skraanende Grusslette mod Sydvest, betegner disse Bakkerækker en smukt udformet Randmorænelinie. Grænse- liniens Fortsættelse fra Hovedlandevejen videre mod Nord- vest er ikke præget af Jordbeskaffenheden, men e:r formet som en Skraaning, der øverst er kronet af en ganske ret- liniet Række af Smaabakker. Paa Strækningen forbi Kirke- vang Gaarde ud for Torpe er Linien endog til begge Sider begrænset af Moræneler og er saaledes udelukkende karak- teriseret ved sin landskabelige Fremtræden. Højlandet mellem Skarresø og Sejerø Bugt med vestligere Forland. er Grænsen over for Lavlandet mod Vest usædvanlig fast formet. Paa den 7 km lange Strækning fra Eskebjærg til et Stykke SØ for Daverup har den et ganske retliniet For- løb. Den sydligste Del af Grænselinien - omkring Dave- rup - er formet som Rækker af Grusbakker, rn.ed indtil fire Rækker bag hinanden. Saaledes som de ligger, begræn- sede af Højlandets Morænelandskab mod Nordøst og af en jævnt skraanende Grusslette mod Sydvest, betegner disse Bakkerækker en smukt udformet Randmorænelinie. Grænse- liniens Fortsættelse fra Hovedlandevejen videre mod Nord- vest er ikke præget af Jordbeskaffenheden, men e:r formet som en Skraaning, der øverst er kronet af en ganske ret- liniet Række af Smaabakker. Paa Strækningen forbi Kirke- vang Gaarde ud for Torpe er Linien endog til begge Sider begrænset af Moræneler og er saaledes udelukkende karak- teriseret ved sin landskabelige Fremtræden. 1 ½ km Sydøst for Daverup bøjer Grænselinien brat om fra Retningen NV- SØ til Retningen NØ- SV. Højlandet Bjærgsted Bakker, imellem Skarresø og Grusterrænet om- kring Stenrand Plantage, ligger som en samlet Gruppe af langstrakte Bakker med dominerende Højde i Forhold til de lave Omgivelser. Bakkeknuden rummer mange store, jordfaste Blokke og viser derved og ved sin øvrige Rigdom paa Sten, sammen med hele sin Stilling i Forhold til Om- givelserne, at den er afsat som Randmoræne langs en stille- staaende Isrand. Det ret omfattende Omraade: Svebølle Station- Eske- bjærg og Viskinge-Daverup er i Hovedsagen en ret plan Grusslette. Det kunde paa Forhaand ligge nær at opfatte denne Grusslette som en ekstramarginal Hedeslette, afsat i nøje Tilslutning til den nu omtalte Israndstilling. Det viser sig dog, at en saadan Betragtning af Slettens Oprindelse ikke lader sig gennemføre. Dels er det kun en Del af Om- raadet - omend det største - der kan opfattes som Hede- slette, og dels maa den allerstørste Del af hele Grusomraadet være afsat i Forbindelse med et Isdække, der har ligget læn- gere fremme, end Bjærgsted-Eskebjærg Israndstillingen er Udtryk for. For at forstaa Omraadets Udformning maa For- 91 bindeisen knyttes med Omtalen af Asnæs- Udby-Svebølle lsrandstadiet (Side 70). bindeisen knyttes med Omtalen af Asnæs- Udby-Svebølle lsrandstadiet (Side 70). Saaledes som det der er nævnt, er der i Bakken Syd for Svebølle Station to S- N gaaende Erosionsdale, af hvilke den største helt gennemskærer Bakkeknuden og kun kan være udskaaret af Smeltevand fra et Isdække fra Sydøst. Højlandet mellem Skarresø og Sejerø Bugt med vestligere Forland. Der er her en smukt formet Skraaflade m ed Toppunk- tet liggende ganske tæt ved Knækket af Israndslinien, hvor der ganske naturligt har været den største Vandtilstrøm- ning. Højdekurverne viser, at Afløbet er gaaet fra Isranden i lige vestlig Retning. Paa Strækningen mellem Lille Breg- ninge og Dejvad Bro har Smeltevandsfloden ikke formaaet at udslette Sporene efter den tidligere Israndstilling, selvom den muligvis her har strømmet ud over Strækningens den- gang begravede Ismark, der nu ligger lavere end den øst- ligere, skraanende Grusslette. Men dennes egentlige Afløb har øjensynlig været længst mod Nord mellem Lille Breg- ninge og Sultenkrog og videre mod Nordvest til Dalen ved Bregninge Kirkeby (se Kortet Fig. 31). Medens det saaledes er foran Israndslinien SvebøHegavn- Dejvad- Sultenkrog, at den største Del af hele Grusfladen er opstaaet, er der dog ogsaa et Par mindre Partier, der er afsat i Forbindelse med den langt mere dominerende Is- randslinie Eskebjærg-Daverup- Bjærgsted. Dette maa saa- ledes være Tilfældet med et lille Parti Sydvest for Eske- bjærg, hvor Isdækket fra Nordøst maaske yderligere har naaet et lille Stykke længere frem end til Hoved.linien, nem- lig omtrent til Gaarden Torpelund. - Den anden Del af Fladen, som er afsat i Forbindelse med den store Isrands- linie, ligger ved dennes Ombøjning imellem Daverup og Bjærg- sted. Der er her en smukt formet Skraaflade m ed Toppunk- tet liggende ganske tæt ved Knækket af Israndslinien, hvor der ganske naturligt har været den største Vandtilstrøm- ning. Højdekurverne viser, at Afløbet er gaaet fra Isranden i lige vestlig Retning. Paa Strækningen mellem Lille Breg- ninge og Dejvad Bro har Smeltevandsfloden ikke formaaet at udslette Sporene efter den tidligere Israndstilling, selvom den muligvis her har strømmet ud over Strækningens den- gang begravede Ismark, der nu ligger lavere end den øst- ligere, skraanende Grusslette. Men dennes egentlige Afløb har øjensynlig været længst mod Nord mellem Lille Breg- ninge og Sultenkrog og videre mod Nordvest til Dalen ved Bregninge Kirkeby (se Kortet Fig. 31). Allerede forud derfor eller senest samtidig med Frem- komsten af den her omtalte, skraanende Hedeslette er Ud- skæringen af Floddalen mellem Dejvad Bro og Bregninge foregaaet. Hele Udformningen og det siksakformede Forløb af Dalen, hvori nu Bregninge Aa har sit Leje fra Jærnbane- dæmningen ved Avnsø til Bregninge vilde være meget ejen- dommelig, hvis Udskæringen i hele Dalens Udstrækning skulde tænkes foregaaet under eet. Højlandet mellem Skarresø og Sejerø Bugt med vestligere Forland. Sporene efter dette Israndsomraades Fortsættelse mod Nord træder tydeligt frem i Stenrand Plantage. Der er her en be- tydelig Mængde af temmelig store Sten, baade i den centrale Del og i Yderkanterne af Plantagen; oftest ses de i Skræn- terne, som der findes adskillige af. Ved Savskæreriet umid- delbart Nord for Plantagen er der en lav Bakke med store, jordfaste Sten. Flere Steder i Nærheden er der Profiler i Morænegrus med Kubikfod-store Sten. Ved Savskæreriet ligger Samlinger af store Stenblokke, og i Omegnen af Dejvad Gaard findes ligeledes mange, meget store Blokke. Land- skabet i Stenrand Plantage er stærkt kuperet med uregel: mæssigt liggende Bakker og med enkelte lange Skrænter, der kan lyde paa en stedfunden, betydelig Erosion i Ret- ningen SSØ- NNV. Det maa anses for sikkert, at Fremkomsten af dette bak- kede, stærkt stenholdige Grusterræn er sket i Forbindelse med en S- N gaaende Isrand, der har strakt sig fra den nærmeste Omegn af Svebøllegavn og har haft sin Yder- grænse nær Vest om de store Moser: Svebølle og Dejvad Moser og nær forbi Dejvad Gaard, muligvis saa langt mod Vest som Løgtved Huse. Sporene af denne Isrand kan yder- ligere følges videre i nordlig Retning forbi L. Bregninge, hvor baade en cirkelrund, flere Meter dyb Iskedel og Ter- rænets øvrige Former antyder Forløbet i Retning mod Sul- tenkrog ved Kanten af Højlandet. De fleste Grussletter Vest for den her omtalte Isrands- linie er da, med en enkelt Undtagelse, at betragte som en ekstramarginal Hedeslette, afsat ud fra denne Jsrand. Den er temmelig plan og uden nogen kendelig Faldretning; des- uden rummer den flere mosefyldte LavningeT, hvori der maa have henligget begravet Is, dengang Sletten blev formet af Smeltevandet. 92 Medens det saaledes er foran Israndslinien SvebøHegavn- Dejvad- Sultenkrog, at den største Del af hele Grusfladen er opstaaet, er der dog ogsaa et Par mindre Partier, der er afsat i Forbindelse med den langt mere dominerende Is- randslinie Eskebjærg-Daverup- Bjærgsted. Dette maa saa- ledes være Tilfældet med et lille Parti Sydvest for Eske- bjærg, hvor Isdækket fra Nordøst maaske yderligere har naaet et lille Stykke længere frem end til Hoved.linien, nem- lig omtrent til Gaarden Torpelund. - Den anden Del af Fladen, som er afsat i Forbindelse med den store Isrands- linie, ligger ved dennes Ombøjning imellem Daverup og Bjærg- sted. Højlandet mellem Skarresø og Sejerø Bugt med vestligere Forland. Anderledes naar Dalen mellem Dejvad Bro og Bregninge allerede er formet i nær Tilslutning til Israndstillingen Svebøllegavn- Sultenkrog. Paa en lang Strækning Nordvest for Dejvad Bro er den 400 m brede Dal skaaret 10-15 m ned i den tilgrænsende Grusslette. Ved Dejvad Gaard, hvor den senglaciale Dal- bund ligger fremme i Dagen, har den ligget bestrøet. med Fig. 31. Israndsomraade, I-Iedesletlcr og Smcltevandsdal. Se Teksten Side 92. Autoriseret Gengivelse efter Maalebordsbladene d 7 Følleslev 1897 og d 6 Bjergsted 1895. 1 : 40.000. ,.c, c,., Fig. 31. Israndsomraade, I-Iedesletlcr og Smcltevandsdal. Se Teksten Side 92. Autoriseret Gengivelse efter Maalebordsbladene d 7 Følleslev 1897 og d 6 Bjergsted 1895. 1 : 40.000. 94 store Blokke. Heraf fremgaar, i Forbindelse med den dybe Nedskæring af den brede Dal, at denne har været gennem- strømmet af en særdeles kraftig Flod. store Blokke. Heraf fremgaar, i Forbindelse med den dybe Nedskæring af den brede Dal, at denne har været gennem- strømmet af en særdeles kraftig Flod. store Blokke. Heraf fremgaar, i Forbindelse med den dybe Nedskæring af den brede Dal, at denne har været gennem- strømmet af en særdeles kraftig Flod. Medens denne Del af Bregninge Aadal saaledes er en ud- præget Erosionsdal, bærer Strækningen oven for Dejvad Bro, mod Syd og Sydvest henimod Svebøllegavn, langtfra saini'ne Præg. Den har ikke nogen tilskaaret Sidebegræns- ning, men rummer skiftevis Udbugtninger og Indsnævringer, og dens Moseflade gaar paa de fleste Steder højdemæssigt ret umærkeligt over i det omgivende Grusterræn. Kun i Dalens nordligste Del er der nogen Sideerosion at spore. Aadalens Plads ser derfor ud til væsentlig at have været betinget af en forud tilstedeværende Sænkning, hvis Længde- retning har stemt nær overens med Retningen af Isranden. - Et yngre Afsnit af Bregninge Aadal er dens øverste Del ved Avnsøgaard (se Kortet Fig. 32), hvori Aaens Udspring ligger, og som først blev udskaaret af Smeltevandsafløbet fra Store Aamose, saaledes som det tidligere er omtalt (Side 85). Det ser ud, som om der en Tid, forinden Løbet blev aabnet igennem den bugtede Dal nærmest Svebølle Station, har været et Tilløb til Fremkomsten af Erosionsdal fra Sydøst mod Nordvest igennem Stenrand Plantage. Det er dog væsentlig kun ved Udformningen af en lang, retliniet Erosionsskrænt, at dette Tilløb til en Daldannelse giver sig til Kende. Højlandet mellem Skarresø og Sejerø Bugt med vestligere Forland. Den her givne Fremstilling af de to Israndstadier, Grus- sletternes Oprindelse samt Tilblivelsen og Udformningen af Bregninge Aadals forskellige Dele, har søgt at fremdrage de enkelte Led i det Sammenspil af Kræfter, der har virket til at frembringe dette sammensatte Landskab. Af de to Is- randstadier er den ældste altsaa den, der fortsatte Asnæs- Udby-Svebøllegavn Stadiet videre mod Nordøst. Ved Sal- tofte overskar den Røsnæs- Rugtved Stadiet og blev selv ved Sultenkrog overskaaret af Eskebjærg- Daverup- Bjærg- sted Stadiet. Hvorledes Forløbet har formet sig videre fra Sultenkrog mod Nordøst maa fuldstændig henstaa i det uvisse. Det kan være fristende at sætte den stærke Afsmelt- ning, der maa være foregaaet fra Isranden imellem Sve- Fig. 32. Israndsomraade og Smeltevandsdale. Se Teksten Side 94. Autoriseret Gengivelse efter Maalebordsblad d 6 Bjergsted 1895. 1 : 40.000. ' ~/ ' S.KAl?JI <-0 c;, Fig. 32. Israndsomraade og Smeltevandsdale. Se Teksten Side 94. Autoriseret Gengivelse efter Maalebordsblad d 6 Bjergsted 1895. 1 : 40.000. Fig. 32. Israndsomraade og Smeltevandsdale. Se Teksten Side 94. Autoriseret Gengivelse efter Maalebordsblad d 6 Bjergsted 1895. 1 : 40.000. 96 bøllegavn og Sultenkrog, i Forbindelse med denne Stræk- nings Beliggenhed i Forhold til Nordenden af Lille Aamose, hvor der maaske kan have været en Tilløbsdal for en Flod under Isen. - Eskebjærg- Daverup- Bjærgsted Linien, der overskærer Svebølle- Sultenkrog Linien er - som nævnt - i Modsætning til denne kun i yderst ringe Grad ledsaget af Sandsletter foran Isranden. Den er derimod Udtryk for et kraftigt Isfremstød, der har medført Fremkomsten af de stærkt bakkede Højdedrag Øst for Linien. Dennes mest ka- rakteristiske Træk er da, at den ligger som det skarpt skaarne Skel mellem dette Bakkelandskab og de tilstødende, forud opstaaede Grus- og Sandflader. Bakkeomraadet Nord for Aamose. I Fortsættelse af Højdedraget mellem Føllenslev og Skarresø følger mod Sydøst og videre mod Øst et Bakke- omraade med betydelige Højder og vekslende Landskab. Til- sammen udgør de to Højdedrag den vestlige og sydlige Del af Rammen om Morænefladen med de hatformige Bakker Syd for Lammefjord. Mod Syd begrænses Højdeomraadet Skarresø- Skamstrup- Søndersted skarpt af Store Aamoses lange Dal. Det er kun i underordnet Grad præget af lang- strakte Bakker eller Kæder af Bakkeformationer, men er fortrinsvis opbygget af massive Bakkekupler af meget va- rierende Højde og Omfang og liggende regelløst spredt i et Landskab med smaakuperet Overfladeform. Dette kommer især til Udtryk i Omraadets centrale og østlige Del. - Lige saa afvekslende som Landskabet er Jordbeskaffenheden. Som tidligere omtalt er det i Omraadet mellem Knabstrup og Kongsdal Fladbakker med Plateauler, der især giver Landskabet dets Karakter. Bakkeomraadets Begrænsning mod Nord og mod Syd er særdeles forskelligt formet. Højdeforskellen er størst mod Nord, hvor den lavtliggende Flade mødes med det kupe- rede Bakkedrags stærkt stigende Højder; derimod er Græn- sens Forløb her meget uregelmæssig. Mod Syd har Højde- dragets Grænse et anderledes formet Præg. Dels staar selve Grænsen med et skarpt skaaret Forløb over for Aamose, og 97 dels er der til Grænseomraadet mod Sydvest og mod Syd- øst knyttet iøjnefaldende Systemer af rækkestillede Bakker, der som Israndsbakker betegner det samlede Højdedrags Afslutning paa disse Steder. Den sydvestlige Afdeling af disse Bakker strækker sig fra Nordspidsen af Lille Aarn.ose mod Sydøst i Retning mod Bromølle Kro ved Nordvestspid- sen af Slore Aamose. Den største samlede Gruppe af Bak- ker ligger Sydvest for den Sænkning i Landskabet, ad hvil- ken Afløbet fra Store Aamose, Halleby Aa, er kommet i Stand. Paa den modsatte Side af Halleby Aa optræder der adskillige fremtrædende Spor efter et Israndstadium; der kan saaledes nævnes en lang Bakkekæde Nordvest for Holm- strup. Her og flere andre SLeder i den nærmeste Egn fore- laa der ved den geologiske Kortlægning i 1894 en kraftig Stenbestrøning med mere end meterstore Sten, foruden hvad der laa samlede i store Stengærder og Stenhobe. I Skoven Bredevang NV for Bromølle fandtes en 7,0 X 4,5 X 2- 3 m stor Blok _af rødlig, stribet Granit. - Østpaa fra Bromølle, forbi Brokøb, henimod Skellingsted findes der mindre Bakkerækker langs Kanten af Højlandet. Det samme er Til- fældet længere mod Øst, ved Tømmerup og Syd for Kongs- dal. Bakkeomraadet Nord for Aamose. Der er ikke ret store indbyrdes Højdefor- skelle, men der optræder dog enkelte smaa Bakkerækker og spredtliggende Bakketoppe. Højderne er stigende fra Vest mod Øst, saaledes at den største Del af Omraadet Syd for Holbæk Fjord har sit Overfladeafløb gaaende i vestlig Ret- ning, trods dets nære Naboskab med Holbæk Fjord, Brams- næs Bugt og den dybt nedskaarne Dal langs Elverdamsaa. Holbæk Fjord. Medens det første har en stærkt udjævnet Overfladeform, er Landskabet Syd for Holbæk Fjord tem- melig ujævnt. Der er ikke ret store indbyrdes Højdefor- skelle, men der optræder dog enkelte smaa Bakkerækker og spredtliggende Bakketoppe. Højderne er stigende fra Vest mod Øst, saaledes at den største Del af Omraadet Syd for Holbæk Fjord har sit Overfladeafløb gaaende i vestlig Ret- ning, trods dets nære Naboskab med Holbæk Fjord, Brams- næs Bugt og den dybt nedskaarne Dal langs Elverdamsaa. Mod Sydvest, midt imellem Kvanløse og Tølløse, ændres Landskabets Karakter i usædvanlig høj Grad. · Der optræ- der her Bakkeformationer og mellemliggende Dalslugter med saadanne Højdeforskelle og et saa sammenslynget Forløb, at man ikke træffer noget Magestykke dertil paa Sjælland og næppe nok i nogen anden Del af det danske Bakkeland- skab. Det højeste Punkt, med sin glimrende Udsigt, Mørke mose Bjærg, naar en Højde af 105 m , medens Vandfladen i den nærliggende Maglesø ligger ved 51 m; og i Grøntved Overdrev paa den modsatte Side af Søen, skilt fra denne ved en høj Bakkeryg og en dyb Dal, naar en anden Bakketop 102 m, skønt Afstanden fra Søen kun er 500 m. Bakkesiderne staar gennemgaaende med uhyre stejle Skraa- ninger over for de snævre, krogede Dalfurer. De største Højder ligger i Bakkekompleksets østlige Del; dets Afvan- ding henimod Slutningen af Istiden har navnlig fundet Sted i Retningerne mod Vest, Nordvest og Nord, hvor der flere Steder findes Afløbsfurer, som ogsaa viser, at Afløbet af Smeltevandet fra Omraadet til Dels er foregaaet hen over isdækket Jordoverflade. Sammen med Omraadet Syd for Holbæk Fjord er det naturligt ogsaa at omtale Halvøen paa Nordsiden af denne Fjord: Tuse Næs. Overfladen er her stærkt vekslende. Visse Dele mod Øst omkring Udby er et meget jævnt Moræneland- skab, ligesom Omraadet længst mod Vest. Derimod er Stræk- ningen imellem Uglerup og Hagested fyldt med høje, kup- lede Bakker af vidt forskelligt Omfang og liggende omgivne af et mere smaakuperet Landskab. Bakkeomraadet Nord for Aamose. Endelig er der midt imellem Kongsdal og Søndersted et ret udpræget Randmoræneomraade, bestaaende af et Sy- stem af lave Grusbakker, hovedsagelig orienteret i Retnin- gen Syd- Nord. dels er der til Grænseomraadet mod Sydvest og mod Syd- øst knyttet iøjnefaldende Systemer af rækkestillede Bakker, der som Israndsbakker betegner det samlede Højdedrags Afslutning paa disse Steder. Den sydvestlige Afdeling af disse Bakker strækker sig fra Nordspidsen af Lille Aarn.ose mod Sydøst i Retning mod Bromølle Kro ved Nordvestspid- sen af Slore Aamose. Den største samlede Gruppe af Bak- ker ligger Sydvest for den Sænkning i Landskabet, ad hvil- ken Afløbet fra Store Aamose, Halleby Aa, er kommet i Stand. Paa den modsatte Side af Halleby Aa optræder der adskillige fremtrædende Spor efter et Israndstadium; der kan saaledes nævnes en lang Bakkekæde Nordvest for Holm- strup. Her og flere andre SLeder i den nærmeste Egn fore- laa der ved den geologiske Kortlægning i 1894 en kraftig Stenbestrøning med mere end meterstore Sten, foruden hvad der laa samlede i store Stengærder og Stenhobe. I Skoven Bredevang NV for Bromølle fandtes en 7,0 X 4,5 X 2- 3 m stor Blok _af rødlig, stribet Granit. - Østpaa fra Bromølle, forbi Brokøb, henimod Skellingsted findes der mindre Bakkerækker langs Kanten af Højlandet. Det samme er Til- fældet længere mod Øst, ved Tømmerup og Syd for Kongs- dal. Endelig er der midt imellem Kongsdal og Søndersted et ret udpræget Randmoræneomraade, bestaaende af et Sy- stem af lave Grusbakker, hovedsagelig orienteret i Retnin- gen Syd- Nord. Saaledes som den her optrukne Sydgrænse for det store Højdedrag optræder, ligger den som en naturlig Fortsæt- telse af Israndslinicn Eskebjærg- Bjærgsted. Der er foran Linien paa Strækningen fra Bromølle til noget forbi Brokøb afsat en skraanende Grusslette, der kan anses for at være opstaaet foran Isranden. Hvor stort et Omfang Afsmelt- ningen fra Isranden iøvrigt har haft, kan ikke afgøres, da Linien paa en lang Strækning ligger tæt op ad Grænsen mod den tørvefyldte Aamose. Omraadet mellem Ugerløse og Holbæk Fjord, samt Tuse Næs. Der er en betydelig Forskel i Landskabskaraktererne i Omraaderne Syd for Lammefjord og det østligere, Syd for 7 Omraadet mellem Ugerløse og Holbæk Fjord, samt Tuse Næs. Der er en betydelig Forskel i Landskabskaraktererne i Omraaderne Syd for Lammefjord og det østligere, Syd for Holbæk Fjord. Medens det første har en stærkt udjævnet Overfladeform, er Landskabet Syd for Holbæk Fjord tem- melig ujævnt. Bakkeomraadet Nord for Aamose. Flere af de større Bakker her har saa stejle Sideskraaninger, at de efter al Sandsyn- Fig. 32. Det bakkede Landskab Soficholm- Møi;kemose Bjærg og vestligere. Se Teksten Side 98. Autoriseret Gengivelse efter Maalebordsblad M 3224 Søndersted 1939. 1 : 40.000. Fig. 32. Det bakkede Landskab Soficholm- Møi;kemose Bjærg og vestligere. Se Teksten Side 98. Autoriseret Gengivelse efter Maalebordsblad M 3224 Søndersted 1939. 1 : 40.000. 100 lighed maa op falles som. Erosionsskrænter. Ligeledes er Landskabet langs Kysten Nordøst for Løserup meget ku- peret. lighed maa op falles som. Erosionsskrænter. Ligeledes er Landskabet langs Kysten Nordøst for Løserup meget ku- peret. En Ting, som særlig er karakteristisk for Egnen Sydvest for Holbæk, er Forekomsterne af paleocæn Mergel. Den største af dem, der kendes, er den ved Hanerup, Sydvest for Hegstrup, hvorfra en stor Del af Holbæk Amts Jorder er blevet forsynet med Mergel, og som er nærmere omtalt Side 11. Kortet Fig. 1 viser Udstrækningen af det Landom- raade, hvor man med det nuværende Kendskab ved, at der i denne Egn optræder Flager af paleocæn Mergel. Baade efter hvad der vides om saadanne Lags Forekomst paa pri- mært Lejested og om Indlandsisens Bevægelsesretninger i denne Del af Sjælland i sidste Afsnit af Istiden, er der nær- mest Grund til at formode, at Flagerne her stammer fra Dybder i den sydligste Del af Isefjord Syd for Orø. Selv om Landskabet omkring Holbæk Fjord og sydligere ikke er rigt paa Linier, der viser Forløbet af Indlandsisens Bortsmeltning fra Egnen, kan der dog uddrages visse Træk deraf i Forbindelse med Jordbeskaffenheden. Det kan saa- ledes anses for sikkert, at den lange Sandbakke imellem Trønninge og Bullerup, Trønninge Aas, viser en Strømret- ning ØSØ- VNV. Hermed stemmer Retningen af Kalveaa og de Smeltevandslag af Sand, der ledsager Aaløbet. Læn- gere østpaa, Øst for Taastrup, Vest for Store Grandløse, ved Vallestrup og Øst for Asmindrup, er der smaa Bakkeræk- ker, der kan afspejle en Israndstilling, svarende til en Vest- Øst gaaende Tilbagerykning af Isranden inden for dette Af- snit af Omraadet. I det sydvestlige Afsnit er der ogsaa en saadan og mere sammenhængende Serie af Bakker. Den strækker sig fra Egnen omkring Vinstrup over Kvanløse, Sdr. Jærnløse og Lille Knabstrup. Lidt bagved denne Linie, Nord for Igelsø og Mogenstrup til Borup, kan man drage en Linie, der marke- rer Mundingerne af de nedskaarne Afløbsrender for Smelte- vandet fra det store Bakkeomraade Sydøst derfor. Bakkeomraadet Nord for Aamose. Isrands- linien kan anses for at have haft sin videre Fortsættelse i Forbindelse med den tidligere (Side 84) omtalte Israndslinie forbi Bonderup (se Israndskortet). Længst mod Nord ved Holbæk Fjord er det meget sand- synligt, at der som en Krans omkring Fjordbassinet har ligget en afsmeltende Isrand. Ved 'Fjordens Sydside er der dog kun et Par Steder, der kan antyde Liniens nærmere Forløb, nemlig den langstrakte Sandbakke »Bjærgene« Syd- vest for Allerup samt en lignende Sandbakke ved Tuse. - Paa Tuse Næs er Linien betegnet af en ret fremtrædende Række Bakker, der strækker sig fra Omegnen af Hagested østpaa henimod Udby og langs Østkysten Nordøst for Løse- rup til Næssets Nordøstspids. De stejle Bakkeskrænter Syd og Vest for Uglerup og vestligere skyldes sikkerlig Smelte- vandserosion i Tilslutning til en Israndstilling. Bakkeomraadet Nord for Aamose. Dens Be- tydning som Grænselil1ie fremgaar af, at den som umiddel- bar Fortsættelse har en udpræget Randmorænelinie, der fra ;,)' ' ·, i' ---- -1"'\:-•~·· "\" " ~ ,, , .. -~-jr•-· ... -- . . -- ·~~. 'f:' \- - ' :< \ ~--·{\J··:? ~ "·-·1 ~2\;t_,- ~ . \ ! N, . --r_-c: ~- · ,;'•~~\,~ "\ I 1, / . , ilt3Itlts ... ··~ t ' , " . L ·7 S'··{.-·l: -~~ .e. ~,.V·. ,r -~ -- ·· ~- r·, ~-»--1~ · •.· ~--s;_~-[:j-- --.. 116Tj· 'ns+ 1';;-: . -?"~ - _ _____...._,_ " ,' -. ~ . . . ' ~:;::,- , '. ,- -. .- ·- ' ',, '\. ~ . . ·-1/,---- . , • ni " I,\ ·:, . . ' fe a. ,,&.,m • ·"' 7 · ""- ..._,, ·-.• , , '-·i<? ·---. ~ " ~,-_ . ' _,, ,:Ji , ) ' ,,, .. - . \l/1 -, J . ._, ' ' \ ' ' C • - • ,,~-""' • • 1 Je,,'!-/,)...,, ,•-:r -" ••1:1 ,\ . '"•• li •- o 7 -' ( ~- •.:fi'--,.-_J • ,J~ ,--_- ~_-•_;_•_l·-l,::':...:11_ ·_1- < -·, -·r~;~f,I, 7 ·-'---·!... _:•¼ '.t' __ • t~.,~'": :~~-~l·:o ,.·, :"t"Pc·1c: • -½)-- - • •.j n· i )_)·i·-. . '. . . ,-, '· "r:'>-"2:;,; ➔--~:-~~ -- ~ ',: ~!~~-: . \'f ',\·· , : ;r- ·., . '.\'-' ~~/; 1:) ~- ·,1 ,:- \.~ \ ·.::: i-~ ,;".';~~li,,fu1__--1-_ -.....,.,.1,.,.,~"""f.~- · I" _ _,I ··,-,__ Fig. 3-1. Glaciale Landskabsformer paa Tuse Næs. Se Teksten Side 98. Autoriseret Gengivelse efter Maalebordsbladene g 8 !lagested 1897 og h 8 Tudse Næs 1896. 1 : 40.000. ~:- '18:J. _,,- 0 ,-,. 0 ,-,. _ Fig. 3-1. Glaciale Landskabsformer paa Tuse Næs. Se Teksten Side 98. Autoriseret Gengivelse efter Maalebordsbladene g 8 !lagested 1897 og h 8 Tudse Næs 1896. 1 : 40.000. 102 Borup strækker sig ca. 3 km videre mod Sydvest og Syd, et Stykke Øst om Søndersted. Denne Linie markerer sig af- gjort som en Randaflejring som Følge af sin Opbygning af et stort Antal smaa Grusbakker liggende i hele Strøgets Længderetning. Forholdene viser saaledes med tilstrækkelig Tydelighed, at den afsmeltende Isrand helt har omkranset det store Bakkekompleks mod Nord og mod Vest. De bueformige Højdedrag i Odsherred mellem Dragsholm og Højby. Saaledes som Omridset af Odsherred fremtraadte fra Na- turens Haand, forinden de forskellige Inddæmninger fandt Sted i forrige Aarhundrede, var det en højst uregelmæssig Landsdel. Omridsets uregelmæssige og fligede Præg var dog endnu mere fremtrædende i Stenalderen, da ikke blot Ise- fjords dybe Indskæringer, Lammefjord med Svinninge Vejle og Sidinge Fjord henlaa som Fjorde, men der ogsaa ved Vestsiden, fra Sejerø Bugt var dybe Havindskæringer ind i det nuværende Landomraade. Det, der nu fremtræder som Ods- herreds Højland Syd for Linien Højby- Nykøbing, udgjorde dengang hele Landomraadet. De fligede Højdedrag var da næsten overalt direkte begrænset af Hav uden noget helt lavtliggende, fladt Forland som Overgangsled. 103 De to medfølgende Overfladekort, Højdekortet og det geo- logiske Kort, viser væsentlige Træk af dette Højlands Ka- rakter. Bakkedragenes øverste Kam ligger som tre toppede Buestykker, der omslutter Indskæringer fra Øst: Lamme- fjord, Sidinge Fjord og Nykøbing Bugt. Buen omkring Lammefjord rummer de største Højder. Højest, 121 m, naar Vejrhøj, ved Buens Sydvestende; men Bakkekammen derfra mod Nordøst til Høve Stræde og vi- dere naar flere Steder 80---90 m og derover, og Højdepunk- terne rummer som Følge af de lave Omgivelser en vidt- strakt Udsigt, naar det er klart Vejr. Fra Vejrhøj ses da Roskilde Domkirkespir og ind i Østjylland. Fra Esterhøj ved Høve har man en særdeles instruktiv Udsigt: mod Syd ud over Indersiden af Bakkebuen, den udtørrede Lamme- fjord og den tilgrænsende Moræneflade med de »hatformige Bakker«, til Højdedragene Syd for Jærnbanen Holbæk- Jy- derup med Toppunktet, Knøsen (99 m) , ved Skams trup. Mod Nordøst og Nord ses ud over Ydersiden af den mellemste og den nordlige Bakkebue, med det foranliggende, flade Land ned imod Sejerø Bugt og Sjællands Odde i Baggrunden. Ved de to nordlige Bakkebuer er Højdepunkterne ikke saa fremtrædende, de naar kun undtagelsesvis over 50 m. Ved de to nordlige Bakkebuer er Højdepunkterne ikke saa fremtrædende, de naar kun undtagelsesvis over 50 m. Den Side af Højlandsbuerne, der vender mod Isefjord og dens Sidefjorde, afviger væsentlig fra den Side, der vender mod Sejerø Bugt. Den første bestaar aldeles overvejende af Moræneler, medens der i Retning mod Sejerø Bugt findes betydelige Strækninger med Sand og Grus. Morænelersom- raadets Overfladeforhold er noget forskellige fra Egn til Egn. Paa Strækningen fra Vejrhøj til et Stykke Øst for Høve, hvor de største Terrænhøjder findes, er Landskabs- formen stærkt præget deraf, med langstrakte Bakker og Bakkeskraaninger i Højdedragets Længderetning. De bueformige Højdedrag i Odsherred mellem Dragsholm og Højby. I den stør- ste Del af det øvrige Omraade har Overfladen uregelmæs- sigt kuperede, men jævnt bølgende Former og med enkelte mere fremtrædende Bakker og Bakkegrupper. Et mindre Parti i Kystlandskabet Syd for ykøbing Bugt har dog smaakuperet Overflade, med lydelig Orientering i samme Retning som Kysten. 104 Fig. 35. Randmoræne og Israndsbakker. Se Teksten Side 105 og Side 113. Autoriseret Gengivelse efter Maalebordsblad g 10 Højby 1897. 1 : 40.000. Fig. 35. Randmoræne og Israndsbakker. Se Teksten Side 105 og Side 113. Autoriseret Gengivelse efter Maalebordsblad g 10 Højby 1897. 1 : 40.000. 10 105 Aflejringerne af Sand og Grus er - med visse karakte- ristiske Afvigelser - især knyttet til den Del af Bakke- buerne, der vender i Retning mod Sejerø Bugt. Materialet bestaar for en væsentlig Del og i et enkelt Omraade ude- lukkende af Grus. Dette sidste er Tilfældet paa Strækningen langs Jærnbanen mellem Højby og Svinninge (Fig. 35). Her findes et Strøg med et stort Antal parallelt liggende, lang- strakte Bakker, hvis Hovedretning er Nord- Syd, men dog med mange Afvigelser. Paa nogle Steder er enkelte Bakker mere fremtrædende, indtil 15 m højere end de øvrige; andre Steder ligger der flere Rækker af omtrent ens høje Bakker Side om Side og slyngede mellem hverandre paa den mest uregelmæssige Maade. Indholdet af Bakkerne kan veksle mellem Grus, Sand og andre Jordlag. Lagene kan være opretstaaende eller ligge skraat eller vandret. Forholdene viser utvetydigt, at saa- ledes som dette Grusstrøg er bygget og ligger placeret mel- lem et højere liggende Morænelerslandskab og en lavtlig- gende Flade, er det at opfatte som et System af Randmo- rænebakker. Det er derved stærkt vejledende for Afgørelsen af, hvordan ikke blot Grusaflejringerne langs de to andre Bakkebuer, men selve de bueformige Højdedrag skal op- fattes, nemlig som et mægtigt Kompleks af Is rands- dan n c 1 ser, opslaact i Forbindelse med en tungeformig lsrand. Nordøst for Hønsinge er der en udpræget Randmoræne- vold, dels Morænegrus med mange Sten, dels finere lagdelt Materiale. Fra Hønsinge strækker der sig mod Syd igen- nem Jyderup Skov et Drag af Grusbakker, der træder for- holdsvis stærkt frem i Terrænet som Følge af, at det bag- ved liggende Grundm.orænelandskab kun naar lave Højder. Paa Strækningen mellem Hønsinge og Ravnsbjærg Bakker ved Nordenden af Jyderup Skov kendetegner de langstrakte Bakker tydelig Grusstrøget som et Randmorænestrøg. De bueformige Højdedrag i Odsherred mellem Dragsholm og Højby. Syd for Ravnsbjærg Bakker overskæres Bakkestrøget af en 300 m bred Dal, frembragt ved glacial Erosion paa tværs af Rand- morænestrøget. Det udskyllede Materiale har nordligst i Fig. 36. Israndslandskab i Odsherred. Se Teksten Side 107- 08. Autoriseret Gengivelse efter Maalebordsblad f 9 Hønsinge 1897. 1 : 40.000. ... 0 cr, Fig. 36. Israndslandskab i Odsherred. Se Teksten Side 107- 08. Autoriseret Gengivelse efter Maalebordsblad f 9 Hønsinge 1897. 1 : 40.000. 107 J yderup Skov frembragt en jævnere, skraanende Overflade end det nordlige Bakkelands. J yderup Skov frembragt en jævnere, skraanende Overflade end det nordlige Bakkelands. Fra Ravnsbjærg ved Sydøsthjørnet af Jyderup Skov strækker der sig videre mod Sydøst til Lynghuse et smalt Grusbælte (Fig. 36). Strækningen her er karakteriseret ved en meget stor Rigdom paa Sten. Ved Opdyrkningen er Markoverfladen for Størstedelen ryddet for Sten, som der- 40 1n over Ha:oei, V SV ONO Mcuile.stolt fo-r Hojckr 10 20 30 40 50 60 70 80 90 100 m 4 5 G 7 9 JOOO Tfl fo r Læ;ngd.n1· Fig. 37. Skematisk Snit ved Lynghuse. a ~roræncler, b ~forænegrus, c lagdelt Sand og Grus, d stenfrit Ler, c Ton. Efter D. G. U. I. R. Nr. 8. Fig. 37. Skematisk Snit ved Lynghuse. a ~roræncler, b ~forænegrus, c lagdelt Sand og Grus, d stenfrit Ler, c Ton. Efter D. G. U. I. R. Nr. 8. efter er samlet i de talrige Stengærder m ed Blokke paa flere Kubikfods Størrelse; men paa adskillige Bakkeloppe og Skraaninger har der indtil nyeste Tid kunnet ses en tæt Stenpakning i Jordskorpen. Gruset i Bakkerne er nogle Ste- der lagdelt og godt rullet, men er paa andre Steder ganske uden Sortering og Lagdeling. En Grusgrav, der viste begge Aflejringsmaader, var i Vinteren 1898- 99 i Anledning af Odsherredbanens Bygning aabnet i en Bakkekam 300 m Syd for Ravnsholm Bakke. Profilet strakte sig fra Bakkens vestlige Del omtrent til dens Top og viste en næsten jævn Overgang fra Moræneler, nederst ved Bakkekammens Vest- rand, igennem Morænegrus med 1æt Stenpakning til lagdelt Grus og Sand, der ved Bakkens Top laa i regelmæssige, uforstyrrede Lag med Hældning mod Øst. efter er samlet i de talrige Stengærder m ed Blokke paa flere Kubikfods Størrelse; men paa adskillige Bakkeloppe og Skraaninger har der indtil nyeste Tid kunnet ses en tæt Stenpakning i Jordskorpen. De bueformige Højdedrag i Odsherred mellem Dragsholm og Højby. Gruset i Bakkerne er nogle Ste- der lagdelt og godt rullet, men er paa andre Steder ganske uden Sortering og Lagdeling. En Grusgrav, der viste begge Aflejringsmaader, var i Vinteren 1898- 99 i Anledning af Odsherredbanens Bygning aabnet i en Bakkekam 300 m Syd for Ravnsholm Bakke. Profilet strakte sig fra Bakkens vestlige Del omtrent til dens Top og viste en næsten jævn Overgang fra Moræneler, nederst ved Bakkekammens Vest- rand, igennem Morænegrus med 1æt Stenpakning til lagdelt Grus og Sand, der ved Bakkens Top laa i regelmæssige, uforstyrrede Lag med Hældning mod Øst. Omkring Lynghuse, Øst for denne Bakke, er der et Om- raade med stenfrit Ler. Nordøst derfor er der igen en Bakkerække, som ind imod Lerbassinet indeholder Sand, medens der paa den modsatte Side, mod Nord og Nordøst, 108 paa Bakkeskraaningerne kan findes en stærk Stenbestrø- ning, og man ved Bakkefoden atter kommer over i Mo- rænelersterræn. Et Snit, som gengiver disse Forhold, ses i Fig. 37. Det stenfri Ler strækker sig mod Syd helt frem til en Sydskraaning, men trods Overfladens Hældning mod Syd har Lerlagene, der her hviler paa Sand, Hældning mod Nord. Det fremgaar deraf, at Leromraadet og de til- stødende Sandbakker ved Tiden for deres Opstaaen har lig- get omgivet af et Isdække ; paa dettes Overflade har den Sø ligget, hvis Bund det stenfri Leromraade nu viser. paa Bakkeskraaningerne kan findes en stærk Stenbestrø- ning, og man ved Bakkefoden atter kommer over i Mo- rænelersterræn. Et Snit, som gengiver disse Forhold, ses i Fig. 37. Det stenfri Ler strækker sig mod Syd helt frem til en Sydskraaning, men trods Overfladens Hældning mod Syd har Lerlagene, der her hviler paa Sand, Hældning mod Nord. Det fremgaar deraf, at Leromraadet og de til- stødende Sandbakker ved Tiden for deres Opstaaen har lig- get omgivet af et Isdække ; paa dettes Overflade har den Sø ligget, hvis Bund det stenfri Leromraade nu viser. Det smalle Grusslrøg Ravnsbjærg- Lynghuse ledsages af Hækker af langstrakte, tørvefyldte Fordybninger, der har Længderetning fælles med Grusstrøget. Landskabet adskil- ler sig derved paa karakteristisk Maade fra det omgivende Morænelerslandskab. Grusbakkerne omkring Lynghuse naar Højder paa indtil 84 m , medens det omgivende Land- skab ligger 20- 30 m lavere. I Grusstrøgels Retning, paa den ca. 4 km lange Strækning fra Lynghuse Lil Sydøsthjør- net af Jyderup Skov, viser de største Terrænhøjder et Fald paa ca. 40 m. De bueformige Højdedrag i Odsherred mellem Dragsholm og Højby. De_r er al god Grund Lil at formode, al der ved Tiden for Indlandsisens Bortsmeltning fra Omraadet her fandtes en SØ-NV gaaende Sænkning i Isdækket fra Lynghuse i Retning mod Sejerø Bugt. De skraanende Overflader af Istungerne fra Sidinge Fjord og fra Lammefjord har mød- tes langs denne Linie, og Afstrømningen af Vand fra Smelt- ningen er derfor her blevet stærkt forøget. Dette har kunnet give sig Udslag i Form af Erosionsrender under Isen. De langstrakte Dalfurer langs Grusbæltet kan være fremkom- met paa den Maade. Pindals Mose, der ligger i Forlængelse af Grusstrøget, har rimeligvis faaet sin Længderetning for- met ved en saadan Erosion. Men selve de højtliggende Grus- volde i den nærmere Omegn af Lynghuse er at betragte som en »interlobat Moræne«, liggende indeklemt mellem Istunger fra begge Sider. I den sydligste og mest dominerende af de tre Højde- dragsbuer, den der omkranser Lammefjord, ligger Grus- partierne mere spredt end ved de andre. Paa nogle Steder 109 findes Gruset aflejret paa Bakkerne, men det almindeligste er, at Bakkerne indeholder Moræneler, medens de lagdelte Istidslag findes aflejret i de mellemliggende Lavninger og da for en væsentlig Del bestaar af Sand. Hvadenten Bak- kerne indeholder Moræneler eller lagdelte Istidslag, er de næsten alle langstrakte af Form og ligger med deres længste Led i Bakkebuens Længderetning. De viser derved paa slaaende Maade Aarsagsforbindelsen med Lammefjords- dalen, hvorfra Istungen har presset Bakkerækkerne op i den buede Bakkekæde. Det er tidligere nævnt (Side 61), at Lammefjord geologisk sel ikke udgør nogen Skillelinie i det nordvestsjællandske Landskab. Sammen med de tilgrænsende, lavtliggende syd- ligere Omraader har det tværtimod været den »Central- depression«, uden om hvilken en mægtig Istunge har afsat og opstablet ikke blot det store Højdedrag Nord om Lamme- fjord, men ogsaa dem Nordvest for Jyderup og Nord for Store Aamose. Disse tre store Højdedrag maa derfor op- faltes som et sammenhørende Hele trods det store adskil- lende Lavland. Den Jsrand, som. har omslutlet den store Bue af Bakkedrag, synes at mangle et Led, som skulde for- binde Odsherredbuen ved Vejrhøj med Daverup- Eske- bjærg Buen ved Eskebjærg. En saadan Forbindelse maa jo have eksisteret, og der er da ogsaa Forhold i Egnen, der antyder dens Plads. Langs Kysten Nordvest for Vejrhøj til Ordrup Næs stræk- ker der sig et smalt Højdedrag, som af en lav Sandflade adskilles fra den store Vejrhøjbue. De bueformige Højdedrag i Odsherred mellem Dragsholm og Højby. Det nævnte lille Højde- drag, der mest bestaar af Sand med pletvis opragende Mo- rænelersbakker, antyder, at her har Vejrhøjbuens Isrand haft sin Fortsættelse. Den modsatte Ende af den manglende Israndslinie er der Grund til at søge Nordøst for Saltbæk Vig. Der strækker sig her, fra Eskebjærg mod Nordvest til Nørrehoved ved Alleshave, et svagt kuperet Morænelandskab, som særdeles vel kan betegne Fortsættelsen af den mere ud- prægede Isrand Daverup-Eskebjærg. Det falder endvidere nalurligt at anse Nekselø som et Forbindelsesled mellem den sandsynlige Linies Punkter ved Ordrup Næs og Nørre- 110 110 hoved. Nekseløs kuperede Overflade og anselige Højde i Forhold til Øens Størrelse kan stemme godt med Formod- ningen om, at den tilhører et Israndsomraade. hoved. Nekseløs kuperede Overflade og anselige Højde i Forhold til Øens Størrelse kan stemme godt med Formod- ningen om, at den tilhører et Israndsomraade. At Isranden har haft en saadan Udposning i Omraadet omkring Nekselø falder ogsaa godt i Traad med Landska- bets Udformning mellem Vejrhøj og Eskebjærg- Føllens- lev. I Modsætning til de betydelige Højder i Vejrhøjbuen og Syd for Føllenslev naar Landskabet · mellem Nekselø Bugt og Vestenden af Svinninge Vejle kun undtagelsesvis op til 30 Meters Højde. Det svarer da højdemæssigt ganske til Odsherreds Kystland langs Isefjord. Men medens Isdæk- kets Bevægelse her er blevet standset, i Sammenhæng med at den store Materialeophobning i Odsherreds Bakke- huer har fundet Sted, har Banen for Isen derimod Syd for Vejrhøj været fri, fordi en tilsvarende Ophobning ikke har været til Stede. I det lave Slip mellem Vejrhøj og Føllens- lev er der kun efterladt kendelige Spor af en saadan Ophob- i1ing ved Israndsbuernes Sidegrene: Ordrup Næs, Nekselø og Alleshave. I Stedet for at være sat op i Bakkedrag er Morænematerialet blevet bredt ud i Fladen NV for den 8 km brede Aabning mellem Vejrhøj og Føllenslev. Efter at Isranden fra denne formodede Nekselølinie var rykket et Stykke tilbage over det lave Landskab Sydøst for Nekselø Bugt, opstod der rimeligvis et Afsmeltningsstadium med en stationær Isrand paa Halvøen Nord for Svinninge Vejle, gaaende fra Dragsmølle, forbi Hørve og Vejleby, Nord om Vallekilde og i en Bue Vest om Svinninge Vejle frem til det sydlige Højland i Omegnen af Føllenslev. Saavel Hørve- Vallekilde Halvøens Terrænforhold som Overfladeafløbene, der næsten alle gaar fra Sydøst mod Nordvest, peger tyde- ligt i Retning af, at en stagnerende Isrand har haft sin Plads her. De bueformige Højdedrag i Odsherred mellem Dragsholm og Højby. Det blev nævnt før, at det lille Bakkedrag Syd for Ordrup Næs er adskilt fra den store Vejrhøjbue ved en lav Sand- flade. Denne Sandflade og dens Fortsættelse mod Nordøst langs Sejerø Bugt er et vigtigt Led i det geologiske Billede af selve Højlandsbuen. Dette jævne, skraanende Sandbælte er nemlig opstaaet ved, at der fra den stagnerende Isrand ud- 111 slæmmedes Sand og Ler, hvoraf Sandet afsattes nærmest foran Isranden, medens det mere finkornede Slam førtes længere bort. Aflejringerne af Sand, opstaaet paa isfri Mark, kan ogsaa følges et Stykke op i Højlandet, hvor Dalene / < \ /,:~:t~~-~··'~~, - ' 11tu ,Mi.,, : llk~ ;C,.p ,'-.:,1;1.> ~ 1/ ' ' ~·:\ ''i o-;.1' \ '\ r ·-7 • BOPLADS \\ ~ \\ \ : , 1ol~Jcl,ul ' \ J Fig. 38. !{ort over Hedeslette Øst for Ordrup Næs. Efter C. J. BEc1rn11 (Aarb. f. no1·d. Oldkyndigherl og Hist. 1939). Fig. 38. !{ort over Hedeslette Øst for Ordrup Næs. Efter C. J. BEc1rn11 (Aarb. f. no1·d. Oldkyndigherl og Hist. 1939). indeholder Sand, medens de omgivende Bakker indeholder grovere Materiale eller Moræneler. Bortset fra en enkelt Afbrydelse Nordvest for Høve kan den smalle, ekstramargi- nale, d. v. s. uden for Isranden afsatte, Hedeslette foran Vejrhøjbuen følges et Stykke videre mod Nordøst, hen foran Israndsbuen omkring Sidinge Fjord. Derimod er der foran Morænelinien Højby- Svinninge kun svage Spor af en saa- dan Sandflade til Syne; den ligger muligvis skjult under de yngre Jordlag i Trundholm. indeholder Sand, medens de omgivende Bakker indeholder grovere Materiale eller Moræneler. Bortset fra en enkelt Afbrydelse Nordvest for Høve kan den smalle, ekstramargi- nale, d. v. s. uden for Isranden afsatte, Hedeslette foran Vejrhøjbuen følges et Stykke videre mod Nordøst, hen foran Israndsbuen omkring Sidinge Fjord. Derimod er der foran Morænelinien Højby- Svinninge kun svage Spor af en saa- dan Sandflade til Syne; den ligger muligvis skjult under de yngre Jordlag i Trundholm. 112 Fremkomsten af det samlede bueformige System af Højde- drag Vest for Isefjord og dens vestlige Sidegrænse kan man forestille sig paa omtrent følgende Maade. Paa et bestemt Tidspunkt under Indlandsisens Forsvinden fra Landet laa der i Isefjordsbassinet et forholdsvis mægtigt Isdække med Tilførsel fra Øst og med Bevægelse frem imod foranliggende Højder. Efterhaanden som Isens Mægtighed formindskedes, blev den relative Forskel mellem Mægtigheden af Isdækket i Bassinets dybeste Dele og Isen over Højdepartierne større og større. De bueformige Højdedrag i Odsherred mellem Dragsholm og Højby. Medens Isens Bevægelse og Erosionsevne kunde holdes vedlige paa de Steder, hvor dens Mægtighed var størst, maatte den aftage, hvor Istykkelsen svandt ind. Isens Moræneindhold afsattes derfor dels langs Randen af den stagnerende Is som Randmorænebakker, dels bagved Ran- den i Form af et bakket Morænelandskab. Foran Isranden aflejredes det ved Smeltningen udskyllede og sorterede Ma- teriale. I de fremskudte Istungers centrale Dele var Isens Bevægelseshastighed og Erosionsevne stor nok til at hindre, at der foregik nogen Aflejring af væsentlig Betydning. Paa ethvert Sted, hvor der var tilslrækkelige Betingelser for, at Isen kunde skyde sig tungeformigt frem, opstod derved Muligheden for Fremkomsten af følgende fire Zoner: en Grus- eller Sandslette foran den stagnerende Isrand, en Randzone med en eller flere Bakkerækker, et uregelmæssigt Morænelandskab bagved Isranden og en centralt liggende Fjord eller lav Moræneflade. en Grus- eller Sandslette foran den stagnerende Isrand, en Randzone med en eller flere Bakkerækker, et uregelmæssigt Morænelandskab bagved Isranden og en centralt liggende Fjord eller lav Moræneflade. Saaledes som Odsherreds store Bakkebuer og de dermed sammenhørende Højdedrag Nord 9g Sydøst for Skarresø fremtræder, kan de anses for at være opstaaet som Følge af et kraftigt Fremstød af Indlandsisen. Efter at Isdækket var begyndt at smelte bort fra Omraadet, fremkom der dog enkelte Steder mindre fremtrædende Afsmeltningszoner langs en trinvis stagnerende Isrand. En saadan Zone gaaende forbi Hørve og Vallekilde er allerede lidligere omtalt. Forinden Isen helt forsvandt fra Odsherred, ser det ud, som om Is- randen yderligere har gjort et kortvarigt Ophold under Til- bagerykningen. En Antydning i den Retning viser Terrænet 113 fra Kysten Syd for Annebjærg Gaard, forbi Brændt og videre mod Sydøst i Retning mod Kongsøre Næbbe. fra Kysten Syd for Annebjærg Gaard, forbi Brændt og videre mod Sydøst i Retning mod Kongsøre Næbbe. Øst for Randmorænestrøget Sydøst for Svinninge og hen- imod Brændt er der et yderst svagt kuperet Morænelers- plateau, der strækker sig videre mod Nord forbi Asmind- rup. Dette jævne Plateau begrænses ved Brændt skarpt af et stærkt bakket Landskab med dominerende, ovale Moræne- lersbakker og talrige mellemliggende, dybe Mosehuller. Bakkestrøget strækker sig fra Brændt mod NNØ gennem Annebjærg Skov til Annebjærg Gaard med en Længde af 4 km og en Bredde af 200-600 m. Højdeforskellen mellem dette og det mod Øst tilgrænsende, jævnere Landskab er 20~30 m. Den fremtrædende Bakkerække strækker sig kun lidt Syd for Brændt. De bueformige Højdedrag i Odsherred mellem Dragsholm og Højby. Videre mod Syd strækker sig et Grus- omraade, der NØ for Eskilstrup er formet som et NNV- SSØ rækkeordnet Landskab, der dog kan anses for at udgøre Fortsættelsen af Randmorænestrøget Højby- Svinninge. Fortsættelsen af Bakkestrøget er derimod snarest at søge i mere lige østlig Retning fra Brændt henimod Kysten imel- lem Strandhuse og Bøsserup. Landskabsformen herfra og mod Syd langs Kysten er smaabakket, med Bakker og Dale der fremtræder med udpræget Længderetning i samme Ret- ning som Kysten og med spredtliggende, fremtrædende ovale Bakker langs hele Strøget til Kongsøre Skov. I Sydsiden af en saadan Bakke, Kildebakker, østligst i Kongsøre Skov, er der Gruslag, liggende som en Kuppel; det er naturligst at tyde Bakken som en Kame afsat i Tilslutning til en Isrand. H d d d li d li d S d i Hvad der yderligere sandsynliggør en saadan Standsning paa Halvøen Øst for Sidinge Fjord, er, at en Opholdslinie her danner en naturlig Fortsættelse af den tidligere (Side 102) omtalte Israndzone igennem Tuse Næs og Syd for Holbæk Fjord. Odsherreds nordligste Del. Istidslagene træder i det nordligste Odsherred frem li mange spredt liggende Dele, der nu af Saltvandsalluviets hævede Havbund er sammenknyttet til et smalt Hele, men med en Længdeudstrækning paa over 30 km, fra Rørvig til Sjællands Oddes yderste Spids, Gniben. 114 Dets største Omraade fra Nykøbing til Ebbeløkke rum- mer et meget forskelligartet Landskab. I sin østlige Del mellem Nykøbing og Stenstrup er det temmelig stærkt ku- peret, men uden fremtrædende Højder undtagen i det store Grus- og Sandparti omkring Troldebjærg imellem Højby og Nykøbing, og ved Sekshøje Nordvest for Højby, som begge Steder naar Højder over 50 m. Et større samlet Højdc- omraade strækker sig fra Sonnerup til Ebbeløkke. Det der i~ær 'karakteriserer dette Bakkedrag, er ikke dels absolute Højde, der ligesom de to førnævnte kun naar lidt over 50 m; det er derimod dets Fremtræden i Forhold til Omgivelserne, især mod Syd; her er dets Grænse en skarpt skaaret, brat Skraaning, der som en 8 km lang Bue med en indtil 40 m stor Højde fra Fod til Bakkekanten skiller Højdedraget fra dets lavtliggende Nabolandskab. Saaledes som denne fast sammenknyttede Bakkekæde ligger, er det vanskeligt at se, i hvilket oprindelsesmæssigt Forhold den staar til sine Om- givelser. Det kan med Sikkerhed formodes, at det Isdække, hvorfra den stammer, maa være kommet til Stedet fra Nordsiden, men hvordan selve den imponerende Bakke- skraaning er opstaaet, maa henstaa i det uvisse. Højlandet paa Sjællands Odde imellem Overby Lyng og Yderby Lyng er et jævnt kuperet Morænelandskab uden Højder over 29 m, men dog med et temmelig stort Antal mosefyldte Fordybninger. De mange skarpt skaarne Strand- skrænter ved saavel Sydsiden som Nordsiden viser, at Land- omraadet i sin Tid maa have strakt sig videre i begge Ret- ninger. Gniben ved Oddens yderste Spids er en nogle Hundrede Meter lang Morænelersbakke. Den maa i Stenalderen have haft en langt større Udstrækning end nu (se Side 169). Det isolerede Bakkeparti omkring Klint, Nord for Højby, er især karakteriseret ved sine N-S gaaende Bakkerygge, hvis Højder stiger mod Nord og ender ved Nordkysten med en indtil 35 m høj Skrænt, Klintebjærg. Her findes mæg- tige Lag af Grus med store Sten og en stor Mængde Blokke af paleocæn Kalk og Saltholmskalk (Side 13). 115 De her nævnte Strækninger fra Højby til Gniben ligger det nærmest at anse for opstaaet i nær Sammenhæng med de store Bakkebuer imellem Højby og Vejrhøj. Odsherreds nordligste Del. At bestemme Pladsen for Israndens Beliggenhed lader sig ikke gøre, man maa nøjes med den Antydning, som Forløbet af Sjællands Odde giver. Det er derefter sandsynligt, at den stagnerende Isrand paa langs med den lange Odde har været den venstre Grænse for en stor Istunge, som er kommet hertil fra Øst sammen med Isen, der frembragte Højby-Vejrhøj Buerne. Til et lidt senere Stadium af samme Isudbredelse hører Fremkomsten af Højlandet Nord og Syd for Rørvig. Syd- ligst i Omraadet omkring Nakke er Landskabet temmelig stærkt kuperet, medens den nordlige Del har en jævnere Overflade. Der er derfor nogen Sandsynlighed for, at Nakke- land har været krydset fra Sydvest mod Nordøst af en Smellningsgrænse imellem Dele af Isen, der er smeltet bort med ulige stor Hastighed. Det er nærliggende at samstille denne Grænse med den formodede Afsmeltningsgrænse Vest for Nykøbing Bugt, hvis da ikke Isranden ved Annebjærg Gaard har bøjet af mod Nordvest og fortsat i Retning mod Klintebjærg. Sejerø. Den Del af Sejerø, der stammer fra Istiden, ligger som et splittet System af Bakker, der rager op over den om- givende Flade af hævet Saltvandsalluvium. Højlandet ligger i det store og hele som to Strøg, knyttet til hver sin Kystside. Under Landets dybes te Nedsænkning i Stenalderen bestod Omraadet af 25 Smaaøer og Holme. En Del af Holmene danner temmelig høje Bakker, liggende i Række med andre Bakker, der som Toppe rager op over det omgivende Flad- land. A<;lskillige af disse opragende Smaabakker har en ud- præget Længdeudstrækning Nordvest- Sydøst, samme Ret- ning som hele Øens Længderetning. Det hele Kompleks gør derved afgjort Indtryk af at være et System af »drumlins«, ogsaa kaldet Radialmoræner, cl. v. s. Bakker, der er afsat paa langs med Isens Bevægelsesretning. Denne har da været Sydøst- Nordvest, dengang Morænebakkerne her blev afsat. 116 stemmelse mellem Indholdet af Ledeblokke paa Sejerø og ved Nordkysten af Røsnæs, saaledes at man deraf maa drage den Slutning, at det er en og samme Isstrøm, der har afsat de Jordlag, hvorfra Stenene stammer. Sammen- sætningen af Stenindholdet viser, at denne Isstrøm er kom- met fra Sydøst, nær Øst om det sydøstlige Sverige. Røsnæs' Bakkedrag er opstaaet som en Sidemoræne ved denne Is- 116 Dette Resultat falder godt i Traad med, hvad man ad anden Vej kan skønne har været Tilfældet. Det er Øens Indhold af Ledeblokke, som her er vejledende. Det er tid- ligere nævnt (Side 72), at der er en meget stor Overens- Fig. 39. Landskabets Udformning paa Sejerø. Autoriseret Gengivelse efter Geodætisk Instituts Atlasblad fra 1904. 1: 80.000. Fig. 39. Landskabets Udformning paa Sejerø. Autoriseret Gengivelse efter Geodætisk Instituts Atlasblad fra 1904. 1: 80.000 stemmelse mellem Indholdet af Ledeblokke paa Sejerø og ved Nordkysten af Røsnæs, saaledes at man deraf maa drage den Slutning, at det er en og samme Isstrøm, der har afsat de Jordlag, hvorfra Stenene stammer. Sammen- sætningen af Stenindholdet viser, at denne Isstrøm er kom- met fra Sydøst, nær Øst om det sydøstlige Sverige. Røsnæs' Bakkedrag er opstaaet som en Sidemoræne ved denne Is- 117 tunges venstre Side, og Sejerøs »drumlins«-Bakker afspejler Bevægelsesretningen i Istungens m.ere centrale Dele. Under det senere Trin i Udviklingen, hvor Odsherreds Bakkebuer og Sjællands Odde blev opbygget, var Isbevægelsens Retning ændret fra Sydøst-Nordvest til Øst-Vest, hvad Indholdet af Ledeblokke viser (Side 60). tunges venstre Side, og Sejerøs »drumlins«-Bakker afspejler Bevægelsesretningen i Istungens m.ere centrale Dele. Under det senere Trin i Udviklingen, hvor Odsherreds Bakkebuer og Sjællands Odde blev opbygget, var Isbevægelsens Retning ændret fra Sydøst-Nordvest til Øst-Vest, hvad Indholdet af Ledeblokke viser (Side 60). Hedesletter. Senglacialtiden er det Tidsrum i Jordlagenes Udviklings- historie, der spænder fra Isdækkets Bortsmeltning indtil Skovtidens Begyndelse. Nedadtil er Tidsrummet vanskeligt at afgrænse. Til de senglaciale Aflejringer ·ligger det dog lige for at henregne Hedesletterne, de Sletter med Sand eller Grus, der af Smeltevandsfloderne fra Isdækket er afsat paa isbar Mark foran Isranden, og som i Nutiden har Mu- lighed for at fremtræde med nøjagtig det samme Overflade- præg, som. da de blev afsat. I Nordvestsjælland forekommer der flere Steder Aflejringer af denne Art. Flere af disse er allerede omtalt i Afsnittene om Landskabets Udformning ved Istidens Slutning. Den mest omfattende af disse Sletter er den, der indtager Størstedelen af det flade Omraade mellem Bjærgsted og Saltbæk Vig; dens mest udprægede Del ligger Vest for Li- nien Viskinge- Bregninge, hvor især visse Partier ved Kald- red og Nord for Viskinge er fladt udformede. Jordmateria- let indeholder i Almindelighed en Del Sten. Tæt Nord for Viskinge findes i en stor Grusgrav øverst 1-2 m gulgraat, horisontalt lagdelt, smaastenet Sand og derunder sammen- pakket Grus med mange hovedstore Sten. Nogle Hundrede Meter Syd for Bregninge er der i 8- 10 m dybe Grusgrave krydslejret Diluvialsand og -grus dækket af et Par Meter Hedegrus med nævestore Sten. Videre mod Syd svinder Præ- get af Hedeslette mere og mere i saavel Overfladens som Jordlagenes Udformning. Derimod er der nærmest Nord- vest for Bakkelandet ved Bjærgsted en smukt udformet, sen- 118 glacial Smeltevandskegle; den har haft Afløb mod Nord og Nordvest ved Lille Bregninge og Sultenkrog, hvor der i 1942 i en Udgravning saas smukt formede, horisontalt lig- gende Lag af smaastenet Hedesand. glacial Smeltevandskegle; den har haft Afløb mod Nord og Nordvest ved Lille Bregninge og Sultenkrog, hvor der i 1942 i en Udgravning saas smukt formede, horisontalt lig- gende Lag af smaastenet Hedesand. Et andet betydningsfuldt Omraade, der er formet som Hedeslette, findes langs den store, bueformede Israndslinie, der strækker sig igennem Odsherred. Slettens bedst udfor- mede Del ligger ned imod Sejerø Bugt foran den store Vejrhøjbakkebue og imellem denne Bue og Ordrup Næs, som det allerede tidligere er omtalt (Fig. 38). Efter en Af- brydelse Nordvest for Høve, hvor Højlandet gaar helt ud til Kysten, fortsætter Hedesletten mod Øst foran den øst- lige Del af Vejrhøjbuen og den følgende Israndsbue. Hedesletter. Dette Afsnit af Hedesletten har ikke en saa udpræget Sletteform i sin nordøstlige Del, men gaar her jævnt og umærkeligt over i det tilgrænsende Randmorænelandskab. - Foran den tredie store Morænebue er der kun ubetydelige Smaapar- tier, der kan opfattes som Hedeslette. Foruden disse Aflejringer af Sand og Grus, der er skyllet ud og afsat som Sletter foran en stagnerende Isrand, maa ogsaa i denne Forbindelse nævnes en Del skraa11ende, sand- fyldte Dale, der findes ved Sydvestsiden af Røsnæs, især Vest for Kongstrup og Skambæks Mølle, hvor disse sen- glaciale Smeltevandsdale skærer sig tværs igennem Strøget af Sandbakker, ned imod Kalundborg Fjord. Langs Store Aamose og Lille Aamose og Øst for Tissø er der lavtliggende Flader med Sand og Grus, der naturligt kan opfattes som senglacialt, dels afsat som Hedesletter og dels som Søaflejringer. Fossilførende, senglaciale L~g. Medens der ingen Vanskeligheder knytter sig til Bestem- melsen af Hedesletternes Sand og Grus som senglacialt, er Forholdet et andet, hvor det drejer sig om Lerlag, der er afsat i Forbindelse med Indlandsisens Bortsmeltning ved Afslutningen af Istiden. Udgangspunktet for Senglacialti- dens Begyndelse bliver her forskelligt efter, om der regnes 119 med Bortsmeltningen fra selve det snævre Omraade, som rummer de paagældende Lag, eller om. man regner Begyn- delsen fra det Tidspunkt, da Israndens Tilbagerykning fra sin Ydergrænse begyndte (det vil her sige: fra Grænsen mellem Vest-. og Østjylland). Hedesletterne maa betegnes som senglaciale, hvilket Udgangspunkt der end vælges. An- derledes med Lerlag, der er afsat i snæver Forbindelse med Isdækket, i Bassiner der har ligget helt eller delvis omgivet af Isen, men hvor Lerlagene ikke senere har været dækkede af Is. Hvis Udgangspunktet regnes fra Tilbagesmeltningen fra det centrale Jylland, saa at Issøleret paa de danske Øer derefter skulde bestemmes som senglacialt (som afsat efter Afsmeltningens Begyndelse) , da burde ogsaa samtidig dan- nede, tilgrænsende Lag af Moræneler, Diluvialsand og -grus henregnes til senglaciale og ikke til glaciale Aflejringer. Ud fra disse Betragtninger er Issøleret i nærværende Skrift ligesom i Nordøstsjællands Geologi (D. G. U. V. R. Nr. 3) regnet for glacialt, saaledes at Grænsen er sat efter, om La- gene er afsat under væsentlig de samme Overfladeforhold som Nutidens, eller naar Lagene er fossilførende. med Bortsmeltningen fra selve det snævre Omraade, som rummer de paagældende Lag, eller om. man regner Begyn- delsen fra det Tidspunkt, da Israndens Tilbagerykning fra sin Ydergrænse begyndte (det vil her sige: fra Grænsen mellem Vest-. og Østjylland). Hedesletterne maa betegnes som senglaciale, hvilket Udgangspunkt der end vælges. An- derledes med Lerlag, der er afsat i snæver Forbindelse med Isdækket, i Bassiner der har ligget helt eller delvis omgivet af Isen, men hvor Lerlagene ikke senere har været dækkede af Is. Hvis Udgangspunktet regnes fra Tilbagesmeltningen fra det centrale Jylland, saa at Issøleret paa de danske Øer derefter skulde bestemmes som senglacialt (som afsat efter Afsmeltningens Begyndelse) , da burde ogsaa samtidig dan- nede, tilgrænsende Lag af Moræneler, Diluvialsand og -grus henregnes til senglaciale og ikke til glaciale Aflejringer. Ud fra disse Betragtninger er Issøleret i nærværende Skrift ligesom i Nordøstsjællands Geologi (D. G. U. V. R. Nr. Fossilførende, senglaciale L~g. 3) regnet for glacialt, saaledes at Grænsen er sat efter, om La- gene er afsat under væsentlig de samme Overfladeforhold som Nutidens, eller naar Lagene er fossilførende. Selv med den anførte Afgrænsning kan Skellet mellem de glaciale og de senglaciale Lerlag ingenlunde drages skarpt. Der er i Nordvestsjælland adskillige Forekomster af Issøler, der ligger saaledes, al det nærmes t bliver en Skønssag, om de skal regnes som hørende til den ene eller den anden Gruppe. Saadanne Forekomster er der allerede gjort op- mærksom paa i Afsnittet om stenfrit Diluvialler (Side 44). Paa nogle Steder overlejres det lagdelte Diluvialler direkte af fossilførende Lag, som ved deres Indhold af Plante- og Dyrelevninger viser, at de stammer fra Senglacialtiden. En Aflejring, der staar tæt ved Grænsen mellem glacialt Smeltevandsler og senglacialt Ler, afgiver Lergravene ved K n a b s t r u p T e g 1 v æ r k Eksempel paa. Som det tid- ligere er nævnt (Side 51), er der her en indtil 16 m mægtig Leraflejring, hvori der er fundet Lag med Levninger af Planter og Dyr. Leret har gennemgaaende en tydelig Lag- deling, der kan være udformet med V arv (Aarslag) . Der er paa Grundlag deraf gjort Forsøg paa at paavise dets Sam- 120 tidighed med Leraflejringer ved Munke-Bjærgby og ved Stenstrup paa Fyn, uden at det dog endnu kan siges, om ikke Ligheden mellem det ringe Antal (og maaske usikre) Varv beror paa Tilfældighed. • tidighed med Leraflejringer ved Munke-Bjærgby og ved Stenstrup paa Fyn, uden at det dog endnu kan siges, om ikke Ligheden mellem det ringe Antal (og maaske usikre) Varv beror paa Tilfældighed. • Stedet for Teglværksgraven er i Forhold til Omgivelserne mod Øst en lavtliggende Flade med svagt Fald mod Nord og Vest. Søen, hvori Lerlagene er afsat, maa ved Dødis have været afspærret fra Afløbet i nordlig og vestlig Ret- ning, dengang de øverste Lerlag blev aflejret. Hosstaaende Tegning (Fig. 40) viser et Snit fra 1895 i den daværende østlige Gravs Vestvæg. Øverst fandtes 1- 1 ½ m rødgult, ensartet Ler med yderst regelmæssig, vandret Lagdeling. Derunder et 3- 4 dm tykt moræneagtigt Lag, dels fuldstændig uden Lagdeling dels med nogen Lagdeling og Sortering. De derunder liggende Lag af Ler og Sand har, saaledes som Tegningen viser, været Genstand for betydelig Forstyrrelse. Fossilførende, senglaciale L~g. Den ensartede Udformning af de forstyrrede Sandlag viser, at disse oprindelig har udgjort et sammen- hængende Lag, der ved Forstyrrelsen er blevet indæltet i de underliggende Lerlag._ - Omkring 35 Aar senere er der betydelig længere mod Nordvest i Graveomraadet set et 5-10 cm tykt morænelignende Lag af samme Art som det ovennævnte, liggende nær under Overfladen, men uden For- styrrelser i de underliggende Lag. Derimod afgav Lerlagene her Mulighed for Varvmaaling, og det viste sig derved, at det tynde Morænelerslag faldt sammen med et V arvs Som - merzone. Dette antyder, at det stenholdige Lag er opstaaet som et Moræneslamlag, der ved Foraarssmeltningens Ind- træden er gledet fra det højere Omraade mod Sydøst, ned paa den isdæmmede Søs Vinteris; men det har kun forstyr- ret de underliggende bløde Lag i den nærmeste Del af Bas- sinet, hvor tillige Laget har sin største Tykkelse. Forinden disse øvre Lerlag blev afsat, var der imidlertid Mulighed for Dyreliv i den isdæmmede Sø. Ved Undersøgel- serne i 1895 fandtes der i den østlige Grav i en Dybde af 5 m og nedad flere Steder i Leret sandede Partier med Skaller af Ferskvandsmollusker (bl. a. Limnæa auricularia og Pisidier). I omtrent samme Dybder som disse skalførende - - - - - -- - - - ~ (L • ~= ~-~~cc=~~~~It~1!~?!1!!ii~~i~!{~': -- - - ::..,....-----;...-::_. ____ ------ It/_:;;..- --- - - - ~ ..__ ..___.,,,,.,...1/_) / -- _ _ :-----____ ..___ ~ ~ ~ - ------""'l~r.(.l'//,1/...-_=_:::::-...:::-...:::-----::: 1i///,--::C::::C---_-::_:::-,.--=:_ ✓----=:-._\':?c.,,--:::- - - - - - _-::__ --- -- -...- - - - - - - -- -'--,,,,. t,,;::--------- - ---0:...J./// //,;;;---------"--:,,-/ --,,, /,, 1/-;- -_ C C ~~1;13~j?l~~~1~-'~~~~~~~~-%~P'?-~;'~= 0 5 JO'n, Fig. 40. Profil i Lergraven ved Knabstrup Teglværk, 1895. a og c Stenfrit Ler, b moræneagtigt Ler, d Sand. Efter D. G. U. I. R. Nr. 8. .. I'.. .... I'-' .... 0 5 JOn, Fig. 40. Profil i Lergraven ved Knabstrup Teglværk, 1895. a og c Stenfrit Ler, b moræneagtigt Ler, d Sand. Efter D. G. U. I. R. Nr. 8. 122 Lag fandtes ogsaa Lag med Plantelevninger. Det var dels Ler, der var mørkplettet af Planterester, og dels centimeter- tykke, planteførende Sandlag. Særlig rige fandtes ved Besøg i 1898 to Lag østligst i Graven 5,5 og 6,6 m under Over- fladen. Der er herfra paavist Levninger af følgende Planter: Lag fandtes ogsaa Lag med Plantelevninger. Fossilførende, senglaciale L~g. Det var dels Ler, der var mørkplettet af Planterester, og dels centimeter- tykke, planteførende Sandlag. Særlig rige fandtes ved Besøg i 1898 to Lag østligst i Graven 5,5 og 6,6 m under Over- fladen. Der er herfra paavist Levninger af følgende Planter: Batracllium, Vandranunkel. Calluna vulgaris, Hedelyng. Dryas octopetala, Rypelyng. Heleoclwris sp. (?), Sumpstraa. Menyanthes, Bukkeblad. Potamogeton sp., Vandaks. Salix sp. (cfr. phylicifolia), Lofarvet Pil. Salix polaris, Polarpil. Salix reticulata, Netaaret Pil. Saxifraga oppositifolia, Stenbræk. Mosser. Potamogeton sp., Vandaks. Salix sp. (cfr. phylicifolia), Lofarvet Pil. Salix reticulata, Netaaret Pil. Saxifraga oppositifolia, Stenbræk. Det viser sig derved, at Søen i den paagældende Tid har været i Stand til at rumme baade Mollusker og højere Plan- ter, og at der paa det tilgrænsende, højere Land fandtes en Bestand af Polarplanter, blandt hvilke Polarpil har været fremherskende efter de fundne Levninger at dømme. I de samme Lag som de nævnte Planter fandtes ogsaa Dæk- vinger af en Bille samt nogle Planterester, rimeligvis Eks- krementer af Lemming. Desuden er der senere (i 1904) fun- det Albueben af en Gaas (Graagaas?) i en Dybde af 5- 10 m, men uden nærmere Bestemmelse af Fundets Plads. Karakteren af de Flora- og Faunaforhold, som de fundne Fossiler bærer Præg af, stemmer saa fuldstændig overens med, hvad der kendes fra Senglacialtidens ældste Fersk- vandslag, det Nedre Dryasler, at man næppe behøver at be- tænke sig paa at regne dette fossilførende Ler for at være senglacialt. Hvad der kunde tyde paa mere rent glaciale Forhold i den efterfølgende Tid skulde være det moræne- agtige Lag i Forbindelse med de samtidigt opstaaede Varv. Maaske drejer det sig dog her kun om et Sammenspil mel- lem rent lokale og øjeblikkelige Forhold, stærkL Foraars- tøbrud og Regnskyl, hvorved dels Slamlaget dels det under- 12 123 liggende Sandlag (se Profilet) er ført ud i Søen fra de høje Bakker Sydøst for Teglværksgraven. AL det Materiale, som har udfyldt Søen, er tilført i Ret- ningen Sydøst- Nordvest, fremgaar dels af Fordelingen af det fede og det mere magre Ler i Lergraven og dels af Lej- ringen af grovere og finere Sandlag i Teglværkets Sandgrav Sydøst for Lergraven. Søtilstanden kan antages at være op- hørt ved, at Dødisen, der spærrede Afløbene mod Nord og Vest, er smeltet bort. Dette er sket, forinden Senglacialtidens milde Klima i Allerødtiden indtraadte. Fossilførende, senglaciale L~g. Som det tidligere er nævnt ( Side 51), har der oven paa en Bakke Øst for Tegl- værket været en ægte Issø, som dog er blevet aftappet efter kort Tids Forløb. Og det var rimeligvis først en betydelig Tid derefter, at Aflejringen af Leret fandt Sted i den Sænk- ning, hvor Teglværkets store Lergrav ligger. Da Lokaliteten her er den eneste af denne Art der kendes fra Nordvestsjæl- land, er den paa det geologiske Kort afsat med samme Sig- natur som de andre Lokaliteter med Issøler. Den Søudvikling, som ved Knabstrup blev afbrudt i Sen- glacialtidens første Afsnit, Ældre Dryastid, har andre Ste- der haft Mulighed for at videreføres igennem hele Sengla- cialtiden. Medens Klimaet i Ældre Dryastid var renl arktisk og Landjorden kun var beklædt med en yderst sparsom Planlevækst, hvor særlig Polarpil og Dværgbirk var frem- trædende og dens højere Dyreverden ogsaa har været arls- faltig, skifter Billedet. i det følgende Afsnit, Allerødtiden. I denne, mellemste Afdeling af Senglacialtiden, der har sit Navn fra en Forekomst i Nærheden af Birkerød, er der ind- traadt en Klimasvingning I Allerød-Oscillationen) med en betydelig Forøgelse af Sommertemperaturen. Dermed er fulgt en anselig Tilvækst i Plantebestanden, som nu rum- mer storbladede Birkearter, Fyr, Enebær, Melbærris og Rev- ling og et stort Antal andre Plantearter. Et rigere Dyreliv er fulgt efter den rigere Plantevækst, saa at det fra de senere Afsnit af Senglacialtiden vides, at der i Danmark har levet Rensdyr, Elsdyr og Bæver, irsk Kæmpehjort og Vildhest, og at der i deres Spor er fulgt store Rovdyr som Ulv og Bjørn. Senglacialtiden afsluttedes efter et nyt Klimaomslag i ark- 124 tisk Retning med fornyede Aflejringer af stenfrit Ler og Sand i Bassiner, hvor der tidligere var afsat Gytje og tynde Tørvelag. Hvor Bassinerne endnu ikke dermed var udfyldt, har der i den efterfølgende Tid kunnet opstaa Tørvemoser med Rester af den til enhver Tid eksisterende Plantevækst. tisk Retning med fornyede Aflejringer af stenfrit Ler og Sand i Bassiner, hvor der tidligere var afsat Gytje og tynde Tørvelag. Hvor Bassinerne endnu ikke dermed var udfyldt, har der i den efterfølgende Tid kunnet opstaa Tørvemoser med Rester af den til enhver Tid eksisterende Plantevækst. Fossilførende, senglaciale L~g. Den fulde Udvikling af Senglacialtidens Lagserie med saavel overliggende Tørvelag som underliggeride Lag af gla- cialt, lagdelt Ler finder man i R u d s - V e db y T e g 1- v ær k s Ler grav ved Jærnbanen umiddelbart Vest for Byen. Lagene har i Tiden omkring 19,30 været synlige paa to Steder i Gravens Sydvæg vestpaa fra Teglværket, dels ved dens Østende og dels ved dens Vestende. I den mellem- ste Del af Lervæggen laa de underliggende Lerlag i flad KuppelfoFm nogenlunde konformt med deres Underlag af Moræneler, medens den øverste, opragende Del af Kuplen var skaaret bort, saa at der paa dens Plads var opstaaet en næsten horisontal Slette, hvor Lerlag af lidt forskellig Alder naaede helt op til Jordoverfladen, saaledes som Tegningen Fig. 41 viser. Den af Nedbøren bortfjernede Del af Kuplen er ført ned over tilgrænsende Lavninger, som oprindelig har ligget udfyldt af lerblandede Isrester, men kom til Syne efterhaanden som Isen smeltede. Denne Bortsmeltning maa have fundet Sted allerede i Løbet af Senglacialtidens første Afsnit, Ældre Dryastid, thi i de ved Isens Smeltning op- staaede Bassiner er der i den efterfølgende Allerødtid afsat forskellige fossilførende Lag. Det egentlige Allerød.lag var dels sort Gytje, dels kalkrig Lergytje med Planterester og mange Skaller af Sphærier, Pisidier, Anodontaer og Snegle. Tykkelsen har været ca. 25 cm eller noget mere i Bassinets dybeste Parti. Derunder fandtes skalholdigt Ler, som indeholdt Skaller af Anodonta ned til 50 cm under Allerødgytjen og danner Overgangslag til Allerødzonen fra det underliggende Nedre Dryasler. Over Allerødzonen fulgte 2- 2½ m Øvre Dryasler, hvis nederste Del var rig paa Skaller af Sphærier, Pisidier og Snegle og saaledes danner Overgang fra det subarktiske Allerødlag til det arktiske Øvre Dryasler. De Bassiner, som endnu ved Senglacialtidens Slutning fandtes i Jordoverfla- 10-.cJ~°:1:_,<-r -~1..:od,l"'"'.tt:" '""=l tli/:it=Jif:°)JHJt~~~~~E~~~=..:_::==_: ____ _ i...JJ_-ur:i -~b3k~lo;fcrli.:hl t~Pr<>ftk!: Lw19.k..,..., ,,s.,Joi----+--+--+--+ 10...., ~Oll!fl~Qrl\~ld. f~~~n:d! tt,~.i~ '"' 1-1oo ~ / - --------- - ---------- - Fig. 41 . Sydvæg i Teglværksgraven ved Ruds-Vedby, ca. 1931. Efter S. A. ,-\NDEHSEN (D. G. F. l:ld . 10. Hefte 1. 1941). - ---------- - Fig. 41 . Sydvæg i Teglværksgraven ved Ruds-Vedby, ca. 1931. Efter S. A. ,-\NDEHSEN (D. G. F. l:ld . 10. Hefte 1. 1941). 126 den, er i den efterfølgende Tid udfyldt med postglaciale Lag saa at sige helt i Højde med den tilgrænsende Overfla- des rent glaciale Jordlag. Fossilførende, senglaciale L~g. I 1942 var Graven udvidet baade mod Nordvest og mod Sydvest fra Vestenden af den i Tegningen viste Sydvæg. I den nordvestlige Del af Graven rager der nederst Moræneler frem, endog højt op i Lervæggen, dækket af lagdelt Ler, hvis højest opragende Del er bortdenuderet, i Lighed med hvad Tegningen viser. Ved MidLen af den henved 300 m lange Vestvæg er der i Landskabet en svag Sænkning, under hvilken Allerødseriens Lagrældrn vedblivende vil være at se, eflerhaanden som Lergravningen skrider frem i vestlig Ret- ning. I Lergraven ved Und 1 øse Te g 1 værk har der og- saa været Allerødlag til Syne. Det var her et decimeterlykt, tørveagtigt Lag, der hvilede næsten umiddelbart paa det varvige Diluvialler og ikke var dækket af Øvre Dryasler, men kun af endnu yngre Lag af ganske ubelydelig Tykkelse. Medens Allerødtidens Aflejringer i Bassinerne ved Ruds- Vedby er opstaaet i Tilslutning Lil tidligere Søaflejringer, har Forholdet været et andet ved en Forekomst i Nærheden af Fa v r bo i det bakkede Højdedrag Nord for Skarresø. Anledningen til, at der i sin Tid blev foretaget Undersøgelser her, var Fundet af et Spoleben (Radius) af Bjørn. Stedet er vist paa Kortet Fig. 42. I den tidligere afgravede og pla- nerede Mose var der følgende Lag paa det Sted, hvor Knog- len fandtes: 20 cm stenfrit Ler, Øvre Dryasler. 20 cm gulbrun, kalk.rig Gytje } Allerødlaa. 0 20 cm gulbrun, kalk.rig Gytje 20-50 cm brunt, sandet Dynd 0 Derunder Moræneler. I det Øvre Dryasler blev der fundet Rester af Dværgbirk (Betula nana) samt Hvidbirk (B. alba), Rypelyng (Dryas octopetala) og Pil (Salix reticulata ); desuden en Del Mosser af nordlige og alpine Arter. I Kalk.gytjen, der ligesom Dryasleret er afsat i aabent Vand, er der fundet Rakleskæl af Dun-Birk (B. pubescens), 127 Lo Arter af Vandaks (Potamogeton) samt Star (Carex); des- uden er der fundet Pollen (Støvkorn) af Birk, Fyr og Pil. I samme Lag var det, at Bjørneknoglen fandtes. Det underliggende, mørkere, brune Dyndlag indeholdt tal- rige afbarkede og bævergnavede Grene af Pil og Birk, baade i den nederste og i den øversle Del af Laget. Det indeholdt Rester af følgende Planter: Arctostaphglos uva ursi, stedsegrøn Melbærris Arctostaphglos uva ursi, stedsegrøn Melbærris. Arctostaphglos uva ursi, stedsegrøn Melbærris. Batrachimn sp., Vandranunkel. Betula pubescens, Dun-Birk. Carex rostrata, tosnablet Slar. Hippuris vulgaris, Vandspir. Pimzs, Fyr (Pollen) . Potamogeton sp., Vandaks. Salix sp., Pil. Urtica dioeca, Tvebo-Nælde. Lycopodium complanatum, flad Ulvefod. Levninger af Insekter, Smaakrebs, Mosdyr, Svamp samt Alger. Batrachimn sp., Vandranunkel. Betula pubescens, Dun-Birk. Carex rostrata, tosnablet Slar. Hippuris vulgaris, Vandspir. Pimzs, Fyr (Pollen) . Potamogeton sp., Vandaks. Urtica dioeca, Tvebo-Nælde. Lycopodium complanatum, flad Ulvefod. Levninger af Insekter, Smaakrebs, Mosdyr, Svampe samt Alger. Den senglaciale Serie er paa delte Sted begyndt med en Bevoksning af Sumpkrat uden noget forudgaaende Søsta- dium. Det fremgaar heraf, at Overfladen af Stedet ikke dengang kan have ligget saa lavt i Forhold til dets Omgi- velser som nu (se Kortet Fig. 42). Man kan da regne med, at den nuværende Sænkning har været udfyldt af Is om- trent i Højde med Omgivelserne. Under Allerødtidens Temperaturstigning er det begravede Islag smeltet saa vidt ned, at der paa den daværende Overflade ikke har været Mulighed for Fremkomsten af et Muldlag (Allerødmuld), saaledes som man kender det fra andre Steder paa Sjælland. Smeltningen har dog heller ikke været saa vidt fremskre- den, at der forinden Allerødtidens varmeste Afsnit har været opstaaet nogen Sø. Derimod har Betingelserne netop været Lil Stede for Fremkomsten af et saadant Sumpkrat, som de talrige efterladte Rester af Plan ler og Spor af Dyreliv viser: Frugter og Frugtsten samt bævergnavede Grene af Pil og Birk. - I Løbet af den forholdsvis tempererede Allerødtid 128 er Stedets Sænkning skredet videre og videre frem, saaledes at Sumpkrattet er blevet efterfulgt af den Sø, i hvis Bundlag Bjørneknoglen er fundet; og Søens Levetid er fortsat op er Stedets Sænkning skredet videre og videre frem, saaledes at Sumpkrattet er blevet efterfulgt af den Sø, i hvis Bundlag Bjørneknoglen er fundet; og Søens Levetid er fortsat op Fig. 42. Kort over Omraade ved Favrbo. Efter KNUD JESSEN (D. G. F. Bd. 6. Nr. 24 i 924). 1. ~und af Bjørneknogle, se Teksten Side 126; 2. Fnnd af fuld- stændigt Skelet af Elsdyr, se Side 152; 3. Fund af Levninger af Elsdyr og Rensdyr, se Side 149. Om Plateanler paa Lindebjærg se Teksten Side 52 og 54. Fig. 42. Kort over Omraade ved Favrbo. Efter KNUD JESSEN (D. G. F. Bd. 6. Nr. 24 i 924). 1. ~und af Bjørneknogle, se Teksten Side 126; 2. Arctostaphglos uva ursi, stedsegrøn Melbærris Fnnd af fuld- stændigt Skelet af Elsdyr, se Side 152; 3. Fund af Levninger af Elsdyr og Rensdyr, se Side 149. Om Plateanler paa Lindebjærg se Teksten Side 52 og 54. 1. ~und af Bjørneknogle, se Teksten Side 126; 2. Fnnd af fuld- stændigt Skelet af Elsdyr, se Side 152; 3. Fund af Levninger af Elsdyr og Rensdyr, se Side 149. Om Plateanler paa Lindebjærg se Teksten Side 52 og 54. gennem Senglacialtidens yngste Afsnit med Afsætning af Øvre Dryasler. igennem Senglacialtidens yngste Afsnit med Afsætning af Øvre Dryasler. I forskellige Moser af Dyreknogler, som glacial Tid. Dette er der i disses Bundlag gjort Fund være bleven lejret her i sen- især Levningerne af Rensdyr. maa gælder 129 Saadanne Fund er gjort paa følgende Steder i Nordvestsjæl- land: Mullerup Teglværk, Undløse Mark, Løvenborg Mose, Kelleklintegaard og en Mose ved Værslev Mølle. Muligvis stammer ogsaa nogle af de mosefundne Elsdyr (Side 152) fra senglaciale Lag. Postglaciale Aflejringer. Postglacialtidens Begyndelse regnes fra det Tidspunkt, da den vedvarende Skovvækst tog Bolig i Landet. I den Tid der er forløbet siden da, har det nordvestsjællandske Land- omraade undergaaet meget betydelige Omskiftelser i Hen- seende til Udstrækning. Ved Senglacialtidens Ophør laa Landet adskilligt højere i Forhold til Havfladen end i Nu- tiden. Sporene deraf finder man paa den nuværende Hav- bund i Form af dybt nedskaarne Flodrender. Dette kommer for Nordvestsjællands Vedkommende klart til Udtryk ved den sydlige Del af Isefjord. Alle tre Sidefjorde fra Vest: Si- dinge Fjord, Lammefjord og Holbæk Fjord har markerede, nedskaarne Udløbsrender, der gør Indtryk af, at de oprinde- lig har været Lejet for Flodløb. Det er særlig Tilfældet ved Mundingen af Holbæk Fjord, hvor der Sydøst for Bognæs er en saadan Flodrende, der er skaaret temmelig skarp ned til en Dybde af 4-5 m under den tilgrænsende Fjordbund. Langt mere markeret er dog en undersøisk Flodrende, Veslerløb, imellem Tuse Næs og Orø. Den har haft sit Tilløb fra Syd, dels fra Inderbredning og dels fra Holbæk Fjords Rende. Skarpest er den formet ud for Hønsehalsen, hvor d en har en Bredde af 300 m og bratte Skrænter paa indtil 15 Meters Højde. Den naar her en Dybde af 17 m under Havfladen. Dette er dog næppe Rendens oprindelige Dybde i Forhold til den tilgrænsende Havbund. Dette turde frem- gaa af, at Isefjord ikke andre Steder synes at naa saa stor en Dybde. Men selv om Renden i sin undersøiske Tid er blevet noget uddybet af ud- og indadgaaende Fjordvand, kan det ikke betvivles, at dens oprindelige Anlæg stammer fra den Tid, da dens Bund laa højere end Havfladen. Dette n 130 har muligvis været Tilfældet baade i Senglacialtiden og langt ind i den postglaciale Tid. har muligvis været Tilfældet baade i Senglacialtiden og langt ind i den postglaciale Tid. har muligvis været Tilfældet baade i Senglacialtiden og langt ind i den postglaciale Tid. I Tiden omkring Begyndelsen af den Ældre Stenalder var der indtraadt en afgørende Ændring i Henseende til Land- omraadets Udstrækning. Fra at være langt mere omfat- tende end i Nutiden, svandt det nu mere og mere ind. Havet trængte sig efterhaanden ind over tidligere Landstræknin- ger, og det nøjedes ikke med at naa de Kystlinier, vi nu ser, men skred paa adskillige Steder langt ind over de nuvæ- rende Landomraader. Postglaciale Aflejringer. Nordvestsjælland gik i Løbet af Sten- alderen fra at være en Del af et udstrakt Fastland i stor Stil over til at blive et Ølandskab. Sporene af de daværende Kysters Plads kommer mange Steder tydeligt til Syne i Form af fremtrædende, bratte Skrænter, der nu ligger langt inde i Landet. Eksempelvis kan nævnes følgende Steder, hvor saadanne, fortidige Kystskrænter findes: Syd for Halleby Aas Udløb i Store-Bælt, flere Steder paa Sejerø, Vest for Gudmindrup i Odsherred, Vest for Ebbeløkke, flere Steder ved Siderne af Sonnerup Skov, Torsbjærg Nordvest for Ny- købing og de markerede Skrænter inden for Flyndersø og Dybesø ved Rørvig. Disse forskellige Skrænter staar som fremtrædende Vidnesbyrd om, at Stenalderhavet her har grebet ind med kraftigt Angreb og Nedskæring langs frem- springende Dele af det daværende Kystland. Ferskvandsaflejringer. Igennem Størstedelen af Senglacialtiden maa det antages, at der endnu har henligget mange begravede Rester af Is, ved hvis endelige Bortsmeltning der paa deres Plads opstod Fordybninger i Jordoverfladen. Disse Fordybninger afgav senere i rigt Maal Plads for postglaciale Ferskvandsaflej- ringer, nogle Steder begyndende som Bundlag i Søer, andre Steder opstaaet paa fugtig Bund uden forudgaaende Søsta- dium. Dette afhang af, hvor hastigt de begravede Islinser smeltede bort og hvor vidt fremskredet Fordybningernes Udformning var ved Overgangen fra Senglacialtiden til Postglacialtiden. De Lag, der afsattes i Løbet af de ca. 10.000 131 Aar, som Postglacialtiden omfatter, giver et klart Billede af den Udvikling, Landets Plantevækst og Klima har gennem- gaaet efter Istidens Ophør. Skovens Præg har ændret sig fra Tid til anden, ligesom tørre og fugtige Perioder har skiftet, og varme Tidsrum har været afløst af køligere og omvendt. Sammen hermed har Lagenes Præg været varierende mel- lem Tørv og Gytje afsat i Søer, dels Tørvegytje og dels kalk- holdig Gytje med yderst forskelligt Indhold af kulsur Kalle Mosekalk og Kildekalk. M o s e k a 1 k en er kalkholdig Gytje, afsat i en Mose- dannelses Søstadium. Oftest har et saadant Søstadium været til Stede i et tidligt Afsnit af Mosens Udviklingshistorie. En Mose har dog ogsaa senere kunnet omdannes til Sø, nemlig i et af Postglacialtidens fugtige Tidsrum med dermed føl- gende Stigning af Grundvandstanden. Mosekalk.en kan der- for træffes baade i den dybere og den højere Del af Mo- serne. I Nordvestsjælland forekommer der Mosekalk i en Del spredte Moser. Fra nogle af disse foreligger der Be- stemmelse af Lagenes Indhold af Skaller af Mollusker. Ved Strand g a ard tæt ved Kysten Vest for Svallerup er der en lang, smal Mose »Søen«, der ad en rendeformig Dal :-~ Hulbæk - har Afløb til Store-Bælt. Her har i Sen- glacialtiden Afløbet været fra en Flade med Plateauler Nord for Svallerup (Side 48). Mosens Overflade ligger nu ved en Højde imellem 14 og 15 m , men har tidligere naaet en Højde af indtil 17 m, Afløbsrendens Tærskelhøjde. Ved en Højde af 16,5 m, nordligst i Mosen, har en Gravning vist følgende Lagforhold: 30 cm Muld og formuldet Tørv. 24 cm sandet Ler og Sneglegytje. 27 cm Sand og sandet Ler uden Mollusker. Derunder Moræneler. Derunder Moræneler. I selve den lavere liggende Mose har den samme Lagfølge været til Stede. Der er fundet det Side 134- 135 anførte betydelige Antal Mollusk.arter. Gytjelaget kan anses for at være opstaaet nær ved Overgangen fra Fastlandstiden til 132 Litorinatiden. Tørvelaget maa stamme fraLitorinatiden, hvad der fremgaar af, at der i Tørven i en nærliggende Grav fandtes Skaller af Litorina litorea, Mytilus og Cardium edule. De maa være bragt til Stedet af Mennesker. Disse har tillige efterladt sig Spor i Form af primitive Flintredskaber i de samme Lag som de fundne Skaller. Nogle af Mol- luskerne i Tørvelaget (Cyclostoma elegans, Helicodonta ob- voluta og Succinea elegans) er ogsaa først indvandret i Slutningen af Stenalderen. Der er ligeledes fra andre Steder i Nordvestsjælland fore- taget Indsamling og Bestemmelse af Land- og Ferskvands- mollusker fra Gytjelag, hvorved disse Lags Alder og Karak- ter oplyses. Til en temmelig ung Del - Planorbis corneus- Zonen - af den postglaciale Mollusk.faunas Udviklings- historie, den første Del af Egeperioden, hører Lagene i en Mose ved Bjørnstrup paa Røsnæs. I samme Lag er der fun- det en Sumpskildpadde (Emys orbicularis). Mosekalk og Kildekalk. Tæt ved Strandkanten ved Svaleklint paa Sejerø - 2 km NNV for Sejerby - er der ved en Terrænhøjde af ca. 0,6 m under et tyndt Lag af skalførende Saltvandsdynd og ned til en Dybde af 2,5 m fundet vekslende Lag af Tørv og Fersk- vandsgytje med Skaller. Ferskvandslagene strækker sig vi- dere ud i Havbunden. Nordøstligst i Overby Lyng, lidt Vest for Tjørneholm, er der ved en Terrænhøjde af 1 m, under marint Grus og Ler, truffet 0,6 m Tørv med Blade og Birkegrene og derunder et tyndt Lag Gytje med Skaller af Ferskvandsmollusk.er. I en Mose 700 m SV for Tengslemark er der over et Tørvelag med mange Fyrrelevninger men næsten ingen Ege- levninger fundet et Gytjelag med en stor Mængde Mollusk- skaller. Tæt ved Tengslemark er der to Moser, hvor der er fundet baade Saltvandslag og Ferskvandslag. Ved Mosen 125 m SV for Byen er der fundet et enkelt, ved Mosen 300 m SØ for Byen er der to marine Lag. Indholdet af Ferskvands- Molluskskaller er opført i Listen Side 134- 135. Lagene med Bløddyrskallerne i de to sidste Moser stammer fra selve Litorinatiden. Ved alle de øvrige Moser 133 stammer Lagene dels fra samme Tid, dels ogsaa fra den nært forudgaaende Tid. Listen viser, at det som Helhed er et stort Antal Arter, der er fundet. Særlig har Lagene ved Strandgaard ved Store-Bælt været righoldige. K i 1 de k a 1 k er afsat i meget betydeligt Omfang paa to Steder, nemlig ved Fristrup Møller og Heslemølle, Nord- øst for Tølløse, samt ved Vintre Møller Øst for Elver- damsaa. Ved det første Sted, A as trup K a 1 kl eje, stræk- ker Kalken sig over et stort Omraade. Et V- 0-gaaende, 3- 400 m langt og indtil 4 m højt Profil viser de bølgede Lag af Kildekalk. Fra det kilderige Omraade omkring Møl- lerne Vest for Kalklejet har det udstrømmende Kildevand afsat Kalklagene som en skraanende Flade i Elverdams- aaens Dal. En dybt nedskaaren, smal, senglacial Erosions- dal Vest for Fristrup Møller har i stor Stil ført Vand ud til Kalklejets vestligste Parti. - Kalken er jordagtig og er i udstrakt Grad anvendt som Gødningskalk. Mosekalk og Kildekalk. Medens det nysomtalte Kalkleje har en meget betydelig horisontal Udstrækning og stammer fra mange Kilder langs Højlandets Bakkerand, har det fra gammel Tid kendte Kalk- leje ved V in tre Mø 11 er kun en ringe Udstrækning i Fladen og synes hovedsagelig at have haft sit Udspring fra et eneste Kildesystem, der ligger ved Kalkforekomstens øver- ste Ende, 100 m Syd for den øverste Mølle. Herfra strækker Kalk.lejet sig ca. 600 m mod NNV langs den By, hvis fem Vandmøller har givet Stedet Navn (rimeligvis fordi de som Følge af Kildevandets Temperatur har kunnet holdes i Drift selv i stærke Frostperioder). Bækken har paa den 600 m lange Strækning et Fald paa 25 m (1 : 24). Kalklejet ved Vintre Møller har to Gange været gjort til Genstand for Undersøgelse med Indsamling af Mollusk- og Plantelevninger som Formaal: første Gang i 1860 og 1862, anden Gang i 1893. Den praktiske Udnyttelse af Kalken har begge Gange drejet sig om de sammenhængende, haarde Kalklag, der i 1860 anvendtes til Kalkbrænding og i 1893 til Bygningssten. Til Bygningsbrug har Kalken været be- nyttet allerede i tidlig Middelalder, hvad der findes Vidnes- byrd om i flere af Omegnens Kirkebygninger. 134 I Stra:dg~a~ Svallerup I! f--~----1 1 ci: Acanthinula aculeata .. . . . . Amphipeplea glutinosa .. . . Ancylus lacustris ......... . Anodonta cygnæa . .... .... . - sp . ........ . ..... I Balea perversa ........... . Bithynia leachi . .. . ....... . - tentaculata ..... . Buliminus obscurus ....... . Carychium minium .. .... . . Clausilia laminata ...... .. . . - bidentata ..... .. . X X X X I l x X X X X X X X X X X X ::, b ,,, §~ .~; .ile<; x l X X X I xl Cochlicopa lubrica ........ j x li _x Cyclostoma elegans .. .. .... , x I Euconulus f ulvus ....... .. . ! x I x Eulota fruticum ........ . ... I I x I H elicella strigella . . . . . . . . . . x Helicodonta obvoluta . . . . . . x , H elix hor tens is ... ... ..... . - nemoralis ...... . . . . Hyalinia nitidula ......... . pura . ........ . . . radiatula . . ..... . sp . . . . .. ........ . Limnæa auricularia ........ Mosekalk og Kildekalk. I - ovata ..... ...... . - palustris ....... . pereger .. ..... .. . stagnalis ......... . truncatula ....... . X X 212 : J: X X X X X Paludestrina Steini . . . . . . . . x X X X Physa fontinalis .......... . - hypnorum . . . . . . ... . . x Pisidium gassiesianum .... . - nitidum . . . . . . . . . . x milium ......... . X subtruncatum ... . Henslowianum ... . obtusale ..... .. . . X X X X X X li X X X X X X X X X X X X X I I X X X X X X X X X 1x I 1 X X X X X X X X 135 Strandgaard I Svall erup bJl bD 0" I "C~ ;~; "'c., I Pisidium sp. ....... . . ..... Planorbis umbilicatus . ..... I X - arcticus . .. . . .... - carinatus . . . . . .. · 1 X - albus . .. . ........ X - stroemi .. . ... . . . I - vortex . . . .. . ... . >< - vorticulus . .. .... - nautileus ... ... .. X I - - var crislalus - contortus ....... · I X - parvus . . .. . .. .. . X - nilidus .......... 1 X - riparius ... .. . .. X - spirorbis .. .. .. .. X - fontanus ... .... . X Punctum pygmæum . . ... ... i X Pupa muscorum . .. .. .. .... Pyramidula rotundata . ..... X - ruderata . . . . . . X Sphærium I corneum ..... .. . :1 X - var pisiodiodes .. - - var draparnaldii 11 Succinea elegans . .. . .. .. .. I Pf eif eri .... . .. . . . I Unio tumidus . . . . . . . . . . . . . x Vallonia pulchella . . . . . . . . x f. costata . Valvata cristata .. . ..... . . . . piscinalis . .. . . . . . . . x macrostoma V erligo antivertigo . .. . .. . . pusilla .. .. .... ... . Vitrea crystallina .. ... . . .. . Mosekalk og Kildekalk. sp. . ...... .. .... .. . Vitrina pellucida . .. . . . ... . Zonitoides nitidus ....... . Cl. :i "" .::: r.ll " 8~ ] "'ø 0 - .,...,0 ..-. c::c:: I X X I X X I X X X X X X X X I X X X X X X I X X I X I X X X I X X X X X X X X 11 X X X 11x X X li ~ " ;..i).., ... ,; ... .,. ~ ... o- ~~ '8 ... ;'il <l) E ....... ~ " " " C: 0 :>-E >E <SlE C. - ..c: "' Q) "' " "'" :i ~ :;;; (I) " C: Er,, E°ro e-:rn ... ; C, ... C: " ~ - " -~:g oc: I(?§ oc: :i C: "·- :,... 0). o" '"· o" .... ~ [/J :/J f-0 ,,f-< -c:-- 00 E-< "" I I X X X X X I X X X X X I X I I I I X I I I X X X X X X X X X X I X I I X X I X I X X X X I X I I X I I I X X X I I I X I X [x X I X I X X X X X X I X 136 I 1860 fandtes tæt Vest for den næstøverste Mølle en om- trent 45 m lang Kalktufvæg. I den nordlige Del af denne Væg saas øverst ca. 1/s m brunlig Jord med adskillige Sten, »men især en Mængde større og mindre Kalktufbrudstyk- ker, der næsten dannede et Lag midt i det brune Jordlag. Paa et Sted noget sydligere laa under den øverste Jord med Kalktufstykker omtrent 1 ½ Fod [5 dm] brun Jord med mange Stene«. Efter denne Beskrivelse at dømme, er dette Lags Kalktufbrudstykker Affald fra gammel, maaske middelalderlig Brydning af »Fraadsten« til Datidens Kirke- byggeri. Derunder laa et 5- 6 dm tykt, fossilrigt Lag af løs, hvid Kalk, hvilende paa en 2 cm tyk, horisontalt liggende »Man- ganstribe«, der kunde følges omtrent 11 m. Under denne Stribe laa henved 2 m , temmelig fast, nederst meget fast Kalk tuf. I 1893 laa Kalkvæggen ca. 30 m Vest for Møllen. Lagføl- gen svarede meget nær til den anførte Lagrække. Mosekalk og Kildekalk. Fyldlaget naaede dog en Meters Tykkelse, og det nederste Kalk.lag var skiftevis fastere og løsere. Kalken naaede en Meter under Gravens Bund, og derunder boredes mindst en Meter i mørkt Dynd, uden at Bunden naaedes. Hele Lagserien, yngre end Istiden, har saaledes haft en Tykkelse af ca. 5 m foruden det Kalklag - maaske et Par Meter eller mere - som Fylden repræsenterer. I 1941 var der Øst for Bækken ud for den næstnederste Gaard (Bryggeri) et 50 m langt, 1 ½ m højt Profil i temme- lig hvid, løs Kalk med et fast Lag ca. 3/4 m under Overfla- den. - Ved Kilden Syd for Byen saas et 2- 3 m højt Snit i skiftevis røde, hvidlige og mørke Lag af Kildekalk. Den er overvejende jordagtig, men indeholder ogsaa haarde, ind- til 15- 20 cm tykke Kalk.lag. Fortegnelse over de i 1860 og 1862 indsamlede Skaller af Mollusker er opført Side 134-135. Den viser, at de fundne Arter hører til inden for samme Tidsrum som de fleste an- førte Arter fra Mosekalk. De undersøgte Kalk.lag ved Vintre Møller skulde derefter senest være afsat omkring Litorina- tidens Begyndelse. At der ikke er fundet yngre Former 137 staar naturligt i Sammenhæng m ed, at de øvre Lag af Kildekalk allerede var fjærnet, dengang Indsamlingen blev udført (1862). staar naturligt i Sammenhæng m ed, at de øvre Lag af Kildekalk allerede var fjærnet, dengang Indsamlingen blev udført (1862). Samtidig med denne Indsamling samt i 1893 blev der ogsaa foretaget Undersøgelse af Kalklagenes Indhold af Plantelevninger. Der blev paavist følgende Plantearter: Angelica silvestris, Skov-Angelik. Angelica silvestris, Skov-Angelik. Betula odorata, Hvidbirk. Betula pendula, Vortebirk. Corylus avellana, almindelig Hasse!. Dryas octopetala'? Rypelyng. Phrngmites vulgaris, almindelig Tagrør. Pinus silvestris, Skovfyr. Populus tremula, Bævreasp. Rubus clwmæmorus, Multebær. Salix aurita, øret Pil. caprea, Vidje-Pil. cinerea, graa Pil (hyppig). nigricans, sort Pil. reticulata, netaaret Pil. Tilia cordata, smaabladet Lind. Ulmus glabra, storbladet Ælm. Marclwntia polymorpha, Levermos. Efter Planternes Optræden at dømme er det sandsynligt, al de undersøgte Kalklag stammer fra Fyrreperioden og nærmeste omliggende Tid. Tørv og Tørvemoser. De Jordlag, som især er i Stand til at give Oplysninger om Postglacialtidens Planteliv og Klimaforhold, er Tørven i vore Tørvemoser. Dette hænger til Dels sammen med, at man ikke som i tidligere Tid er henvist til at nøjes med Under- søgelsen af de grovere efterladte Plantedele: Frø, Frugter, Bladrester o. I. Det vigtigste Emne for Undersøgelserne er nu Planternes uhyre smaa Støvkorn (Pollen), som i For- aars- og Sommertiden ved Vinden spredes til Bestøvning af Hunplanterne. Da de forekommer i utallig Mængde og 138 hører til de bedst bevarede Plantedele, har de kunnet gøres til Genstand for statistisk Undersøgelse. En Optælling under Mikroskop af de forskellige Plantearters Pollen i Præpara- ter, fremstillede af smaa Boreprøver fra Mosernes Lagserier, kan umiddelbart vise Planternes relative Mængdeforhold Mosens hele Dannelsestid igennem. Ved Undersøgelser af Tørveprøver, hvis arkæologiske Alder er kendt, er det gjort muligt at bestemme Karakteren af Omegnens Plantevækst paa disse Tidspunktet. Og ud fra Kendskabet til, hvorledes Landets Plantevækst har været igennem hele Postglacial- tiden, er det saa atter muligt ved Hjælp af Pollentællinger at tidsfæste arkæologiske Fund, hvis Alder man ikke har Mid- ler til at fastslaa ad anden Vej. Der har her været et frugt- bart, gensidigt Sammenspil. hører til de bedst bevarede Plantedele, har de kunnet gøres til Genstand for statistisk Undersøgelse. En Optælling under Mikroskop af de forskellige Plantearters Pollen i Præpara- ter, fremstillede af smaa Boreprøver fra Mosernes Lagserier, kan umiddelbart vise Planternes relative Mængdeforhold Mosens hele Dannelsestid igennem. Ved Undersøgelser af Tørveprøver, hvis arkæologiske Alder er kendt, er det gjort muligt at bestemme Karakteren af Omegnens Plantevækst paa disse Tidspunktet. Og ud fra Kendskabet til, hvorledes Landets Plantevækst har været igennem hele Postglacial- tiden, er det saa atter muligt ved Hjælp af Pollentællinger at tidsfæste arkæologiske Fund, hvis Alder man ikke har Mid- ler til at fastslaa ad anden Vej. Der har her været et frugt- bart, gensidigt Sammenspil. Side om Side hermed har andre Undersøgelser af Moser- nes Planteindhold og Dannelsesvilkaar givet Grundlag for Kundskaben om, hvilke Ændringer ikke blot Plantelivet men ogsaa Landets Klimaforhold har undergaaet i Løbet af Postglacialtiden. Dette er gengivet i omstaaende skematiske Oversigt, hvoraf det fremgaar, hvilken Skiften der har fun- det Sted mellem kolde og varme, tørre og fugtige Perioder i Løbet af den postglaciale Tid. Tørv og Tørvemoser. Samtidig har Landets Be- liggenhed i Forhold til Havet været underkastet Ændrin- ger, idet den nordlige Del af Nordvestsjælland først har ligget højere end i Nutiden og derefter i en stor Del af Stenalderen ligget lavere og endelig tilsidst er hævet op til sin nuværende Højdebeliggenhed. I de tørre Tidsafsnit (bo- real Tid og subboreal Tid) , har Grundvandet staaet lavt, Skoven kan være vandret ud paa Moserne og have efterladt Stubbe (henholdsvis bl. a. Fyrrestubbe og Egestubbe). Om- vendt har Moserne i de fugtige Afsnit kunnet omdannes til Søer med Afsætning af Tørvegytje eller Mosekalk. I Kyst- egnene har Havet i Litorinatiden kunnet brede sig ind over Tørvemoserne, medens de marine Lag senere efter Havets Tilbagetræden har kunnet overdækkes af nye Tørvelag. I Løbet af de omtrent ti Tusinde Aar, som Postglacialtiden omfatter, har Moserne saaledes kunnet være Genstand for en Række forskelligartede Omskiftelser. 139 I<ystfor- Klima- I Skov- Kultur- ca. skydninger perioder perioder perioder Aar: I Nutid, I Historisk I temmelig Tid tør -I Nu tids- Bøge- 1000 forhold periode - -- Subatlanlisk Tid, .færnalder 1:>D I kølig og fugtig ---- ----;- 400 c:: ·-c:: ~ Broncealder '-- 0) t:C Subboreal '" ..0 Tid, - Ege- ----;- 1500 ~ ~ varm og tør 0) Yngre > '" I Stenalder :r: > blandings- co / --,- 2300 ~ ~ / c:: / '-- .3 skovens / c:: :::i / 0) Allanlisk I/ I oc Erle- I c:: Tid, æ l:: varm og Periode bølle- 'u fugtig ku ltur c:: - til '-- 0) 0) 'u > ~ ro :r: c:: -- B - - --,- 5000 V) 0) '-- 'u - Boreal Tid, Fyrre- r..e I tør og \ Hasse!- 1 Mull erup- Fastlandstid efterhaanden periode ku ltu r temmelig varm I Fyne: n;,•k I I -:- 8000 periode 140 Der findes i Nordvestsjælland Tørvemoser uregelmæssigt spredt over hele Omraadet. I de bakkede Egne er der en rig Mængde smaa Moser, og der findes inden for Omraadet flere af Sjællands største Moser. Den største er Store Aa- mose, der naar en Længde af 13 km (med østlige Udløbere: 20 km) og har en Bredde af mellem 2 og 4 km. De Areale•r, der paa det medfølgende geologiske Kort er afsat som post- glaciale Ferskvandsaflejringer, omfatter dog ogsaa andre Jordlag end Tørv, saasom Kildekalk, samt udskyllede Lag af Ler og Sand i Aadalene. Tørv og Tørvemoser. En Undersøgelse af særlig Interesse er foretaget i en ganske lille Mose 700 m SS V for Jyderup By i V i g Sogn i Odsherred. Undersøgelsen fandt Sted i 1905, da der i betydelig Dybde var fundet S k e 1 et te t a f e n U ro k s e, der var »saaret og dræbt med Flintvaaben«. Over Tørvelaget laa et 75- 90 cm tykt Lag af stenblandet Muld. Moseprofilet var derefter følgende. 65 cm »Sort Tørv«. 90cm »Svampetørv«. 65 cm »Brun Tørv« (Gytje). 10cm Sand. 20cm Brun Gytje. 40cm Sort Gytje. 10 cm Brun, sandet Gytje. 65 cm »Sort Tørv«. 90cm »Svampetørv«. 65 cm »Brun Tørv« (Gytje). 10cm Sand. 10cm Sand. 20cm Brun Gytje. 20cm Brun Gytje. 40cm Sort Gytje. 10 cm Brun, sandet Gytje. Den sorte Tørv indeholdt talrige Stammer og Rødder af Æl samt en Del Egestammer. Under en kraftig Ællestub, der stod paa Roden, laa en Ophobning af musegnavede Hassel- nødder. I de nederste 5 cm af den sorte Tørv fandtes endelig en Del Rester af Skovfyr (Pi nus silvestris ), baade Kogler, Grene og Naale. - Tørven selv kan opfattes som en Sump- tørv dannet af fugtigt Ællekrat. »Svampetørven« var dannet af Tørvemos (Sphagnum); den var i frisk Tilstand lys, men fik ved Iltningen i Luften en mørk Farve. Paa den svampede Mose har der vokset talrige Tranebær ( Oxycoccus palustris), enkelte Skovfyr og lidt Kæruld '(Erioplwmm vaginatum ). Der fandtes ogsaa en Del velbevarede Biller i Tørven. / Fig. 43. Skelet af » Uroksen fra Vig«. Efter Fotografi, laant fra Nationalmuseet. Jvfr. Jotts. BRØNDSTED: Danmarks Oldtid. I. ...... .... ...... ...... .... ...... Fig. 43. Skelet af » Uroksen fra Vig«. Efter Fotografi, laant fra Nationalmuseet. Jvfr. Jotts. BRØNDSTED: Danmarks Oldtid. I. 142 Den øvre Del af det næste Lag, den »brune Tørv«, var en fed, gytjeagtig Masse, dannet i en tæt Rørsump og gen- nemvævet af Rødder og Rodstokke af Tagrør (Phragmites vulgaris), hvis Mængde aftog nedefter. Den nedre Del af den brune Tørv var en blød, fed Gytje, aflejret i aabent Vand; den indeholdt talrige Rester af Vandplanter: hvid og gul Aakande (Nymphæa alba og Nuplwr luteum), Vand- aks (Potamogeton natans) og Bukkeblad (Menyanthes tri- foliata). Rester af Skovfyr blev færre og fæn-e nedad igen- nem Laget, medens Levninger af Birk (Betula odorata) og Bævreasp (Populus tremula ) blev hyppigere og hyppigere. Tørv og Tørvemoser. Ved selve Underkanten af det brune Tørvelag laa Skelettet af Uroksen, delvis i Gytjen og delvis i det underliggende, tynde Sandlag. Den derunder liggende brune Gytje indeholdt endnu tal- rige Blade af Bævreasp og faa Rester af Fyr. I det følgende Lag af sort Gytje, som indeholdt mange Skaller af Fersk- vandsmollusker, fandtes derimod hverken Fyr, Birk eller Bævreasp. I det nederste Lag, som det ved Undersøgelsen var muligt at faa Prøver af, en brun, sandet Gytje, fandtes en Del Lev- ninger af Polarplanter, nemlig: Batrachium, cfr. confervoides, Vandranunkel, 1 Frugt. Betula nana, Dværgbirk, et Grenstykke og enkelte Frugter. Dryas octopetala, Rypelyng, 7 smaa, smalle Blade og nogle Frugter. Dryas octopetala, Rypelyng, 7 smaa, smalle Blade og nogle Frugter. Empetrum nigrum, Revling, 1 (gnavet) Frugtste Empetrum nigrum, Revling, 1 (gnavet) Frugtsten. Potamogeton sp., Vandaks, 1 lille Frugtsten. Salix polaris? Polarpil, 1 lille Gren. Salix polaris? Polarpil, 1 lille Gren. Det er da en Række vigtige Skifter i Egnens Plantevækst, den lille Mose afgiver Vidnesbyrd om. I den ældste Del af sin Tilværelse har den ligget som en Sø, først under et Tundraklima, omgivet af en temmelig arktisk Plantevækst, som derefter afløstes af Skove med mere varmekrævende Træarter som Hvidbirk og Bævreasp og begyndende Spor af Skovfyr. Ved denne Tid - for omtrent 9- 10.000 Aar 143 siden - fandt den velvoksne, midaldrende Uroksetyr her sin Død, saaret af Datidens Jægere. - I den efterfølgende Tid nærmede Mosen sig mere og mere Afslutningen af Sø- stadiet, gik først over til Rørsump og derefter til Højmose hele den sidste Del af Fyrreperioden igennem. I Egeblan- dingsskovens Tid vandrede endelig Ællekrattet ud paa den svampede Mosebund. Den største Ejendommelighed ved denne lille Mose er dog Fundet af Uroksen og dets Vidnesbyrd om Menneskets Færden her ved Overgangen fra Birke-Bævreaspeperioden til Fyrreperioden. Sit særlige Værd har Skelettet nemlig ved at vise to Saar frembragt af Flintpile; det ene Saar har ved Dyrets Død været ganske friskt, medens det andet dengang forlængst var lægt. Begge Saar fandtes paa Ribben i højre Side og bar Rester af de afskudte Pile; ved det friske Saar var det 4 mm tykke, faste Ben helt gennemboret, og Lungen var sikkert beskadiget. Som saaret har Oksen søgt den lille Sø for at lindres og er her død. Tørv og Tørvemoser. En anden Mose, som ogsaa paa Grund af særlige For- hold har været Genstand for Undersøgelse, er M u 11 er up Mose, der ligger længst mod Sydvest paa det foreliggende Kortomraade. Det var her Fund af Levninger fra en Sten- alders-Boplads, der gav Anledning til Undersøgelse. Det var kun den nedre Del af Mosens oprindelige Lag, der forelaa til Undersøgelse. Dette fremgaar af, at der i dens Overflade fandtes et Antal Fyrrestubbe staaende paa Roden. Ved Si- den af en saadan Stub er følgende Lag blevet gennemboret: Ca. 20 cm Sphagnumtørv. Ca. 20 cm Sphagnumtørv. Ca. 50 cm Svampet Tørv, dannet af en Vegetation af Tagrør (Phragmites vulgaris) og Avneknippe (Cladimn mariscus). Ca. 15 cm »Vandtørv«, opstaaet af Algeplankton i lavvandet Sø. Ca. 15 cm »Vandtørv«, opstaaet af Algeplankton i lavvandet Sø. Ca. 25 cm Snegledynd. Ca. 125 cm Detritusgytje. Ca. 185 cm Senglacialt Ler. Derunder Moræneler. 144 144 Denne Lagfølge viser, at der her i den længste Del af den Tid, dens Opstaaen strækker sig over, har været Sø. I en Del af dette Tidsrum er der afsat Snegledynd i Søen; de fundne Arter af Mollusker er opført Side 134- 135; de viser hen til Postglacialtidens boreale Afsnit, Fyrreperioden. Optalte Træpollen fra Snegledyndet og underliggende Lag viste ogsaa, at den omgivende Skov næsten udelukkende har bestaaet af Birk og Fyr; først ved Overgangen til det overliggende Lag af Algegytje tager Hassel til i Mængde, og Tilsynekomsten af Egeblandingsskov (Ælm, Eg og Lind) begynder at vise sig. Samtidig hermed indfandt der sig Mennesker og slog sig ned paa en lille Holm, Toppen af en langstrakt Højning i Søens Undergrund. De medførte en Kulturform, som efter Stedet har faaet Navnet Mullerup Kulturen, en Indlands- kultur, ældre end Ældre Stenalders kendte Kystkulturfor- mer. Beboerne har været en Jægerbefolkning, hvem det rige Dyreliv har kunnet forsyne med de nødvendige Fornøden- heder til Føde, Klæder, Redskaber og Vaaben. De neden- nævnte Hvirveldyr, hvoraf der er fundet Knogler, giver et Indtryk af den Dyrerigdom, der har været til Stede : Gedde (Esox lucius), adskillige Knogler; (ogsaa andre Fisk). Sumpskildpadde (Emys orbicularis). Et Eksemplar. Sumpskildpadde (Emys orbicularis). Et Eksemplar. Spidsand (Anas acuta). Enkelte. p p ( y ) p Spidsand (Anas acuta). Enkelte. Stokand (Anas boscas). Nogle, ogsaa af Ællinger. Knopsvane (Cygnus olor). Adskillige, ogsaa af unge Fugle. Havlit (Pagonetta glacial is). To Eksemplarer. ( g g ) p Toppet Lappedykker ( Podicipes cristatus). Enkelte. Sortstrubet Lom ( Colymbus arcticus). Et Par. Trane ( Grus cinerea). Et Par. Hættemaage (Lams ridibundus) . Et Eksemplar. Hejre (Ardea cinerca). Et Eksemplar. Rørdrum (Botaurus stellaris). Et Par Stykker. Skarv (Phalacrocorax carbo). Enkelte. Glente (Milvus ictinus). To Knogler af ung Fugl. Toppet Lappedykker ( Podicipes cristatus). Enkelte. Sortstrubet Lom ( Colymbus arcticus). Et Par. Toppet Lappedykker ( Podicipes cristatus). Enkelte. Toppet Lappedykker ( Podicipes cristatus). Enkelte. Sortstrubet Lom ( Colymbus arcticus). Et Par. Trane ( Grus cinerea). Et Par. Hættemaage (Lams ridibundus) Et Eksemplar Sortstrubet Lom ( Colymbus arcticus). Et Par. Trane ( Grus cinerea). Et Par. Hættemaage (Lams ridibundus) . Et Eksemplar. Hejre (Ardea cinerca). Et Eksemplar. Rørdrum (Botaurus stellaris). Et Par Stykker. Glente (Milvus ictinus). To Knogler af ung Fugl. 145 Havørn (Haliaetus albicilla). Enkelte Knogler. Sortspætte (Picus martius). En hel Mellemhaand. Skovskade (Garrulus glandarius). Knogle af en ung Fugl. Hare (Lepus europæus). En Knogle. Bæver ( Castor fiber). Enkelte Knogler. Bæver ( Castor fiber). Enkelte Knogler. Egern (Sciurus uulgaris). En Knogle. Egern (Sciurus uulgaris). En Knogle. Pindsvin (Erinaceus europæus). Pindsvin (Erinaceus europæus). Vildkat (Fe/is siluestris). Enkelte Knogler. Vildkat (Fe/is siluestris). Enkelte Knogler. Ræv (Vulpes uulpes) . Enkelte Knogler. Ræv (Vulpes uulpes) . Enkelte Knogler. Hund (Canis familiaris). Enkelte Knogler. Hund (Canis familiaris). Enkelte Knogler. Bjørn (Ursus arctus). Enkelte Knogler. Skovmaar (Martes martes). Et lille Antal Knogler. Ilder (M ustela putorius). Grævling (Meles meles). Knogler af nogle faa Dyr. Vildsvin (Sus scrofa ferus). Knogler i Mængde. Raadyr (Capreolus capreolus). Mange Knogler. Krondyr (Ceruus elaphus). Knogler i Mængde. Elsdyr (Al ces al ces). Mange Knogler. Urokse (Bos primigenius). Mange Knogler. Denne store Mængde Dyreknogler er især fundet paa en Boplads, der laa Syd for og lavere end den først beboede Holm, men som kun har kunnet beboes ved Sommertid, naar Søbunden var tørlagt. Hermed stemmer, at en Del af de fundne Knogler stammede fra Unger saavel af Pattedyr som af Fugle; det boreale Tidsrums Somre anses for at have været tørre og ligesaa varme som Nutidens, medens Vintrene har været koldere og har haft Hovedmængden af Nedbøren, saa at det er rimeligt, at Søen har haft ret for- skellig Sommer- og Vintervandsland. En Undersøgelse af »Vandtørven«, der afsattes Side om Side med Bopladsen, samt af de nærmeste under- og over- liggende Lag, viser et Udsnit af den samtidige Plantevækst: Betula, cfr. pubescens, dunet Birk. Carex pseudocyperus, Knippe-Star. Carex sp., Star. Cladium mariscus, hvas Avneknippe. Betula, cfr. pubescens, dunet Birk. Carex pseudocyperus, Knippe-Star. 146 C orylus cwellana, almindelig Hassel. Frangula alnus, Tørstetræ. M enyanthes trifoliata, Bukkeblad. Najas marina, enfrøet Najade. Nymphæa alba, hvid Aakande (Nøkkerose). Peucedanum palustre, Kær-Svovlrod. Phragmites vulgaris, almindelig Tagrør. Pinus silvestris, Skovfyr. Populus tremula, Bævreasp. Potamogeton natans, svømmende Vandaks. Potamogeton sp., Vandaks. Rubus saxatilis, Fruebær. Rumex hydrolapathum, Skræppe. Sambucus nigra, almindelig Hyld. Scirpus Tabernæmontani, blaagrøn Kogleaks. Ulmus glabra, storbladet Ælm. Fra en Boplads fra Mulleruptid i Vinde-Helsinge Mose Nord for Mullerup foreligger der foruden Knogler af mange store Pattedyr ogsaa Knogler af Vildhest (Equus caballus ferus). Der er her tillige fundet et Menneskealbueben, som har været bearbejdet ved Skrabning. Den Moseflade, der benævnes Store A a mose, stræk- ker sig fra Bromølle Kro til Undløse Bro over et Omraade paa ca. Sortspætte (Picus martius). En hel Mellemhaand. 36 km 2 • Den 13 km lange Mosestrækning har sin største Højde, 27 m , i sin østlige Del; den laveste Højde, 24 m, findes imellem Kajemose og Sønderød, hvor der endnu før Aarhundredskiftet var enkelte, ikke tilgroede Smaa- søer. Det kunde maaske ligge nær paa Forhaand al formode, at Mosens Underlag udgjorde en lige saa plan Slette som dens Overflade. Dette er dog ikke Tilfældet, i hvert Fald ikke for den østlige Dels Vedkommende. Der er her i For- bindelse med den geologiske Kortlægning i 1895 udført en Serie paa 11 Boringer ned til Mosens Bundlag. Boringerne udførtes i et Omraade Syd for Halleby Aa paa Strækningen mellem l½ km Vest for Magleø og 2½ km Øst for denne Bakke i Mosen. Højden af Mosens Overflade varierer her mellem 26 og 27 m, men Mosens Bund naaedes i saa veks- lende Dybder som. 2 og 5½ m, og dens Højde varierer mel- 147 lem 20½ og 25 m. Bundlagene bes lod skiftevis af stenfrit Ler, Sand og Grus. Ved syv af de otte Boringer, hvor Moselagene havde en Tykkelse af mere end 2 m , var der nederst skalholdig Gytje, hvilket falder godt i Traad med, at den slore Mosestrækning i Tiden efter Isdækkets Borlsmeltning maa have været en Sø med lavere Bundhøjde end Afløbet ved Bromølle Kro. Søtilslanden kan da antages i hvert Fald al falde sammen med Postglacialtidens boreale Afsnit, ældste Del af Fyrre- tiden. I Aarene omkring 1940 er der i Forbindelse med arkæo- logiske Undersøgelser ved nogle Stenalders Bopladser i Aa- mose ogsaa foretaget Undersøgelse af Pollenindholdet i Mo- sens Lag til Hjælp for Aldersbestemmelsen af de deri fundne Kulturrester. Ved en enkelt af Bopladserne /Øgaarde) er der tæt over Mosens Bund fundet skalførende Gytje og derover et Lag med Oldsager og Trækul. Nærmest ind imod Land var dette Tørvelag en Sumptørv, længere ude derimod et Gytjelag med Frø af hvid og gul Aakande. Der har altsaa her været Søtilstand. Den omliggende Skov har bestaaet af Fyr og Birk samt Hassel, og Tidsrummet er Borealtiden. Først derefter kom. Indvandringen af Egeblandingsskoven i Begyndelsen af den atlantiske Tid, sammenfaldende med }Eldre Stenalders Ærtebøllekultur. I denne fugtige Periode har Mosen i Følge de foreliggende Undersøgelser ikke egnet sig for Beboelse. Sortspætte (Picus martius). En hel Mellemhaand. Derimod er der baade ved Øgaarde og ved andre Bopladser (Magleø og Kildegaard) fundet betydelige Kulturlag fra det følgende Afsnit af Postglacialtiden, det subboreale, svarende til Yngre Stenalder. I denne Tid har Klimaet været tørt og varmt, Mosen har plelvis været be- vokset med Star og Æ llekrat, saa den har kunnet egne sig til Beboelse. Ved de Bopladser i Aamosen, som stammer fra Mullerup- tiden, er der fundet en nær tilsvarende Fauna til den i Mullerup Mose. Af Dyr, som ikke fandtes der, kan nævnes: Vandrolle (Arvicola terrestris). Los (Lynx lynx) . Ulv (Canis lupus ). Odder (Lutra lutra) . Los (Lynx lynx) . 148 Af Fugle: Af Fisk: Malle (Silurus glanis) . Af Fisk: Mere enkeltvis end i Stenalderens Bopladsfund er der i adskillige nordvestsjællandske Moser fundet Knogler af Dyr, som nu forlængst er uddøde i Danmark. Ved Ved ding e ved Faarevejle fandtes i 1917 ved Tørvegravning i en tidligere dyrket, lille Mose tre Skeletter af Sumpskildpadde. Nærved Fundstedet maaltes denne Lag- følge: 0- 82 cm Muld og derunder Ælleskovtørv med Hasselnødder, og nederst en Stamme af Fyr. 0- 82 cm Muld og derunder Ælleskovtørv med Hasselnødder, og nederst en Stamme af Fyr. 82-147 cm Mosrig Sumptørv af Avneknippe (Cla- dium mariscus) og Tagrør m. m. Heri en Stamme og Frø af Fyr, en Hassel- nød, Blade af Lind, Hassel og Pil (Salix cinerea). 82-147 cm Mosrig Sumptørv af Avneknippe (Cla- dium mariscus) og Tagrør m. m. Heri en Stamme og Frø af Fyr, en Hassel- nød, Blade af Lind, Hassel og Pil (Salix cinerea). 82-147 cm Mosrig Sumptørv af Avneknippe (Cla- dium mariscus) og Tagrør m. m. Heri en Stamme og Frø af Fyr, en Hassel- nød, Blade af Lind, Hassel og Pil (Salix cinerea). 147-200 cm Brun Detritusgytje; talrige Naale og Kogler af Fyr, Blade af Lind og Birk, Frugter af Cladium, Vandplanter. 200--250 cm Graagul Sneglegytje. 250--387 cm Graablaat, fedt, nederst noget sandet, stenfrit Ler (Øvre Dryasler). 387- 490 cm Gulbrun Gytje med enkelte Skalresler over gulbrun Sneglegytje (Allerødgytje). 490- 555 cm Brun, sandet Muld med mange Ved- rester over muldet Sand. Tørvelaget afgravedes ned til Sneglegytjen, og Skildpadde- resterne fandtes i det nederste Spadestik af Sumptørven. De tidsfæstes til en fremskreden Del af Fyrreperioden eller maaske en meget tidlig Del af Egeblandingsskovens Periode 149 (Periode VII), idet der har staaet meget Fyr sammen med Lind omkring Bassinet, da de levede. Sortspætte (Picus martius). En hel Mellemhaand. Nordvest for Esk e bjærg (Øst for Saltbæk Vig) ligger der en temmelig stor Mose, Eng hav e, som er skilt fra Havet ved en Bræmme af flyvesandsdækket Strandmateriale. Fra denne Bræmme er der blæst lidt Sand ind over Mosens Randparti. Dens Overflade ligger ved en Højde af 2- 3 m , og den har igennem Langholm Rende Afløb mod Nordvest til Havet. Et noget simplificeret Profil igennem Mosen viser Lagfølgen : 0 - 0,5 m Kærtørv. 0,5-3,9 m Sumptørv, væsentlig dannet af Cladium og Tagrør. 3,9- 7,6 m Brakvands-Tagrørstørv, delvis sandet. I denne anselige Mose er der i Tidens Løb fundet en betydelig Mængde Dyreknogler, hvis Plads i Lagene dog ikke har kunnet oplyses, da Tørven i Almindelighed er taget op m ed Ketsjer. Herfra stammer bl. a. flere Kæber af Bæver, Dele af Sumpskildpadder og en Del Knogler af Urokse. I Li torinatiden h ar der h er været en Lagune, der afspærredes fra Havet ved Strandvolde, og i hvilken der gennem hele Perioden har staaet en tæt Bevoksning af Tagrør. Der er i denne Mose fundet Skeletdele af ikke mindre end 9 Bævere, deraf to Unger i Tandskifte. I F a v r b o Mose (Kortet Fig. 42, Tr . 3) fandtes i l920 paa et og samme Sted en Hovedskal m ed Takker af Elsdyr, nogle andre Knogler af Elsdyr samt den nedre Del af en Rensdyrtak. Fundstedet laa ca. 30 m Syd for Mosens Nord- rand paa et Sted, hvor Mosens Lag tilsammen havde haft en Mægtighed af h enved 2,3 m . De n edre Lag her bestod af Gytje (ca. 60 cm) over et Lag Sumptørv, væsentlig Tag- rørstørv (10--15 cm), der hvilede paa stenet Sand. I Sump- tørven fandtes talrige Knopskæl af Bævreasp og Blade af dunet Birk, m edens der i Gytjen saas meget Bark af Fyr, et Blad af Vorte~irk og en Frugt af Najas marina. I et noget I F a v r b o Mose (Kortet Fig. 42, Tr . 3) fandtes i l920 paa et og samme Sted en Hovedskal m ed Takker af Elsdyr, nogle andre Knogler af Elsdyr samt den nedre Del af en Rensdyrtak. Fundstedet laa ca. 30 m Syd for Mosens Nord- rand paa et Sted, hvor Mosens Lag tilsammen havde haft en Mægtighed af h enved 2,3 m . De n edre Lag her bestod af Gytje (ca. Sortspætte (Picus martius). En hel Mellemhaand. 60 cm) over et Lag Sumptørv, væsentlig Tag- rørstørv (10--15 cm), der hvilede paa stenet Sand. I Sump- tørven fandtes talrige Knopskæl af Bævreasp og Blade af dunet Birk, m edens der i Gytjen saas meget Bark af Fyr, et Blad af Vorte~irk og en Frugt af Najas marina. I et noget ~ .... Fig. 44. Skelet af Elsdyr fra Bregnemose ved Grevinge. Efter HERLUF WrnGE : Om jordfundne Pattedyr fra Danmark. 1904. .. i: 0 ...... i:.,, 0 Fig. 44. Skelet af Elsdyr fra Bregnemose ved Grevinge. Efter HERLUF WrnGE : Om jordfundne Pattedyr fra Danmark. 1904. Fig. 44. Skelet af Elsdyr fra Bregnemose ved Grevinge. Efter HERLUF WrnGE : Om jordfundne Pattedyr fra Danmark. 1904 151 yngre Lag et andet Sted i Mosen fandtes en Frugt af Lind (Tilia cordata). En Del af Knoglerne laa endnu in situ, dengang den geologiske Undersøgelse fandt Sted; deres Plads var ved Overgangen mellem Sumptørven og Gytjen. yngre Lag et andet Sted i Mosen fandtes en Frugt af Lind (Tilia cordata). En Del af Knoglerne laa endnu in situ, dengang den geologiske Undersøgelse fandt Sted; deres Plads var ved Overgangen mellem Sumptørven og Gytjen. Fig. 45. Hovedskal af Elsdyr fra l{astrupgaard, Nord for Haraldsted. Hornenes Spændvidde 1470 111111. Efter HERL UF W1,-.GE (1904). Fig. 45. Hovedskal af Elsdyr fra l{astrupgaard, Nord for Haraldsted. Hornenes Spændvidde 1470 111111. Efter HERL UF W1,-.GE (1904). Den ejendommelige Lagfølge: Gytje over Sumptørv har kunnet følges igennem en stor Del af Mosen. Kun i et be- grænset, dybere Parti af Bassinet fandtes ældre Lag, nemlig den sen glaciale Serie: Yngre Dryasler, Allerødgytje og Ældre Dryasler. Hele Lagfølgen og Planteindholdet viser, at Knoglefundet maa henføres til en meget tidlig Del af Post- glacialtiden. Fundet af Rensdyr i postglaciale Lag - saaledes som her - hører til de meget sjældne Fund. Rensdyrene for- svandt i det store og hele, da Skovtiden indtraadte. - Et tilsvarende Fund stammer fra submarin Tørv ved Svale- klint paa Sejerø. 152 I en lille Tørvemose ved Snavhuse Sydøst for D'ragsholm er der i 19,15 fundet et Elsdyrkranium med paasiddende Tak- ker. Kraniet laa paa Moræneleret under et 1,2 m mægtigt Tørvelag. Kraniet blev opbevaret paa Dragsholm. Her har ogsaa Kraniet af en Urokse fra Omegnen været opbevaret. Sortspætte (Picus martius). En hel Mellemhaand. I en lille Tørveprøve fra en Hulhed i dette Kranium er der fundet Pollen af Birk, Fyr og Hassel i et Mængdeforhold, der viser, at Dyret har levet paa Stedet i Tiden lidt før Bosættelsen i Mullerup Mose fandt Sted. p Fra Tiden forud for 1900 foreligger der Fund af et stort Antal Dyreknogler fra nordvestsjællandske Moser. Heraf skal kun nævnes Findestederne for de nu uddøde Pattedyr: Bæver, Vildsvin og Elsdyr. Det er for Bæver: Aldersro ved Værslev, Ørslev Mose ved Kragerupgaard, Mose ved Raklev og Aamose ved Skellingsted. Vildsvin: Aldersro (Dyret har været saaret med Flintvaaben, men ikke dræbt), Agersvold, Vognserup Mose, Tranemose Nord for Kalundborg samt en Mose ved Alleshave. Knogler af Elsdyr er fundet i Moser ved følgende Steder: Overby paa Sjællands Odde, Højby, Sejstrup, Aastofte, Sneglerup (fuldstændigt Skelet), Gre- vinge (se Fig. 44), Hagestedgaard og Gurede Mark paa Tuse Næs, Favrbo (fuldstændigt Skelet, se Kortet Fig. 42, Nr. 2) , Agersvold, Vognserup, Løvenborg, Aagerup, samt Kastrup- gaard Nord for Haraldsted (se Fig. 45). Af de mosefundne Dyr, som der er fremkommet Rester af i indeværende Aarhundrede, skal følgende yderligere nævnes: I Dyrehøjgaards Mose i Kongstrup paa Røsnæs er i 19·42 fundet et næsten helt Skelet af Landbjørn (U rsus arctus) , der stammer fra boreal Tid. Af en Urokse er der i Mose ved Knabstrup Hovedgaard fundet Hjernekasse med paa- siddende Stejler; dens Alder ligeledes boreal Tid. Et helt Skelet af Elsdyr er i 1925 udgravet i en Mose ved Lunte- bjærg, tilhørende Grevinge Præstegaard. Af Elsdyr er der ogsaa fundet Knoglerester i Ærtemosen ved Vig i Ods- herred. Af Kronhjort er der baade i ældre og nyere Tid gjort mange Fund i Nordvestsjællands Moser. Et næsten fuldstæn- digt Skelet af en gammel Han er i 1942 fundet paa Sejerø. 153 Samtidig med dette indkom der fra Sejerø mosefundne Knogler af Gedde og Dele af Skjold af Sumpskildpadde. Fra en Køkkenmødding paa Sejerø kendes desuden Graasæl, Marsvin, Kronhjort, Vildsvin, Odder og Ræv. Af Fugle maa fremhæves Gejrfugl og Svane; af Fisk: Pighaj. Af den i Danmark yderst sjældne Svømmefugl, Pelikan, er der i 1940 fundet væsentlige Dele af et Skelet i en Mose ved Syvhøje imellem Højby og Nykøbing. Pollenanalyse af den omgivende Tørv h ar vist, at den har levet h er i den at- lantiske Periode (se Skemaet Side 139). Sortspætte (Picus martius). En hel Mellemhaand. I en Mose i Nyrup, Højby Sogn, er ogsaa i 1940 fundet Knogler af Trane, hvis Alder er bestemt til Slutningen af subboreal Tid, Bronce- alder eller m eget tidlig Jærnalder. Som det er nævnt tidligere, har der inden for Postglacial- tiden været et Tidsrum, da Havet, Litorinahavet, strakte sig ind over Kystegne, som nu er tørt Land. I disse Egne har der i den forudgaaende Tid, Fastlandstiden, paa flere Steder været T ørvemoser. Dette h ar saaledes været Tilfældet inden for Saltbæk Vig, i Trundholm i Odsherred og paa Sejerø, hvorfra det vides, a l der ogsaa findes Tørvelag paa Havbunden uden for den nuværende Kyst (se senere under Saltvandsaflejringer). Disse Tørvelag er hovedsagelig op- staaet fra Begyndelsen af Skovtiden til ind i den ældste Del af Egeperioden. Hvor saadanne Tørvemoser forekommer paa den nuværende Havbund, er der saaledes Mulighed for, at Fyrrestammer, der staar paa Roden, kan rage op over de omgivende Tørvelag og volde Forstyrrelse for Fiskeriet. Saa- danne Vanskeligheder kan selve T ørven ogsaa volde for Ned- ramningen af Bundgarnspæle. Tørvelag af denne Art er ogsaa blevet afsat i den nu ud- tørrede Del af L a m m e f j o r d. Der er h er fundet Ege- stammer ved Fjordens nordlige Side og i en Sænkning, Skjoldshule, 2½ km SØ for Faarevejle Station. Paa en Plads 1300 m NØ for Gislinge Bro er der sket en begyndende Tørvedannelse, som dog hurtigt er blevet afbrudt ved Sten- aldershavets Indtrængen i tidlig atlantisk Tid. Et Ildsted og Rester fra en omlejret Køkkenmødding oven paa de marine Lag viser, at Havet atter har trukket sig noget tilbage, inden 154 Ældre Stenalders højatlantiske Stigning tog Fart og Lito- rinahavet indtog sit mest omfattende Omraade. Ældre Stenalders højatlantiske Stigning tog Fart og Lito- rinahavet indtog sit mest omfattende Omraade. Saltvandsaflejringer. Medens der i et Tidsrum efter Istiden (Fastlandstiden) var tørt Land i betydelige Dele af Nordvestsjælland og til- grænsende Havomraader, som nu ligger under Havets Ni- veau, skete der i Stenalderen en Sænkning, hvorved Havet bredte sig over Landstrækninger, som ogsaa i Nutiden ligger højere end Havfladen. Man finder Sporene af denne Land- sænkning dels som højtliggende Strandvolde langs Kysterne og dels som skalførende Lag af Ler og Sand paa Steder, der nu ligger hævet over Havet, men som dengang var Fjorde og Havbugter. Nordvestsjælland er særlig rig paa Spor efter denne Sænkningsperiode, der med flere Vekslinger først blev afbrudt af en varig Landhævning i den sidste Del af Sten- alderen. Forinden denne Hævning indtraadte, var det nord- ligste Odsherred delt i større og mindre Øer, og Sejerø bestod af mere end en Snes Smaaøer og Holme. Havindskæringerne og deres Fauna. Havindskæringerne og deres Fauna. Fra Store-Bælt har der ord for Reersø været et Par Ind- skæringer, som under den senere Landhævning er bleven udfyldt med Strandvolde og Tørvelag. y g g Den sydligste var ved Udløbet af Halleby Aa, hvis Løb under Landets Tilvækst blev forskudt saa langt mod Syd som Indskæringen strakte sig. Ind i Landet langs Halleby Aa har Bugten næppe naaet længere end til en Morænelerstange Vest for Hovedlandevejen. Ved Boring i Engen tæt ved Vejen til en Dybde af 3 m er der truffet Tørv, hvori der 2 m under Overfladen var et 15 cm tykt Sandlag. Det var uden Spor af Skaller, men da det laa ca. ½ m under Havets Niveau, kan det muligvis være afsat i Forbindelse med Havbugten. Ved Halleby Aa 1 ¼ km Vest for Landevejen er der taget Prøver af Skallagene, der viser den sparsomme Fauna, som har levet her (se Tabellen). - I den mindre Vig læn- 155 gere mod Nord har der levet en lignende mager Mollusk- fauna. K a 1 und borg Fjord har strakt sig 3,5 km længere ind i Landet end nu. I Fjordens inderste Del, Rørmose, fore- kommer der under Strandsand Cardiumler med Skaller. Et langt tykkere Lag findes dog i Munkesø, umiddelbart Nord- vest for Kalundborg, hvor der ved Boring er truffet over (j m Cardiumler, medens der i Rørmose kun er iagttaget et Par Meter. Paa G i s s e 1 ø r e er der i 1928 for Kalundborg Radio udført en 76 m dyb Boring ned i det Plastiske Ler. Efter at der til en Dybde af 32 m var boret i vekslende marine Lag af Sand, Grus og Ler, blev der fra 32 til 4 7 Meters Dybde gennemboret et Lag af graat Dynd med fint Sand og mange Skalstumper. Der er fundet Skaller af Abra alba (?), Bittium reticulatum, Corbula gibba, Lacuna diuari- cata, Mya sp., Mytilus edulis, Nucula sp., Ostrea edulis, Ris- soa inconspicua, Tellina baltica og Echinocardium sp. Dette marine Lag var underlejret af Sand, af Brøndboreren beteg- ne! som »Strandsand«. - Forsaavidt det 15 m mægtige, dybt- liggende, skalholdige Dyndlag stammer fra Litorinatiden - hvad det anførte Skalindhold ikke modsiger - maa det vel :antages, at den usædvanlige Fordybning i Fjordbunden er opstaaet efter langsom Bortsmeltning af en Isklump, der har ligget her som en Rest af Indlandsisen, begravet under Smeltevandssand foran den stagnerende Isrand paa Røsnæs. Havindskæringerne og deres Fauna. S alt bæk V i g havde i Tiden for den maksimale Land- sænkning en betydelig større Udstrækning, end da den i 186() stod foran sin Inddæmning og delvise Udtørring. Den havde Udløbere baade ved Vestsiden og Østsiden og strakte sig mod Sydøst ind i Aaenge til 2 km Vest for Bregninge By. Bundlagene bestaar næsten udelukkende af Sand. Som Prøver fra Saltmade ved Vigens Vestside viser, har Faunaen ikke været righoldig. Ved Vigens Sydøstende i Bregninge Aaenge er der, under 2/3 m Tørv, 1/3 111 Saltvandsdynd med Skaller af Cardium edule (Hjærtemusling), og derunder er dPr et mere end 3 m tykt Tørvelag. Paa G i s s e 1 ø r e er der i 1928 for Kalundborg Radio udført en 76 m dyb Boring ned i det Plastiske Ler. Efter at der til en Dybde af 32 m var boret i vekslende marine Lag af Sand, Grus og Ler, blev der fra 32 til 4 7 Meters Dybde gennemboret et Lag af graat Dynd med fint Sand og mange Skalstumper. Der er fundet Skaller af Abra alba (?), Bittium reticulatum, Corbula gibba, Lacuna diuari- cata, Mya sp., Mytilus edulis, Nucula sp., Ostrea edulis, Ris- soa inconspicua, Tellina baltica og Echinocardium sp. Dette marine Lag var underlejret af Sand, af Brøndboreren beteg- ne! som »Strandsand«. - Forsaavidt det 15 m mægtige, dybt- liggende, skalholdige Dyndlag stammer fra Litorinatiden - hvad det anførte Skalindhold ikke modsiger - maa det vel :antages, at den usædvanlige Fordybning i Fjordbunden er opstaaet efter langsom Bortsmeltning af en Isklump, der har ligget her som en Rest af Indlandsisen, begravet under Smeltevandssand foran den stagnerende Isrand paa Røsnæs. S alt bæk V i g havde i Tiden for den maksimale Land- sænkning en betydelig større Udstrækning, end da den i 186() stod foran sin Inddæmning og delvise Udtørring. Den havde Udløbere baade ved Vestsiden og Østsiden og strakte sig mod Sydøst ind i Aaenge til 2 km Vest for Bregninge By. Bundlagene bestaar næsten udelukkende af Sand. Som Prøver fra Saltmade ved Vigens Vestside viser, har Faunaen ikke været righoldig. Ved Vigens Sydøstende i Bregninge Aaenge er der, under 2/3 m Tørv, 1/3 111 Saltvandsdynd med Skaller af Cardium edule (Hjærtemusling), og derunder er dPr et mere end 3 m tykt Tørvelag. Havindskæringerne og deres Fauna. 156 Vest og Syd om D r a g s h o 1 m har der fra Sejerø Bugt været en Indskæring ind i Dalen Øst for Dragsholm. Den naaede her tæt ind til Lammefjord, som den forblev adskilt fra af en Morænelersvold med en Mindstebredde af 100 m. I den vestlige og den centrale Del af den lille Fjord har deT levet en temmelig rig Fauna; længst mod Øst har Faunaen derimod været yderlig fattig, hvilket understreger, at der her ingen Forbindelse har været med Lammefjord, hvis Fauna har været righoldig til Fjordens inderste Grænse. Ved Vestsiden af Odsherred har der været en stor Hav- indskæring, der langs Kysten har strakt sig fra Høve Strand til Lumsaas og har naaet 4½ km ind i Landet fra den nuværende Kyst. Medens det ved det yderste Par Kilo- meter væsentligst er skalfri Sandaflejringer der findes, fore- kommer der i T r u n d h o· 1 m i Vigens inderste Del en Veksling mellem Saltvandsdynd og Fersk vandsdynd eller Tørv. Der er saaledes 1 km Vest for Svinninge, ved Grav- ning og Boring truffet nedennævnte Jordlag: 0,3 m Tørv. 2,8 m Ferskvandsdynd. 1,3 m Saltvandsdynd. 1,6 m Tørv. 1,6 m Tørv. 0,3 m stenfrit Ler. 0,3 m stenfrit Ler. En lignende Rækkefølge er fundet andre Steder i det for- dums Fjordomraade. Faunaen har ikke været righoldig paa Arter, der er ikke fundet Østers ved nogen af Stederne. Allerøverst i det øvre Tørvelag var det, at den berømte Broncealders Solvogn fandtes i Efteraaret 1902 ved Første- pløjningen paa Stedet. S ej er ø har efter Istiden været underkastet gennemgri- bende Ændringer i Omrids og Udseende. Det flade Land, som forbinder de mange opragende Lerbakker, bestaar næ- sten udelukkende af Saltvandsalluvium og var i Stenalderen Hav. Sejerø var dengang en Samling af 25 Smaaøer og Holme. Men i den forudgaaende Tid, Fastlandstiden, var denne Øgruppe Dele af et større Landomraade end Nutidens Sejerø, hvilket direkte fremgaar af, at der mange Steder 157 langs Øens Kyster forekommer Tørvelag paa den nuværende Havbund. Paa adskillige Steder inde i Landet forekom1ner der Tørv under tilsvarende Forhold, nemlig dækket af Salt- vandslag af Dynd eller Sand. Havindskæringerne og deres Fauna. Der er paa et enkelt Sted ved Haloverbjærg NØ for Sejerby fundet et næsten 2 m tykt Tørvelag, et andet Sted (ved Svaleklint) er der under Salt- vandsdynd truffet Ferskvandslag med en lignende samlet Tykkelse, men med en tre Gange gentagen Veksling mellem Tørv og skalførende Ferskvandsdynd. - De dækkende Salt- vandslag bestaar paa nogle Steder af Ler (Gytje) , men Over- fladelagene er dog mest Sand. En udtagen Prøve ved Hal- overbjærg viser, at der i det derværende Hav har levet en kraftig udviklet Fauna, selvom den udtagne Prøve ikke inde- holdt mange Arter. Paa S j æ 11 a n d s O d de er der fundet Cardiumdynd, som her optræder paa en temmelig sjælden Maade. Stedet er Strandkanten ud for den lille Morænelersbakke Rævehøj, der ligger 1 ¼ km Sydøst for Oddens yderste Spids, Gniben. Det System af Strandvolde, som har strakt sig langs Nord- kysten af Yderby Lyng, men som nu er bortgravede, havde deres nordvestligste Ende ved Rævehøj, hvor de med en Højde af ca. 5 m løber skraat ud mod Stranden og her staar med brat afskaaret Skrænt. Under Strandvoldens Grus fore- kommer der Saltvandsdynd, hvoraf det fremgaar, at der her forinden Strandvoldens Aflejring har været en beskyttet Fjord med roligt Vand, hvori det skalholdige Dynd og Ler kunde afsættes. Ved en Gravning i Tilslutning til den geo- logiske Kortlægning blev der her i Saltvandsdyndet fundet Kraniet af en Delfin, Lagenorhynclws albirostris (Hvid- næse), som ogsaa i Nu tiden kan træffes i vore Farvande. - I den sydlige Del af Y derby Lyng skal der lejlighedsvis være truffet Tørv under de marine Sandlag. Paa den lave Strækning mellem Overby og Ebbeløkke har der under Litorinatidens Landsænkning været et Sund tværs over Sjællands Odde. I Sandlagene i den østlige Del af Lav- ningen forekommer der Skaller adskillige Steder. Særlig Interesse har en Forekomst i Strandvolden længst mod Nord- vest i Overby Lyng, hvor der er truffet skalførende, marint 158 Sand og Dynd ved en Højde af indtil 3,2 m over den nuvæ- rende Hav flade (Side 170). I samme Tidsrum var Højlandet ved Klintebjærg en 0, der var adskilt fra Hovedlandet ved et Sund over Sonnerup Skov og Klintsø. Havindskæringerne og deres Fauna. Denne sidste udgjorde Sundets dybeste Del, der ved Strandvolde afspærredes fra direkte Forbin- delse med Havet og derved blev en lukket Fjord med en temmelig rig Fauna; delle fremgaar af Skalindholdet i en Køkkenmødding Vest for Tengslemark ved Kanten af Klintsø (se Tabellen). Ved Boring i K 1 in t s ø ned til en Dybde af 5 m igen- nem Gytje er derimod kun truffet Skaller af Cardium edule og Mytilus edulis. Ved Landhævningen blev den afspærrede Fjord, hvis Bund nu ligger indtil 1 m under Havfladen, om- dannet til en Sø, Klintsø, der i 184Oerne blev tørlagt og gjort til en frugtbar Eng. Ved T en g s 1 em ark ved Sydsiden af Klintsø fandtes der ved den geologiske Kortlægning i 1896 to Moser, hen- holdsvis Sydvest og Sydøst for Byen, som indeholdt Skallag med baade Saltvands- og Ferskvandsmollusker. Moserne var ved en Strandvold adskilt fra den marine Klint Fjord. I den vestlige Mose var der et enkelt Lag med Saltvandsfor- mer og saavel dækket som underlejret af Ferskvandslag; i den østlige Mose var der to saadanne Lag, adskilte af et Ferskvandslag. Denne sidste Mose har senere været gjort til Genstand for en speciel Gennemboring og Undersøgelse, som sammen med Undersøgelser andre Steder viser, at Sten- aldersænkningen ikke strakte sig over en enkelt sammen- hængende Periode, men at Stenalderen rummede flere, gen- tagne Sænkninger og Hævninger. Fig. 46 gengiver et Profil gennem Mosen, der viser den skiftende Lagfølge. I Følge Undersøgelsen i 1896 var det øverste marine Lag det, der var rigest paa Mollusker; i det nedre Lag har Arterne om- fattet Carclium edule (Hjærtemusling), Hydrobia ulvæ (begge talrige), samt enkelte Mytilus edulis (Blaamusling). - Fig. 4 7 gengiver et Pollendiagram fra Mosen (Punkt 5 i Tvær- profilet). De i dette Diagram angivne relative Mængder af Træpollen (Støvkorn) viser, i hvilke Afsnit af Stenalderen 159 og den postglaciale Skovudvikling Havets Indbrud i Søen har fundet Sted. Det fremgaar deraf, at det første fandt Sted i sidste Del af Ældre Stenalder, og at den anden Fjord- periode ligger i Begyndelsen af Yngre Stenalder, begge temmelig centralt i Egetiden i Østdanmark. I Tilslutning til Landsænkningen i den sidste Fjordperiode maa da den og den postglaciale Skovudvikling Havets Indbrud i Søen har fundet Sted. Havindskæringerne og deres Fauna. Det fremgaar deraf, at det første fandt Sted i sidste Del af Ældre Stenalder, og at den anden Fjord- periode ligger i Begyndelsen af Yngre Stenalder, begge temmelig centralt i Egetiden i Østdanmark. I Tilslutning til Landsænkningen i den sidste Fjordperiode maa da den "'NV "'NV SØ 2 3 4 5 G, 7 8 ~ Q " '<:l "' l "'i s~·en to -4 MO 70 100 220 300 G, ±o -lf M \!uld Phrag1ni- Claclium- 1Fll es11mp- J<c.dk- tes Tørv Tør,· Tørv liytjc Gy lje C.:,rdium- Diluvit11n gy tj e Fig . .i6. Profil gt'nnem Mose verl Tengslemark. Efter Iben .J"sSEN (D. G. F. 19:l7). SØ Fig . .i6. Profil gt'nnem Mose verl Tengslemark. Efter Iben .J"sSEN (D. G. F. 19:l7). tilgrænsende Strandvold være afsat, hvorved Mosen for be- standig afspærredes fra Havet. En tilsvarende Udvikling som her ved Tengslemark har samtidig fundet Sted ved Sø- borg Sø i Nordøstsjælland og andre Steder (se Noterne). Øst for Nykøbing var der i Tiden for den dybeste Land- sænkning et bredt Sund. Den sydligste Del deraf laa indtil 1869 og 1871 som to flade Vige, som dengang blev inddiget og tørlagt. Deres Bund bestaar mest af Sand; i den østligste, Hovvig, levede en ret sparsom, marin Fauna. S i di n g e F j o r d er en fra Lammefjord af dæmmet og ved Udpumpning tørlagt Havvig, hvis dybeste Del ligger 3 m under Havfladen. Dens Tørlægning paabegyndtes i 160 0 M 2 3 4 s A 8 I I -- -- C f-- ------ -- E ---- F ~-,r. - ----- - --- -- G -- -- ---- -- -- - - 10 20 30 40 50 GO 70 80 90 100 150 X I I I I Meget lidt Po/len, \_ 7t-L I ,r - , 7----- ,W -r--r-- ~ ----@-- Pil --o- Birk ---- > VIlI - -Cl-- ------ vm -0--- Fyr Hasse! 1Elm I.ind Eg .'Elm + Lind + Eg _,.__ Bøg 200 250 300 350¾ Vlla. VI Fig. 4 7. Po liendiagram fra Mose ved Tengslemark. Efter KNUD JESSEN (D. G. F. 1937). Fig. 4 7. Po liendiagram fra Mose ved Tengslemark. Efter KNUD JESSEN (D. G. F. 1937). 161 1842. Langs Fjordens sandede, fordums Strandbred findes Levninger af den recente Fauna, hvori Mya arenaria, men dog navnlig smaa Cardium edule dominerer. Havindskæringerne og deres Fauna. Længere ude i Fjorden optræder der større Eksemplarer, og her bliver ogsaa Scrobicularia piperata hyppig, ligesom der tillige fin- des adskillige andre Arter, endog en enkelt lille Østers. Skal- lerne af Østers tiltager baade i Antal og Størrelse indad i Fjorden, hvor der ogsaa findes enkelte Østersbank.er. Langs Fjordens Midtlinie fra Udløbet til Vestenden er der frugtbar Dyndjord, og Dyndlaget skal i Nærheden af Dæmningen have en Tykkelse af 6 m. Lamme fjord, der sammen med Svinninge Vejle lig- ger ved Sydgrænsen af Odsherred, er den største af Ind- skæringerne fra Isefjord. I Stenalderen har den som før nævnt kun været afspærret fra Forbindelse med Sejerø Bugt ved en smal Landstrimmel »Draget« ved Dragsmølle Nord- vest for Hørve. Men det havdækkede Omraade var i Sten- alderen ikke væsentlig større end i Tiden forud for Fjor- dens Tørlægning. De dybeste Indskæringer var Øst for Gis- linge og langs Gislinge Aa. Svinninge Vejle blev udtørret i 1852- 54, og i Aarene 1873- 77 fandt Udtørringen af den ca. 5000 ha store Lammefjord Sted, saaledes at Vandstan- den sænkedes ca. 4 m. I den vestlige Del bestaar Fjordbun- den af Dyndjord, og der findes i den øvrige Del af Fjorden spredte Pletter med Dyndjord, medens Overfladelagene her iøvrigt er Sand, paa nogle Steder dog kun som et tyndt Lag over Moræneler. l Litorinatidens Lammefjord har der levet et righoldigt Mollusksamfund, navnlig i Fjordens vestlige Del. Baade i Svinninge Vejle og i Lammefjord har der været rige Østers- banker med stor~ Skaller. Især er der Grund til at bemærke den rige Fauna, der har levet i Fjorden paa det Sted, hvor Landvandskanalen omkring den inddæmmede Fjord ved en særlig Kanal har Forbindelse med Sejerø Bugt. Som Tabellen viser, har der umiddelbart Øst for Kanalforbindelsen levet en særdeles artsrig Fauna med store Eksemplarer. I den lille Indskæring fra Sejerø Bugt ved Dragsholm har Fau- naen baade været mindre artsrig og bestaaet af mindre Dyr. 11 162 Denne Forskel stemmer godt overens med, at Jordbunds- forholdene ogsaa taler imod, at der har været nogen marin Forbindelse over det lave og smalle »Drag«, som nu er gennemskaaret af Kanalen mod Vest til Sejerø Bugt. - I de Prøver af Skallagene, der er udtaget i de inddæmmede Fjorde, forefindes ogsaa recente Former som Mya arenaria og andre, saaledes som Faunalisten viser. Havindskæringerne og deres Fauna. I »Fastlandstiden« forud for Litorinahavets Lammefjord var der her tørt Land med Skov og Tørvemoser og Mulig- hed for Beboelse (se Side 153) . I Kisserup Enge ved Nordsiden af Tuse Næs har der været en Indskæring med en Fauna, der dog ikke var saa rig som den i den indre Del af Lammefjord. Ved Orø har der været adskillige mindre Indskæringer. Den største fandtes ved Salvig, Nord for Øens Kirkeby, men der har dog ikke her levet nogen artsrig Fauna. H o l b æ k F j o r d har i Litorinatiden strakt sig be- tydelig længere mod Vest end i Nutiden, nemlig langs Tuse Aa til ca. 1 ½ km Sydvest for Tuse Bro, og som et bredt Sandbælte til 3 km Vest for Tuse Aas Udløb i Fjorden. Den lave Sandflade har som vestlig Fortsættelse en smal Lav- ning, som man har formodet dannede Saltvandsforbindelse mellem Holbæk Fjord og Lammefjord i Litorinatiden. En saadan Forbindelse er dog ikke direkte paavist, og det maa anses for tvivlsomt, at den har eksisteret. - Langs den ydre Del af Holbæk Fjord har der ingen egentlige Indskæ- ringer været, men der er dog enkelte Steder fossilførende Lag, saaledes ved Bognæs paa Tuse Næs og ved Langehage 2 km Øst for Holbæk. Syd for Orø strækker Isefjords sydligste Udløber, Brams- n æ s B u g t og Te 111 p el k ro g, sig forbi Eriksholm til Aagerup. I Litorinatiden naaede Fjorden endnu henved 2 km længere mod Syd, til nogle Smaaholme Øst for Marup. Her er fundet Sand- og Dyndlag med en temmelig righoldig Fauna og med veludviklede Former. Listen over fundne Molluskskaller giver en Oversigt over Indholdet af Mollusker i Litorinahavets Indskæringer. For- skelligheden fra Forholdene i Nutiden viser sig i, at der 163 dengang forefandtes Arter, som nu ikke lever i disse Egnes Farvande; blandt disse er der særlig Grund til at nævne Østers ( Ostrea edulis), som dengang optraadte i rigelig Mængde og veludviklede Eksemplarer. Foruden Østers fandtes et stort Antal andre spiselige Muslinger: Blaamusling (M ytilus edulis), Hjærtemusling (Cardium edule), samt Strandsnegl (Litorina litorea). Til mange af Litorinahavets Kyster er derfor knyttet Forekom- sten af Skaldynger som Levninger efter Stenalderfolkenes Bopladser og Maallidspladser. De findes i størst Antal om- kring de inderste Dele af Isefjords Indskæringer, men der forekommer ogsaa betydelige Skaldynger nordligst i Ods- herred, f. Eks. ved Klintsø og andre Steder. Strandaflejringerne og deres Højde. Den Tilvækst, det nordvestsjællandske Landskab har faaet Tiden fra dels dybeste Nedsænkning indtil Nutiden, giver sig ikke blot Udtryk i, at tidligere Havomraade er overgaaet til Landjord. Udjævningen af Kystlinierne og Forbindelserne mellem Landets daværende Øer og Fremspring, hvorved Landomraadet har naaet sit nuværende Udseende, er det ogsaa muligt at følge den gradvise Udvikling af. Formen for Tilvækstens Forløb kan adskillige Steder direkte aflæses ved Hjælp af Slrandvolde, der afsattes efter som Kystfor- skydningerne fandt Sted; disse Serier af Strandvolde afspej- ler paa oplysende Maade Tilvækstens gradvise Fremadskri- den. Sammen med andre Forhold langs Kyslerne viser de endvidere, at Landhævningens Størrelse ikke var den samme overalt, m en var tiltagende fra Sydvest mod Nordøst. I den foreliggende Del af Sjælland har Landhævningen siden den dybeste Nedsænkning rimeligvis været 1 ½ - 2 m længst mod Sydvest og 4½-5 111 længst mod Nordøst. Ved Storebæltskysten Syd for Asnæs er der to Indskæ- ringer fra Litorinahavet, lukkede af smukke, regelmæssige Systemer af lave Strandvolde, der naar en Maksimums- højde af omkring 3 m. Tilførselen af Materialet er foregaaet fra Nord mod Syd. Systemet ved den nordlige Indskæring, 164 Sydvest for Svallerup, rummer ialt 15 Enkeltvolde. Ved den sydlige har Opbygningen været mere kompliceret, Strand- voldene har her dannet en Spærring for Udløbet af Balleby Aa og har efterhaanden tvunget Aaens Løb mere og mere sydpaa til dets nuværende faste Plads. Forløbet af de ældste og inderste Strandvolde ved Systemets Nordende viser, at det nordligere Højland ved Bjærge Klint i Tidens Løb har været Genstand for en betydelig Beskæring ved Havets Angreb, og at Kysten her siden Stenalderen er rykket flere Hundrede Meter tilbage. Sydvest for Svallerup, rummer ialt 15 Enkeltvolde. Ved den sydlige har Opbygningen været mere kompliceret, Strand- voldene har her dannet en Spærring for Udløbet af Balleby Aa og har efterhaanden tvunget Aaens Løb mere og mere sydpaa til dets nuværende faste Plads. Forløbet af de ældste og inderste Strandvolde ved Systemets Nordende viser, at det nordligere Højland ved Bjærge Klint i Tidens Løb har været Genstand for en betydelig Beskæring ved Havets Angreb, og at Kysten her siden Stenalderen er rykket flere Hundrede Meter tilbage. R e er s ø har i Litorinahavets Tid bestaaet af to Øer og to ganske smaa Holme. Den nuværende Halvøs nordligste Del og Forbindelsen med Hovedlandet er bygget op af lave Strandvolde. Strandaflejringerne og deres Højde. Det samlede System af Strandvolde her har et ejendommeligt Forløb og viser, at Landomridset stærkt har skiftet Udseende i Løbet af hele Systemets Dannelsestid. - Forløbet af den vestlige, ældste Halvdel af Strandvoldene, indtil den lille, 6 m høje Grusbakke, Hesselbjærg, viser, at de maa være afsat ud fra et Højland, der naaede nogle Hundrede Meter længere mod Nord end Reersøs nuværende Nordvesthjørne. I Læ mellem de inderste Strandvolde og det nu bortskaarne Højland opstod en Lagune, hvis sidste Rest er Store Saltsø, 1 km Nordvest for Reersø By; dens Over- flade ligger lavere end Havfladen. Under fortsat Afsætning af Strandvolde og Kystens Forskydning mod Øst, rykkede Højlandets Nordende ved Havets stadige Nedbrydning til- bage imod dens nuværende Plads. Denne Udvikling har fort- sat sig, indtil Kysten var naaet frem mod Øst til Hessel- bjærg, som Strandvoldene fra Vest støttede sig til. Paa dette Tidspunkt kan Havstrømmen endnu have haft fuld Passage igennem Vejlen, mellem Højlandet ved Reersø By mod Vest og Hovedlandets høje Fremspring »Hals« mod Øst. Ved Vestenden og ved Nordsiden af dette Højland har Havets Erosion frembragt høje Skrænter med mange store Sten ved Foden, og i Tilslutning til Skrænten ved Nordsiden af Højlandet er der mod Øst afsat en Strandvold. I den efter- følgende Tid indsnævredes Gennemløbet ved Vejlen mere og mere, efter som Afsætningen af Strandvolde Øst for Hes- Molluskerne Nordvestsjællands Fjordaflejringer fra Litorinatiden. D. G. U. V. R. Nr. 6. Til Side 154- 163. Store-Bælt Sejerø Bugt Kattegat Isefjord ...; "O "O bl) ..§ i> t/l .... ...; ...; i> .... "' .... ~ 8 0 i> .... ~ c:I -a " ~ 8 0 z +l 8 >, "" 8 8 i> ~ z > Sl ... .... .E .... 'o ..q "" .8 .!I( 8 "Cl 8 ~ ...; z z ,"'I til os ., z ~ o.> .... i> 8 i> ~ 8 i> ,"'I .... 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X X X X X X X X X X X X X 1 Ekspl. X X X X X X X X X X X X ta . .... . ......... . . X X X nspicua • .. ... .. ... 1 Ekspl. X X X X X X X X 1 Ekspl. X X X X X X X X X 2Ekspl. X X mbranacea . .... ..... X X X X X X X X X X X 1 Ekspl. 1 Ekspl. X X X X X X uncatulus . ......... 1 E kspl. X X X X 1Ekspl. btusus .. ..... . ..... X X ta . .. . .... . ....... . X undatum .... ..... .. X X uiatilis . ........... X ..... ' ...... .. .... I I X X X X 165 selbjærg og den dermed sammenhængende Kystforskydning mod Øst skred frem. Forinden Afsætningen af Strandvolde naaede frem til Hes- selbjærg, laa denne Bakke som en Holm. Svage Antydninger af Strandvolde Syd derfor antyder dog, at der her var duk- ket tørt Land frem, dengang Holmen gik ind i Kystlinien. Strandaflejringerne og deres Højde. Hesselbjærg kom nu til at ligge som et Hjørnepunkt i en Kystlinie, hvis Fortsættelse h erfra gik i sydlig Retning, gan- ske svarende til den Stilling, Skæreby Bakke (se Side 172) havde, forinden Sundet imellem Nykøbing og Rørvig var begyndt at udfyldes med det derværende, vifteformige Sy- stem af Strandvolde. Men medens Skæreby Bakke vedblev at være Drejningspunkt under hele Forløbet af dette Systems Opstaaen, blev Hesselbjærg straks forladt som Kystens Drejningspunkt. Dette har fra en Beliggenhed nogle Hun- drede Meter Nordvest for Hesselbjærg været Genstand for en stadig Forskydning i østlig Retning, medens det 800 m brede Bælte af Strandvolde ØNØ for Hesselbjærg opstod. Samtidig med at disse Strandvolde afsattes, indsnævredes - og lukkedes tilsidst - Vandforbindelsen mellem Vejlen Øst for Reersø og den lille Vig, Flasken, hvori Halleby Aa har sit Udløb. Et Forsøg paa at indtegne hele Forløbet af Kystændrin- gen ved Reersøs Nordside paa et Kort vil vise, at Havet - som efterhaandein bortskar mere og mere ved Vestsiden af Reersøs vestlige Højland - i Tilknytning dertil opbyggede en mod Øst stadig voksende Odde, og stadigvæk Nord for den nuværende Kysts Plads. Samtidig med Oddens Vækst mod Øst rykkede Højlandets Nordspids automatisk tilbage som Følge af Nedbrydningen langs Vestkysten. I Sammen- hæng hermed maatte ogsaa de ældste Strandvoldes nord- vestligste Dele blive bortskaaret, efterhaanden som Landets Tilvækst mod Øst fortsattes. Opbygning og efterfølgende Bortskæringer gav da Kystlinien en stadig skiftende Plads, inden den fik sin nuværende Form. Men Beskæringen ved Vestkysten af Reersø er stadigvæk i Gang, saa Landet her er ikke i Ro. 166 Langs Kysten foran Højlandet fra Reersø til Vestpynten af Asnæs er der kun en smal Forstrand bag en ung Ero- sionsskrænt; først ved Sydvestspidsen af Asnæs er der en Terrasse med en Strandvold, som dog kun naar en Højde af 3 m. Ved Havnemark paa den nordre Side af Næsset naar en Strandvold Højden 3,5 m. I Litorinahavets Indskæ- ring Øst for Kalundborg Fjord naar en Strandvold Højden 3 m, og den samme Højde 11aas paa Gisseløre ved Kalund- borg. Langs Sydkysten af Røsnæs er der omtrent overalt unge Erosionsskrænter, og Sydvest for Ulstrup er der en til- svarende lav Strandterrasse. Ved Nordkysten af Røsnæs er der Nord for Bjørnstrup set et tyndt Lag Strandgrus paa Moræneler ved en Højde af 4,7 m. ') I Kortbladsbeskrivelsen slaar fejlagtigt: N f. Aagerup. 1) En i Kortbladsbeskrivelsen anført Højde 5,8 m for en enkelt Strand- vold er en Trykfejl for: 3,8 m. Strandaflejringerne og deres Højde. Nordøst for Aagerup1 ) og Nord for Kallerup er der Strandvolde med Højderne hen- holdsvis 3,6 og 3,8 m. Videre mod Øst findes der Nord for Saltbæk Vig enkelte, ret fremtrædende Strandvolde. Det er ved Store Vrøj, hvor- fra der mod Sydøst strækker sig et voldformigt Grusbælte; det deler sig i Lo parallelt løbende Volde, af hvilke den yderste kun er 1½ km lang, medens den inderste har en Længde af 3- 4 km. Der kan maaske være Grund til at for- mode, at de fra første Færd har haft deres Udspring fra en Diluvialø Nordvest for Store Vrøj. I Indskæringen fra Sejerø Bugt ind i Odsherred har Li- torinahavet efterladt et bredt Bælte med Sand og Grus, der i Tidsrummet for Landhævningen er afsat langs Kysten af det tilbagevigende Hav. I store Dele af Omraadet er de ma- rine Lag dog blevet dækket af Flyvesand. Strandvolde er ikke meget fremtrædende, men der forekommer dog en- kelte; deraf naar de inderste gennemgaaende Højder lidt over 5 m. At Havets Angreb lokalt har været stærkt, ses Sydvest for Gudmindrup, hvor der findes en Erosionsskrænt, som er indtil 15 m høj. Sejerø s Overflade bestaar for Størstedelen af Salt- vandsalluvium, der ved Landhævningen efter Stenalderen kom til at forbinde de tidligere Øer og Holme til et samlet 167 Fig. 48. Gissclørc ved Kalllnclborg. Fig. 48. Gissclørc ved Kalllnclborg. 168 Hele. Det indre Bælte af marine Lag, der strækker sig igen- nem Landet, bestaar af Sand og Ler. Hvor Saltvandsallu- viet grænser op til Kysten, er det paa de fleste Steder i form af Strandvolde, hvis Højder almindeligst ligger imel- lem 3,5 m og 5 m. 1 ) Kun paa et enkelt Sted, nemlig om- kring Mastrup ved Sejerøs Sydøstende, strækker der sig Strandvolde ind i Landet. Deres Beliggenhed viser, at de maa have strakt sig flere Hundrede Meter længere mod Sydvest og Syd uden for den nuværende Kyst, hvor de senere atter er skaaret bort af Kyststrømmen. Denne maa saaledes i Tidens Løb have faaet en stærkt ændret Retning. Det er i Forbindelse med en saadan Retningsændring, at den lange Landspids, Knoben, har kunnet opstaa. Under Dannelsen af Strandvoldene ved Mastrup maa Strømmen være kommet fra Sydøst, medens Dannelsen af Knoben forudsætter en modsat Retning. Hele. Det indre Bælte af marine Lag, der strækker sig igen- nem Landet, bestaar af Sand og Ler. Strandaflejringerne og deres Højde. Hvor Saltvandsallu- viet grænser op til Kysten, er det paa de fleste Steder i form af Strandvolde, hvis Højder almindeligst ligger imel- lem 3,5 m og 5 m. 1 ) Kun paa et enkelt Sted, nemlig om- kring Mastrup ved Sejerøs Sydøstende, strækker der sig Strandvolde ind i Landet. Deres Beliggenhed viser, at de maa have strakt sig flere Hundrede Meter længere mod Sydvest og Syd uden for den nuværende Kyst, hvor de senere atter er skaaret bort af Kyststrømmen. Denne maa saaledes i Tidens Løb have faaet en stærkt ændret Retning. Det er i Forbindelse med en saadan Retningsændring, at den lange Landspids, Knoben, har kunnet opstaa. Under Dannelsen af Strandvoldene ved Mastrup maa Strømmen være kommet fra Sydøst, medens Dannelsen af Knoben forudsætter en modsat Retning. Paa S j æ 11 ands O d de giver Strandaflejringerne et vigtigt ~idrag til Forstaaelsen af, hvorledes Kystens Forløb har ændret sig fra Litorinatiden til nu. De har paa to Steder bidraget til at gøre Odden til et Hele, nemlig mellem Gniben og Y derby og mellem Over by og Ebbeløkke. Det særlig karakteristiske for det første Omraade var det smukt udviklede System af Strandvolde, hvis Naturpræg nu er fuldstændig forsvundet ved Bortgravningen og Ud- nyttelsen af Strandvoldenes Sten til Skærver. Strandvoldene fandtes som et flere Hundrede Meter bredt Bælte langs Od- dens Nordside fra Rævehøj til Brokkebjærg ved Yderby. De største Sten fandtes mod Nordvest ved Rævehøj, hvor de gennemgaaende naaede 1 Kubikfod. Størrelsen aftog grad- vis mod Sydøst, hvor Stenene jævnlig erstattedes af Sand. De største Højder, Strandvoldene har naaet, var 7 m ved Brokkebjærg og 6 m Sydøst for Rævehøj. Udformningen af hele Systemet er foregaaet fra Nord- vest mod Sydøst. Voldenes Retning og Underlag ved Ræve- høj viser, at det oprindelige Udgangspunkt for Dannelsen 169 af dem har ligget adskilligt østligere end Sjællands Oddes nuværende Yderspids Gniben. Ved Rævehøj ligger de ældste Strandvolde skraat ud imod Kysten og ender brat med en Skrænt ved en Højde af omkring 5 m. En endnu ældre Af- deling af Systemet har krydset den nuværende Kystlinie 2- 300 m længere mod Sydøst. Det fremgaar deraf, at Kyst- relningen har skiftet stærkt i Tidens Løb. Dertil kommer, som det før er omtalt (Side 157), at Strandvoldenes Underlag ud for Rævehøj er Fjorddynd. Strandaflejringerne og deres Højde. Der har her været en Fjord mellem Rævehøj og Strandvoldene; disses oprindelige Plads bar ligget mindst et Par Hundrede Meter østligere end Ræve- høj. Medens Kystretningen nu er NV- SØ, maa den oprinde- lig have været N- S. Udgangspunktet for Strandvoldene maa derfor have ligget adskillige Hundrede Meter Øst for Gniben. Af denne ændrede Kystretning og af Størrelsen af Materiale- mængden i Strandvoldene mellem Rævehøj og Brokkebjærg kan man drage den Slutning, at den lille Morænelersbakke, som nu danner Spidsen af Sjællands Odde, kun udgør 1/10- ½o af det Højland, hvis sidste Rest den er. Medens denne nuværende Rest maaler omkring 9 ha, maa Størrelsen af det oprindelige Istidsland i Stenalderen have ligget imellem 1 og 2 km2 • Saaledes som Kortet viser, er der ved Sydsiden af Y derby Lyng, Sydvest for Y derby og vestligere, smaa Partier med Strandvolde. Ved det østlige Parti viser Stillingen i Forhold til Kysten, at Afsætningen - til en Højde af 5 m - op- rindelig skyldtes en vestgaaende Kyststrøm, men at Strøm- retningen er ændret til østgaaende, hvorefter der er fore- gaaet baade en Bortskæring og en fornyet Afsætning af la- vere Strandvolde. Ved det vestlige Parti og ved et Sted 1 km længere mod Nordvest viser Forholdene, at Kystretningen efter Afsætningen af Strandvolde paa disse Steder er ændret i Sammenhæng med en Beskæring. Bortset fra de nævnte Systemer af Strandvolde skyldes Y derby Lyngs øvrige Sandflade den almindelige Landhæv- ning op til en Beliggenhed af 2- 3 m over Havfladen. Ved Over by Lyng har Stenalderhavet tværs igen- nem Landet haft et Sund, som har omsluttet en lille Mo- 170 rænelersø. Den væsentligste Gennemstrømning er foregaaet Vest for denne 0 og har her afsat et System af Strandvolde, hvis Forløb viser, hvorledes Sundet er blevet snævret ind og tilsidst ganske lukkedes. Derefter afsattes langs de ny Kyster Bælter af Strandvolde - fra Vest - til en Højde af 6,9 111 ved Nordkysten og 5 m ved Sydkysten. - Hosstaaende er afbildet et Snit fra en Gennemskæring af den yderste Strandvold langs Nordkysten, længst mod Vest (Fig. 49). Snittet viser, at der her, forinden Strandvolden blev bygget op, var smult Vand, hvori Mollusker kunde trives. Strandaflejringerne og deres Højde. Det skal- førende Sandlag naar en Højde af 3,2 mover den nuværende Havflade, Strandvoldens største Højde er 6,9 m, og det kan derefter paaregnes, at Landet her i Tiden for dets dybeste Sænkning har ligget omkring 4½ m lavere end i Nutiden. Østpaa fra Overby Lyng, N o r d o m E b b e 1 ø k k e, er der Sænlmingsperioden igennem sket Bortskæring langs Ky- sten. Erosionen har fra den nuværende Kystskrænt strakt sig 2- 3 km videre ind i Landet ved Vestsiden af Sonnerup Skov. Herfra er Strømmen gaaet videre i østlig Retning gennem Sundet mellem Tengslemark og Klint og har derfor ogsaa paa sin Vej fremad stødt imod Vestsiden af Klintøen og har her frembragt en 8- 16 m høj Stejlskrænt af nogle Hundrede Meters Længde. Man kan maaske ogsaa formode, at det var paa samme Tid, at der ved Tengslemark blev op- kastet en Strandvold, foran de tidligere (Side 158) nævnte Bassiner med skiftevis Ferskvandslag og marine Lag, for- inden Klintsø blev afspærret fra sin direkte Forbindelse med Havet mod Nordøst. Kun i den yderste Del af S o n n er u p S k o v er der fremtrædende Strandvolde, som viser Forløbet af Landets Tilvækst. Højden af den tidligere Havbund tiltager jævnt fra Klintsø mod Nordvest ud imod Kysten, hvor Strand- voldene naar de største Højder. Det maa da antages, at der først er sket nogen Tilsanding af Sundet i Sonnerup Skovs sydøstlige Del, forinden de inderste Strandvolde kunde frem- komme, støttet til den skarptskaarne Kystskrænt ved Ebbe- løkke. Højderne stiger fra 3- 4 m til 7 m længst mod Nord- vest; Retningen af Strandvoldene er omtrent parallel med 7'i'N 6 :i t, :1 2 () ~--.---------------,-----,------,--------------,--------,------------ 0 JO 20 30 40 50 60 70 80 90 Fig. 49. Tværsnit i Strandvold NV i Overby Lyng. 1896. R '· 100"' et Strandgrus, b Sand, nedcrsl med Skaller a[ Saltvandsmollusker, c Dynd og »Tanglørv« med Skaller og Planlclcvninger, d Sand. Efter D. G. U. I. R. Nr. 8. >-' -.J >-' R '· >-' -.J >-' Fig. 49. Tværsnit i Strandvold NV i Overby Lyng. 1896. et Strandgrus, b Sand, nedcrsl med Skaller a[ Saltvandsmollusker, c Dynd og »Tanglørv« med Skaller og Planlclcvninger, d Sand. Efter D. G. U. I. R. Nr. 8. Strandaflejringerne og deres Højde. 172 Kysten, som de dog skærer under en meget spids Vinkel og støder op til med en stejl Skrænt, der er Genstand for Bort- skæring. Den Ændring, der saaledes er foregaaet ved denne Kyst fra Aflejring til Bortskæring, skyldes sikkert, at Høj- landets Kyst ved Klintebjærg som Følge af Erosionen er blevet rykket tilbage til sin nuværende Plads fra en tidligere, nordligere Beliggenhed. Derved skabtes der Mulighed for, at Kyststrømmen ogsaa ved Sonnerup Skov kunde gaa over til at angribe Landet, hvor den før selv havde bygget op. Mod Nordøst er K 1 i n t s ø adskilt fra det aabne Hav ved et Bælte af Strandvolde. Nærmest ved Klintebjærg naas Højden 5,7 m og ved Sydøstenden 7 m, hvorimod Højden i den midterste Del ikke naar 5 m. Materialet er af Strøm- men taget fra Klintebjærg Bakkerne; efter Mængden at dømme kan man regne, at disse Bakker forud for Strand- voldenes Dannelse har strakt sig adskillige Hundrede Meter længere mod Nord end nu. De kan ved denne fremskudte Beliggenhed have været stærkt medvirkende til, at der ved Nordsiden af Sonnerup Skov fandt en Tilvækst af Landet Sted, hvor der nu skæres bort. Strandaflejringerne i V es ter 1 y n g og Ø s ter ly n g, de lave Strækninger Nordvest, Nord og Nor·døst for Nykø- bing, viser særdeles anskueligt, hvorledes Kystforskydnin- gen og den gradvise Indsnævring af det brede Sund Øst for Nykøbing er foregaaet. Den første Lukning er sket mellem Skæreby Bakke og Højlandet Nord for Nykøbing, hvor en Strandvold med stærkt buet Forløb har hindret Kyststrøm- men fra Nord vest i at trænge ind over den nyskabte Bar- riere, der ligger ved en Højde af 6 m. Der er paa Forhaand kun Grund til at antage, at denne Lukning fandt Sted, kort forinden de inderste Strandvolde ved Vesterlyng blev afsat. I Tilslutning til disse sidstes Opstaaen er der frembragt en skarp, indtil 5 m høj Erosionsskrænt ved Nordvestsiden af Torsbjærg, 1 km NV for Nykøbing, hvorimod der ved Høj- landets Nordøstside ingen Erosion er at spore i Forbindelse med den først opstaaede Spærrevold. De inderste Strandvolde i Vesterlyng har støttet sig til Erosionsskrænten ved Torsbjærg og har derfra strakt sig vi- 173 dere mod Nordøst til Skæreby Bakke. Deres Vestender peger vinkelret mod den nuværende Kyst og viser, at Kystliniens Forløb har været vidt forskelligt fra det nutidige. Strandaflejringerne og deres Højde. Højlan- det mellem Klintsø og Vesterlyng maa tydeligvis have naaet omkring 1 km længere mod Nordøst end nu, saa at Havet herfra har fjærnet mindst ½ - 1 km2 Højland, som det har opbygget Strandlagene i Vesterlyng med. Samtidig med den ældre Afdeling af Strandvoldene i Ve- sterlyng er ogsaa det ejendommeligt slyngede System af Volde i Østerlyng opstaaet. For hele dette System har Ud- gangspunktet været Skæreby Bakke i dens nuværende Om- fang. Materialet til Opbygningen maa derfor være hentet længere borte fra. Forbindelsen mellem Kattegat og Nykø- bing Bugt er af dette System af Strandvolde blevet indsnæv- ret til Indskæringen Hovvig. Først ved Udfyldningen af de nordligere Flader, Nykøbing Lyng og Nakke Lyng med Sand, i Sammenhæng med Afsætningen af de ydre Rækker Strandvolde i Vesterlyng, er Sundet helt blevet lukket. Paa Strækningerne omkring R ø r vi g og N ak k e er der efter Litorinatiden baade sket Beskæringer langs Ky- sterne og foregaaet Tilvækst af Landet. Begge Dele har især Nord for Rørvig efterladt sig fremtrædende Spor, bl. a. om- kring Lagunesøerne Dybesø og Flyndersø, hvor der er en 10 m høj Erosionsskrænt og Strandvolde med Højden 6 m. Fælles for hele Odsherreds Nordkyst er, at Erosion og Tilvækst overalt er sket fra Vest. Dette har trods alle For- skellighederne givet Strandvoldenes Orientering og Aflejrin- gernes uensartede Kornstørrelse et fremtrædende Fællespræg med grovere Materiale mod Vest og finere mod Øst. Paa Strækningerne omkring R ø r vi g og N ak k e er der efter Litorinatiden baade sket Beskæringer langs Ky- sterne og foregaaet Tilvækst af Landet. Begge Dele har især Nord for Rørvig efterladt sig fremtrædende Spor, bl. a. om- kring Lagunesøerne Dybesø og Flyndersø, hvor der er en 10 m høj Erosionsskrænt og Strandvolde med Højden 6 m. Fælles for hele Odsherreds Nordkyst er, at Erosion og Tilvækst overalt er sket fra Vest. Dette har trods alle For- skellighederne givet Strandvoldenes Orientering og Aflejrin- gernes uensartede Kornstørrelse et fremtrædende Fællespræg med grovere Materiale mod Vest og finere mod Øst. Flyvesand. Der findes i Nordvestsjælland adskillige, større og mindre Arealer med Flyvesand. Det er udelukkende i Kystegnene, det forekommer, og det er overalt Strandsand, som har været Genstand for Sandflugt af nævneværdig Betydning. Prøver, som er udtaget i Forbindelse med den geologiske Kortlægning, har vist, at .dette Sand ved Vindsorteringen er 174 blevet saa ensartet, at Kornstørrelsen ved mere end 98 0/o deraf laa mellem saa snævre Grænser som 0,5 og 0,25 mm. blevet saa ensartet, at Kornstørrelsen ved mere end 98 0/o deraf laa mellem saa snævre Grænser som 0,5 og 0,25 mm. Fig. 50. Flyvesandsbakken Højesand og Kystskrænter fra Litorinahavet. Autoriseret Gengivelse efter Maalebordsblad h 11 Rørvig 1897. 1 : 40.000. Fig. 50. Flyvesandsbakken Højesand og Kystskrænter fra Litorinahavet. Autoriseret Gengivelse efter Maalebordsblad h 11 Rørvig 1897. 1 : 40.000. De største af Flyvesandsomraaderne ligger ved Kysterne af Sejerø Bugt og Kattegat. Store Dele af Odsherreds lave Flade ned imod Sejerø Bugt har Flyvesand som øverste Jordlag og derunder mere eller mindre stenet Strandsand eller Strandgrus. Særlig karakteristisk er Flyvesandets Optræden i Forbindelse med de smukt udformede Systemer af Strand- 175 volde langs Nordkysten af Odsherred. Som det er nævnt i foregaaende Afsnit, er alle disse Systemer afsat i Retning fra Vest mod Øst, og har i Forbindelse hermed aftagende Kornstørrelse i samme Retning. Dette bliver saa meget stær- kere fremhævet derved, at de forskellige Systemers Strand- volde alle er dækkede af Flyvesand i deres østlige Afsnit, hvor Strandmaterialet i Forvejen har sin mindste Kornstør- relse. Det største af Sandflugtsomraaderne ligger Nordvest for Rørvig. Det dækker ikke blot hele den nordlige Del af Strandaflejringerne imellem Nykøbing og Rørvig, men strækker sig ogsaa ind over den største Del af Højlandet Øst for Rørvig Kirke. Særlig fremtrædende er her den mar- kerede, nu tilplantede Flyvesandsvold, Højesand, der stræk- ker sig fra Rørvig omtrent lige nordpaa i Retning mod Dybesø. Den naar en Højde af 20 m og rager indtil 15 m op over Omgivelserne. Denne Vold kan ikke anses for at være opstaaet ad naturlig Vej, men skyldes Menneskets Ind- griben. For at hindre Sandets videre Vandring mod Øst skal der langs Højesand i sin Tid være opført Risgærder for at fange det. Kort før Aarhundredskiftet var der endnu gamle Beboere ved Rørvig, som mindedes, at der 50-70 Aar tidligere var Sandflugt, som Folk fra andre Sogne blev I il- sagt for at dæmpe ved Paakørsel af Tang. Flyvesand. Vest for Rørvig Kirke er der set et betydeligt Lag Flyve- sand over Tørv, og tilsvarende Forhold er set andre Steder f. Eks. paa Overby Lyng og Vig Lyng og ligeledes ved en sanddækket Mose Vest for Saltbæk. Her og andre Steder har Pollentællinger i Tørven Mulighed for at kunne give Oplysning om, hvornaar Sandflugten er begyndt, om den er ung eller meget gammel. Ved Retskrivningen i Teksten af Stednavnene er Stednavne- udvalgets »Fortegnelse over Sjællands Stednavne« af 1932 fulgt. S. 7- 19. V. MrLTHERS: Det ældre Tertiærs Udbredelse i det nordvestlige Sjælland. D. G. F. 1907. THEODOR SoRGENFREr: Et geologisk Kort over Danmarks prækvartære Undergrund. D. G. F. 1939. Den største Del af Oplysningerne om de anførte Boringer stammer fra D. G. U.'s Borearkiv. FORKORTELSER: FORKORTELSER: . D. G. U.: Danmarks Geologiske Undersøgelse. D. G. F.: Meddelelser fra Dansk Geologisk Forening. D. G. U.: Danmarks Geologiske Undersøgelse. D. G. F.: Meddelelser fra Dansk Geologisk Forening. D. G. U.: Danmarks Geologiske Undersøgelse. D. G. F.: Meddelelser fra Dansk Geologisk Forening. Den geologiske Kortlægning i det nordvestlige Sjælland blev udført i Somrene 1892- 96, og i 1900 udkom den tilsvarende geologiske Beskrivelse: K. RøRDAM og V. MrLTHERS: Kortbladene Sejrø, Nykjøbing, Kalundborg og Holbæk. D. G. U. I. Række Nr. 8. Den da udførte Kortlægning udgør i det store og hele Grund- laget for det geologiske Overfladekort, som medfølger nærvæ- rende Skrift. Kun paa enkelte Steder har der maattet indføres Ændringer. Dette er sket, efter at det ved Revision i Somrene 1941 og 1942 skønnedes, hvor Kortet rummede væsentlige Mang- ler, enten som Følge af urigtig Bedømmelse af Jordlagenes Art, eller fordi den daværende Vurdering af forskellige Enkelthe- der ikke svarede til den nuværende Opfattelse af de geologiske Forhold. Der er Grund til her at gøre opmærksom paa, at Kortblads- beskrivelsens to Forfattere var fælles om Ansvaret for dens Tekst med Undtagelse af Afsnittet om Randmoræner (S. 69-83), som kun nærværende Forfatter hæfter for. I Løbet af de ca. 45 Aar der er gaaet, siden den geologiske Beskrivelse til de nordvestsjællandske Kortblade udarbejdedes, er der fremkommet en Række Arbejder vedrørende forskellige specielle Emner. Derved er der skabt Mulighed for at give en nogenlunde fyldig Oversigt over Nordvestsjællands geologiske Forhold, hvad Kortbladsbeskrivelsen ikke kunde gøre Krav paa at være. g g j g S. 7- 19. V. MrLTHERS: Det ældre Tertiærs Udbredelse i det nordvestlige Sjælland. D. G. F. 1907. THEODOR SoRGENFREr: Et geologisk Kort over Danmarks prækvartære Undergrund. D. G. F. 1939. Den største Del af Oplysningerne om de anførte Boringer stammer fra D. G. U.'s Borearkiv. 177 S. 9. H. ØouM: Marint Interglacial paa Sjælland, Hven, Møn og Rugen. D. G. U. IV. R. Bd. 2. Nr. 10. 1933, indeholder Oplys- ninger om Boringerne ned i Paleocænel ved Holbæk. HELGE GRY: Petrology of the Paleocene Sedimentary Rocks of Denmark. D. G. U. II. R. Nr. 61. 1935. S. 11. Om Flager af Paleocæn se: V. M1LTHERS: Kalk og Mergel paa Sjælland. D. G. U. III. R. Nr. 23. 1923. Srnuno HANSEN: Om Forekomster af Glacialflager af paleocæn Mergel paa Sjælland. D. G. U. IV. R. Bd. 2. Nr. 7. 1930. S. 13. FORKORTELSER: Om Blokke ved Klintebjærg se: K. A. GRi:iNWALL: For- steningsførende Blokke fra Langeland, Sydfyn og Ærø. D. G. U. II. R. Nr. 15. 1904. S. 13- 19. Om vulkanske Askelag paa Røsnæs se: D. G. F. 1913. S. 219. 0. B. BøGGILD: Den vulkanske Aske i Moleret. D. G. U. II. R. Nr. 33. 1918. S. A. ANDERSEN: De vulkanske Aske- lag i Vejgennemskæringen ved 01st og deres Udbredelse i Dan- mark. D. G. U. II. R. Nr. 59. 1937. S. 16. I ELLEN LOUISE MERTZ: Geologiske Profiler gennem danske Sunde og Fjorde. D. G. U. II. R. Nr. 60. 1937, er Side 135 an ført Resultatet af Slæmning af Plastisk Ler fra Røsnæs, som vise1· dette Lers overordentlige Finkornethed. S. 19. R1cHARD BøGvAo: Jarosit i Danmark. D. G. F. 1942. S. 20- 26. Om de interglaciale Lag se: H. Øou~1: Marint Inter- glacial (se ovenfor). D. G. U. IV. R. Bd. 2. Nr. 10. 1933, og V. M1LTHEHS: Tellina calcaria Leret ved Høve i Odsherred. D. Li. F. 1900. Faunaen fra Boringen i 1905 for Høng Vandværk er be- skrevet hos V. NoRDMANN: La Position stratigraphique des De- pots d'Eern. D. G. U. II. R. Nr. 47. 1928. Meddelelserne om de to nyere Boringer er velvilligst givet mig underhaanden af Dr. NonDMANN. Den fuldstændige Bearbejdelse af Skalmaterialet fra de to Boringer er endnu ikke tilendebragt. Foreløbig er følgende Mollusker bestemt: Tellina tenuis. Tlwcia papyracea. Haminea nauicula. Lilorina litorea. Nassa pygmæa. Natica intermedia. Rissoa membranacea. Trif oris peruersa. Troclms ( cinerarius?). Turbonilla· ruf a. T11rrilella lerebra. Tellina tenuis. Tlwcia papyracea. Haminea nauicula. Lilorina litorea. Nassa pygmæa. Natica intermedia. Rissoa membranacea. Trif oris peruersa. Troclms ( cinerarius?). Turbonilla· ruf a. T11rrilella lerebra. ''Tapes senescens. T11rrilella lerebra. Tapes decussatus. T ellina ballica. 178 De Arter, hvis Navne er betegnet med '\ er Ledefossiler for Eem-Lagene. S. 42- 56. I S1GURD HANSEN: Varvighed i danske og skaanske senglaciale Aflejringer. D. G. U. II. R. Nr. 63. 1940, er omtrent alle de Lokaliteter i Diluvialler nærmere omtalt, hvor der er Profiler. Begrundelsen for, at disse Leraflejringer i nærværende Afhandling er kaldt »g,laciale«, medens SrGURD HANSEN regner dem for »senglaciale«, er givet under Afsnittet: Fossi lførende, senglaciale Lag, Side 118- 19. g g S. 58. V. NoRDMANN: Nyere Fund af Elefant-Levninger i Dan- mark. D. G. F. 1921. S. 59- 61. V. M1LTHERS: Israndens Tilbagerykning fra Østjyl- land til Sjælland- Fyn, belyst ved Ledeblokke. D. FORKORTELSER: G. U. IV. R. Bd. 2. Nr. 9. 1932. KELD M1LTHERS: Ledeblokke og Landskabs- former i Danmark. D. G. U. II. R. Nr. 69. 1942. S. 62- 7 4. V. M1LTHEHS: Grundlinjer i Isens Bortsmeltning fra Sjælland. Forb. ved 16. skand. naturforskermøte 1916. Chra. 1918. V. MrLTHERS: Is randens Tilbagerykning o. s. v ., se ovenfor. Her er det Spørgsmaal behandlet, fra hvilken Side Isdækket h ar frembragt Røsnæs. I sidstnævnte Afhandling er den herhenhø- rende Litteratur anført. S. 80. S. A. ANDERSEN: Om Aase og Terrasser inden for Sus- aa's Vandomraade. D. G. U. II. R. Nr. 54. 1931. Der er her om- talt en Del Enkeltheder fra Egnen mellem Stenlille og Harald- sted. Et Omraade Vest for Haraldsted, der af ANDERSEN (1. c. p. 80) er opfattet som »aflejret i en isdæmmet Sø« med Strand- linie ved 37½ m Kurven, maa dog anses for at være af rent glacigen Oprindelse. Den af ham ved nævnte Højde antagne »Strandlinie« ved Kærnehøj er en ved Kunst (Grusgravning) fremkommen Terrænform. S. 124- 25. S. A. ANDERSEN: Om Aarsvarvene ved Ruds Vedby og nogle Bemærkninger om Aarsvarvene i Danmark. D. G. F. 1941. S. 126- 28. KNUD JESSEN: Et Bjørnefund i Allerødgytje. D. G. F. 1924. cfr. N. HARTz: Allerød-Muld. D. G. F. 1912. S. 131- 32. A. C. JOHANSEN og HERM• LYNGE: Om Land- og Ferskvandsmolluskerne i holocæne Lag ved Strandgaarden SSO for Kalundborg, og deres Vidnesbyrd om Klimaforandringer. D.G.F. 1917. S. 132. De skalførende Lag i Mosen med Sumpskildpadde ved Bjørnstrup paa Røsnæs er omtalt af A. C. JOHANSEN: Om den fossile kvartære Molluskfauna i Danmark og dens Relationer til Forandringer i Klimaet. 1904. S. 106-7. Bestemmelsen af Mol- luskerne fra de øvrige Steder er i sin Tid udført af A. C. JOHANSEN. 179 S. 133- 37. Om Indsamlingerne al' Skaller og Plantelevninger ved Vintre Møller henvises Lil CAHL ELBEHLING: Undersøgelser over nogle danske J(alktufdannelser. Vidsk. Medd. fra cl. na- turh. Forening i Kbhvn. 1870. J. P .. J. RAVN: Om Kildekalken ved Vintremøllerne paa Sjæ lland. D. G. F. 189G. S. 140- 43. N. HAHTZ og HEnLUP WrNGE: Om Uroxen fra Vig, saaret og dræbt med Flintvaaben. Aarb. f. Nord. Oldkyndighed og Historie. 1906. S. 143- 46. Der er foretaget Undersøgelse af Lagene i Mulle- rup Mose i Aarene l!J00 (G. F. L. SARAuw), 1904 (C. NEEHGAArw), 1915 (LAUGE KoCH og THERKEL :'llATHIASSE;-.;), 1915 og 1933 (KNUD JESSEN). S. 153. Om Bopladsen paa Lammefjord se: J. TnoELS-S~11TH: Geologisk Datering af Dyrholm-Fundet. Del kgl. danske Vid. Selsk. Arkæologisk-kunsthist. Skrifter. Bel. I. Nr. 1. 1942. Cfr. EnIK vVESTERBY: Nogle Stenalderfund fra tørlagt Havbund. D. G. F. 1933. FORKORTELSER: Derom henvises Lil: GEORG F. L. SARAUW: En Stenalders 13oplads i Maglemose ved Mullerup. Aarb. f. nord. Oldkyndighed og Historie. Il. R. 18. Bd. 1903. LAUGE KocH: Nye Bidrag Lil Mullerupkullurens geologiske Alder. D. G. F. 191G. KNUD JESSEN: The Composilion of lhe Foresls in Korlhern Europe in Epipa- la:olilhie Time. Vidensk. Selsk. Bio!. Meddelelser. XII. 1935. Jo11ANNES BnøNDSTED: Danmarks Oldtid. I. Stenalderen. 1938. S. 146- 48. Om Forholdene i Aamosc i Forbindelse med de arkæologiske Undersøgelser henvises Lil .I. TnoELS-S~11n-i: Geolo- giske Dateringer af Bopladser i Aamoscn, samt MAGNUS DEGEHBøL: Dyrelivet i Aamosen i Stenalderen. Nordiske Fol'tidsminder. Bel. III. 1943. De forskellige Indlandsboplaclser fra Stenalderen er venligst indtegnet paa Korlet ar Hr. Museumsinspektør, Dr. phil. THERKEL i\lATHIASSEN. S. 148- 52. Oplysningerne om Mosefundene ved Vedclinge, Eskebjærg, Favrbo og Snavhuse saml Uroksen fra Dragsholm skylder jeg Hr. Prof., Dr. phil. KNUD JESSEN. De fleste af Under- søgelserne skyldes i\Iedclelelser fra daværende Læge TuLINIUS i Snertinge. Rensdyrfundet ved Favrbo er bestemt af Dr. V. No1rn- MANN. Om Fundet af Rensdyr ved Svaleklint paa Sejerø se: V. NonDMANN: On Remains of Reincleer anci BeaYer from the com- mencemenl ol' lhe Postglacial Forest Period in Denmark. D. G. U. Il. R. Nr. 28. 1915. S. 152- 53. Oplysningerne om i\fosefundene af Palledyr forud for 1900 er hentet fra HERLUF W1NGE: Om jordfundne Pattedyr fra Danmark. Vid. Meddel. fra cl. naturh. Foren. i Kbhvn. 1904. Oplysningerne om de senere gjorte I<noglefund er velvilligsl rneclclelt al' Hr. Dr. phil. MAGNUS DEGERBOL. S. 153. Om Bopladsen paa Lammefjord se: J. TnoELS-S~11TH: Geologisk Datering af Dyrholm-Fundet. Del kgl. danske Vid. Selsk. Arkæologisk-kunsthist. Skrifter. Bel. I. Nr. 1. 1942. Cfr. EnIK vVESTERBY: Nogle Stenalderfund fra tørlagt Havbund. D. G. F. 1933. 12• 180 S. 155. Om Skallagene ved Gisseløre se: H. ØDUM. D. (i. J•. 1932, s. 190. S. 158- 59. Om Undersøgelsen af Mosen SØ for Tengslemark se: KNUD JESSEN: Litorinasænkningen ved Klinte sø i pollenflori- stisk Belysning. D. G. F. 1937. Cfr. JoHs. IvERSEN: Undersøgelser over Litorinatransgressioner i Danmark. D. G. F. 1937. Denne sidste viser, at Søborg Sø i Nordøstsjælland har haft fire Fjord- perioder i Tidsrummet fra Overgangen mellem Fyrre- og Ege- tid til et Stykke ind i Yngre Stenalder. Og i Ordrup Mose ved Klampenborg og Korup Sø paa Grenaa Halvøen har der været tre tilsvarende Trangressioner fra Stenalderhavet, nemlig en tidlig-atlantisk, en højatlantisk og en senatlantisk Fjordperiode. FORKORTELSER: De to første af disse er tillige paavist i Vendsyssel (JoHS. IVERSEN: Et Litorinaprofil ved Dybvad i Vendsyssel. D. G. F. 1943). Ved Tengslemark har de to Trangressioner været den højatlantiske og den senatlantiske. Denne sidste er ogsaa repræ- senteret i sydligere Dele af Danmark, nemlig paa Amager, Lange- land og Als (JoHs. IVERSEN 1943). Den kortvarige Beboelse paa Lammefjord (S. 153) laa imellem den første og den anden af de nævnte tre Fjordperioder eller Transgressions-Maksima. S. 164- 65. Udformningen af Strandvoldene paa Reersø er indgaaende behandlet af Adjunkt, cand. mag. AXEL Scr-rou i et Arbejde om Kystfcirmer, som jeg har set et Afsnit af i Manu- skript. S. 170- 71. Fundet i 1896 af den højtliggende Strandvold i Overby Lyng, med underliggende, fossilførende Lag fra Litorina- tiden, bragte Afgørelsen af et Aldersspørgsmaal, som VrcTOR MADSEN fornylig har henledt Opmærksomheden paa (»Strand- voldene ved Hornbæk og Alderen af dem«. D. G. F. 1942), idet han her omtaler, at man i Begyndelsen af Halvfemserne i for- rige Aarh. ansaa de højeste Strandvolde langs Nordsjællands Kattegatskyst for at være af senglacial Alder. VrcTOR MADSEN undrer sig over, hvorfor N. V. UssrnG i 2. Udgave af sin Dan- marks Geologi (1904) har anset disse Strandvolde for at være alluviale og ikke senglaciale saaledes som i 1. Udg. (1899). Svaret er, at i Mellemtiden (1900) var den geologiske Kortblads- beskrivelse til Nordvestsjælland (D. G. U. I. R. Nr. 8) udkommet, og her var Profilet fra Strandvolden i Overby Lyng gengivet (S. 113-14). Uss1NG har i Noterne i al Almindelighed henvist til de geologiske Kortbladsbeskrivelser uden at anføre Enkelt- heder. - Et Ordskifte om det samme Spørgsmaal ved et Møde i Dansk Geologisk Forening 22/ 2 1943 er gengivet i D. G. F. 1943. Stedfortegnelse. Eskilstrup (Vig Sogn) 113. Eskilstrup Sø 79. Borup 100, 102. Bramsnæs Bugt 98, 162. Bredevang 57, 97. Bregnemose 150. Bregninge 52, 71, 85, 89, 92, 117, 155. Brcgninge Aa 85, 92, 94. Brokkebjærg 168, 169. Brokøb 97. Bromme 53, 77, 79, 84. Bromølle 57, 85, 97. Brændt 113. Butterup 42, 100. Bøgebjærg 68. Bøsserup 113. Borup 100, 102. Bramsnæs Bugt 98, 162. Bredevang 57, 97. Bregnemose 150. Bregninge 52, 71, 85, 89, 92, 117, 155 Brcgninge Aa 85, 92, 94. Brokkebjærg 168, 169. Brokøb 97. Bromme 53, 77, 79, 84. Bromølle 57, 85, 97. Brændt 113. Butterup 42, 100. Bøgebjærg 68. Bøsserup 113. Aaenge 155. Aagaard 42, 73. Aagerup (Røsnæs) 45, 166. Aagcrup (ved Holbæk) 152, 162. Aamosc (Lille) 73, 74, 85, 96, 97, 118. Aamosc (Store) 37, 53, 54, 60, 62, 75, 76, 78, 79, 84, 85, 86, 89, 94, 96, 97, 109, 118, 140, 146-48, 152, 179, Aarby 57, 69. Aastofte 152. Aastrup 133. Agersvold 89, 152. Aldersro 152. Algestrup 89. Allerup 102. Alleshave 89, 109, 110, 152. Allindelille 27, 82. Allindelille Fredskov 8, 80. Annebjærg Gaard 113, 115. Annebjærg Skov 113. Arnakkegaard 86. Asmindrup 100. Asnæs (Halvø) 61, 62, 69, 71, 72, 74, 163, 166. Asnæs (By) 9, 14, 43, 52. Asnæs Teglværk 52. Assentorp 44. Avdcbo 10. Avnsø 92. Avnsøgaard 14, 34, 35, 57, 85, 94. Baarup 84. Bjærge Klint 74, 164. Bjærgene 102. Bjærgsted 89, 90, 92, 117. Bjærgsted Bakker 90. Bjørnstrup 132, 134, 135, 166, 178. Bodal 53. Rognæs 129, 162. Bonderup 82, 87, 102. Aagaard 42, 73. Aagerup (Røsnæs) 45, 166. Aagerup (Røsnæs) 45, 166. Aagcrup (ved Holbæk) 152, 162. Aagcrup (ved Holbæk) 152, 162. Aamosc (Lille) 73, 74, 85, 96, 97, 118. Aamosc (Lille) 73, 74, 85, 96, 97, 118. Aamosc (Store) 37, 53, 54, 60, 62, 75, 76, 78, 79, 84, 85, 86, 89, 94, 96, 97, 109, 118, 140, 146-48, 152, 179, Bromme 53, 77, 79, 84. y Aastofte 152. Aastofte 152. Bromølle 57, 85, 97. Aastrup 133. Butterup 42, 100. Aldersro 152. Bøgebjærg 68. Algestrup 89. Bøsserup 113. Allerup 102. Daverup 89, 90, 92, 109. Dejvad Bro 92, 94. Dejvad Gaard 91, 92. Dejvad Mose 91. Dragsholm 102, 152, 156. Dragsmølle 110, 161. Drengager Huse 48. Drusebjærg 86, 89. Dybesø 130, 173. Dyrehaven (Asnæs) 69. Dyrehøjgaard 152. Dyresten 57. Dysselodden 48. Døjringe 27, 77, 79. Annebjærg Skov 113. Arnakkegaard 86. Stedfortegnelse. Aaenge 155. Aagaard 42, 73. Aagerup (Røsnæs) 45, 166. Aagcrup (ved Holbæk) 152, 162. Aamosc (Lille) 73, 74, 85, 96, 97, 118. Aamosc (Store) 37, 53, 54, 60, 62, 75, 76, 78, 79, 84, 85, 86, 89, 94, 96, 97, 109, 118, 140, 146-48, 152, 179, Aarby 57, 69. Aastofte 152. Aastrup 133. Agersvold 89, 152. Aldersro 152. Algestrup 89. Allerup 102. Alleshave 89, 109, 110, 152. Allindelille 27, 82. Allindelille Fredskov 8, 80. Annebjærg Gaard 113, 115. Annebjærg Skov 113. Arnakkegaard 86. Asmindrup 100. Asnæs (Halvø) 61, 62, 69, 71, 72, 74, 163, 166. Asnæs (By) 9, 14, 43, 52. Asnæs Teglværk 52. Assentorp 44. Avdcbo 10. Avnsø 92. Avnsøgaard 14, 34, 35, 57, 85, 94. Baarup 84. Bjærge Klint 74, 164. Bjærgene 102. Bjærgsted 89, 90, 92, 117. Bjærgsted Bakker 90. Bjørnstrup 132, 134, 135, 166, 178. Bodal 53. Rognæs 129, 162. Bonderup 82, 87, 102. Borup 100, 102. Bramsnæs Bugt 98, 162. Bredevang 57, 97. Bregnemose 150. Bregninge 52, 71, 85, 89, 92, 117, 155. Brcgninge Aa 85, 92, 94. Brokkebjærg 168, 169. Brokøb 97. Bromme 53, 77, 79, 84. Bromølle 57, 85, 97. Brændt 113. Butterup 42, 100. Bøgebjærg 68. Bøsserup 113. Daverup 89, 90, 92, 109. Dejvad Bro 92, 94. Dejvad Gaard 91, 92. Dejvad Mose 91. Dragsholm 102, 152, 156. Dragsmølle 110, 161. Drengager Huse 48. Drusebjærg 86, 89. Dybesø 130, 173. Dyrehaven (Asnæs) 69. Dyrehøjgaard 152. Dyresten 57. Dysselodden 48. Døjringe 27, 77, 79. Ebbeløkke 114, 130, 157, 167, 170. Ellingegaard 7, 8. Elverdamsaa 84, 98, 133. Eriksholm 162. Eskebjærg 89, 90, 92, 109, 110, 149, 179. Eskebjærg Enghavc 149. Eskilstrup (Vig Sogn) 113. Eskilstrup Sø 79. Esterhøj 103. . , , Borup 100, 102. Bramsnæs Bugt 98, 162. Bredevang 57, 97. Bregnemose 150. Bregninge 52, 71, 85, 89, 92, 117, 155. Brcgninge Aa 85, 92, 94. Brokkebjærg 168, 169. Brokøb 97. Bromme 53, 77, 79, 84. Bromølle 57, 85, 97. Brændt 113. Butterup 42, 100. Bøgebjærg 68. Bøsserup 113. Daverup 89, 90, 92, 109. Dejvad Bro 92, 94. Dejvad Gaard 91, 92. Dejvad Mose 91. Dragsholm 102, 152, 156. Dragsmølle 110, 161. Drengager Huse 48. Drusebjærg 86, 89. Dybesø 130, 173. Dyrehaven (Asnæs) 69. Dyrehøjgaard 152. Dyresten 57. Dysselodden 48. Døjringe 27, 77, 79. Ebbeløkke 114, 130, 157, 167, 170. Ellingegaard 7, 8. Elverdamsaa 84, 98, 133. Eriksholm 162. Eskebjærg 89, 90, 92, 109, 110, 149, 179. Eskebjærg Enghavc 149. Stedfortegnelse. Asnæs (Halvø) 61, 62, 69, 71, 72, 74, 163, 166. Asnæs Teglværk 52. Assentorp 44. Avdcbo 10. Avnsø 92. Avnsøgaard 14, 34, 35, 57, 85, 94. Aagaard 42, 73. Avnsøgaard 14, 34, 35, 57, 85, 94. Ebbeløkke 114, 130, 157, 167, 170. Ellingegaard 7, 8. Elverdamsaa 84, 98, 133. Eriksholm 162. Eskebjærg 89, 90, 92, 109, 110, 149, 179. Eskebjærg Enghavc 149. Eskilstrup (Vig Sogn) 113. Eskilstrup Sø 79. Esterhøj 103. Baarup 84. Bjærge Klint 74, 164. Bjærgene 102. Bjærgsted 89, 90, 92, 117. Bjærgsted Bakker 90. Bjørnstrup 132, 134, 135, 166, 178. Bodal 53. Rognæs 129, 162. Bonderup 82, 87, 102. p Bjærge Klint 74, 164. Bjærgene 102. Bjærgsted 89, 90, 92, 117. 182 Faarevcjlc 13, 14, 52. Favrbo 52, 51, 55, 89, 126- 28, 119, 152, 179. Flasken 165. Flinlerup 27. Flyndersø 130, 173. Forsinge 46, 73. Frankerup 70. Frederikslund (Store) 79. Frislrup ~folier 133. Føllenslev 89, 96, 110. Geclebjærg 40. Gis! inge 11. Gislinge Aa 1Gl. Gislinge Bro 153. Gisselore 14, 155, 166, 167, 179. Gniben 113, 114, 115, 157, 167, 169. Grevinge 35, 150, 152. Grydemølle 85. Grønnebjærg Gaard 84. Gront ved Overdrev 98. Gudminclrup 130, 166. Gureclc 152. Gylclen lo\'cshøj 51. Gyrstinge So 3-l, 41, 42, 80, 84. Gørlev 9, 43. H ages ted 98, 102. Hagestedgaard 152. Halleby Aa -14, 50, 80, 81, 84, 85, 97, 130, 116, 151, 164. llallebygaard 9, 14. llaloverbjærg 159. IIals 161. IIanerup 11, 13, 100. IIaraldsLed 80, 178. Ilaraldsled Aas 41. lla ralclslecl Sø 41, 80. Havnemark 166. Ilellesklint 16, 31. llerrestrup 8, 9, 10. IIeslemølle 133. llesselbjærg 164, 165. Hesselø 61. IIjembæk 39, 52. Holbæk 8, 9, 10, 23, 21 , 25, 177. Hyrdehøj 37, 38, 86. Iløjbjærg 51. Iløjby 35, 102, 105, 114, 115, 152. !Iøjesand 174, 175. Jiøng 10, 22, 25, 177. Hønsehalsen 129. llønsinge 105. !Iørve 110, 112. Høve 25, 2G, 29, 103, 111, 118, 177. 1 [øve Strand 13. Igclsø 100. Isefjord 100, 102, 110, 129, 163. J yderup 43, 89, 109. Jyderup (Vig Sogn) 1 I0--13. Jyderup Skov 105, 107, 108. K aastrup 67. Kajemose 85. Kaldred 117. Kallerup 45, 68, 166. Kallerup Teglværk 45. Kalundborg 5, 14, 67. Kalundborg Fjord 60, 62, 155, 166. Kalveaa 42, 100. Kastrupgaarcl 151, 152. Kastrup Sko\· 8. Katholm •18. Kaltrup 85. Keldbjærg 3 1, 36, 68, 69. Kelleklin te 70. Kelleklintegaard 129. Kildebakker 113. Kildegaard 1•17. Kirkebjærg 40, 86, 88. Kirke-Eskilstrup 4 l. Kirke-Helsinge 14. Kirkevang Gaarde 90. Kisserup 162. Klint 114, 170. Klintebjærg 13, 58, 11 I, 115, 158, 172, 177. Klintsø 158, 163, 170, 172, 173, 180. Klovby 46. Kløveshøj 74. Knabstrup 50; 51, 96, 119- 23, 152. Avnsøgaard 14, 34, 35, 57, 85, 94. Faarevcjlc 13, 14, 52. Favrbo 52, 51, 55, 89, 126- 28, 119, 152, 179. Flasken 165. Flinlerup 27. Flyndersø 130, 173. Forsinge 46, 73. Frankerup 70. Frederikslund (Store) 79. Frislrup ~folier 133. Føllenslev 89, 96, 110. Geclebjærg 40. Gis! inge 11. Gislinge Aa 1Gl. Gislinge Bro 153. Gisselore 14, 155, 166, 167, 179. Gniben 113, 114, 115, 157, 167, 169. Grevinge 35, 150, 152. Grydemølle 85. Grønnebjærg Gaard 84. Gront ved Overdrev 98. Gudminclrup 130, 166. Gureclc 152. Gylclen lo\'cshøj 51. Gyrstinge So 3-l, 41, 42, 80, 84. Gørlev 9, 43. H ages ted 98, 102. Hagestedgaard 152. Halleby Aa -14, 50, 80, 81, 84, 85, 97, 130, 116, 151, 164. llallebygaard 9, 14. llaloverbjærg 159. IIals 161. IIanerup 11, 13, 100. IIaraldsLed 80, 178. Hyrdehøj 37, 38, 86. Iløjbjærg 51. Iløjby 35, 102, 105, 114, 115, 152. !Iøjesand 174, 175. Jiøng 10, 22, 25, 177. Hønsehalsen 129. llønsinge 105. !Iørve 110, 112. Høve 25, 2G, 29, 103, 111, 118, 177. 1 [øve Strand 13. Hyrdehøj 37, 38, 86. Iløjbjærg 51. Iløjby 35, 102, 105, 114, 115, 152. !Iøjesand 174, 175. Jiøng 10, 22, 25, 177. Hønsehalsen 129. llønsinge 105. !Iørve 110, 112. Høve 25, 2G, 29, 103, 111, 118, 177. 1 [øve Strand 13. Igclsø 100. Isefjord 100, 102, 110, 129, 163. J yderup 43, 89, 109. Jyderup (Vig Sogn) 1 I0--13. Jyderup Skov 105, 107, 108. K aastrup 67. Kajemose 85. Kaldred 117. Kallerup 45, 68, 166. Kallerup Teglværk 45. Kalundborg 5, 14, 67. Kalundborg Fjord 60, 62, 155, 166. Kalveaa 42, 100. Kastrupgaarcl 151, 152. Kastrup Sko\· 8. Katholm •18. Kaltrup 85. Keldbjærg 3 1, 36, 68, 69. Kelleklin te 70. Kelleklintegaard 129. Kildebakker 113. Kildegaard 1•17. Kirkebjærg 40, 86, 88. Kirke-Eskilstrup 4 l. Kirke-Helsinge 14. Kirkevang Gaarde 90. Kisserup 162. Klint 114, 170. Klintebjærg 13, 58, 11 I, 115, 158, 172 177. Klintsø 158, 163, 170, 172, 173, 180. Klovby 46. Kløveshøj 74. Knabstrup 50; 51, 96, 119- 23, 152. Knoben 168. Knøsen 103. Kongsdal 50 51 96 97 Flasken 165. Flinlerup 27. Flyndersø 130, 173. Forsinge 46, 73. Frankerup 70. Frederikslund (Store) 79. Frislrup ~folier 133. Føllenslev 89, 96, 110. Geclebjærg 40. Gis! inge 11. Gislinge Aa 1Gl. Gislinge Bro 153. Gisselore 14, 155, 166, 167, 179. Gniben 113, 114, 115, 157, 167, 169. Grevinge 35, 150, 152. Grydemølle 85. Grønnebjærg Gaard 84. Gront ved Overdrev 98. Gudminclrup 130, 166. Gureclc 152. Gylclen lo\'cshøj 51. Avnsøgaard 14, 34, 35, 57, 85, 94. Kundby 30, 32, 39, 86. Kvanløse 98, 100. Kyringe 27. Kærby 70. Kærnehøj 178. Kongsøre Næbbe 113. Kvanløse 98, 100. Kyringe 27. Kyringe 27. Kærby 70. Kærby 70. Kærnehøj 178. Lammefjord 61 , 82, 102, 103, 108, 109, 129, 153, HH , 162, 179. Langebjærg 42. Langedams Gaard 45. Langehage 162. Langholm Rende 149. Lerbjærg 46. Lerchenborg 69. Lille Bregninge 91 , 92, 118. Lille Knabslrup 100. Lillcsø 79. Lindebjærg 52, 54, 55, 128. Lumsaas 7, 8, 30. Lundebjærg 39, 40, 86. Lun lebjærg 152. Lynghuse 53, 107, 108. Løgt Yed Huse 91. Loserup 100, 102. LoYcnborg 129, 152. Magleø 116, 147. .\lagkso (Gronl ved Overdre,') 97. \Iaglesø (S f. Bromme) 79. ~Iarnp 162. \Iastrup 168. \Ielby 37, 69. i\Ierlose (Store-) 10, 82, 83. \[ogenslrup 100. \Iullerup 9, 13, 14, 129. \Iullerup ll[ose 13-1, 135, 113-46, 147, 179, Munke-Bjærghy 4:1. 53, 58, 74, 79, 120. ~lunkPsø 155 Møllebakken 67. Møller\'ænge 85. l\Iorkemose Bjærg 98, 99. Nakke 115, 173. Nakkeland :W. Langedams Gaard 45. Langehage 162. Langehage 162. Langholm Rende 149. Oddens Mej eri 8, 9, 10. Ordrup :--;æs 109, 110, 111, 118. Orebo Teglværk 4'1. Orø 7, 100, 129, 162. Ostrnp 41. Ovcrby 7, 152, 157, 168. Overby Lyng 11-1, 132, 157, 169, 170. 171, 175, 180. Lerbjærg 46. Lerchenborg 69. Lille Bregninge 91 , 92, 118. Lille Knabslrup 100. Lillcsø 79. Lindebjærg 52, 54, 55, 128. Lumsaas 7, 8, 30. Lundebjærg 39, 40, 86. Lun lebjærg 152. P edersborg 79. Pind als l\lose 108. Lynghuse 53, 107, 108. Løgt Yed Huse 91. Loserup 100, 102. Raklev 62. Ranglemølle 85. Ravnsbjærg 108. Havnsbjærg Bakker 105 . Havnsholm Bakke 107. Havnshøj 45. Reerslev 74. Rce rso 71, 74, 15,t, 164-65, 166, 180. Hegslrnp 11. His 52. l~isbakke 29. Hudc-Eskilstrup 53, 79. Ruds-Yeclby 9, 10, 43, 121 - 26, 178. Rugtved 67, 70. RæYchøj 157, 168, 169. Rorby 73. Rørmose 155. RørYig 8, 113, 115, 130, 173, 175. Høryig Kirke 175. Rosnæs 5, 6, 13, 11, 2-1, 30, 60, 61, 62, 71, 72, 74, 116, 118, 166, 177, 178. Røsnæsgaa rd 16, 67. LoYcnborg 129, 152. LoYcnborg 129, 152. Magleø 116, 147. .\lagkso (Gronl ved Overdre,') 97. \Iaglesø (S f. Bromme) 79. ~Iarnp 162. \Iastrup 168. \Ielby 37, 69. i\Ierlose (Store-) 10, 82, 83. \[ogenslrup 100. \Iullerup 9, 13, 14, 129. \Iullerup ll[ose 13-1, 135, 113-46, 147, 179, Munke-Bjærghy 4:1. 53, 58, 74, 79, 120. ~lunkPsø 155 Møllebakken 67. Møller\'ænge 85. Avnsøgaard 14, 34, 35, 57, 85, 94. Gyrstinge So 3-l, 41, 42, 80, 84. Gørlev 9, 43. H ages ted 98, 102. Hagestedgaard 152. Halleby Aa -14, 50, 80, 81, 84, 85, 97, 130, 116, 151, 164. llallebygaard 9, 14. llaloverbjærg 159. IIals 161. IIanerup 11, 13, 100. IIaraldsLed 80, 178. Ilaraldsled Aas 41. lla ralclslecl Sø 41, 80. Havnemark 166. Ilellesklint 16, 31. llerrestrup 8, 9, 10. IIeslemølle 133. llesselbjærg 164, 165. Hesselø 61. IIjembæk 39, 52. Holbæk 8, 9, 10, 23, 21 , 25, 177. Holbæk Fjord 86, 97, 98, 100, 102, 113, 129, 162. Ilolmstrup 39, 85, 97. Ilovvig 173. Geclebjærg 40. Gis! inge 11. Gislinge Aa 1Gl. Gislinge Bro 153. Gisselore 14, 155, 166, 167, 179. Gniben 113, 114, 115, 157, 167, 169. Grevinge 35, 150, 152. Grydemølle 85. Grønnebjærg Gaard 84. Gront ved Overdrev 98. Gudminclrup 130, 166. Gureclc 152. Gylclen lo\'cshøj 51. Gyrstinge So 3-l, 41, 42, 80, 84. Gørlev 9, 43. H ages ted 98, 102. Hagestedgaard 152. Halleby Aa -14, 50, 80, 81, 84, 85, 97, 130, 116, 151, 164. llallebygaard 9, 14. llaloverbjærg 159. IIals 161. IIanerup 11, 13, 100. IIaraldsLed 80, 178. Ilaraldsled Aas 41. lla ralclslecl Sø 41, 80. Havnemark 166. Ilellesklint 16, 31. llerrestrup 8, 9, 10. IIeslemølle 133. llesselbjærg 164, 165. Hesselø 61. IIjembæk 39, 52. Holbæk 8, 9, 10, 23, 21 , 25, 177. Holbæk Fjord 86, 97, 98, 100, 102, 113, 129, 162. Ilolmstrup 39, 85, 97. Ilaraldsled Aas 41. lla ralclslecl Sø 41, 80. Havnemark 166. Ilellesklint 16, 31. llerrestrup 8, 9, 10. IIeslemølle 133. llesselbjærg 164, 165. Hesselø 61. IIjembæk 39, 52. Knabstrup 50; 51, 96, 119- 23, 152. Holbæk 8, 9, 10, 23, 21 , 25, 177. Holbæk Fjord 86, 97, 98, 100, 102, 113, 129, 162. Holbæk Fjord 86, 97, 98, 100, 102, 113, 129, 162. Knøsen 103. Kongsdal 50, 51, 96, 97. Ilolmstrup 39, 85, 97. Kongsted 76. Ilovvig 173. 183 Kongslrup 16, 19, 2,1, 31, 63, 6 1, 65, 72, 118, 152. Xakke Lyng 173. Nekselø 109, 110. Xeksclo Bugt 110. 1'iløse 76. Nordrupluncl 10, 24, 25. :'sostrup 16, 19, 67. :'.\osln1p Gaard ,15, 72. :\'ygaard ,18. :\'ykobing 8, l H, 172. Nykøbing Bugt 103. Nykobing Lyng 173. Nyrup (Rosnæs) 66, 67. :--;yrup (l 'gerlosc Sogn) 84. "-'yrup (llojby Sogn) 153. Nørrehoved 109- 10. Kongslrup 16, 19, 2,1, 31, 63, 6 1, 65, 72, 118, 152. Kongsøre Næbbe 113. Kongsø re Skov 113. Kragerupga::m.l 152. Avnsøgaard 14, 34, 35, 57, 85, 94. l\Iorkemose Bjærg 98, 99. Nakke 115, 173. Nakkeland :W. Magleø 116, 147. Havnsbjærg Bakker 105 . .\lagkso (Gronl ved Overdre,') 97. \Iaglesø (S f. Bromme) 79. ~Iarnp 162. \Iastrup 168. Møllebakken 67. RørYig 8, 113, 115, 130, 173, 175. l\Iorkemose Bjærg 98, 99. Rosnæs 5, 6, 13, 11, 2-1, 30, 60, 61, 62, Nakke 115, 173. Nakkeland :W. Røsnæsgaa rd 16, 67. 184 Saltbæk 175. Saltbæk Vig 62, 80, 85, 117, 153, 155, 166. Saltmade 155. Saltofte (Stenlille Sogn) 79. Saltofte "(Udby Sogn) 73, 94. Saltofte Udsigtsbakke 71. Salvig 162. Sandlyng Aa 42, 76, 78, 79, 82. Sandlynggaard 53. Sejerby 13, 14. Sejerø 6, 60, 61, 71, 115, 116, 130, 132, 151, 152, 153, 156, 166, 168. Sejerø Bugt 13, 62, 86, 89, 102, 103, 105, 108, 110, 161, 162. Sejstrup 152. Sekshøje 114. Sidinge Fjord 8, 102, 103, 108, 111, 113, 129, 159. Sjællands Odde 6, 61, 103, 114, 117, 157, 168. Skambæks Mølle 24, 25, 118. Skamslrup 39, 96, 103. Skarresø 89, 90, 96, 112. Skee 10, 27, 80, 84. Skee Mose 80. Skee Taastrup 80. Skellingsted 97. Skellingsted Bro 53. Skjoldshule 153. Skuerup 76. Skæreby Bakke 165, 172, 173. Snavhuse 152, 179. Sneglerup 152. Snogekær Gaard 16, 19. Sobjærg 74. Sofieholm 11, 99. Sonnerup 114. Sonnerup Skov 130, 158, 170, 172. Stenlille 42, 74, 77, 78, 79, 80, 82, 84. Stenmagle 76, 78, 82. Stenrand Plantage 90, 91, 94. Stensbjærg 68. Stenstrup 114. Stestrup 80, 84. Stestrup Bjærg 51, 80. Stestrup Mejeri 10. Store Grandløse 100. Store Merløse 10, 82, 83. S S l 164 Strandgaard 131, 134, 135, 178. Strandhuse 113. Sultenkrog 91, 92, 94, 96, 118. Svaleklint 132, 134, 135, 151, 157, 179. Svallerup 34, 35, 43, 48, 50, 70, 72, 131, 164. Svebølle 70. Svebøllegavn 91, 94. Svebølle Mose 91. Svebølle Station 71, 90, 91, 94. Svenstrup 31. Svinningc (Odsherred) 35, 105. Svinninge Vejle 89, 102, 110, 161. Syvhøje 153. Særslev 89. Sønder Jærnløse 100. Søndersted 96, 97, 102. Sønderød 76. Taastrup 100. Tempelkrog 162. Tengslemark 25, 26, 29, 132, 134, 135, 158-60, 170, 180. Tersløse 79. Timeglasklint 31. Tindingebanke 42. Tissø 70, 73, 74, 85, 86, 118. Tjørneholm 132, 134, 135. Tjørntved 42, 78, 79. Tornved Bjærg 40, 88. Torpe 90. Torpelund 92. Torsbjærg 130, 172. Tranemose 152. Troldebjærg 114. Trundholm 111, 153, 156. Trønninge 42, 100. Tudeaa 79, 84. Tuse 102. Tuse Aa 162. Avnsøgaard 14, 34, 35, 57, 85, 94. Tuse Næs 7, 97, 98, 101, 102, 113, 129, 162. Tysinge 34, 35, 44, 84. Tølløse 43, 80, 84. Tømmerup (Undløse Sogn) 97. Tømmerup 73. Tømmerup Banke 68. Ubberup 35, 46, 68, 69. Ubberup Højskole 33 34 Saltbæk 175. Saltbæk Vig 62, 80, 85, 117, 153, 155, 166. Saltmade 155. Saltofte (Stenlille Sogn) 79. Saltofte "(Udby Sogn) 73, 94. Saltofte Udsigtsbakke 71. Salvig 162. Sandlyng Aa 42, 76, 78, 79, 82. Sandlynggaard 53. Sejerby 13, 14. Sejerø 6, 60, 61, 71, 115, 116, 130, 132, 151, 152, 153, 156, 166, 168. Sejerø Bugt 13, 62, 86, 89, 102, 103, 105, 108, 110, 161, 162. Sejstrup 152. Sekshøje 114. Sidinge Fjord 8, 102, 103, 108, 111, 113, 129, 159. Sjællands Odde 6, 61, 103, 114, 117, 157, 168. Skambæks Mølle 24, 25, 118. Skamslrup 39, 96, 103. Skarresø 89, 90, 96, 112. Skee 10, 27, 80, 84. Skee Mose 80. Skee Taastrup 80. Skellingsted 97. Skellingsted Bro 53. Skjoldshule 153. Skuerup 76. Skæreby Bakke 165, 172, 173. Snavhuse 152, 179. Sneglerup 152. Snogekær Gaard 16, 19. Sobjærg 74. Sofieholm 11, 99. Sonnerup 114. Sonnerup Skov 130, 158, 170, 172. Stenlille 42, 74, 77, 78, 79, 80, 82, 84. Stenmagle 76, 78, 82. Stenrand Plantage 90, 91, 94. Stensbjærg 68. Stenstrup 114. Stestrup 80, 84. Stestrup Bjærg 51, 80. Stestrup Mejeri 10. Store Grandløse 100. Store Merløse 10, 82, 83. Store Saltsø 164. Store Vrøj 166. Strandgaard 131, 134, 135, 178. Strandhuse 113. Sultenkrog 91, 92, 94, 96, 118. Svaleklint 132, 134, 135, 151, 157, 179. Svallerup 34, 35, 43, 48, 50, 70, 72, 131, 164. Svebølle 70. Svebøllegavn 91, 94. Svebølle Mose 91. Svebølle Station 71, 90, 91, 94. Svenstrup 31. Svinningc (Odsherred) 35, 105. Svinninge Vejle 89, 102, 110, 161. Syvhøje 153. Særslev 89. Sønder Jærnløse 100. Søndersted 96, 97, 102. Sønderød 76. Taastrup 100. Tempelkrog 162. Tengslemark 25, 26, 29, 132, 134, 135, 158-60, 170, 180. Tersløse 79. Timeglasklint 31. Tindingebanke 42. Tissø 70, 73, 74, 85, 86, 118. Tjørneholm 132, 134, 135. Tjørntved 42, 78, 79. Tornved Bjærg 40, 88. Torpe 90. Torpelund 92. Torsbjærg 130, 172. Tranemose 152. Troldebjærg 114. Trundholm 111, 153, 156. Trønninge 42, 100. Tudeaa 79, 84. Tuse 102. Tuse Aa 162. Tuse Næs 7, 97, 98, 101, 102, 113, 129, 162. Tysinge 34, 35, 44, 84. Tølløse 43, 80, 84. Tømmerup (Undløse Sogn) 97. Tømmerup 73. Tømmerup Banke 68. Avnsøgaard 14, 34, 35, 57, 85, 94. Ubberup 35, 46, 68, 69. Ubberup Højskole 33, 34. Udby 37, 43, 4~, 50, 70, 72. Strandgaard 131, 134, 135, 178. g Svaleklint 132, 134, 135, 151, 157, 179. Saltofte (Stenlille Sogn) 79. Saltofte "(Udby Sogn) 73, 94. Saltofte Udsigtsbakke 71. Sandlyng Aa 42, 76, 78, 79, 82. Sidinge Fjord 8, 102, 103, 108, 111, 113, 129, 159. Sidinge Fjord 8, 102, 103, 108, 111, 113, 129, 159. Sjællands Odde 6, 61, 103, 114, 117, 157, 168. Sjællands Odde 6, 61, 103, 114, 117, 157, 168. Skambæks Mølle 24, 25, 118. Skamslrup 39, 96, 103. Skarresø 89, 90, 96, 112. Skarresø 89, 90, 96, 112. Skee 10, 27, 80, 84. Skee Mose 80. Skee Taastrup 80. Skellingsted 97. Skellingsted Bro 53. Skjoldshule 153. Skuerup 76. p Skæreby Bakke 165, 172, 173. Snavhuse 152, 179. Sneglerup 152. Snogekær Gaard 16, 19. Trønninge 42, 100. Tudeaa 79, 84. Sonnerup Skov 130, 158, 170, 172. Stenlille 42, 74, 77, 78, 79, 80, 82, 84. Tuse Aa 162. Stenmagle 76, 78, 82. Tuse Næs 7, 97, 98, 101, 102, 113, 129, 162. Stenrand Plantage 90, 91, 94. Tysinge 34, 35, 44, 84. Tølløse 43, 80, 84. Tømmerup (Undløse Sogn) 97. Tømmerup 73. Tømmerup Banke 68. Udby 37, 43, 4~, 50, 70, 72. 185 Udby (Tuse Næs) 98, 102. Udstrup 80, 84. Uger løse 97. Ugerløse (Rørby Sogn) 50, 69. Uglerup 98, 102. Ulstrup 16, 19, 62, 67, 166. Ulvebjærge 31, 68. Unclløse 51, 55, 82, 126, 129. Vallek ilde 110, 112. Valleslrup 100. Vanløse 44, 82. Vanløse Teglværk 43. Veddinge 29, 52, 148. Vejleby 110. Vejlen 164. Vejrhøj 103, 109, 115. Vcrup 53, 78. Vesterbygaard 14. Vesterlyng 172, 173. Vesterløb 129. Vesterskov 69. Vielsled 41, 80, 82. Vig 7, 10, 140, 152, 180. Vinde-Helsinge Mose 146. Vintre Møller 133-37, 178, 179. Viskinge 90, 117. Vognserup 9, 152. Vollerup 45. Værslev 46, 70, 129. Værslev Gaard 46. Yderby 168, 169. Yderby Lyng 114, 157, 169. Yppenbjærg Huse 48. Ærtemose 152. Øgaarde 54. Ørslev Mose 152. Østerlyng 172, 173. Vesterskov 69. Vielsled 41, 80, 82. Vig 7, 10, 140, 152, 180. Vinde-Helsinge Mose 146. Vintre Møller 133-37, 178, 179. Viskinge 90, 117. Vognserup 9, 152. Vollerup 45. Værslev 46, 70, 129. Værslev Gaard 46. Yderby 168, 169. Yderby Lyng 114, 157, 169. Yppenbjærg Huse 48. Ærtemose 152. Øgaarde 54. Ørslev Mose 152. Østerlyng 172, 173. Udby (Tuse Næs) 98, 102. Udstrup 80, 84. Uger løse 97. X X X Sten.alders Skala'!p~9er og .Boplads,,;• Avnsøgaard 14, 34, 35, 57, 85, 94. Ugerløse (Rørby Sogn) 50, 69. Uglerup 98, 102. Ulstrup 16, 19, 62, 67, 166. Ulvebjærge 31, 68. Unclløse 51, 55, 82, 126, 129. Vallek ilde 110, 112. Valleslrup 100. Vanløse 44, 82. Vanløse Teglværk 43. Veddinge 29, 52, 148. Vejleby 110. Vejlen 164. Vejrhøj 103, 109, 115. Vcrup 53, 78. Vesterbygaard 14. Vesterlyng 172, 173. Vesterløb 129. Udby (Tuse Næs) 98, 102. Udstrup 80, 84. Uger løse 97. Ugerløse (Rørby Sogn) 50, 69. Uglerup 98, 102. Ulstrup 16, 19, 62, 67, 166. Ulvebjærge 31, 68. Unclløse 51, 55, 82, 126, 129. Udby (Tuse Næs) 98, 102. Udstrup 80, 84. Uger løse 97. Ugerløse (Rørby Sogn) 50, 69. Uglerup 98, 102. Ulstrup 16, 19, 62, 67, 166. Ulvebjærge 31, 68. Unclløse 51, 55, 82, 126, 129. Vallek ilde 110, 112. Valleslrup 100. Vanløse 44, 82. Vanløse Teglværk 43. Veddinge 29, 52, 148. Vejleby 110. Vejlen 164. 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https://openalex.org/W3126193933	https://zenodo.org/records/5880006/files/25%2018319.pdf	English	null	Dialogue management using reinforcement learning	Telkomnika	2,021	cc-by-sa	6,197	TELKOMNIKA Telecommunication, Computing, Electronics and Control Vol. 19, No. 3, June 2021, pp. 931~938 ISSN: 1693-6930, accredited First Grade by Kemenristekdikti, Decree No: 21/E/KPT/2018 DOI: 10.12928/TELKOMNIKA.v19i3.18319 TELKOMNIKA Telecommunication, Computing, Electronics and Control Vol. 19, No. 3, June 2021, pp. 931~938 ISSN: 1693-6930, accredited First Grade by Kemenristekdikti, Decree No: 21/E/KPT/2018 DOI: 10.12928/TELKOMNIKA.v19i3.18319 931  Corresponding Author: Hanif Fakhrurroja School of Electrical Engineering and Informatics (SEEI) Institut Teknologi Bandung Ganesha No.10, Coblong, Bandung, Jawa Barat 40132, Indonesia Email: hani002@lipi.go.id Dialogue management using reinforcement learning Binashir Rofi’ah, Hanif Fakhrurroja, Carmadi Machbub School of Electrical Engineering and Informatics, Institut Teknologi Bandung, Indonesia ABSTRACT Dialogue has been widely used for verbal communication between human and robot interaction, such as assistant robot in hospital. However, this robot was usually limited by predetermined dialogue, so it will be difficult to understand new words for new desired goal. In this paper, we discussed conversation in Indonesian on entertainment, motivation, emergency, and helping with knowledge growing method. We provided mp3 audio for music, fairy tale, comedy request, and motivation. The execution time for this request was 3.74 ms on average. In emergency situation, patient able to ask robot to call the nurse. Robot will record complaint of pain and inform nurse. From 7 emergency reports, all complaints were successfully saved on database. In helping conversation, robot will walk to pick up belongings of patient. Once the robot did not understand with patient’s conversation, robot will ask until it understands. From asking conversation, knowledge expands from 2 to 10, with learning execution from 1405 ms to 3490 ms. SARSA was faster towards steady state because of higher cumulative rewards. Q-learning and SARSA were achieved desired object within 200 episodes. It concludes that reinforcement learning (RL) method to overcome robot knowledge limitation in achieving new dialogue goal for patient assistant were achieved. Received Jul 14, 2020 Revised Oct 3, 2020 Accepted Oct 14, 2020 Keywords: Dialogue management Human-robot interaction Knowledge growing Reinforcement learning This is an open access article under the CC BY-SA license. This is an open access article under the CC BY-SA license. This is an open access article under the CC BY-SA license. Corresponding Author: Hanif Fakhrurroja School of Electrical Engineering and Informatics (SEEI) Institut Teknologi Bandung Ganesha No.10, Coblong, Bandung, Jawa Barat 40132, Indonesia Email: hani002@lipi.go.id This is an open access article under the CC BY-SA license. TELKOMNIKA Telecommunication, Computing, Electronics and Control Vol. 19, No. 3, June 2021, pp. 931~938 ISSN: 1693-6930, accredited First Grade by Kemenristekdikti, Decree No: 21/E/KPT/2018 DOI: 10.12928/TELKOMNIKA.v19i3.18319 Corresponding Author: Hanif Fakhrurroja School of Electrical Engineering and Informatics (SEEI) Institut Teknologi Bandung Ganesha No.10, Coblong, Bandung, Jawa Barat 40132, Indonesia Email: hani002@lipi.go.id 1. INTRODUCTION Betacoronavirus SARS-CoV-2 or Covid-19 outbreak is currently affecting almost all of the world with a total of 503,203 people infected or confirmed positive, and 22,340 of them died as of March 26, 2020 [1]. Since 11 March 2020, its status has changed to a pandemic. One of the conditions for a pandemic is the explosion of spread or the high number of cases that occur in a short period [2]. To reduce significant transmission rate, the mild-symptom patient must be isolated in the hospital until healed, which leads to stress [3]. The latest developments regarding companion/assistantship robots have been carried out [4], in order to support the mental health of patients, one of them is Silbot which has 6 activities such as waking the patient, checking the mood, reminding during meditation, checking safety, helping therapy, and emergencies. Silbot will care for mild-dementia patients [5]. p Human-robot interaction (HRI) aims for extending robot functionality by making natural communication with human [6]. They [7] introduce algorithm from learning reward and human synchronously. The flexibility would gain from simultaneous learning; it gives a trainer ability to go in as desired and update reinforcement learning reward whereas it is still in progress. They [8] introduce learning scenario combination between practice and end-user critique, practice gives actual-world experience and Journal homepage: http://journal.uad.ac.id/index.php/TELKOMNIKA  932  ISSN: 1693-6930 end-user/human critique whether good or bad label as input for loss function or unexpected value of candidate policies. end-user/human critique whether good or bad label as input for loss function or unexpected value of candidate policies. To build natural communication between human and robot for assistantships, our research team has prior works such as speech recognition [9], unclear pronunciation [10], robot walking and pattern generator [11, 12], robot path planning [13], speech and gesture recognition[14], multimodal interaction [15] and rule-based/scenario dialogue management [16]. However the problem of rule-based is unable to follow dialogue development, so the possibility of scenario mismatch is getting bigger, once the robot does not understand, no other choice for this method besides end the dialogue and gives generic answers like "I don't understand your commands" [17]. This paper takes part on overcome robot knowledge limitation to achieve new goal through flexible dialogue. We propose robot asking method to gather new knowledge from human feedback through conversation. 1. INTRODUCTION The robot does not stop immediately because of not understand, but it will ask first and gather new knowledge with goal to take patient belongings. We also provide entertainment and emergency request to complement patient needs. This paper would discuss natural language processing both in understanding and generation with different sub-section, dialogue management method, also the hardware set-up for robot. 2.1. Natural language understanding 2.1. Natural language understanding This paper would focus on goal-driven dialogue. The function of natural language understanding (NLU) are extracting the raw voice until system got the information needed, and provide dialogue information for dialogue management. NLU includes identification of domain and intent, also semantic parsing [18]. Text will get several processes: Stopword process to remove unnecessary such as common words. Part of speech (POS) tagging process to get grammar tag with POStag_idn and use Indonesian tag set at [19]. We focused on tag VB (verb), NN (noun), and CD (cardinal number). We use Indonesia Tnt-Tagger for POS tag method and Indonesia IDPOSTAG corpus from [20]. Followed by stemming process to get root word by removing the affix [21]. Ended with storing process using JavaScript Object Notation (JSON) format. 2.2. Dialogue management 3, June 2021: 931 - 938 TELKOMNIKA Telecommun Comput El Control 933  𝑉𝑛𝑒𝑤(𝑠) = (1 −𝛼)𝑉𝑜𝑙𝑑(𝑠) + 𝛼[𝑟+ 𝛾𝑉𝑜𝑙𝑑(𝑠′)] ⏟ 𝑇𝐷 𝑇𝑎𝑟𝑔𝑒𝑡 (3) 𝑉𝑛𝑒𝑤(𝑠) = 𝑉𝑜𝑙𝑑(𝑠) + 𝛼[𝑟+ 𝛾𝑉𝑜𝑙𝑑(𝑠′) −𝑉𝑜𝑙𝑑(𝑠)] ⏟ 𝑇𝐷 𝐸𝑟𝑟𝑜𝑟 (4) 𝑉𝑛𝑒𝑤(𝑠) = (1 −𝛼)𝑉𝑜𝑙𝑑(𝑠) + 𝛼[𝑟+ 𝛾𝑉𝑜𝑙𝑑(𝑠′)] ⏟ 𝑇𝐷 𝑇𝑎𝑟𝑔𝑒𝑡 (3) (3) (4) The value function usually also called as state-value function 𝑉(𝑠) is the total amount of expected rewards that an agent can collect from that state to the end of the episode. The action-value function 𝑄(𝑠, 𝑎) is total amount of expected rewards of taking an action from the state until the end of the episode. The way agent learns the best policy called update policy, and the way agent behaves called behavior policy. In this paper, we also implement two TD learning methods that are off and on policy (Q-learning and SARSA). 2.2.2. State-action-reward-state-action (SARSA) Pseudocode of RL with TD-policy: (a) Q-learning pseudocode and (b) SARSA pseudocode Estimate 𝑄≈𝑞∗ with SARSA (on-policy TD control) Initialize 𝑄(𝑠, 𝑎), for all 𝑠∈𝑆, 𝑎∈𝐴(𝑠) Repeat (each episode): Initialize 𝑆 Choose 𝐴 from 𝑆 using 𝜋 derived from 𝑄 Repeat (each step): Take action 𝐴, observe 𝑅, 𝑆′ Choose 𝐴′ from 𝑆′ using 𝜋 derived from 𝑄 𝑄(𝑆, 𝐴) ←𝑄(𝑆, 𝐴) + 𝛼[𝑅+ 𝛾𝑄(𝑆′, 𝐴′) −𝑄(𝑆, 𝐴)] 𝑆←𝑆′ 𝐴←𝐴′ Until 𝑆 is terminal (b) Estimate 𝜋≈𝜋∗ with Q-learning (off-policy TD control) Initialize 𝑄(𝑠, 𝑎), for all 𝑠∈𝑆, 𝑎∈𝐴(𝑠) Repeat (each episode): Initialize 𝑆 Repeat (each step): Choose 𝐴 from 𝑆 using 𝜋 derived from 𝑄 Take action 𝐴, observe 𝑅, 𝑆′ 𝑄(𝑆, 𝐴) ←𝑄(𝑆, 𝐴) + 𝛼[𝑅+ 𝛾𝑚𝑎𝑥𝑎𝑄(𝑆′, 𝑎) −𝑄(𝑆, 𝐴)] 𝑆←𝑆′ Until 𝑆 is terminal (a) Figure 1. Pseudocode of RL with TD-policy: (a) Q-learning pseudocode and (b) SARSA pseudocode 2.2.2. State-action-reward-state-action (SARSA) 2.2.2. State-action-reward-state-action (SARSA) Agent in SARSA learns optimal policy and behaves with the same policy. Because the update policy and behavior policy are similar, so SARSA is categorized as on-policy. Q-Value of SARSA is shown in (6). ( ) Agent in SARSA learns optimal policy and behaves with the same policy. Because the update policy and behavior policy are similar, so SARSA is categorized as on-policy. Q-Value of SARSA is shown in (6). 𝑄(𝑆, 𝐴) ←𝑄(𝑆, 𝐴) + 𝛼[𝑅+ 𝛾𝑄(𝑆′, 𝐴′) −𝑄(𝑆, 𝐴)] (6) (6) (6) From (6) we know that update policy 𝛾𝑄(𝑆′, 𝐴′) and behavior policy 𝑄(𝑆, 𝐴) also from pseudocode below, we know that 𝑆←𝑆′ and 𝐴←𝐴′ means update policy is the behavior policy. We use pseudocode [26] to implement SARSA in our python code as shown in Figure 1 (b). From (6) we know that update policy 𝛾𝑄(𝑆′, 𝐴′) and behavior policy 𝑄(𝑆, 𝐴) also from pseudocode below, we know that 𝑆←𝑆′ and 𝐴←𝐴′ means update policy is the behavior policy. We use pseudocode [26] to implement SARSA in our python code as shown in Figure 1 (b). Estimate 𝜋≈𝜋∗ with Q-learning (off-policy TD control) Estimate 𝑄≈𝑞∗ with SARSA (on-policy TD control) Initialize 𝑄(𝑠, 𝑎), for all 𝑠∈𝑆, 𝑎∈𝐴(𝑠) Repeat (each episode): Initialize 𝑆 Repeat (each step): Choose 𝐴 from 𝑆 using 𝜋 derived from 𝑄 Take action 𝐴, observe 𝑅, 𝑆′ 𝑄(𝑆, 𝐴) ←𝑄(𝑆, 𝐴) + 𝛼[𝑅+ 𝛾𝑚𝑎𝑥𝑎𝑄(𝑆′, 𝑎) −𝑄(𝑆, 𝐴)] 𝑆←𝑆′ Until 𝑆 is terminal Initialize 𝑄(𝑠, 𝑎), for all 𝑠∈𝑆, 𝑎∈𝐴(𝑠) Repeat (each episode): Initialize 𝑆 Choose 𝐴 from 𝑆 using 𝜋 derived from 𝑄 Repeat (each step): Take action 𝐴, observe 𝑅, 𝑆′ Choose 𝐴′ from 𝑆′ using 𝜋 derived from 𝑄 𝑄(𝑆, 𝐴) ←𝑄(𝑆, 𝐴) + 𝛼[𝑅+ 𝛾𝑄(𝑆′, 𝐴′) −𝑄(𝑆, 𝐴)] 𝑆←𝑆′ 𝐴←𝐴′ Until 𝑆 is terminal (a) (b) Figure 1. 2.2.1. Q-learning Q g Absolute policy is used by agent in Q-learning to learn optimal policy, on the other hand, agent behaves with other policy. Because the behavior policy is different from update policy, so Q-learning is categorized as off-policy TD control. Q-value of Q-learning is shown in (5). 𝑄(𝑆, 𝐴) ←𝑄(𝑆, 𝐴) + 𝛼[𝑅+ 𝛾𝑚𝑎𝑥𝑎𝑄(𝑆′, 𝑎) −𝑄(𝑆, 𝐴)] (5) (5) From (5) we have known that update policy 𝛾𝑚𝑎𝑥𝑎𝑄(𝑆′, 𝑎) is different from behavior policy 𝑄(𝑆, 𝐴). We use pseudocode from [26] to implement Q-learning in our python code as shown in Figure 1 (a). 2.2. Dialogue management Dialogue management (DM) consists of state tracking and generates action. Approaches for dialogue management problems are graph-based dialogue, frame-based dialogue, statistical approach [22]. Human involvement in DM framework has been successfully carried out in previous studies [23]. The reward as feedback from expertise (can be formed in negative or positive rewards) was given to optimize policy on reinforcement learning (RL) in [24]. RL was still the main instrument for DM. RL is current mainstream technology in order to solve real-world problem with large-scale belief state space [18]. Before RL can be explained, it necessary to understand basic components used. A learner called an agent in RL studies its behavior by select actions in an environment [25]. At each time, the agent receives a representation of state 𝑠, while 𝑠∈𝑆, where 𝑆 is states. The agent pickups an action 𝑎, while 𝑎∈𝐴, where 𝐴 is a set of possible actions that the agent can take. As the return of its action, the agent receives reward 𝑟, while 𝑟∈𝑅, and goes to new state s′. 𝛼 is learning rate, γ is a discount factor, and 𝜋 is policy that defines how an agent response from a specific state. The aim of an agent is selecting the optimal actions by maximizing its cumulative discounted reward. g In this paper, we use RL with temporal difference (TD) learning method. TD learning is a fusion of two benefits from Monte Carlo and dynamic programming as shown in (3), and (4). On one side, Monte Carlo methods have no model of environment’s dynamics as shown in (1), so TD learns from raw experience. On the other side, dynamic programming (2) that no need waiting until the final outcome, so TD able to update estimates based on partially learned estimation [26]. Recall Monte Carlo: 𝑀𝐶: 𝑉𝜋(𝑠) ←𝑉𝜋(𝑠) + 𝛼[𝑅(𝑠) −𝑉𝜋(𝑠)], 𝛼= 1 𝑛𝑅(𝑠) (1) (1) Recall dynamic programming: 𝐷𝑃: 𝑉𝜋(𝑠) = 𝔼𝜋[𝑟𝑡+1 + 𝛾𝑉𝜋(𝑠𝑡+1)\|𝑠𝑡= 𝑠], (2) 𝐷𝑃: 𝑉𝜋(𝑠) = 𝔼𝜋[𝑟𝑡+1 + 𝛾𝑉𝜋(𝑠𝑡+1)\|𝑠𝑡= 𝑠], (2) TD to make an update 𝑉𝑛𝑒𝑤(𝑠) ←𝑉𝑜𝑙𝑑(𝑠), Given (𝑠, 𝑎, 𝑟, 𝑠′): TD to make an update 𝑉𝑛𝑒𝑤(𝑠) ←𝑉𝑜𝑙𝑑(𝑠), Given (𝑠, 𝑎, 𝑟, 𝑠′): TELKOMNIKA Telecommun Comput El Control, Vol. 19, No. 2.5. Humanoid robot Bioloid grand prix (GP) is a humanoid robot equipped with CM-530 controller, and lithium battery for power supply [30]. We use modified Bioloid GP from [13] as previous project with an additional speaker mounted on top of the robot. Analog voltages from Arduino Mega 2560 [31] are converted to digital values by analog to digital converter (ADC) as a reference command for CM-530 that will be translated into robot movement. Robot movement consists of forward, backward, left, and right with its iteration. 2.3. Knowledge growing Entertainment purpose consists of: musik/playing music audio, dongeng/playing fairy tale audio, komedi/playing comedy audio. Motivation purpose, consist of motivasi/Playing motivation audio. Emergency purpose: memanggil perawat/calling nurse, keluhan/reporting complaint of pain. Helping purpose, taking an object/patient’s belongings. Researchers have emphasized on implementing robots that can imitate owning memory/knowledge to mitigate many social-robot challenges [27], some studies have exploit data, based on user profile [28, 29] to make memory-based adaptations. We implement robot asking during interaction to gather new information from human feedback, Figure 2 (a) is an example of additional knowledge. Dialogue management using reinforcement learning (Binashir Rofi’ah)  934  ISSN: 1693-6930 Entertainment, motivation, and emergency need back-end intervention from admin to add appropriate content manually. In emergency, the robot behavior (calling nurse in fix place, where robot moves, and what robot talk) cannot be changed by user/patient. Meanwhile, helping is moving action from robot that depends on user/patient habit on locating his/her belongings, so it can be useful to use end-user experience. Only for this kind of action robot will grow up its knowledge. The helping conversation can be seen in Figure 2 (a). Grey shades show the unknown of robot, then from the conversations that we proposed then appear words in cyan, yellow, and orange shades, that is new knowledge. New knowledge will be saved at Q-Table shown in Figure 2 (b) for further training, then RL both Q-learning and SARSA takes part on build new environment by calculating rewards at each action and state. (a) (b) Figure 2. (a) Additional knowledge gathers from dialogue and (b) New knowledge stored in database (a) (b) (b) (a) Figure 2. (a) Additional knowledge gathers from dialogue and (b) New knowledge stored in database 2.4. Natural language generation N l l i Natural language generation (NLG) is responsible to generate linguistic realization of the system's dialogue. The goal of NLG is to produce spoken that is easy for humans to understand. In this paper we had 3 response systems there are rejection, asking, and aborting. Once system found that all word in a sentence has no verb (listed on corpus) or unique words, system will reject and request to change with other new words until there is verb or unique word in that sentence. Asking response is started with searching verb in system database knowledge, if there is no similar verb then system will categorize it as new verb with no relation to object. The system will ask for object then searching the word in corpus, if there is object in the corpus then system will search in database. That is why some verbs can have one same object. After the system has new verb and new object, then system will ask for place, if system able to fulfill direction and iteration, then it will save as new knowledge. Aborting response is where the system will able to abort mission if user says terima kasih/thank you in the middle of asking conversation. TELKOMNIKA Telecommun Comput El Control, Vol. 19, No. 3, June 2021: 931 - 938 2.6. System implementation The hardware needed for this system is a microphone input (Kinect 2.0), processor (Laptop), controller (Arduino and CM-530), and output in the form of speakers and robots as shown in Figure 3 (a). In the hardware implementation, robot able to move everywhere without wire on cable as shown in Figure 3 (b). Speech output and robot movement control are sent from laptop to Arduino via bluetooth. We used Google speech recognition with id-ID (Indonesian language) to recognize and adjust ambient noise. Laptop powered by the Intel Core i5 processor, 8GB of memory. We use python language and RL algorithm builds on it. We equipped robot voice with speech registry from Windows called Microsoft Andika to give Indonesian voice and accent, also pronounce cardinal number in Indonesian. We set robot to talk 150 words per minute (WPM). The average speech rate for conversational is 120-150 WPM [31]. ELKOMNIKA Telecommun Comput El Control, Vol. 19, No. 3, June 2021: 931 - 938 TELKOMNIKA Telecommun Comput El Control 935  935  (a) (b) Figure 3. System configuration: (a) design, and (b) implementation (b) (a) (b) Figure 3. System configuration: (a) design, and (b) implementation 3.1. Entertainment execution This experiment gived us insight on execution time for single request. We implemented using 1 m fixed distance. Time counted right after translation from speech to text, we did it because the length of dialogues and the speed of people’s speech rates varied. An average time was 3.74 ms. The slowest time occurs on purple shade with 8.23 ms. The fastest time was 0.86 ms with pink shade shown in Table 1. Table 1. Time for entertainment execution Dialogue Musik/Music Dongeng/Fairy tale Motivasi/Motivation Komedi/Comedy [request] 2.99 2.00 3.92 3.91 nyalakanlah [request] (please turn on [request]) 5.76 1.99 5.18 3.60 mainkanlah [request] (please play [request]) 2.00 2.01 5.34 4.16 mainkan [request]nya (play the [request]) 2.01 2.00 2.79 0.86 saya ingin mendengarkan [request] (I want to listen to [request]) 2.00 1.03 7.47 4.90 [request] yang bagus (please the best [request]) 8.23 2.00 6.35 6.81 saya bosan ingin [request] (I am bored, want to [request]) 2.55 2.03 1.99 5.85 saya butuh [request] (I need [request]) 3.91 2.00 7.10 3.91 [request]nya, tolong diputar (the [request], please play) 2.00 2.03 3.99 6.60 minta tolong [request] (please [request]) 7.48 1.03 3.36 4.31 Average time level 3.74 Table 1. Time for entertainment execution Dialogue Musik/Music Dongeng/Fairy tale Motivasi/Mot 3.2. Emergency execution g y In emergency situation, we asked robot to call nurse by talk unique word “perawat (nurse)”. We use sentence “panggilkan perawat (call the nurse)”, then robot will ask for complaint of pain. After conversation, robot will walk to the place where the nurse usually standby and describe complaint of pain to the nurse. On the other hand, the complaint will record on a report shown in Table 2. 3. RESULTS AND ANALYSIS We conducted several experiments to see the performance of system. System configuration and environment as shown in Figure 3 (b). The performance will represent how fast robot execution, how accurate, and how knowledge growing. The experiment consists of entertainment execution, emergency execution, helping conversation with knowledge growing, policy behavior, and reward convergence. Dialogue management using reinforcement learning (Binashir Rofi’ah) 3.3. Knowledge growing Q-Table for SARSA Wallet Pencil Blanket Puzzle Remote Book Barbell Tissue L R L R L R L R L R L R L R L R Save None 100 99 0 89 0 80 0 72 0 65 0 59 0 53 None Write None 93 100 100 99 0 89 0 80 0 72 0 65 0 59 None Note None 91 100 100 99 0 89 0 80 0 72 0 65 0 59 None Sleep None 85 0 98 100 100 99 0 89 0 80 0 72 0 65 None Play None 80 0 89 0 99 100 100 99 0 89 0 80 0 72 None Watch None 72 0 80 0 89 0 99 100 100 99 0 89 0 80 None Read None 65 0 72 0 80 0 89 0 99 100 100 99 0 88 None See None 72 0 80 0 89 0 99 100 100 99 0 89 0 80 None Exercise None 59 0 65 0 72 0 80 0 89 0 99 100 100 81 None Clean None 53 0 59 0 65 0 72 0 80 0 89 0 99 100 None Table 4. Q-Table for SARSA Wallet Pencil Blanket Puzzle Remote Book Barbell Tissue L R L R L R L R L R L R L R L R Save None 100 99 0 89 0 80 0 72 0 65 0 59 0 53 None Write None 93 100 100 99 0 89 0 80 0 72 0 65 0 59 None Note None 91 100 100 99 0 89 0 80 0 72 0 65 0 59 None Sleep None 85 0 98 100 100 99 0 89 0 80 0 72 0 65 None Play None 80 0 89 0 99 100 100 99 0 89 0 80 0 72 None Watch None 72 0 80 0 89 0 99 100 100 99 0 89 0 80 None Read None 65 0 72 0 80 0 89 0 99 100 100 99 0 88 None See None 72 0 80 0 89 0 99 100 100 99 0 89 0 80 None Exercise None 59 0 65 0 72 0 80 0 89 0 99 100 100 81 None Clean None 53 0 59 0 65 0 72 0 80 0 89 0 99 100 None Figure 4. 3.3. Knowledge growing Q-Table for Q-Learning Wallet Pencil Blanket Puzzle Remote Book Barbell Tissue L R L R L R L R L R L R L R L R Save None 100 99 80 89 72 80 65 72 59 65 53 59 47 53 None Write None 92 100 100 99 80 89 72 80 65 72 59 65 53 59 None Note None 89 100 100 99 80 89 72 80 65 72 59 65 53 59 None Sleep None 88 73 99 100 100 99 80 89 72 80 65 72 59 65 None Play None 80 72 89 80 99 100 100 99 80 89 72 80 65 72 None Watch None 72 65 80 72 89 80 99 100 100 99 80 89 72 80 None Read None 65 59 72 65 80 72 89 80 99 100 100 99 64 85 None See None 72 65 80 72 89 80 99 100 100 99 79 89 71 80 None Exercise None 59 53 65 59 72 66 81 73 89 80 99 100 100 96 None Clean None 53 47 59 53 65 59 72 65 80 72 89 80 99 100 None Table 3. Q-Table for Q-Learning Wallet Pencil Blanket Puzzle Remote Book Barbell Tissue L R L R L R L R L R L R L R L R Save None 100 99 80 89 72 80 65 72 59 65 53 59 47 53 None Write None 92 100 100 99 80 89 72 80 65 72 59 65 53 59 None Note None 89 100 100 99 80 89 72 80 65 72 59 65 53 59 None Sleep None 88 73 99 100 100 99 80 89 72 80 65 72 59 65 None Play None 80 72 89 80 99 100 100 99 80 89 72 80 65 72 None Watch None 72 65 80 72 89 80 99 100 100 99 80 89 72 80 None Read None 65 59 72 65 80 72 89 80 99 100 100 99 64 85 None See None 72 65 80 72 89 80 99 100 100 99 79 89 71 80 None Exercise None 59 53 65 59 72 66 81 73 89 80 99 100 100 96 None Clean None 53 47 59 53 65 59 72 65 80 72 89 80 99 100 None Table 4. 3.3. Knowledge growing In beginning there were only 2 verbs and 2 objects, then during this experiment, human gives unknown knowledge to robot. From the conversations, knowledge expanded to 10 verbs and 8 objects. We also tried different verbs related to same object. Tonton/watch and lihat/see have the same object that was remote. Tulis/write and catat/record have same object, that was pencil. j p We separated Q-Table for Q-learning and SARSA because the rewards are different. Q-learning d is shown in Table 3, the blue shades mean the highest reward that was connected between verb and Dialogue management using reinforcement learning (Binashir Rofi’ah) We separated Q-Table for Q-learning and SARSA because the rewards are different. Q-learning reward is shown in Table 3, the blue shades mean the highest reward that was connected between verb and Dialogue management using reinforcement learning (Binashir Rofi’ah) Q-learning and SARSA because the rewards are different. Q-learning e shades mean the highest reward that was connected between verb and Dialogue management using reinforcement learning (Binashir Rofi’ah) Dialogue management using reinforcement learning (Binashir Rofi’a  936 936  ISSN: 1693-6930 object on the Q-table. Whereas Table 4 was the final SARSA Q-Table which has more null/zero values. In SARSA policy, when state is terminal then reward will be grounded to zero. The highest rewards are shown in orange shades. We also did an experiment to execute training from 1 knowledge to 10 knowledge in 200 episodes. Every execution iterates 3 times for Q-learning and SARSA. From knowledge 1 until 5, time was varying, however from 6 knowledge, time consistently ramp up from 1405 until 3490 ms, and Q-learning needs more time than SARSA as shown in Figure 4. Table 2. Report of emergency/complaint of Pain Date Time Complaint of Pain 8/11/2020 22:44 saya pusing mual (I feel dizzy and nausea) 8/12/2020 16:21 saya nyeri (I am in pain) 8/12/2020 16:21 infus saya lepas (My infusion peels off) 8/12/2020 16:22 saya batuk darah (I coughed up blood) 8/12/2020 16:32 saya sesak nafas (I am short of breath) 8/12/2020 16:33 saya meriang (I feel light-headed) 8/12/2020 16:33 saya sakit perut tiba tiba (I have a sudden stomachache) Table 3. 3.3. Knowledge growing Q-Table for SARSA Wallet Pencil Blanket Puzzle Remote Book Barbell Tissue L R L R L R L R L R L R L R L R Save None 100 99 0 89 0 80 0 72 0 65 0 59 0 53 None Write None 93 100 100 99 0 89 0 80 0 72 0 65 0 59 None Note None 91 100 100 99 0 89 0 80 0 72 0 65 0 59 None Sleep None 85 0 98 100 100 99 0 89 0 80 0 72 0 65 None Play None 80 0 89 0 99 100 100 99 0 89 0 80 0 72 None Watch None 72 0 80 0 89 0 99 100 100 99 0 89 0 80 None Read None 65 0 72 0 80 0 89 0 99 100 100 99 0 88 None See None 72 0 80 0 89 0 99 100 100 99 0 89 0 80 None Exercise None 59 0 65 0 72 0 80 0 89 0 99 100 100 81 None Clean None 53 0 59 0 65 0 72 0 80 0 89 0 99 100 None Figure 4. Execution time as knowledge increase Note None 89 100 100 99 80 89 72 80 65 72 59 65 53 59 None Sleep None 88 73 99 100 100 99 80 89 72 80 65 72 59 65 None Play None 80 72 89 80 99 100 100 99 80 89 72 80 65 72 None Watch None 72 65 80 72 89 80 99 100 100 99 80 89 72 80 None Read None 65 59 72 65 80 72 89 80 99 100 100 99 64 85 None See None 72 65 80 72 89 80 99 100 100 99 79 89 71 80 None Exercise None 59 53 65 59 72 66 81 73 89 80 99 100 100 96 None Clean None 53 47 59 53 65 59 72 65 80 72 89 80 99 100 None Table 4. 3.3. Knowledge growing Execution time as knowledge increase Figure 4. Execution time as knowledge increase TELKOMNIKA Telecommun Comput El Control, Vol. 19, No. 3, June 2021: 931 - 938 937 TELKOMNIKA Telecommun Comput El Control  3.4. Policy behavior y To know the movement of policy and actions taken in a certain state to reach appropriate object, we also taking plots for reward value at the end of episode (200th episode). As shown in Figure 5 reward shift towards remot/remote in the middle. Red shade means the lowest reward, where green shade is the highest reward (goal). The yellow shades describe the transition of reward value from the lowest to the highest. Figure 5. Left and right policy direction to object “remot/remote” left right left right left right left right left right left right left right left right Q-Learning 0.00 72.89 65.59 80.99 72.88 90.00 80.94 99.99 100.00 100.00 99.91 79.40 89.73 71.11 80.22 0.00 SARSA 0.00 72.86 0.00 80.98 0.00 89.99 80.94 99.99 100.00 100.00 99.97 0.00 89.94 0.00 80.87 0.00 left left right right right right done left Barbells Tissue Dompet Pensil Selimut Puzzle Remot Buku Barbel Tisu Wallet Pencil Blanket Puzzle Remote Book Barbells Barbel Pensil Pencil Figure 5. Left and right policy direction to object “remot/remote” 3.5. Reward convergence g On this implementation, we want to know the performance of Q-learning and SARSA for every object in each final reward for 200 episodes. Start from 1 to 7 objects. It can be seen in Figure 6 that the SARSA cumulative reward was slightly higher than Q-Learning, which means that its algorithm was faster towards steady states because SARSA's policy does not explore all actions at each step so that it was focused to get the goal. Figure 6. Cumulative reward between Q-learning and SARSA Figure 6. Cumulative reward between Q-learning and SARSA 4. CONCLUSION From the experiment in previous section, it could be shown that the proposed system has the ability to expand from 2 to 10 knowledge. Additional knowledge affected to the time for learning execution that was getting longer from 1405 ms to 3490 ms. SARSA was faster towards steady state because of higher cumulative rewards. However, the difference between off and on learning can still be implemented, and the policy moves the action accordingly to achieve the desired object in 200 episodes. Equipped with entertainment feature to play music, fairy tale, motivation, and comedy request in fast average execution time of 3.74 ms. During emergency situation system able to call nurse and save 7 complaints of pain. It could be concluded that the method proposed in this paper successfully achieved the objective to overcome robot knowledge limitation in achieving new dialogue goal for patient assistant. For further research, dialogue classification and knowledge growing can be extended for chit-chat dialogue or non-goal driven. REFERENCES [1] C. F. Santos, “Reflections about the impact of the SARS-COV-2/COVID-19 pandemic on mental health,” Rev. Bras. Psiquiatr., vol. 42, no. 3, p. 329, 2020, doi: 10.1590/1516-4446-2020-0981. [2] D. M. Morens, G. K. Folkers, and A. S. Fauci, “What Is a Pandemic?,” J. Infect. 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https://openalex.org/W2941293496	https://sciencepubco.com/index.php/ijet/article/download/28986/15401	English	null	Implementation of missed call for making a password and controlling devices	International journal of engineering & technology	2,018	cc-by	3,372	2. AT Commands This paper addressed the control system which is emphasizing on utilizing of the cellular phone services i.e. missed call service. A missed calling is one of the features of the telephone that for some people perhaps it could annoy but for others, it could be utilized, it depends on the condition and usage. The missed call is an activity to call a number of the mobile phone/cellular phone and hang up before that mobile phone number can pick up the call. It could be meaning if there is a commitment before such as requesting to call back, picking up now, etc. [2]. The missed call is an intentional use by older mobile phone users and it also could be known with another term as deliberately missed call (DMC) [3]. The missed call is no charge communication, but it could be charged by the provider if the user intends to miss call to such number of mobile phones but it gets the answer by the provider. At the beginning of the emerging, this feature of the mobile phone in some countries has been widely used and spread. The mechanism of the mobile phones works can be written as following when the sender dials the mobile phone numbers and thus press the call key. The mobile phone will select the available radio channels and sends a radio One of the control commands of the cellular phone modula- tor/demodulator (modem) is known as an AT command. The AT itself is an abbreviation of Attention [4]. Every command to con- trol the modem starts with “AT” so that’s why it is called AT commands e.g. ATA (used to answer), ATD (used to dial), ATH (used to hook control), and ATO (used to return to online data). So the command of “AT” is the prefix that instructs the modem to do something or specific tasks. These command sets are supported by the general packet radio service (GPRS) / global system for mo- bile communication (GSM), besides for the modems, the mobile phones are also supporting these command sets with the specific to the technology of GSM e.g. AT+CMGL (used to list short mes- sage service (SMS)), AT+CMGR (used to read an SMS messages), AT+CMGS (used to send SMS message), and AT+CMSS (used to send SMS message from the storage). 1. Introduction The communication data is a field that has an intersection between the field of communication and information technology. The com- prising of the information technology has a wide contribution to the human life while the communication in the worldwide has a portion to share the information and make a network. The tech- nology has a rapid innovation with the development of the devic- es, products, and theories. This also has an impact on the other sectors. The industrial revolution has also grown more over to balance with the rising of the knowledge [1]. Many applications could be categorized into some definitions. The categories of the human-machine interaction (HCI) has 3 definitions which are the interaction between person and person, the interaction between person and machine, and the interaction between machine and machine. Abstract The cellular phones do not use telephone cables. The cellular phones send radio signals into the air to the nearest transmitter tower called the base station. The transmitter tower will emit the signal to the next station and so on until it reaches the phone that is called. When a new call arrives on the phone number, it will generate a tone until it is picked up, rejected, or the time has been ended to receive this tone (each telephone network provider has different policies related to the time used for this telephone connection so that this causes the num- ber of rings raised to vary). This paper focuses on the missed call services which is one of the features found on the telephone. The tone that has been generated by this calling can emerge ring. This ring can be detected and manipulated to be something useful for controlling devices or making some password. Keywords: Control, Missed call, Password, Ring, Telephone. Keywords: Control, Missed call, Password, Ring, Telephone. signal containing a mobile phone/telephone numbers that will be called to the nearest base station. So then the base station will send a signal to the nearest base station network until it finds the phone that is called. The phone that is called sends a message back through the base station, which states whether the phone is availa- ble or not. At that time, it can be heard the dial tone. This will continue until the telephone is picked up or disconnected by the recipient. The dial tone sound has a number that varies from one provider to another provider. The amount of information that can be sent by the modem is limited by its speed in processing infor- mation. The data compression can speed up the process by cutting up information that is not important. The amount of information that can be processed every second by a telephone is called the bandwidth. Keywords: Control, Missed call, Password, Ring, Telephone. Copyright © 2018 Authors. This is an open access article distributed under the Creative Commons Attribution License, which permits use, distribution, and reproduction in any medium, provided the original work is properly cited. International Journal of Engineering & Technology, 7 (4.36) (2018) 1357-1360 International Journal of Engineering & Technology, 7 (4.36) (2018) 1357-1360 3. Methodology The GSM mobile phone for control has been implemented to con- trol a direct current (DC) motor [5], intelligent home security [6], irrigation system [7], etc. This paper uses AT89S51 microcontrol- ler from the microcontroller family of MCS-51 having 4-Kbyte Flash programmable erasable read-only memory (PEROM) which is packaged within 40 pins. This microcontroller is using a direct current (DC) power supply of 5 Volt. The AT89S51 microcontrol- ler has an on-chip which is utilized as a clock generator to the central processing unit (CPU) or also known as the core. The clock generator in this paper is using a crystal resonator of 11.0592 MHz which is connected to the pins of xtal1 (pin 19) and xtal2 (pin 18) with two grounded capacitors of 33 pF. So the time, , for executing the program instruction program can be written using (1). shown in Figure 1. Fig. 1: PDU Spy While for its conversion, it is applying Implementation of the 7-bit schema as shown in Table 1.   = (1)   = (1) Where  is a cycle number and  is a frequency within Hertz (Hz). So if the frequency of the clock used is 11.0592 MHz, it will have 0.09 s. The mini development system schematic of this micro- controller is shown in Figure 2. Where  is a cycle number and  is a frequency within Hertz (Hz). So if the frequency of the clock used is 11.0592 MHz, it will have 0.09 s. The mini development system schematic of this micro- controller is shown in Figure 2. Fig. 1: PDU Spy While for its conversion, it is applying Implementation of the 7-bit schema as shown in Table 1. While for its conversion, it is applying Implementation of the 7-bit schema as shown in Table 1. Fig. 2: Schematic of a mini development system Table 1: 7-bit schema b 7 0 0 0 0 1 1 1 1 b 6 0 0 1 1 0 0 1 1 b 5 0 1 0 1 0 1 0 1 b 4 b 3 b 2 b 1 0 1 2 3 4 5 6 7 0 0 0 0 0 @  S P 0 - P . . p 0 0 0 1 1 ! 2. AT Commands From this definition, it could be withdrawn a conclusion of the categories which are basic commands and extended commands. The first example mentioned is categorized as basic commands while the second example is International Journal of Engineering & Technology 1358 categorized as extended commands. This AT command is using a conversion from the text to the format of a protocol data unit (PDU) code and otherwise. In sending or receiving the short mes- sage service (SMS), there are two modes i.e. a text mode and PDU mode. For some text mode, it is unavailable on some cellular phones. It is the only encoding of the bit stream data represented by the PDU mode. Many encoding alternatives that can be imple- mented when it is displayed on the cellular phone as an SMS mes- sage but the cellular phone will select the proper encoding. The lack of the use of the text mode is of this mode limited by the set of preset encoding options while if the PDU mode is chosen, any encoding can be implemented. The PDU contains the hexadecimal numbers and has some headers. The tool to make a conversion of the PDU code could be employed the software of PDU spy as shown in Figure 1. categorized as extended commands. This AT command is using a conversion from the text to the format of a protocol data unit (PDU) code and otherwise. In sending or receiving the short mes- sage service (SMS), there are two modes i.e. a text mode and PDU mode. For some text mode, it is unavailable on some cellular phones. It is the only encoding of the bit stream data represented by the PDU mode. Many encoding alternatives that can be imple- mented when it is displayed on the cellular phone as an SMS mes- sage but the cellular phone will select the proper encoding. The lack of the use of the text mode is of this mode limited by the set of preset encoding options while if the PDU mode is chosen, any encoding can be implemented. The PDU contains the hexadecimal numbers and has some headers. The tool to make a conversion of the PDU code could be employed the software of PDU spy as shown in Figure 1. 2. AT Commands o l l e h 00000 110 1111 110 1 100 110 11 00 110 010 1 110 1000 0 6 F D 9 B 3 2 E 8 Because the total of 7-bit multiply by 5 characters is 35-bit, whereas the number needed of 8-bit multiply by 5 characters is 40-bit, so it needs a 5-bit dummy with fulfilling with zero (‘0’). Every 1 byte or 8 bits represents a hexadecimal pair which is of 4- bit represents a hexadecimal number obviously 24=16. So the word “hello”, its conversion result is E8329BFD06. And then change the 7-bit into 8-bit as shown below, 3. Methodology 1 A Q a q 0 0 1 0 2 $  “ 2 B R b r 0 0 1 1 3  # 3 C S c s 0 1 0 0 4  4 D T d t 0 1 0 1 5  % 5 E U e u 0 1 1 0 6  & 6 F V f v 0 1 1 1 7  ‘ 7 G W g w 1 0 0 0 8  ( 8 H X h x 1 0 0 1 9  ) 9 I Y i y 1 0 1 0 1 0 L F  * : J Z j z 1 0 1 1 1 1 + ; K A k a 1 1 0 0 1 2 , < L U l o 1 1 0 1 1 3 C R - = M m 1 1 1 0 1 4  . > N U n u 1 1 1 1 1 5 / ? O o So that, in conversion 7-bit code of word “hello” it will convert into bellow, Fig. 2: Schematic of a mini development system This mini development system as shown in Figure 2 is also con- nected to the RS232 circuit by implementing an integrated circuit (IC) of MAX232. The RS232 communication is an asynchronous meaning that the clock signal is not sent along with the data. This paper has implemented the RS232 with the level set of the 8N1 data structure. The 8N1 means that there are 8 data bits, No parity bit, and 1 stop bit. For communicating between the microcontrol- ler mini development system and the mobile phone, it implements a baud rate of 19200 bits per second (bps). The communication with the serial ports needs to be checked to know what kind of data form that has been sent so it needs another device to monitor. To monitor this communication, it applies a wiring connection as shown in Figure 4. 3. Methodology So that, in conversion 7-bit code of word “hello” it will convert into bellow, Bit 7 6 5 4 3 2 1 h 1 1 0 1 0 0 0 e 1 1 0 0 1 0 1 l 1 1 0 1 1 0 0 l 1 1 0 1 1 0 0 o 1 1 0 1 1 1 1 And then change the 7-bit into 8-bit as shown below, International Journal of Engineering & Technology 1359 Fig. 3: Schematic of the monitoring device Fig. 5: The device of missed call with monitoring test Fig. 5: The device of missed call with monitoring test The serial watcher software is applied to monitor each instruction given by the microcontroller and the response data of the mobile phone. The interruption signal is always checked until the value of register R0 is changing into ‘1’. If this register value has been set into ‘1’ so it will make a delay about 1.5 minutes and then turn the serial interrupt off. When in the delay time, the count program of the missed call will be enabled to count the tone. Since every pro- vider has a different time to end the connection of the telephone so the microcontroller has been programmed for 1.5 minutes. The process of monitoring this connection can be shown in Figure 6. Fig. 3: Schematic of the monitoring device The connector of DB9-1 is connected to the data cable of the mo- bile phone and the connector of DB9-2 is connected to the micro- controller mini development system, whereas the connector of DB9 is connected to the computer. To make monitoring, the com- puter should be installed a program application of serial watcher as shown in Figure 4. Fig. 4: Serial Watcher p g g Fig. 6: Conversation monitoring of the devices Fig. 4: Serial Watcher Fig. 4: Serial Watcher 4. Result and Discussion Fig. 6: Conversation monitoring of the devices The first thing to do in programming this microcontroller is to synchronize the baud rate to match the baud rate on the mobile phone so that communication occurs. The baud rate program for this microcontroller is configured as 19200 bps. After it sets, thus enables the serial interruption and fill the register R0 with 00H. In this case, to detect the missed call signal it could be formed as Figure 4. The signal missed call will be detected as American standard code for information interchange (ASCII) code as <CR><LF>RING<CR><LF>. For the reason of flexibility in pro- gramming, to count the number of the missed call tone it is using <CR><LF>R only as a reference to be detected and programmed to the microcontroller. The result obtained after execution, the counting of the missed call can be counted well that is known from the program to control the DC motor (see Figure 5). For taking control of the DC motor turns on, it can be activated using two times ring that is generated from the missed call tone and to turn the DC motor off can be generated four times of missed call tone. From Figure 6, it shows that the conversation monitoring has been done by both devices. This conversation means that after it has detected the tone of the missed call twice, the microcontroller will send the response to the mobile phone to call the other mobile phone number using a command of ATD, if the mobile phone has accepted the instruction, it will send “OK” to the microcontroller. Then the microcontroller will send an instruction to make a mobile phone is hooked. This program is intended to convince the other mobile phone number that the calls made are correct by making a call back to that mobile phone number for a few seconds until the connection will be turned off by hooking the mobile phone. For the purpose of using the password, it can be used that the missed call tone that is enumerated is correct and the mobile phone will wait for the next count of missed call tone. This program is also included to turn all interruptions off when the count of the tones of missed call has a wrong counting so the password entry will be locked until it is restarted so the program will normally run again. 4. Result and Discussion From Figure 6, it shows that the conversation monitoring has been done by both devices. This conversation means that after it has detected the tone of the missed call twice, the microcontroller will send the response to the mobile phone to call the other mobile phone number using a command of ATD, if the mobile phone has accepted the instruction, it will send “OK” to the microcontroller. Then the microcontroller will send an instruction to make a mobile phone is hooked. This program is intended to convince the other mobile phone number that the calls made are correct by making a call back to that mobile phone number for a few seconds until the connection will be turned off by hooking the mobile phone. For the purpose of using the password, it can be used that the missed call tone that is enumerated is correct and the mobile phone will wait for the next count of missed call tone. This program is also included to turn all interruptions off when the count of the tones of missed call has a wrong counting so the password entry will be locked until it is restarted so the program will normally run again. International Journal of Engineering & Technology 1360 5. Conclusion A device has been designed and implemented to detect a missed call and count the number of tones produced to function as pass- words and control devices. The thing that needs to be considered in the utilization of this device is remembering the counts done when doing a missed call. If it is unable to remember and cause an error in entering the missed call tone count, it will cause the de- vice to turn off all serial interruptions that appear, so that commu- nication can no longer be performed. Acknowledgment Thank you for my supervisor who has guided me, Prof. Dr. Jazi Eko Istiyanto. From his hands and thought, a lot of research that I can do. References [1] Wibowo FW, “A dynamic intelligent control analysis on the wire- less smart machine environments in the industry 4.0”, Proceedings of the 2018 international seminar on research of information tech- nology and intelligent systems (ISRITI), (2018), pp: 389-393. [2] Donner J, “The rules of beeping: exchanging messages via inten- tional “missed calls” on mobile phones”, Journal of Computer- Mediated Communication, Vol. 13, (2008), pp: 1-22. DOI: 10.1111/j.1083-6101.2007.00383.x [3] Fernández-Ardèvol M, “Missed calls: Barcelona and Los Angeles compared”, Proceedings of the Technology & Emerging Media Track – Annual Conference of the Canadian Communication Asso- ciation, (2012), pp: 1-14. pp [4] Maqbool S & Lone SA, “Monitoring system using GSM”, Interna- tional Journal of Computer Applications, Vol. 117, No. 10, (2015), pp: 28-31. [5] Istiyanto JE & Wibowo FW, “A DC Motor Control Based-on Cel- lular Phone”, Proceedings of 2nd Jogja International Physics Con- ference, (2007), pp. EI072-EI075. pp [6] Eseosa O & Promise E, “GSM based intelligent home security sys- tem for intrusion detection”, International Journal of Engineering and Technology, Vol. 4, No. 10, (2014), pp: 595-605. [7] Sangme PB, Khatal RB, Patil OM & Patil AG, “GSM based motor control for irrigation system”, International Journal of Innovative Research in Computer and Communication Engineering, Vol. 6, Is- sue 3, (2018), pp: 2138-2142. DOI: 10.15680/IJIRCCE.2018.0603064
https://openalex.org/W2952547594	https://escholarship.org/content/qt9652h1dp/qt9652h1dp.pdf?t=pgsj48	English	null	A Dynamic Model of Social Network Formation	Oxford University Press eBooks	2,014	cc-by	11,067	Peer reviewed Peer reviewed UC Irvine Copyright Information This work is made available under the terms of a Creative Commons Attribution License, availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewed UC Irvine UC Irvine Previously Published Works Title A dynamic model of social network formation Permalink https://escholarship.org/uc/item/9652h1dp Journal Proceedings of the National Academy of Sciences of the United States of America, 97(16) ISSN 0027-8424 Authors Skyrms, Brian Pemantle, Robin Publication Date 2000-08-01 DOI 10.1073/pnas.97.16.9340 Copyright Information This work is made available under the terms of a Creative Commons Attribution License, availalbe at https://creativecommons.org/licenses/by/4.0/ P i d UC Irvine UC Irvine Previously Published Works Title A dynamic model of social network formation Permalink https://escholarship.org/uc/item/9652h1dp Journal Proceedings of the National Academy of Sciences of the United States of America, 97(16) ISSN 0027-8424 Authors Skyrms, Brian Pemantle, Robin Publication Date 2000-08-01 DOI 10.1073/pnas.97.16.9340 Copyright Information This work is made available under the terms of a Creative Commons Attribution License, availalbe at https://creativecommons.org/licenses/by/4.0/ P i d UC Irvine UC Irvine Previously Published Works Title A dynamic model of social network formation Permalink https://escholarship.org/uc/item/9652h1dp Journal Proceedings of the National Academy of Sciences of the United States of America, 97(16) ISSN 0027-8424 Authors Skyrms, Brian Pemantle, Robin Publication Date 2000-08-01 DOI 10.1073/pnas.97.16.9340 Copyright Information This work is made available under the terms of a Creative Commons Attribution License, availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewed A dynamic model of social network formation Brian Skyrms† and Robin Pemantle‡ School of Social Sciences, University of California, Irvine, CA 92607; and ‡Department of Mathematics, Ohio State University, Columbus, OH 43210 This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected on April 27, 1999. Contributed by Brian Skyrms, May 9, 2000 We consider a dynamic social network model in which agents play repeated games in pairings determined by a stochastically evolv- ing social network. Individual agents begin to interact at random, with the interactions modeled as games. The game payoffs deter- mine which interactions are reinforced, and the network structure emerges as a consequence of the dynamics of the agents’ learning behavior. We study this in a variety of game-theoretic conditions and show that the behavior is complex and sometimes dissimilar to behavior in the absence of structural dynamics. We argue that modeling network structure as dynamic increases realism without rendering the problem of analysis intractable. as in Sewall Wright’s models of assortative mating (4). Wright (5) already realized that positive correlation of encounters could provide an account of evolution of altruism. Thus, the need for social network models has been long recognized. When the social network is modeled, it is almost always static.§ Interactions, for example, may be posited to occur only between players whose locations are close, according to some given spatial data. Biological models in which encounters are governed by spatial structure have become increasingly frequent in the 1990s; see, for example, the work of Durrett, Levin, and Neuhauser (7–9). A similar hypothesis of spatial structure, in a game theory context, arises in ref. 10. Here, technology from statistical mechanics is adapted to the analysis of games whose interactions take place between neighbors in a grid. P airs from among a population of 10 individuals interact re- peatedly. Perhaps they are cooperating to hunt stags and rabbits, or coordinating on which concert to attend together; perhaps they are involved in the somewhat more antagonistic situation of bargaining to split a ﬁxed payoff, or attempting to escape the undesirable but compelling equilibrium of a Pris- oner’s Dilemma. As time progresses, the players adapt their strategies, perhaps incorporating randomness in their decision rules, to suit their environment. But they may also exert con- trol over their environment. The players may have choice over the pairings but not perfect information about the other play- ers. They may improve their lot in two different ways. A child who is being bullied learns either to ﬁght better or to run away. Similarly, a player who obtains unsatisfactory results may choose either to change strategies or to change associates. A dynamic model of social network formation Brian Skyrms† and Robin Pemantle‡ School of Social Sciences, University of California, Irvine, CA 92607; and ‡Department of Mathematics, Ohio State University, Columbus, OH 43210 This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected on April 27, 1999. Contributed by Brian Skyrms, May 9, 2000 Regardless of whether the interactions are mostly cooperative or mostly an- tagonistic, it is natural and desirable to allow evolution of the social network (the propensity for each pair to interact) as well as the individuals’ strategies. A number of recent investigations by game theorists, some directly inspired by biological models, have shown that the dy- namics of strategic interaction can be strikingly different if inter- action is governed by some spatial structure, or more generally, some graph structure (11–13). For instance, one-shot Prisoner’s Dilemma games played with neighbors on a circle or torus al- lows cooperation to evolve in a way that the random encounter model does not. The spatial or graph structure can be important to determine which equilibria are possible, whether repeated in- teractions can be expected to converge to equilibrium, and, if so, how quickly convergence takes place (14). ( ) Because the outcome of a repeated game may vary with the choice of network model, it is important to get the network model right. Further progress in the theory of games and adap- tive strategies would be greatly enhanced by a theory of net- works of social interaction. In particular, it would be desirable to have a framework within which models may be developed that are both tractable and plausible as a mechanism governing interactions among a population of agents seeking to improve their lot. We build a model that incorporates both of these modes of evolution. The idea is simple. When the network changes much more slowly than do the strategies of individuals, it is reasonable to model the social net- work by a structure that is ﬁxed, though possibly random. The question of realistically modeling the randomness in such a case is taken up in a number of papers, of which a recent and well known example is the “small world” model (15). In the other ex- treme (16–18), evolution of social structure is modeled by agents moving on a ﬁxed graph in the absence of strategy dynamics. ( ) Individual agents begin to interact at random. The in- teractions are modeled as games. The game payoffs determine which interactions are reinforced, and the social network structure emerges as a consequence of the dynamics of the agents’ learning behavior. As the details of the speciﬁc game and the reinforcement dy- namics vary, we then obtain a class of models. 9340-9346 \| PNAS \| August 1, 2000 \| vol. 97 \| no. 16 †To whom reprint requests should be addressed. E-mail: bskyrms@uci.edu. §An exception, perhaps, is a preprint we have recently encountered by Jackson and Watts (6). Powered by the California Digital Library University of California eScholarship.org §An exception, perhaps, is a preprint we have recently encountered by Jackson and Watts (6). †To whom reprint requests should be addressed. E-mail: bskyrms@uci.edu. A dynamic model of social network formation Brian Skyrms† and Robin Pemantle‡ School of Social Sciences, University of California, Irvine, CA 92607; and ‡Department of Mathematics, Ohio State University, Columbus, OH 43210 This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected on April 27, 1999. Contributed by Brian Skyrms, May 9, 2000 This type of simple model has been used before in the economics literature to explain the stabilization of market shares at seemingly ran- dom equilibria, due to random reinforcement in the early phases of growth of an industry (20). We remark that the choices made by each agent are independent of the choices made by each other agent, so the social aspect of the model is somewhat de- generate and the model may be viewed as a model of individ- ual choice. Nevertheless, it ﬁts our deﬁnition of social network model in that it gives a probabilistic structure to interactions; one may then extend the model so the interactions are nontriv- ial games. We ﬁrst consider a baseline case of uniform reinforcement. Here, any choice of partner is reinforced as strongly as any al- ternative choice would have been. In other words, the interac- tion game between any pair of players always produces a con- stant reward or punishment. One might expect that such cases would not lead to interesting dynamics, but that is far from the truth. We show both by computer simulation and analytically how structure emerges spontaneously even in these cases. Be- cause the strategic dynamics here are trivial, the baseline case is intended mostly as a building block on which more inter- esting strategic dynamics are to be grafted. We note, however, that the constant reward game is not completely unreasonable. Studies have shown that in the absence of other environmen- tal attributes, sheer familiarity brings about positive attitudinal change (19). In fact, an abstract model of network evolution under uniform positive reweighting has appeared before under the name of “Reinforced Random Walk” (D. Coppersmith and P. Diaconis, unpublished work). Friends II: Symmetrized Reinforcement. Suppose now that the inter- action is as pleasant to the host as the visitor. Thus when agent i visits agent j, we add 1 to both wij and wji. A typical outcome for 10 agents after 1,000 rounds of visits looks similar to the table for Friends I, except that the entries are nearly symmet- ric. There are, however, subtle differences that may cause the two models to act very differently when strategic dynamics are introduced. A dynamic model of social network formation Brian Skyrms† and Robin Pemantle‡ School of Social Sciences, University of California, Irvine, CA 92607; and ‡Department of Mathematics, Ohio State University, Columbus, OH 43210 This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected on April 27, 1999. Contributed by Brian Skyrms, May 9, 2000 In this paper, we treat some simple reinforcement dynamics, which may serve as a base for future investigation. In the general case, however, interaction structures are ﬂuid and evolve in tandem with strategy. What is required here is a dynamics of interaction structure to model how social networks are formed and modiﬁed. We distinguish this structure dynamics from the strategic dynamics by which individuals change their individual behaviors or strategies. g The idea of simultaneous evolution of strategy and social net- work appears to be almost completely unexplored. Indeed, the most thoroughly studied models of evolutionary game theory assume mean-ﬁeld interactions, where each individual is always equally likely to interact with each other. Standard treatments of evolutionary game dynamics 1; 2 operate entirely in this paradigm. This is due, to a large extent, to considerations of theoretical tractability of the model. Models have been intro- duced that allow the agents some control over their choice of partner (3), but the control is still exerted in a mean-ﬁeld set- ting: one chooses between the present partner and a new pick at random from the whole population. In this paper, we introduce a simple, additive model for struc- ture dynamics, and we explore the resulting system under sev- eral conditions: with or without discounting of the past, with or without added noise, and in the presence or absence of strategic dynamics. Common to all our models is a stochastic evolution p p Evolutionary biologists know that evolutionary dynamics can be affected by nonrandom encounters or population structure, from a (usually symmetric) initial state. Individuals in a popu- lation start out choosing whom to interact with at random and then modify their choices according to how their choice is re- inforced, and then the process is repeated. An inﬁnite variety of such models is possible. We will consider only a few basic models, meant to illustrate that rigorous results on structure dy- namics are not out of reach, and that further inquiry will be proﬁtable. Theorem 1. The probability matrix for Friends I with n play- ers will converge to a random limit p as time goes to inﬁnity. The distribution of the limit is that the rows of p are independent, each having Dirichlet distribution (ignoring the zero entry on the diago- nal) whose parameters are n −1 ones. Thus we see spontaneous emergence of structure. Making Friends: A Baseline Model of Uniform Reinforcement g g What may not be apparent from such data is that the limiting weights for Friends II are always 1/2. Only a small fraction of sample outcomes decisively exhibit the proven limiting behavior. The data, in other words, show that after 1,000 iterations, the weights may still be far from their limiting values; when this is the case, one of the three agents is largely ignored by the other two and visits each of the other two herself equally often. Be- cause the lifetime of many adaptive games is 1,000 rounds or fewer, we see that limiting behavior may not be a good guide to behavior of the system on time scales we are interested in. The analysis section discusses both limiting results for this model and ﬁnite time behavior. When the population size is more than 3, the weights will always converge, but the limit is random and restricted to the subspace of symmetric matrices. Again, conver- gence of the weights to their limiting values is slower than in the nonreciprocal game of Friends I. Friends I: Asymmetric Weights. Each morning, each agent goes out to visit some other agent. The choice of whom to visit is made by chance, with the chances being determined by the relative weights each agent has assigned to the others. For this purpose, agent number i has a vector of weights wi1; : : : ; win that she assigns to other players (assume wii = 0). Then she visits agent j with probability Probagent i visits j = wij P k wik : [1] [1] Here we are interested in a symmetric baseline model, so we will assume that all initial weights are 1. Initially, for all agents, all possible visits are equiprobable. Every agent is treated nicely on her visit and all are treated equally nicely. They each get a reinforcement of 1. Each agent then updates her weight vector by adding 1 to the weight as- sociated with the agent that she visited. Her probabilities for the next round of visits are modiﬁed accordingly. At each stage, we have a matrix pij of probabilities for i to visit j. Do these probabilities converge, and if so to what? Theorem 2. The probability matrix pij for Friends II with n players converges to random limit p as time goes to inﬁnity. Skyrms and Pemantle A dynamic model of social network formation Brian Skyrms† and Robin Pemantle‡ School of Social Sciences, University of California, Irvine, CA 92607; and ‡Department of Mathematics, Ohio State University, Columbus, OH 43210 This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected on April 27, 1999. Contributed by Brian Skyrms, May 9, 2000 To see these differences, we describe what is typi- cally observed after 10 runs of a simulation of Friends II to time 1,000 for a set of three agents, this being the minimum popu- lation size for which structural dynamics are interesting. What we see typically is one or two runs in which each players vis- its are split evenly (to two decimal places) between the others. We see another several runs that are close to this. We see one run or so in which two agents nearly always visit the third agent, which splits its time among the other two. The remaining runs give something between these extreme outcomes. SOCIAL SCIENCES Next, we move to the case where players of different types play a nontrivial game and are reinforced by the payoffs of the game. Here, we examine the coevolution of behavior and structure when the structural dynamics and strategic dynamics are both operative. The relative speeds of structural dynamics and strategic dynamics affect which equilibrium is selected in the game. In particular, this can determine whether the risk- dominant or payoff-dominant equilibrium is selected. Making Enemies Let us change the “Making Friends” model in just one way. Instead of being rewarded, agents are punished; instead of uni- formly positive interactions, we have uniformly negative ones: Enemies I Host Visitor −1; 0 Enemies II Host Visitor −1; −1 Etpijt + 1 = Et n X k=1 pikt wij + δjk 1 + Pn l=1 wilt = wijt + pijt 1 + Pn l=1 wilt = pijt: Instead of interactions being reinforcing, we take them as in- hibiting. The dynamics of inhibition might be modeled in a num- ber of ways. Continuing to use the update Eq. 2 will not work because the weights will end up becoming negative and the visi- tation probabilities in Eq. 1 will be meaningless. In this section, we explore two other possible rules for updating the weights so as to inhibit past behavior. With negative reinforcement, it is easy to predict what will happen: the social network always be- comes uniform, and the dynamics are not sensitive to the partic- ular updating mechanism. Indeed, this is what happens. Because there are no surprises, and because this model is just a building block for a model with both structural and strategic dynamics, we keep the discussion brief. = pijt: = pijt: Even without exchangeability, the martingale convergence the- orem (ref. 23, section 4.2) implies convergence of the quantities pij, though it says very little about the limit. Even without exchangeability, the martingale convergence the- orem (ref. 23, section 4.2) implies convergence of the quantities pij, though it says very little about the limit. pij g y y For Friends II, a complete analysis may be obtained (R.P. and B.S., unpublished work). Here is an outline of what is found there. A computation similar to the one for Friends I shows that Etpt + 1 = pt + 1 t Fpt; The Transfer Model. Consider a three-player model with the fol- lowing update rule on the weights. Initial weights are all positive integers. When i visits j, the weight wij is diminished by 1 and the weight wik, k ̸= i; j, is increased by 1. This is equivalent to the Ehrenfest model of heat exchange between two bodies (28). In the original Ehrenfest model, there are two urns. A ball is drawn at random from among all balls in both urns and trans- ferred to the other urn. Making Friends: A Baseline Model of Uniform Reinforcement That they converge to the uniform vector, where each agent has equal probability to visit each other, has prior probability zero. These correspond to cases where one of the three agents is en- tirely ignored and splits her visits equally between the other two. The probability that pt is within ϵ of one of these traps is roughly 3ϵt−1/3, so with t = 1;000; we ﬁnd a reasonably high probability that p1;000 is not near the uniform probability matrix but is instead still near one of the unstable equilibria. This persists with reasonable probability well beyond t = 106. For greater population sizes, similar phenomena apply. Conver- gence to the invariant set is relatively slow. However, for large populations, say 20 or more, another phenomenon takes place. The portion of the space of possible p matrices that are within ϵ of the possible limits goes to 1; this is known as the con- centration of measure phenomenon (27). Thus it becomes very unlikely to get stuck initially far away from the limit, simply be- cause the initial randomness will very likely lead to a point very near a possible limit. Thus for large populations, the dynamics appear very similar to the dynamics for Friends I. These correspond to cases where one of the three agents is en- tirely ignored and splits her visits equally between the other two. The probability that pt is within ϵ of one of these traps is roughly 3ϵt−1/3, so with t = 1;000; we ﬁnd a reasonably high probability that p1;000 is not near the uniform probability matrix but is instead still near one of the unstable equilibria. This persists with reasonable probability well beyond t = 106. For greater population sizes, similar phenomena apply. Conver- gence to the invariant set is relatively slow. However, for large populations, say 20 or more, another phenomenon takes place. The portion of the space of possible p matrices that are within ϵ of the possible limits goes to 1; this is known as the con- centration of measure phenomenon (27). Thus it becomes very unlikely to get stuck initially far away from the limit, simply be- cause the initial randomness will very likely lead to a point very near a possible limit. Thus for large populations, the dynamics appear very similar to the dynamics for Friends I. Furthermore, convergence to the limiting probability matrix is quite rapid. Making Friends: A Baseline Model of Uniform Reinforcement The remainder of Theorem 2 is then established by showing the only stable ze- ros of the vector ﬁeld F are the symmetric matrices with row sums all equal to 1, and that the possible limit points of pt are exactly the stable equilibria of the ﬂow determined by F. Determination of the rate of convergence of pt to its limit is somewhat different in this case. Because of the presence of unstable equilibria from the ﬂow determined by F, there is a possibility of being stuck near one of these equilibria for a long time before eventually following the ﬂow to one of the stable equilibria. For the three player game, the unstable equilibria are the following three matrices: For Friends I, the updating of the weights for any one agent is the same as a P´olya urn process (21). Each agent can be thought of as having an urn with balls of n −1 colors, one color representing each other agent. Initially there is one ball of each color in the urn. The agent picks a ball at random, indicating whom she should visit, then returns it to the urn along with an extra ball of the same color. The urns belonging to different agents are statistically independent.   0 1 2 1 2 0 0 1 0 1 0     0 0 1 1 2 0 1 2 1 0 0     0 1 0 1 0 0 1 2 1 2 0  :   0 1 2 1 2 0 0 1 0 1 0     0 0 1 1 2 0 1 2 1 0 0     0 1 0 1 0 0 1 2 1 2 0  : The analysis of this process is well known (ref. 22, chapter 4). It is easy to show that the sequence of draws for each agent is exchangeable, that is, permuting a sequence does not change its probability. Hence by the de Finetti representation theorem, the random sequence of draws from an urn is equivalent to a mix- ture of multinomial processes, that is, of sequences of indepen- dent draws. The mixing measure is easily seen to be Dirichlet. Consequently, the visiting probabilities converge with probabil- ity one, but they can converge to anything. Making Friends: A Baseline Model of Uniform Reinforcement If n = 3, the limit is the matrix all of whose off-diagonal entries are 1/2. In general, the limit may be any symmetric matrix whose rows sum to 1y that is, the closed support of the random limit is the entire subspace of symmetric stochastic matrices. Given all the symmetry built into the starting point and the reinforcement, it is perhaps surprising that all sorts of struc- tures emerge. Here is a description of a simulated sample run of length 1,000. The probabilities, to two decimal places, seem to converge after a few hundred rounds of visits, to a matrix that is anything but uniform (and to a different matrix each time the process is run from the initial, symmetric weights). There is one agent, A, who visits another agent, B, more than half the time. There is no reciprocation, so this has no bearing on how often B visits A, and in fact most agents will not visit any one agent more than a third of the time. Analysis of Friends I and II. To ﬁt this in the framework of ∗, con- struct the following degenerate games. Each of the two players has only one strategy, and the payoff matrix is as follows. Friends I Host Visitor 1; 0 Friends II Host Visitor 1; 1 Friends I Host Visitor 1; 0 Friends II Host Visitor 1; 1 The weights wij are initialized to 1 for i ̸= j, and are then updated according to wijt + 1 = wijt + ui; jy t; [2] In the analysis section, we show that this outcome is typical. PNAS \| August 1, 2000 \| vol. 97 \| no. 16 \| 9341 PNAS \| August 1, 2000 \| vol. 97 \| no. 16 \| 9341 PNAS \| August 1, 2000 \| vol. 97 \| no. 16 \| 9341 where wijt is the weight agent i gives to agent j at time t and ui; jy t is the payoff of the game played at time t between visitor i and host j (and zero if this visit did not occur at time t). This, together with speciﬁcation of the visitation probabilities in Eq. 1, deﬁnes the model. Changing the initial weights does not affect the qualitative behavior of any model, so there is no need to vary the initialization. ninety degrees), establish convergence of p. Making Friends: A Baseline Model of Uniform Reinforcement Let pt denote the matrix whose i; j-entry is pijt. Then exchangeability implies that, conditional on the limit matrix p = limt→: pt, the sequence of visits is a sequence of independent, identically distributed draws from the limit dis- tribution. Thus at time t, the central limit theorem implies that pt −p is t−1/2 times a multivariate normal. The portion of the space of possible p matrices that are within ϵ of the possible limits goes to 1; this is known as the con- centration of measure phenomenon (27). Thus it becomes very unlikely to get stuck initially far away from the limit, simply be- cause the initial randomness will very likely lead to a point very near a possible limit. Thus for large populations, the dynamics appear very similar to the dynamics for Friends I. For Friends II, exchangeability fails. This is not surprising, because the property of exchangeability is not very robust. More surprising, however, is that the sequence of probability matrices pt does not form a martingale. To explain this terminology, let Et denote the expectation conditioned on the values at time t. A simple computation shows that for Friends I, the expected value of pijt + 1 conditioned on the time t value is equal to pijt: because wij increases only when i visits j, we have 9342 \| www.pnas.org Perturbations of the Models SOCIAL SCIENCES In this section, we add two features, noise and discounting, com- monly used to create more realistic models. We examine the effects on social structure. In particular, these lead to varying degrees of subgroup formation. Discounting the Past. In the foregoing models, a positive (or nega- tive) payoff in the distant past contributes equally to the weight (or resistance) assigned to an edge as does a like payoff in the immediate past. This is implausible, both psychologically and methodologically. As a matter of psychology, memories fade. From the standpoint of inductive logic, it is not at all certain that the learner is dealing with stationary probabilities—indeed, in cases of prime interest, she is not. For this reason, recent ex- perience may have a better chance of being a relevant guide to future action than the remote past. The Resistance Model. The transfer model allows for a ﬁnite cu- mulative amount of negative reinforcement, and indeed yields a ﬁnite Markov chain. Let us explore a rather different model, termed the resistance model, in which negative payoffs generate resistance. Initially every choice has resistance 1. The magnitude of a negative payoff is added to its associated resistance, so the Eq. 2 becomes p A simple and standard way to modify the models to reﬂect this concern is to introduce discounting of the past. We will concentrate here on the models of Making Friends. After each interaction, we will now multiply the weights of the previous stage by a discount factor, d, between 0 and 1. The we add the undiscounted payoffs from the present interaction to get new weights. The modiﬁcation of the dynamics has a dramatic effect on the Making Friends models. wijt + 1 = wijt + ui; jy t : In the case at hand, when all payoffs are negative, the probability of i visiting j is proportional to the reciprocal of the resistance: pij = Probagent i visits j = 1/wij Pn k=1 1/wik with 1/wii = 0 by convention. The dynamics of Enemies I and Enemies II under resistance dynamics are easy to describe. For Friends I, it is immediately evident from simulations with d = 0:9, say, and 10 players, that the probabilities pij converge to 0 or 1. In other words, each individual ends up always visiting the same other individual. y y Theorem 3. Making Enemies In contrast to the P´olya urn, we do not have convergence of the conditional probabilities of visits at each stage given the present: at any time, given the present composition, the probability of a given visit may be 0; 1/2; or 1; depending on the composition of the urns belonging to the visitor. However, if the number of balls, N is large, approximately equal visiting probabilities are very likely in the following sense. The invariant distribution is binomial, which is concentrated around nearly even distributions when the number of balls is large. Thus, with high probability, no matter what the initial state, after roughly N log N/2 steps (29), the composition of an urn with N balls will be close to a draw from a binomial distribution. The conditional probability of either of the two possible visits will therefore be close to 1/2 and will tend to remain there with high probability. Kac (30) uses these properties to resolve the apparent paradoxes that beset Bolzmann’s discussion of irreversibility in statistical mechanics. and the unique stationary vector is 1/4; 1/2; 1/4. In contrast to the P´olya urn, we do not have convergence of the conditional probabilities of visits at each stage given the present: at any time, given the present composition, the probability of a given visit may be 0; 1/2; or 1; depending on the composition of the urns belonging to the visitor. However, if the number of balls, N is large, approximately equal visiting probabilities are very likely in the following sense. The invariant distribution is binomial, which is concentrated around nearly even distributions when the number of balls is large. Thus, with high probability, no matter what the initial state, after roughly N log N/2 steps (29), the composition of an urn with N balls will be close to a draw from a binomial distribution. The conditional probability of either of the two possible visits will therefore be close to 1/2 and will tend to remain there with high probability. Kac (30) uses these properties to resolve the apparent paradoxes that beset Bolzmann’s discussion of irreversibility in statistical mechanics. [3] In the next section, we will see a property this rule has in com- mon with rules that discount the past, namely that it leads to being trapped in a deterministic state where i always visits the same j. Question 1. Making Enemies Is there a model incorporating both positive and negative reinforcement, that is realistic, tractable, and nontrapping? Skyrms and Pemantle Making Enemies The distribution of balls tends to the binomial distribution, where each ball is independently equally likely to be in either urn. In Making Enemies, with transfer where F is a certain function on symmetric n by n matrices. In other words, the random sequence of matrices pt x t = 1; 2; : : : is a stochastic approximation in the sense of Robbins and Monro (24), driven by the vector ﬁeld F. General results of refs. 25 and 26 now imply that pt converges to the set where F vanishes. To show that pt always converges to a single point, Pemantle and Skyrms (unpublished work) compute a Lya- punov function for F, that is, a function V for which ∇V ·F + 0 with equality only when F = 0. This, together with an efﬁciency inequality (bounding the angle between f and ∇V away from 9342 \| www.pnas.org Skyrms and Pemantle dynamics and three players, each player may be thought of as having such a pair of urns. The urns are independent. While uniform positive reinforcement breeds structure from unstructured initial conditions, uniform negative reinforcement evidently breeds uniformity even from structured initial condi- tions. It would appear, therefore, that the customary random encounter (mean-ﬁeld) model is more suitable for Making En- emies than Making Friends. g p p Because the number of balls is ﬁxed, an Ehrenfest urn is a Markov chain with a ﬁnite number of states, where the states consist of distributions over the two urns. For example, if there are only two balls, then there are three states, S1, S2 and S3, corresponding to urn cardinalities of 2; 0, 1; 1, and 0; 2. The transition matrix for this Markov chain is A Better Model? We would like a model that allows for both pos- itive and negative reinforcement. A natural choice is to let wij keep track of the log-likelihood for i to visit j, so that probability of i visiting j is given by   0 1 0 1 2 0 1 2 0 1 0   pij = Probagent i visits j = expwij Pn k=1 expwik: [3] and the unique stationary vector is 1/4; 1/2; 1/4. Perturbations of the Models For Making Friends, let us modify the probability rule (1) so that in the n-player game, the probability of i visiting j is now some ﬁxed positive number ϵ/n −1, plus 1 −ϵ times what it was before: The principle behind the analysis of discounted Friends is that the future behavior of p is largely determined by the present Gp. In particular, we ﬁnd a δ , 0 such that from any state p, for each subgraph H of Gp such that H S, there is a probability at least δ2 that for all sufﬁciently large t, Gpt = H. We show this in two steps: (step i) with probability at least δ, there is some t for which Gpt = H; (step ii) from any state p such that Gp = H, there is probability at least δ that Gpt is equal to H for all later times, t. pij = ϵ n −1 + 1 −ϵ wij P k wik : The effect of this is to push the system by ϵ toward the uniform point p. Neither Friends I nor Friends II is now a martingale, and the stable set of each is reduced to the single point p. Be- cause this is true at any noise level ϵ , 0, we see that there is only one asymptotically stable point. Because the qualitative outcome is sensitive to the existence of a noise term, it is in- cumbent to ask with regard to speciﬁc models whether a noise term is natural and realistic. p q , To see why step i is true, for H S, let fH be any function on vertices of H for which each value fi is a neighbor of i. Observe that there is a number k such that from any state p with H Gp, if each vertex i visits fi for the next k rounds, then Gpk = H. For each round of visits, this probability is at least ϵn, where n is the number of vertices, so taking δ < ϵkn establishes (step i). For (step ii), it sufﬁces to show that with probability δ each agent visits a neighbor in H at all later times. For each agent i, the sum over j not neighboring i in H of pij is at most nϵ + 1/2 by the deﬁnition of Gp = H. Perturbations of the Models Alternatively, an agent may purposefully add noise to her strategy in order to avoid becoming wedded to a less than optimally efﬁcient strategy or structure. This may stem from an agent’s uncertainty, from agent error, or from circumstances beyond an agent’s control. Alternatively, an agent may purposefully add noise to her strategy in order to avoid becoming wedded to a less than optimally efﬁcient strategy or structure. Theorem 4. In Friends II with discount rate d + 1, there is always a partition into pairs and stars and a random time after which each member of a pair visits only the other member of the pair and each noncentral member of a star visits only the center. In Friends I, there is a random function f and a random time after which each player i always visits fi. From a methodological point of view, noise that does not go to zero with time transforms the model into an ergodic Markov chain. No state is then trapping. To the extent that the trap- ping states produced by discounting or linear log-likelihood are unrealistic, we may hope to mitigate the problem by adding a noise component. Because dynamics with a noise term do not lead to a single state, the outcome is usually phrased in terms of stochastically stable states (32). A state is termed stochastically stable if the chance of ﬁnding the system near that state does not go to zero as the magnitude of the noise term goes to zero. p y y f Sketch of Proof: The analysis for Friends I is similar but eas- ier, so we prove the statement only for Friends II. With each probability matrix p we associate a graph Gp as follows. The edge i; j is in the graph G if the probability pij , ϵ, where ϵ + 1/2n is some ﬁxed positive number. Among those graphs having at least one edge incident to each vertex, let S denote the minimal such graphs, that is, ones for which deleting any edge results in an isolated vertex. It is easy to see that S is the set Gp for all p satisfying the conclusion of the theorem. Neither discounting nor noise will affect the limiting behav- ior of Making Enemies. Perturbations of the Models After k rounds of visits where agents only visit their neighbors in H, this must decrease to at most 1/2dk. Thus the probability of N rounds of visits only to neighbors in H is at least Noise and Discounting. In the presence of a discount d + 1 and a noise term ϵ , 0, if 1 −d is much smaller than ϵ; then the dis- count is so low that the noise term wipes out any effect the dis- counting might have had. In the other case, where d is held ﬁxed and ϵ tends to zero, we may ask about the asymptotically stable states of system with past discounting dynamics. For Friends I, nothing much interesting happens: discounting causes the lim- iting state to be degenerate; with noise, the system may jump from one such state to the other, which does not change which states are stochastically stable. N−1 Y k=0 1 −1 2dk n : Sending N to inﬁnity yields a convergent inﬁnite product, since 1/2dk is summable. Taking δ to be less than the inﬁnite prod- uct proves (step ii). For Friends II, as long as the number of players n is at least 4, the introduction of noise does indeed change the set of stochastically stable states: it gets rid of stars. Simulations show that pairings are by far the most prevalent states in discounted Friends II, with a star of size 3 forming when necessitated by an odd number of players. We now show that states with more than one star, or a star of size greater than 3, are not stochastically stable. With steps i and ii, the rest is a standard tail argument. The constraints on evolution are such that Gpt always contains at least one graph in S. As long as it contains more than one graph in S, there is always a probability of at least δ of perma- nently settling into each one. Thus, with probability 1, eventu- ally Gpt is equal to some H S for all future times. This is equivalent to the conclusion of the theorem. QED y Theorem 5. In Friends II, with discounting, with n players, and with noise tending to zero, the stochastically stable states are those that are either unions of pairs (if n is even) or pairs plus a single star of size 3 (if n is odd). Perturbations of the Models For Enemies I or Enemies II, from any initial conditions, the probability matrix pt converges to the uniform probability matrix p where pij = 1/n −1 for any i ̸= j. The of convergence is rapid: of order N log N if the initial resistances are of order N. The deviations from uniform obey a central limit theorem: In Friends II, simulations show the group breaking into pairs, with each member of a pair always visiting his or her “part- ner.” Which pairs form depends on the randomness in the early rounds of visits, but pairs always form. In fact, there are other possible limit states, but their frequency is low except at more extreme discount rates. The set of possible limit states may be described as follows. Some agents are grouped in pairs, each member of a pair always visiting the other. Other agents are grouped in stars. These are clusters of size at least three, in which one agent, called the center, visits each of the others with positive frequency, while the others always visit the center. t1/2p −p →X where X is a multivariate normal with covariance matrix of rank nn −1 in Enemies I and nn −1/2 in Enemies II. In other words, deviations from uniformity are independent normals, subject to the constraints of adding up to zero for each individual and, in the case of Enemies II, the constraints of symmetry. f f y y The central limit theorem may be derived from a stronger, functional central limit theorem, linearizing the system near the uniform probability to see that the paths Analysis of Discounting the Past. It is worth giving a rigorous deriva- tion of the above behavior, because it will shed some light on a defect in the most obvious log-likelihood model to incorporate positive and negative reinforcement. Our derivation highlights this, although the results for discounted Friends I may also be derived from a theorem of H. Rubin (see ref. 31, page 227). t 7→N−1/2pNt −p converge in distribution as N →: to a multivariate Ornstein– Uhlenbeck process. The rate of convergence follows from stan- dard coupling arguments. PNAS \| August 1, 2000 \| vol. 97 \| no. 16 \| 9343 This may stem from an agent’s uncertainty, from agent error, or from circumstances beyond an agent’s control. Skyrms and Pemantle 9344 \| www.pnas.org Reinforcement by Games of Nontrivial Strategy It takes two persons cooperating to effectively hunt a stag, while one person acting independently can hunt a rabbit. Bagging a stag brings a greater payoff. It should be noted that, although the stochastically stable states include those that are not optimally efﬁcient, the op- timally efﬁcient states (those states where rabbit hunters visit rabbit hunters) will have an edge. Because of the possibility of reciprocal reinforcement, it will be easier for a rabbit hunter to switch from visiting a stag hunter to visiting a rabbit hunter than vice versa. Second, when the discount rate is near 1, the model behaves like the undiscounted model for a long enough time that it is very unlikely for a rabbit hunter to get locked into visiting a stag hunter in the ﬁrst place. Simulations of Stag Hunting with 10 players and d = 0:9, seem to show that rab- bit hunters “always” visit rabbit hunters. Because of both of the effects mentioned above, the system is nearly always found in an optimally efﬁcient state, even though there are stochastically stable states that are not optimally efﬁcient. Hunt Stag Hunt Rabbit Hunt Stag 1; 1 0; :75 Hunt Rabbit :75; 0 :75; :75 There are two equilibria in this game: both hunt stag and both hunt rabbit. The ﬁrst carries the higher payoff and is said to be payoff dominant; the second carries the least risk and is said to be risk dominant (35). In models without structural dynam- ics, Kandori, Mailath and Rob (36) have shown that only the risk dominant equilibrium of a two player coordination game is stochastically stable. In the presence of structural dynamics, we will describe a more optimistic conclusion. Coevolution of Structure and Strategy. To the previous model, we now add the possibility of an agent switching states: a stag hunter may decide to become a rabbit hunter, or a rabbit hunter may become bold and hunt stag. When this kind of strategic evolution is faster than the structural evolution, we know from studies of random encounter models that the risk dominant equilibrium of everyone hunting rabbits will be ar- rived at while the network is still near its initial state of uniform visitation probabilities. Theorem 6. Suppose Stag Hunt is played by 2n players, with structural dynamics given by Eq. 1 and cumulative weighting dy- namics (2) with no noise or discounting. Reinforcement by Games of Nontrivial Strategy Second, when the discount rate is near 1, the model behaves like the undiscounted model for a long enough time that it is very unlikely for a rabbit hunter to get locked into visiting a stag hunter in the ﬁrst place. Simulations of Stag Hunting with 10 players and d = 0:9, seem to show that rab- bit hunters “always” visit rabbit hunters. Because of both of the effects mentioned above, the system is nearly always found in an optimally efﬁcient state, even though there are stochastically stable states that are not optimally efﬁcient. g Introduction of a discount rate changes this outcome. Stag hunters still end up visiting stag hunters, because even dis- counted reinforcement beats a reinforcement of zero, but now rabbit hunters will get locked either into pairs and stars as in Making Friends, or into repeated visits to a single stag hunter. h l ll d d g Introduction of a discount rate changes this outcome. Stag hunters still end up visiting stag hunters, because even dis- counted reinforcement beats a reinforcement of zero, but now rabbit hunters will get locked either into pairs and stars as in Making Friends, or into repeated visits to a single stag hunter. These limit states are all invariant under introduction of noise. When a rabbit hunter visits a stag hunter the loss to society is the 0:75 that another rabbit hunter would have proﬁted from the visit. The model is evidently weak here, because it allows only one visit by each agent but any number of visits to each agent in a round of visits. That is, a more realistic loss would be the stag hunter’s wasted time when visited by the rabbit hunter. h ld b d h l h h h h ll bl When a rabbit hunter visits a stag hunter the loss to society is the 0:75 that another rabbit hunter would have proﬁted from the visit. The model is evidently weak here, because it allows only one visit by each agent but any number of visits to each agent in a round of visits. That is, a more realistic loss would be the stag hunter’s wasted time when visited by the rabbit hunter. Rousseau’s Stag Hunt. Consider a two-player version of Rousseau’s Stag Hunt (34). The choices are either to hunt stag or to hunt rabbit (hare, in the original). Perturbations of the Models 9344 \| www.pnas.org Skyrms and Pemantle starting from H = G1 leads in ﬁnite time (because the number of edges decreases each time) to an element of S0. that the probability of a stag hunter visiting a rabbit hunter goes to zero. g ) 0 We now follow the usual method for determining stochastic stability (33). Let the probability ρ of disobeying the structural dynamics Eq. 1 be very small. If ϵ (in the deﬁnition of S) is very small, then a state p with Gp = G S will have Gpt = G for all later times with high probability, until there is a disobey- ing move. After a single disobedience, the graph Gp will be the union of G with one extra edge. By the remark after the proof of Theorem 4, we see that after a disobedience, the graph will then relax to some subgraph in S. By property i, if G S0 then this subgraph is again in S0. Thus, a single disobedience followed by relaxation back to S will never escape S0. Hence, the probability of jumping to S \ S0 is of order ρ2, which implies that states in S0 stay in S0 for time at least ρ−2. On the other hand, by property ii, from any state in S \ S0, there is a chain of single disobediences, such that allowing the system to relax after each may with positive probability land you back in S0. Thus, the expected time spent in S \ S0 before returning to S0 is at most of order ρ. Thus, the process spends 1 −ρ portion of the time in S0, and sending ρ to zero, we see that only states in S0 are stochastically stable. It is easy to see that all of these are indeed stochastically stable. QED Next, consider the subpopulation of rabbit hunters, call it A. For i A, let Zi; t = P j /A wij Pn j=1 wij denote the probability of visiting a given rabbit hunter visiting a stag hunter on the next turn. Perturbations of the Models The expected value of Zi; t + 1 changes according to the formula EZi; t + 1Zi; t = Zi; t + t−1Yi; t; where Yi; t is the proportion of increase in expected weight wij due to j / A: Yi; t = P j /A pij + pji Pn j=1 pij + pji : Ignoring the terms pji in both the numerator and denominator of the above expression would lead to exactly Zi; t. The terms pji for j / A are known to be small, while the total from the terms pji for j A cannot be small. Consequently, Yi; t + 1 −ϵZi; t for some ϵ , 0, whence SOCIAL SCIENCES E Zi; t + 1 −Zi; t Zi; t < −ϵZi; t t : Skyrms and Pemantle Perturbations of the Models Remark: It is actually shown that in (step ii), if we choose ϵ sufﬁciently small, we can choose δ arbitrarily close to 1. We now also see why the log-likelihood rule (3) leads to ﬁxation of a degenerate structure. Under these dynamics, an equivalent phenomenon occurs to step i in the proof of Theo- rem 4. For a pair i; j whose interaction has a positive mean, if the pair plays repeatedly, we will see wijt/t →µ , 0. The probability the i will ever switch partners, once having tried j a few times is at most on the order of P: k=0 B exp−kµ, where B = expP l̸=j wil. From here it is easy to construct an argu- ment parallel to the proof of Theorem 4, to show that in pres- ence of a game with positive mean payoff, discounted structural dynamics lead with probability 1 to ﬁxation at a pairing. g f ( f ) Sketch of Proof: Let S denote the graphs corresponding to possible limit states as in the proof of Theorem 4, and let S0 S denote those graphs with no stars (perfect pairings) or with a single star of size 3. The important properties of the rela- tion of S to S0 are as follows. (Property i) If G is the result of adding a single edge to a graph in S0, then G contains no graph in S \ S0. (Property ii) For any G S there is a chain G = G1; G2; : : : ; Gk leading to S0, where each Gj+1 may be ob- tained from Gj be adding an edge and then deleting two edges. Property i is apparent. To verify property ii, note that if H S and i and j are noncentral vertices in stars of H, and they are not both in the same star of size 3, then adding the edge be- tween i and j and removing the two edges previously incident to i and j produces a new graph in S. Iterating this procedure Introduction of Noise. A common feature in models of adaptation is the introduction of noise: a small chance of a behavior other than the one chosen by the dynamical equation for the model. Reinforcement by Games of Nontrivial Strategy So far we have only considered a baseline model of uniform reinforcement, which turned out still to have nontrivial struc- tural behavior. Now we examine a reinforcement scheme re- sulting from the payoff of a nontrivial game. We will consider the case where evolution of strategy is slower than evolution of structure. Thus, we will consider the agents as divided into types, each type always playing a ﬁxed strategy, and see what sort of interaction structure emerges. We then extend this by al- lowing strategic switching of types. We ﬁnd that coordination of strategy occurs, though whether players coordinate on the risk- dominant or payoff-dominant strategy depends on parameters of the model such as the rate of strategic evolution. Depending on conditions of the model, the social network may or may not split up into pairs. Because the increments in Zi; t are bounded by C/t, there are a λ; µ , 0 for which expλZi; t+µ log t is a supermartingale, which implies that Zi; t converges to zero exponentially fast in log t. QED in log t. QED Introduction of a discount rate changes this outcome. Stag hunters still end up visiting stag hunters, because even dis- counted reinforcement beats a reinforcement of zero, but now rabbit hunters will get locked either into pairs and stars as in Making Friends, or into repeated visits to a single stag hunter. These limit states are all invariant under introduction of noise. When a rabbit hunter visits a stag hunter the loss to society is the 0:75 that another rabbit hunter would have proﬁted from the visit. The model is evidently weak here, because it allows only one visit by each agent but any number of visits to each agent in a round of visits. That is, a more realistic loss would be the stag hunter’s wasted time when visited by the rabbit hunter. It should be noted that, although the stochastically stable states include those that are not optimally efﬁcient, the op- timally efﬁcient states (those states where rabbit hunters visit rabbit hunters) will have an edge. Because of the possibility of reciprocal reinforcement, it will be easier for a rabbit hunter to switch from visiting a stag hunter to visiting a rabbit hunter than vice versa. Reinforcement by Games of Nontrivial Strategy Varying between these ex- tremes can give one or the other result with different probabili- ties, or may leave the group in a state where both strategies are used. We expect to see structure dynamics making a difference in other games as well. Indeed, we have some preliminary sim- ulation evidence showing this to be true for a bargaining game (“split the dollar”), and for a simple coordination game. We describe here the results of simulations of Stag Hunting for 1,000 time steps, where with some probability q at any given time, an individual changes type to whichever type was most successful in the previous round. When q = 0:1, we found that in 22% of the cases all hunters ended up hunting stag, while in 78% of the cases, all hunters hunted rabbit. Thus there was perfect coordination, but usually not to the most efﬁcient equi- librium. On the other hand, when q = 0:01, the majority (71%) of the cases ended in the optimal state of all hunting stag, while 29% ended up all hunting rabbit. Increasing the initial edge weights made it far less likely to reach the stag hunting equi- librium, since stag hunters took a long time to perfectly align, and without alignment, the previous round’s best strategy was almost always rabbit hunting. For instance, if the initial weights were 1,000 for each visit, under 1% of the cases ended up all stag hunting, whether q was 0:1 or 0:01. There are many more avenues to pursue. As mentioned in Making Enemies, it would be desirable to ﬁnd a model in which positive and negative reinforcement are present, but trapping does not occur. We have not modeled any interaction among three or more players. We also have yet to model any explicit interaction between strategy and structure: the choice of a part- ner to play with and a strategy to play against that partner need not be independent. g g q Once hunters largely cease to visit hunters of opposite type, the structural evolution within each of the two subpopulations is a version of Friends II. The resulting social structure will not be a perfect pairing, but will have each rabbit (stag) hunter visiting each other rabbit (stag) hunter, but with varying probabilities. Reinforcement by Games of Nontrivial Strategy Then in the limit, stag hunters always visit stag hunters and rabbit hunters visit rabbit hunters. Sketch of Proof: First note that no visit of a stag hunter to a rabbit hunter is ever reinforced. Thus, wijt = 1 for all t if i is a stag hunter and j is a rabbit hunter. Observing that the weights wijt go to inﬁnity when i and j are both stag hunters, we see PNAS \| August 1, 2000 \| vol. 97 \| no. 16 \| 9345 Whether strategic dynamics are faster or slower than struc- tural dynamics depends, of course, on the activity being mod- eled; sometimes interaction structure is externally imposed, while sometimes it is more easily modiﬁed than strategy or char- acter. Let us suppose that the investment in re-training as a dif- ferent kind of hunter is great, so between each round of visits there is only a small chance that one of the hunters will change types. Then we have seen that hunters always (with no noise or discounting) or nearly always (in discounted models) hunt with others of like type. This eliminates the risk inherent in random encounters and allows hunters to proﬁt from switching to stag hunting after an initial period where they ﬁnd another stag hunter. Slow strategic adaptation gradually converts rabbit hunters to stag hunters and the payoff dominant strategy domi- nates. and surprising results. Even in baseline models where the game being played is degenerate, we ﬁnd spontaneous emergence of structure from uniformity and spontaneous emergence of uni- formity from structure. We ﬁnd processes with extremely long transient modes, where limiting behavior is not a good guide for predicting behavior after thousands of trials. The social interaction structures that emerge tend to separate the population into small interaction groups within which there is coordination of strategy. This separation may be complete, as in discounted Friends II, or may be only a tendency, as in the nondiscounted versions of Friends and Stag Hunting. When we combine structure and strategy dynamics for a non- trivial game, the Stag Hunt, we ﬁnd that the probable outcomes depend on the timing. Where structure is frozen in a random encounter conﬁguration we get the expected risk-dominant equi- librium outcome. But when structure is ﬂuid relative to strategy, structural adaptation neutralizes the risk and we get the socially efﬁcient payoff dominant equilibrium. Conclusion We have taken some basic steps in exploring dynamics of evo- lution of interaction structures and coevolution of structure and strategy. The ultimate goals are to create models that are more true to life, and to ﬁnd theoretical bases for observed behaviors of systems, including prediction of selection between multiple equilibria. We wish to thank Persi Diaconis, Joel Sobel, and Glenn Ellison for bringing us together, for helpful discussions, and for greatly improving our awareness of the relevant literature. This work was supported in part by National Science Foundation Grant DMS 9803249. q The particular dynamics we use here are only examples, but it turns out that the simplest of these may deliver interesting 20. Arthur, W. B. (1989) Econ. J. 99, 116–131. 22. Johnson, N. & Kotz, S. (1977) Urn Models and Their Application (Wiley, New York). 23. Durrett, R. (1996) Probability: Theory and Examples (Duxbury Press, Wadsworth Publishing Company, Belmont, CA), 2nd Ed. 24. Robbins, H. & Monro, S. (1951) Ann. Math. Stat. 22, 400–407. g p p 7. Durrett, R. & Neuhauser, C. (1997) Ann. Appl. Prob. 7, 10–45 25. Pemantle, R. (1990) Ann. Probab. 18, 698–712. 26. Bena¨ım, M. & Hirsch, M. (1995) Ergodic Theory Dyn. 9. Durrett, R. & Levin, S. (1994) Theor. Pop. Biol. 46, 363–394. 27. Talagrand, M. (1995) IHES Publ. Math. 81, 73–205. 10. Blume, L. (1993) Games Econ. Behav. 5, 387–423. 28. Ehrenfest, P. & Ehrenfest, T. (1907) Phys. Zeit. 8, 311–314. 11. Pollack, G. B. (1989) Social Networks 11, 175–212. 29. Diaconis, P. & Stroock, D. (1991) Ann. Appl. Prob. 1, 39–61. 12. Lindgren, K. & Nordahl, M. (1994) Physica D 75, 292–309 30. Kac, M. (1947) Am. Math. Monthly 54, 369–391. 13. Anderlini, L. & Ianni, A. (1997) in The Dynamics of Norms, eds. Bicchieri, C., Jeffrey, R. & Skyrms, B. (Cambridge Univ. Press, Cambridge, U.K.), pp. 87–107. 31. Davis, B. (1990) Prob. Theor. Rel. Fields 84, 203–229. ( ) , 32. Foster, D. & Young, H. P. (1990) Theor. Pop. Biol. 38, 219–232. 32. Foster, D. & Young, H. P. (1990) Theor. Pop. Biol. 3 33. Ellison, G. (2000) Rev. Econ. Studies, in press. 14. Ellison, G. (1993) Econometrica 61, 1047–1071. 34. Rousseau, J.-J. (1984) A Discourse on Inequality; trans. Cranston, M. (Penguin, London). 15. Watts, D. & Strogatz, S. (1998) Nature (London) 393, 440–442. 16. Schelling, T. (1969) Am. Econ. Rev. Papers Proc. 59, 488–493 35. Reinforcement by Games of Nontrivial Strategy One could continue adding complexity so as to allow infor- mation to affect structural evolution, to include communication between players, and so forth. Our main point is this. Struc- tural change is a common feature of the real world. A theory of strategic interaction must take account of it. There is a mathe- matically rich theory which develops relevant tools. We believe that explicit modeling of structural dynamics, and the interac- tion of structure and strategy, will generate new insights for the theory of adaptive behavior. 1. Hofbauer, J. & Sigmund, K. (1988) The Theory of Evolution and Dynamical Systems (Cambridge Univ. Press, Cambridge, U.K.). 2. Weibull, J. (1997) Evolutionary Game Theory (MIT Press, Cambridge, MA). 3. Feldman, M. & Thomas, E. (1987) J. Theor. Biol. 128, 297–315. 4. Wright, S. (1921) Genetics 6, 144–161. 5. Wright, S. (1945) Ecology 26, 415–419. 6. Jackson, M. & Watts, A. (1999) On the Formation of Interaction Networks in Social Coordination Games, working paper. 7. Durrett, R. & Neuhauser, C. (1997) Ann. Appl. Prob. 7, 10–45. 8. Kang, H.-C., Krone, S. & Neuhauser, C. (1995) Ann. Appl. Prob. 5, 1025–1060. 9. Durrett, R. & Levin, S. (1994) Theor. Pop. Biol. 46, 363–394. 10. Blume, L. (1993) Games Econ. Behav. 5, 387–423. 11. Pollack, G. B. (1989) Social Networks 11, 175–212. 12. Lindgren, K. & Nordahl, M. (1994) Physica D 75, 292–309. 13. Anderlini, L. & Ianni, A. (1997) in The Dynamics of Norms, eds. Bicchieri, C., Jeffrey, R. & Skyrms, B. (Cambridge Univ. Press, Cambridge, U.K.), pp. 87–107. 14. Ellison, G. (1993) Econometrica 61, 1047–1071. 15 Watts D & Strogatz S (1998) Nature (London) 393 440 442 21. Eggenberger, F. & P´olya, G. (1923) Zeit. Angew. Math. Mech. 3, 279–289. 1. Hofbauer, J. & Sigmund, K. (1988) The Theory of Evolution and Dynamical Systems (Cambridge Univ. Press, Cambridge, U.K.). 2. Weibull, J. (1997) Evolutionary Game Theory (MIT Press, Cambridge, MA). 3. Feldman, M. & Thomas, E. (1987) J. Theor. Biol. 128, 297–315. 4. Wright, S. (1921) Genetics 6, 144–161. 5. Wright, S. (1945) Ecology 26, 415–419. 6. Jackson, M. & Watts, A. (1999) On the Formation of Interaction Networks in Social Coordination Games, working paper. 7. Durrett, R. & Neuhauser, C. (1997) Ann. Appl. Prob. 7, 10–45. 8. Kang, H.-C., Krone, S. & Neuhauser, C. (1995) Ann. Appl. Prob. 5, 1025–1060. 9. Durrett, R. & Levin, S. (1994) Theor. Pop. Biol. 46, 363–394. 20. Arthur, W. B. (1989) Econ. J. 99, 116–131. Conclusion Harsanyi, J. & Selten, R. (1988) A General Theory of Equilibrium in Games (MIT Press, Cambridge, MA). 17. Schelling, T. (1971) J. Math. Sociol. 1, 143–86. (MIT Press, Cambridge, MA). 18. Epstein, J. & Axtell, R. (1996) Growing Artiﬁcial Societies (MIT/Brookings, Cambridge, MA). ( g ) 36. Kandori, M., Mailath, G. & Rob, R. (1993) Econometrica 61, 29–56. Skyrms and Pemantle 9346 \| www.pnas.org www.pnas.org
https://openalex.org/W2314413844	https://www.biodiversitylibrary.org/partpdf/86267	English	null	The Hawaiian Linnet, Carpodacus Mutans Grinnell	The Auk/The auk	1,912	public-domain	1,235	336 336 ale The Hawaiian 72. Hylocichla guttata auduboni. Avupuson’s Hermit TorusH.— Two taken in Rocky Canyon, Oct. 11, 1906. 73. Hylocichla guttata nanus. Dwarr Hermit THrusH.— One taken on the Mimbres — Black Canyon Divide, Oct. 5, 1908. 74. Planesticus migratorius propinquus. WersTERN Rospin.— Common over all the area, but not noted above 8000 feet. 75. Sialia mexicana bairdi. CHmrSTNUT-BACKED BLUEBIRD.— A number seen at the Alum Camp Oct. 16, 1907. 76. Sialia currucoides. Mountain BuuEBIRD— Common over all the area, Jatest date being Oct. 17, 1908. BY JOHN C. PHILLIPS. In ‘The Auk’ for June, 1912, Mr. Grinnell gives a new name to the introduced linnet of the Hawaiian Isles. I propose to discuss briefly both the name itself and the specific value of the form named. In the first place, the word itself, mutans, implies a very definite condition, namely, a sudden germinal variation expressed in the soma as a Mendelian dominant, dominant because it is not possible to conceive of a recessive character, getting the upper hand in the wild unless it is of marked selectional value. The word mutation means “the act or process of changing” but in the biological sense which it has had since the time of de Vries, a very definite meaning has been given to it, often theoretical perhaps, but nevertheless quite clear. Unfortunately the word has been misapplied to little understood types of variation,— for instance, to rare Mendelian combinations, to the loss of one or more characters from the germ cells, to the products of disease, ete., etc. The name Carpodacus mutans, then, would imply that the following experimental conditions must hold. First the new form must breed true, or as true as the old, even when taken back to its original continental range. Second, in crosses with typical orange oa \| oa \| oot uures, The Hawaiian birds of ancestral stocks, it must behave as a dominant, or at least as a partial dominant. Neither of the above premises is likely to hold good. Now as to the value of these imported Hawaiian Linnets in a racial sense. Mr. Grinnell discusses the change in caged Linnets (University of Cal. Pub., Vol. 7, p. 179) and shows that yellow birds result from red, after a short period of confinement. This appears to be a well known fact with various red birds. The case of the European Linnet is mentioned by Finn in the Avicultural Magazine for June, 1906. Mr. Bangs informs me of like cases from his own experience with Crossbills, Redpolls and Purple Finches and with a Cardinal in confinement. The Cardinal did not go so far off color as the Crossbills. The so-called Japanese Robin (Liothrix luteus) which is brilliantly colored on the chest, throat, and pri- maries with orange and scarlet-vermilion, turns more or less olivaceous all over after a few moults according to Mr. Bang’s experience. BY JOHN C. PHILLIPS. We have all seen the dirty white color which American Flamingoes take on in zodlogical gardens. Is it not thus highly probable, as Mr. Grinnell himself has suggested, that a diminished tyrosine oxidation is responsible for both the caged and the Hawaiian birds. The general environ- mental complex of the Hawaiian Islands is an intricate problem, but the fact that other American insular areas, Guadalupe and San Benito Islands, show a rather high percentage of yellow or orange birds is suggestive as Mr. Grinnell says of some special insular effects. The occurrence of some yellow or orange birds (percent hard to estimate for reasons given Mr. Grinnell on p. 182) in the normal habitat of the species, shows that its color elaborator is in a state of delicate adjustment, that is, it is not always allowed to continue its action up to the ultimate crimson color. We can well imagine, then, that a very slight diminution in the activity of the enzyme might produce the observed results in Hawai, and might not these results be of entirely extrinsic origin? It is certainly easier to apply a theory of this sort than to hunt about for a deeper one. On page 184 Mr. Grinnell calls attention to the fact that eight or ten years ago there were many red or crimson linnets in the islands. The data upon which this suggestion is founded are not quite 338 es s, The Hawatian sufficient for the purpose, but if this is so there has been a change since that time, and the case is a very curious one. In the Museum of Comparative Zodlogy there is a series of twelve male and six female Linnets collected by Flood Bros. on Molokai in 1895 fifteen years before Mr. Grinnell’s series was taken by Miss Alexander on Molokai, Oahu and Maui. This series shows (provided it was taken at random) that the species was at that time nowhere near stability, and that there was almost as great a range of color as in birds from Arizona and other parts of the southwest, only the average color is of a much lighter shade. Three of the males are poppy red to orange vermilion (Ridgway’s Manual of Colors, 1886), three of them are orange to orange ver- milion, five are orange to cadmium-orange, and one is pure cad- mium yellow. BY JOHN C. PHILLIPS. In most of these birds the feathers are of mixed tints, that is, at least two colors occur on one bird. Most of these birds, then, are well off color. There is no individual of the deep crimson type. As a whole this series does not differ very markedly from Mr. Grinnell’s as far as one can judge without comparison. The colors, being so subtle and mixed are not easily described. In the last paragraph of his paper Mr. Grinnell says that at the present time “to assert emphatically any particular factor or group of factors as the prime stimulus does not seem justifiable.” The name however, C. mutans, does assert or at least imply a definite variation of an intrinsic, or germinal nature. The question as to whether this slight alteration in a character already unstable is deserving of special recognition in our nomenclature is an open one, and certainly not for the writer to decide. It was the name itself which first appeared to him as inappropriate. Holding Institution Smithsonian Libraries and Archives This file was generated 1 April 2024 at 12:04 UTC Phillips, John C. 1912. "The Hawaiian Linnet, Carpodacus Mutans Grinnell." The Auk 29, 336–338. https://doi.org/10.2307/4071040. View This Item Online: https://www.biodiversitylibrary.org/item/54327 DOI: https://doi.org/10.2307/4071040 Permalink: https://www.biodiversitylibrary.org/partpdf/86267 Holding Institution Smithsonian Libraries and Archives Sponsored by Smithsonian Copyright & Reuse Copyright Status: Public domain. The BHL considers that this work is no longer under copyright protection. This document was created from content at the Biodiversity Heritage Library, the world's largest open access digital library for biodiversity literature and archives. Visit BHL at https://www.biodiversitylibrary.org. Phillips, John C. 1912. "The Hawaiian Linnet, Carpodacus Mutans Grinnell." The Auk 29, 336–338. https://doi.org/10.2307/4071040. View This Item Online: https://www.biodiversitylibrary.org/item/54327 DOI: https://doi.org/10.2307/4071040 View This Item Online: https://www.biodiversitylibrary.org/item/54327 DOI: https://doi.org/10.2307/4071040 Permalink: https://www.biodiversitylibrary.org/partpdf/86267 Permalink: https://www.biodiversitylibrary.org/partpdf/86267 Copyright & Reuse Copyright Status: Public domain. The BHL considers that this work is no longer under copyright protection. This document was created from content at the Biodiversity Heritage Library, the world's largest open access digital library for biodiversity literature and archives. Visit BHL at https://www.biodiversitylibrary.org. This file was generated 1 April 2024 at 12:04 UTC
https://openalex.org/W2791749859	https://eprints.gla.ac.uk/159482/1/159482.pdf	English	null	The roles of resident, central and effector memory <scp>CD</scp>4 T‐cells in protective immunity following infection or vaccination	Immunology	2,018	cc-by	9,326	I M M U N O L OG Y REV I E W A RT IC L E I M M U N O L OG Y REV I E W A RT IC L E Introduction Vaccines are the most cost-effective form of healthcare worldwide. Most current vaccines act by generating pro- tective antibodies that inactivate the pathogen or its tox- ins.1 We do not, however, have effective vaccines against pathogens responsible for many millions of deaths each year, such as human immunodeﬁciency virus (HIV), malaria and tuberculosis. These pathogens present difﬁ- cult challenges to the immune system either through their complex life cycles and/or via their ability to impair or subvert the host’s immune response. Protective immunity to these infections requires a more diverse immune response than antibodies alone. CD4 T-cells are central to all adaptive immune responses, coordinating pathogen control and clearance by both immune and local stromal cells. Harnessing their diverse functions has the potential to provide novel vaccine strategies that generate effective immunity against even complex infections. MacLeod1 doi:10.1111/imm.12929 Received 11 February 2018; revised 2 March 2018; accepted 6 March 2018. Correspondence: Megan K. L. MacLeod, Centre for Immunobiology, Institute of Infection, Immunity and Inﬂammation, University of Glasgow, 120 University PlaceSGDB, Glasgow G12 8TA, UK. Email: Megan.Macleod@glasgow.ac.uk Senior author: Megan K.L. MacLeod Keywords: CD4 T cell; cytokine; infection; memory; vaccine. secondary lymphoid organs where they amplify the anti- pathogen response by driving B-cell germinal responses and supporting CD8 T-cell activation. Activated CD4 T- cells also migrate from secondary lymphoid organs to inﬂamed sites where they participate in controlling and/or clearing the pathogen. Following pathogen control or clear- ance, the vast majority of activated CD4 T-cells undergo apoptosis with the remainder, usually around 10%, form- ing a population of long-lived memory cells. These memory cells retain knowledge about the initial immune response, enabling them to respond more effectively following a sec- ondary infection. This enhanced response forms the basis for the success of vaccines. Understanding the signals that drive the generation of protective memory CD4 T-cells and the mechanisms by which they act will facilitate the design and development of improved vaccines. The roles of resident, central and effector memory CD4 T-cells in protective immunity following infection or vaccination Joshua I. Gray,1 Lotus M. Westerhof1,2 and Megan K. L. ª 2018 The Authors. Immunology Published by John Wiley & Sons Ltd., Immunology, 154, 574–581 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. doi:10.1111/imm.12929 Received 11 February 2018; revised 2 March 2018; accepted 6 March 2018. Correspondence: Megan K. L. MacLeod, Centre for Immunobiology, Institute of Infection, Immunity and Inﬂammation, University of Glasgow, 120 University PlaceSGDB, Glasgow G12 8TA, UK. Email: Megan.Macleod@glasgow.ac.uk Senior author: Megan K.L. MacLeod This article accompanies the following article: Ahrends T, Borst J. The opposing roles of CD4+ T cells in anti-tumour immunity, doi:10.1111/ imm.12941 doi:10.1111/imm.12929 Received 11 February 2018; revised 2 March 2018; accepted 6 March 2018. Correspondence: Megan K. L. MacLeod, Centre for Immunobiology, Institute of Infection, Immunity and Inﬂammation, University of Glasgow, 120 University PlaceSGDB, Glasgow G12 8TA, UK. Email: Megan.Macleod@glasgow.ac.uk Senior author: Megan K.L. MacLeod Summary Immunological memory provides rapid protection to pathogens previously encountered through infection or vaccination. CD4 T-cells play a central role in all adaptive immune responses. Vaccines must, therefore, activate CD4 T-cells if they are to generate protective immunity. For many dis- eases, we do not have effective vaccines. These include human immunode- ﬁciency virus (HIV), tuberculosis and malaria, which are responsible for many millions of deaths each year across the globe. CD4 T-cells play many different roles during the immune response coordinating the actions of many other cells. In order to harness the diverse protective effects of memory CD4 T-cells, we need to understand how memory CD4 T-cells are generated and how they protect the host. Here we review recent ﬁndings on the location of different subsets of memory CD4 T-cells that are found in peripheral tissues (tissue resident memory T- cells) and in the circulation (central and effector memory T-cells). We dis- cuss the generation of these cells, and the evidence that demonstrates how they provide immune protection in animal and human challenge models. MacLeod1 1Centre for Immunobiology, Institute of Infec- tion, Immunity and Inﬂammation, University of Glasgow, Glasgow, and 2GLAZgo Discovery Centre, Institute of Infection, Immunity and Inﬂammation, University of Glasgow, Glasgow, UK This article accompanies the following article: Ahrends T, Borst J. The opposing roles of CD4+ T cells in anti-tumour immunity, doi:10.1111/ imm 12941 Trm T-cells The anti-pathogen responses coordinated by na€ıve T-cells are delayed until information about the infection is trans- ferred from the infection site to the draining lymph node. Trm cells can provide much more immediate protection, and various studies have demonstrated that they provide the most effective immune protection to the host.14,15,17,30 Tissue resident memory cells are identiﬁed as CD69+ cells that remain within peripheral tissues following patho- gen clearance.30–32 CD69 is thought to act as a retention signal as it inhibits the surface expression of the sphin- gosine-1-phosphate (S1P) receptor 1. S1P is a signalling phospholipid that regulates the migration of immune cells out of lymph nodes into efferent lymphatics, and also guides cells out of tissues towards draining lymph nodes.33–35 Expression of the integrin CD103 by CD8 Trm cells also contributes to their retention, tethering them to skin or mucosal epithelial cells; it is, however, more rare for CD4 Trm cells to express this molecule.36 Immune protection by cytokine-producing CD4 T-cells correlates with their production of several related cytoki- nes, with cells producing IFN-c, tumour necrosis factor (TNF)-a and IL-2 most commonly studied. These multifunctional memory CD4 T-cells are found following vaccination or infection in animal models and in humans.24–29 In infection models of Leishmania major and Mycobacterium tuberculosis, multifunctional CD4 T-cells provide the most effective immune protection, and in humans they correlate with successful recovery from infection with Japanese encephalitis virus.25,27,29 It is currently unclear why multifunctional memory CD4 T-cells offer enhanced protection in comparison to single cytokine-producing cells. The enhanced protective func- tions of these cells could be because they produce higher levels of the individual cytokines on a per cell basis.25,27 Alternatively, or in addition, as multifunctional memory T- cells can simultaneously drive effector responses, for exam- ple via IFN-c, and T-cell survival and proliferation via IL-2 production, they offer a sustained and protective response. Currently we have limited understanding of the priming signals that lead to the generation of multifunctional mem- ory cells. This information will be key to the development There are clear differences between CD4 and CD8 Trm cells ﬁrst revealed by Gebhardt et al. Cytokine production is key to CD4 T-cell protective immunity CD4 T-cells act in concert with innate and other adaptive immune cells to protect the host from pathogens. During primary immune responses, CD4 T-cells are activated in Na€ıve CD4 T-cells can differentiate into a number of dis- tinct functional subsets.2,3 This enables them to tailor the This article accompanies the following article: Ahrends T, Borst J. The opposing roles of CD4+ T cells in anti-tumour immunity, doi:10.1111/ ª 2018 The Authors. Immunology Published by John Wiley & Sons Ltd., Immunology, 154, 574–581 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. 574 CD4 memory T cell diversity immune response depending on the type of pathogen, and to perform multiple functions at different sites during a single infection. Memory CD4 T-cells retain characteristics of the activated CD4 T-cells from which they are derived and can, therefore, also be divided based on their func- tional responses.4,5 This cellular memory is thought to be retained by epigenetic changes to the cell’s DNA or associ- ated histone proteins that keep genes in an open or closed state depending on their expression during the primary immune response.3,6,7 Many genes are thought to be main- tained in a poised state in memory T-cells, enabling rapid re-expression of effector molecules following T-cell reacti- vation by antigen-presenting cells (APCs). However, epige- netic alterations to the genome are not ﬁxed and memory cells can display plasticity.8–11 The level of this plasticity is likely dependent on the extent of differentiation during the primary response and on the reactivation environment. of more effective vaccines capable of producing protective multifunctional CD4 T-cells. Memory CD4 T-cells are found throughout the body The consequences of rapid cytokine production by memory CD4 T-cells depend on the location of the cell. Na€ıve T- cells patrol through lymphoid organs as they have no prior knowledge about where in the body the pathogen they rec- ognize may cause an infection. Memory T-cells have learnt this information during the primary response, and some memory CD4 T-cells, tissue resident memory (Trm) cells, continue to reside at the infection site. Other memory T- cells recirculate through the body. These memory cells can be split into central memory cells (Tcm) and effector mem- ory cells (Tem). Tcm are largely restricted to lymphoid organs and the blood. In contrast, Tem are present in the blood and have the ability to trafﬁc through peripheral organs. All types of memory CD4 T-cells are important. This is because they can provide protection in distinct ways, with recirculating cells acting as reinforcements should resident cells fail to contain the infection (Fig. 1). Cytokine responses by memory CD4 T-cells are key to their ability to protect the host from infectious disease. Rapid production of the most appropriate cytokine enables CD4 T-cells to quickly control the pathogen. For example, interferon (IFN)-c from CD4 T-cells protects against viral infections, while interleukin (IL)-17 aids con- trols of bacterial and fungal infections, and IL-4 protects against infection by parasitic worms.12–20 Most immune protection studies are carried out in animal models where challenge studies are feasible and mechanisms of protec- tion can be identiﬁed by loss or gain of function. Human challenge studies are, however, becoming more frequent and have also demonstrated that cytokine-producing memory CD4 T-cells correlate with reduced symptoms following pathogen challenge.21–23 ª 2018 The Authors. Immunology Published by John Wiley & Sons Ltd., Immunology, 154, 574–581 Trm T-cells in mice infected with herpes simplex virus (HSV).14 While CD8 Trm cells become dendritic-like with limited motility in the epithe- lium of the skin, CD4 Trm cells are mainly found in the dermis and display a more motile behaviour.14 In humans, Trm cells have been found in multiple organs, with elegant studies from Farber and colleagues tracking memory T- cells in individuals of various ages. Human and mouse Trm cells share many characteristics, including CD69 expression and reduced expression of S1P receptor 1.37,38 Similarly, 575 2018 The Authors. Immunology Published by John Wiley & Sons Ltd., Immunology, 154, 574–581 J. I. Gray et al. Macrophage CD4+ Time scale of response Dendritic cell Epithelial cells Pathogen Lymph node Blood vessel Tissue resident memory CD4 T cells can be found in clusters with other immune cells. They can be rapidly reactivated within tissues releasing inflammatory chemokines and cytokines to recruit and activate immune cells T 1 Circulating memory CD4 T cells can be rapidly recruited into inflamed tissues and reactivated by local APCs C 2 Antigen presenting cells transport information about the pathogen to lymphoid organs A 3 Reactivated T follicular helper memory T cells can provide rapid assistance to B cells to generate new germinal centres 5 Central memory CD4 T cells can be reactivated in lymphoid organs and differentiate into different subsets of CD4 T cells 4 Lymphatic vessel B cell Circulating memory CD4 T cells can be rapidly recruited into inflamed tissues and reactivated by local APCs C Reactivated T follicular helper memory T cells can provide rapid assistance to B cells to generate new germinal centres Reactivated T follicular helper memory T cells can provide rapid assistance to B cells to generate new germinal centres Macrophage Lymphatic vessel Tissue resident memory CD4 T cells can be found in clusters with other immune cells. They can be rapidly reactivated within tissues releasing inflammatory chemokines and cytokines to recruit and activate immune cells T Tissue resident memory CD4 T cells can be found in clusters with other immune cells. ª 2018 The Authors. Immunology Published by John Wiley & Sons Ltd., Immunology, 154, 574–581 Trm T-cells Immunology Published by John Wiley & Sons Ltd., Immunology, 154, 574–581 576 CD4 memory T cell diversity circulating cells, so a stable population of Trm cells in trea- ted mice demonstrates that these cells are neither replen- ished by, nor lost to, circulating populations.15,17,40 However, FTY720 may also inhibit egress of cells from peripheral tissues to draining lymph nodes and/or reduce cell survival.35,41–43 Despite these potential caveats, results from FTY720-treated animals reﬂect those from more ele- gant parabiosis experiments that demonstrate that Trm cells are a distinct population neither leaving the tissue nor being replenished by circulating cells.17,30 low levels of persistent antigen, in turn maintains macro- phage CCL5 expression. Similarly, CCL5 produced by CD8 T-cells and macrophages in the skin is responsible for maintaining CD4 T-cells in perifollicular clusters.44 In this case, however, antigen presentation was not required for memory T-cells to be incorporated into these immune cell clusters. Clusters of Trm and APCs may represent nascent ver- sions of ectopic lymphoid structures (ELS), which range from organized clusters of immune cells to lymph node- like structures with designated T- and B-cell zones. ELS are often found in sites of chronic inﬂammation, and have been associated with the generation of autoreactive anti- bodies.49,50 Whether Trm immune cell clusters represent an early stage in ELS development is unclear. They do pro- vide an excellent location for the rapid reactivation of CD4 Trm cells by local APCs. For example, inﬂuenza virus-spe- ciﬁc Trm cells, which can be found in clusters with B-cells in the infected lung, provide effective and rapid immune protection at least in part by providing rapid assistance to B-cells to make neutralizing antibodies.18 g p y g In contrast, data from Collins et al. suggest that CD4 Trm cells are a more dynamic population.44 CD4 Trm cells in mice that express the photoconvertible molecule, Kaede, were found to migrate from the skin of HSV- infected mice to draining lymph nodes. These cells lost expression of CD69 following migration out of the skin. The migrating cells represented about half of the Trm cell population photoconverted at the start of the study and then examined 3 days later. These ﬁndings question the reliability of CD69 as a marker of persistently resident memory T-cells within tissues. Trm T-cells They suggest that at least two distinct populations may be present – a more static, true Trm population and a second more dynamic popu- lation that alter CD69 expression depending on location. In this study, the speciﬁcity of the cells was not deter- mined. These two populations may, therefore, reﬂect resi- dent T-cells that respond to local antigen and remain in the tissue versus cells that are recruited but are not main- tained because they are not re-activated by antigen. Future studies will need to address the functional proper- ties of these two populations and compare the behaviours of T-cells exposed to local antigen versus those recruited by inﬂammation alone. In addition, CD4 Trm cells can enhance the actions of cells that are recruited into the infected tissue. Inﬂuenza- speciﬁc memory CD4 T-cells in the lung drive the pro- duction of chemokines that attract innate immune cells that rapidly control viral spread.51 Similarly, IFN-c pro- duction by L. major-speciﬁc CD4 Trm cells drives the recruitment of inﬂammatory monocytes to the infection site.15 These recruited monocytes produce nitric oxide and reactive oxygen species that are toxic to the parasite. Trm T-cells They can be rapidly reactivated within tissues releasing inflammatory chemokines and cytokines to recruit and activate immune cells T Antigen presenting cells transport information about the pathogen to lymphoid organs A Antigen presenting cells transport information about the pathogen to lymphoid organs A Central memory CD4 T cells can be reactivated in lymphoid organs and differentiate into different subsets of CD4 T cells Figure 1. Protective roles of memory CD4 T-cell subsets. The protective response to previously encountered pathogens is dependent on a number of T-cell subsets. Upon pathogen encounter, resident memory CD4 T-cells (Trm) that can be found in clusters with macrophages or dendritic cells respond rapidly by proliferating and releasing inﬂammatory cytokines and chemokines (1). This stimulates the recruitment of circulating effector memory CD4 T-cells (Tem) to the inﬂamed tissue to augment the immune response (2). Local antigen-presenting cells (APCs) can subsequently transport antigen to lymphoid organs where they activate central memory CD4 T-cells (Tcm) (3–4). These central memory T-cells then expand and recirculate to confer systemic protection or further amplify the response at inﬂamed tissues. Memory follicular helper T-cells (Tfh) reactivated by either dendritic cells or B-cells can enter B-cell zones and induce rapid production of class-switched antibodies, which are then released into the circulation (5). These different memory T-cell subsets work in concert to provide long-lasting protection upon re-exposure to the same pathogen. intravenously shortly before the animal is killed. This demonstrates that these cells are not directly in contact with the vasculature, at least at the time of analysis. Three additional key approaches are used in mice to investigate whether Trm cells are truly resident: in vivo treatment with the S1P functional antagonist, FTY720; parabiosis in which the circulatory systems of two animals are surgically con- nected; and fate mapping using photoconvertible cells. Treatment with FTY720 restricts the migration of CD4 Trm cells in skin are, like their mouse equivalent, mainly found in the dermis.39 Transcriptional analyses comparing circulating cells and Trm cells from the same donor indicate that CD69+ and CD69 CD4 T-cells express overlapping sets of T-cell receptor genes.37 These data indicate that environment, rather than epitope speci- ﬁcity, is the key driver of Trm cell development. In mice, memory T-cell residency is often identiﬁed by their failure to bind ﬂuorescently labelled antibody injected ª 2018 The Authors. ª 2018 The Authors. Immunology Published by John Wiley & Sons Ltd., Immunology, 154, 574–581 Memory CD4 T-cells in secondary lymphoid organs Central memory cells are most likely to be reactivated in secondary lymphoid organs as they lack the chemokine receptors and adhesion molecules necessary to enter peripheral tissues.45 Instead they, like na€ıve T-cells, express high levels of CD62L, which enables entry into lymph nodes from the blood via high endothelial venules and CCR7, the chemokine receptor that is also involved in trafﬁcking to and within lymphoid organs.45,56 CXCR5+ memory cells with an increased ability to help B-cells are commonly referred to as Tfh memory cells,7 even though they may be found in circulation rather than contained within a B-cell follicle. How they are distinct from the general CD4 Tcm pool is a complicated question. Indeed, Tfh memory cells do express lower levels of CXCR5 than Tfh cells present during the primary immune response62,69 and, as Pepper et al. describe, are difﬁcult to distinguish from Tcm cells.57 Reactivation of CXCR5+ memory cells can occur in the absence of B-cells,63 but reactivation by B-cells consolidates the T-cell’s expression of Bcl6, the transcription factor associated with Tfh cell function.72 These data suggest that if there are Tfh memory cells, their function upon reactivation is likely dependent on the context in which they are reactivated. While Tcm cells may not rapidly produce protective cytokines, they proliferate upon reactivation, quickly increasing the number of antigen-speciﬁc CD4 T-cells. These cells can then either remain in the lymphoid organ to help B-cells or migrate to the site of infection to help tackle the infection directly. As Tcm cells are uncommit- ted to any particular effector cytokine production, they can differentiate under the inﬂuence of the cytokine milieu triggered by the challenge infection.57 Vaccines that drive the generation of Tcm cells may provide less immediate protection than those designed to induce Trm cells; however, they offer an adaptable pool of memory CD4 T-cells that can protect via multiple pathways. A further key question that remains to be addressed is the consequences of reactivation of memory CD4 T-cells by B- cells within either ELS or immune cell clusters at the infec- tion site itself. Recirculating memory T-cells To develop vaccines that generate protective CD4 Trm cells, it is imperative that we understand the signals that drive the development and maintenance of these cells. During immune responses, activated T-cells migrate into inﬂamed tissues under the guidance of chemokines, inte- grins and adhesion molecules.45 T-cells that encounter their antigen at the infection site are likely to receive tis- sue-speciﬁc cues that inﬂuence their function and mem- ory potential. The presentation of speciﬁc antigen at the infected tissue is required for the formation of CD4 Trm cells, but may not be necessary for CD8 Trm cells.31,46–48 These data indicate that vaccines aimed at generating pro- tective Trm CD4 T-cells must drive antigen presentation within the tissue targeted by the pathogen. Circulating antigen-speciﬁc CD4 memory T-cells can par- ticipate in protective immune responses either acting as reinforcements to Trm cells32 or protecting tissues not challenged in the initial infection or vaccination.14,52 Immune protection by circulating memory CD4 T-cells is delayed compared with Trm-mediated protection as they must be ﬁrst recruited to the site of infection and reacti- vated by local APCs. Immune protection by circulating cells can require collaboration with other components of the memory immune response. Iijima et al. found that while recirculating HSV-2-speciﬁc memory CD4 T-cells cannot prevent viral replication at the challenge site, they could prevent virus entry into the dorsal route ganglia.52 This protection required the entry of virus-speciﬁc anti- body into the nervous system, which was dependent on increased vascularization mediated by IFN-c derived from reactivated memory CD4 T-cells. Similarly, in mice vacci- nated with the yellow fever vaccine, transfer of CD4 T- cells and immune serum provide the most optimal immune protection.20 Whether persistent antigen presentation following pathogen control is required to maintain CD4 Trm cells is still unclear. CD4 Trm cells in the skin and mucosa are located in clusters of cells with macrophages and/or den- dritic cells that express chemokines that maintain the cells at the site. Macrophage-derived CCL5 maintains CD4 T- cells in the vagina following HSV-2 infection.17 IFN-c produced by CD4 Trm cells, potentially in response to The effector response of recruited antigen-speciﬁc memory CD4 T-cells is likely to be inﬂuenced by the level ª 2018 The Authors. Immunology Published by John Wiley & Sons Ltd., Immunology, 154, 574–581 577 J. I. Gray et al. Memory CD4 T-cells in secondary lymphoid organs In lymphoid organs, secondary germinal cen- tres are thought to be formed by reactivated IgM+ memory B-cells, providing a blank canvas for antibody class switching relevant to the pathogen.73 In inﬂuenza virus-infected mice, CD4 T-cells and B-cells can be found in clusters within the lung, and many virus-speciﬁc B-cells in the lung are class switched.74,75 This suggests that germinal centres formed in ELS in peripheral tissues may follow different rules to those in lymphoid organs following re-infection. Careful studies that dissect the contribution of the reactivation of B-cells in the tissue versus those in lymph nodes to immune protec- tion are needed to establish their relative importance in pro- tecting the host from reinfection. In primary immune responses, activated CD4 T-cells drive B-cell germinal centre reactions leading to the pro- duction of high-afﬁnity class-switched antibody. The cells that coordinate this response are classed as T follicular helper (Tfh) cells that are initially formed in the T-cell zone of the lymphoid organ before moving to the devel- oping germinal centre.7 Depending on the type of infec- tion, Tfh cells can produce T helper type 1 or 2 cytokines to drive appropriate antibody class switching.7 Whether Tfh cells can differentiate into specialized mem- ory cells has been an area of recent debate.7 Within the pool of Tcm cells, a proportion of cells express higher levels of CXCR5, the chemokine receptor that allows cells to move towards or into B-cell follicles.7 Tfh cells can remain for many months in the original draining lymph node where persistent antigen is likely to maintain them in an active state. These cells rapidly expand upon re-challenge and may represent a population of lymph node resident memory cells as they can express CD69.58–60 However, this reliance on antigen suggests that these lymph node resident Tfh cells may not represent ‘true resting memory cells’. Moreover, Pepper et al. found that the number of antigen-speciﬁc Recirculating memory T-cells memory cells that expressed Tfh markers declined over time, suggesting that Tfh cells fail to differentiate into long-lived memory cells.57 However, we and others have shown that both mouse and human CXCR5+ memory CD4 T-cells pro- vide rapid assistance to B-cells upon reactivation.61–69 Importantly, Alexander et al. demonstrated that DNA vacci- nation generated memory CD4 T-cells that, via their rapid assistance to B-cells, protected mice from inﬂuenza virus infection.70 This rapid assistance to generate high-afﬁnity class-switched antibody is particularly relevant to infections such as inﬂuenza virus where regions of the virus targeted by antibody alter much more rapidly than epitopes recognized by CD4 T-cells.71 memory cells that expressed Tfh markers declined over time, suggesting that Tfh cells fail to differentiate into long-lived memory cells.57 However, we and others have shown that both mouse and human CXCR5+ memory CD4 T-cells pro- vide rapid assistance to B-cells upon reactivation.61–69 of MHC II and co-stimulatory molecules displayed by the local APC that reactivates the memory cell.53–55 Further- more, this response is likely to be distinct to that from the same cell reactivated in the very different environ- ment of a lymphoid organ. It is key, therefore, to con- sider not just the type of memory CD4 T-cell that a vaccine should aim to generate but where it is likely to be reactivated, which APCs are involved in this, and the downstream consequences of these interactions. ª 2018 The Authors. Immunology Published by John Wiley & Sons Ltd., Immunology, 154, 574–581 Human vaccines: recent progress and continuing challenges Experimental sporozoite vaccines have either been deliv- ered as irradiated parasites that can infect host cells but fail to differentiate and cause disease, or as live parasites.21,77,78 In this instance, anti-malarial drugs must be given to pre- vent active disease. Protection in these studies is associated with high levels of multifunctional, antigen-speciﬁc mem- ory CD4 T-cells in peripheral blood, although mouse and non-human primate studies point to key roles for liver resi- dent memory CD8 T-cells in preventing parasite growth.78,79 The logistical challenges of sporozoite vaccines are signiﬁcant; current ﬁeld trials are likely to reveal whether or not these approaches are feasible.80 In this instance, anti-malarial drugs must be given to pre- vent active disease. Protection in these studies is associated with high levels of multifunctional, antigen-speciﬁc mem- ory CD4 T-cells in peripheral blood, although mouse and non-human primate studies point to key roles for liver resi- dent memory CD8 T-cells in preventing parasite growth.78,79 The logistical challenges of sporozoite vaccines are signiﬁcant; current ﬁeld trials are likely to reveal whether or not these approaches are feasible.80 Immune protection to this highly diverse virus is thought to require broadly neutralizing antibodies (bnAbs) that recognize more conserved regions of the virus.93 The development of bnAbs likely requires repeated exposure to the antigen and continued input from Tfh cells.93 Suc- cessful HIV vaccines will probably, therefore, require the induction of effective Tfh memory cells that can coordi- nate the development of protective bnAbs. The most advanced vaccine for malaria is GlaxoSmithKline’s RTS,S vaccine, which contains the cir- cumsporozoite protein from the pre-erythrocyte stage of the parasite.81,82 Antibodies and CD4 T-cells speciﬁc to the circumsporozoite protein are correlates of immune protection following RTS,S immunization, which has an efﬁcacy of about 30%.22,81,83 While a positive advance, this relatively low efﬁcacy and the short duration of pro- tection means that more robust vaccines are still required.84 A key contrast between sporozoite vaccines and the recombinant protein RTS,S vaccine is the need for adjuvants to boost the immune response to the sub- unit vaccine. Human vaccines: recent progress and continuing challenges Dissecting the relative contributions of different popula- tions of memory T-cells to immune protection in mice provides mechanistic understanding of immunological memory. Animal vaccine studies often do not, however, easily translate into protective vaccines for humans. Human challenge studies, therefore, play important roles in evaluating vaccines at early stages of development. 578 ª 2018 The Authors. Immunology Published by John Wiley & Sons Ltd., Immunology, 154, 574–581 CD4 memory T cell diversity They often cut the costs of large-scale ﬁeld vaccine trials and reduce tests of non-effective vaccines on large num- bers of individuals.76 This is especially the case in dis- eases, including malaria and tuberculosis, in which our understanding of the correlates and mechanisms of immune protection are limited. compared with more traditional subcutaneous or intra- dermal injection in a non-human primate model.88 Potentially these injection routes provide enhanced pro- tection as they are better at driving the development of CD4 Trm cells in the lung. Human immunodeﬁciency virus presents bigger hurdles still as immune protection does not develop in the vast majority of infected individuals and human challenge studies are not possible. The RV144 HIV vaccine trial has offered a number of key insights into immunity to HIV. The vaccine demonstrated an efﬁcacy of 312%.89 Protec- tion correlated with high levels of anti-viral IgG, depen- dent on CD4 T-cell responses, while vaccine-driven IgA was associated with an increased risk of infection.90–92 Immune protection to this highly diverse virus is thought to require broadly neutralizing antibodies (bnAbs) that recognize more conserved regions of the virus.93 The development of bnAbs likely requires repeated exposure to the antigen and continued input from Tfh cells.93 Suc- cessful HIV vaccines will probably, therefore, require the induction of effective Tfh memory cells that can coordi- nate the development of protective bnAbs. Human immunodeﬁciency virus presents bigger hurdles still as immune protection does not develop in the vast majority of infected individuals and human challenge studies are not possible. The RV144 HIV vaccine trial has offered a number of key insights into immunity to HIV. The vaccine demonstrated an efﬁcacy of 312%.89 Protec- tion correlated with high levels of anti-viral IgG, depen- dent on CD4 T-cell responses, while vaccine-driven IgA was associated with an increased risk of infection.90–92 Human challenge studies in malaria have demonstrated the relative safety and efﬁcacy of sporozoite vaccines. Human vaccines: recent progress and continuing challenges RTS,S contains a combined adjuvant with a saponin and toll-like receptor agonist.82 This adjuvant combination is one of only a handful approved for use in humans, with few able to induce the size and correctly tailored immune response driven by either natural infec- tion or vaccination with an attenuated or inactivated pathogen.82 The live vaccines are, however, more likely to lead to adverse effects or negative side-effects.82,85 Improved understanding of the mechanisms of action of current and experimental adjuvants is likely to lead to further improvements allowing us to strike the right bal- ance between immunogenicity and vaccine safety. ª 2018 The Authors. Immunology Published by John Wiley & Sons Ltd., Immunology, 154, 574–581 References Front Immunol 2017; 8:204. 6 Bevington SL, Cauchy P, Withers DR, Lane PJ, Cockerill PN. T ce 33 Cyster JG, Schwab SR. Sphingosine-1-phosphate and lymphocyte egress from lymphoid organs. 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Immunity 2009; 30:155–67. 12 Anthony RM, Urban JF Jr, Alem F, Hamed HA, Rozo CT, Boucher JL, et al. Memory T(H)2 cells induce alternatively activated macrophages to mediate protection against nematode parasites. Nat Med 2006; 12:955–60. Future perspectives We have appreciated the concept of protective immuno- logical memory for thousands of years and have been manipulating it via vaccination since the 10th century.85 The majority of successful vaccines act by generating neu- tralizing class-switched antibodies produced by long-lived plasma cells.1 These vaccines must, therefore, drive activa- tion of helper CD4 T-cells that act, at the very least, dur- ing the primary response to protect the host. We are now beginning to understand the potential myriad roles mem- ory CD4 T-cells can themselves play in protection from infectious diseases. The recent studies discussed here have revealed the complexity of the memory CD4 T-cell pool. We now need to understand how vaccine formation and delivery can be altered to bias the development of protec- tive memory CD4 T-cells. As our fundamental understanding of memory CD4 T- cells improves, we must put this into a real-world context. Studies in wild or pet-shop mice have highlighted the major differences between memory compartments and responses to infections in these animals versus their labo- ratory equivalent.94,95 The microbiome plays a major role in these differences, but the exposure to varied and multi- ple pathogens throughout life is also likely to affect the generation and function of immune memory cells.94,95 Human challenge studies offer a valuable extra arm to vaccine studies, but most are carried out on healthy west- ern volunteers. Evidence from the recently introduced rotavirus vaccine, and also polio and cholera vaccine stud- ies, demonstrates reduced vaccine-effectiveness in major at-risk populations in low–middle-income countries.96–98 Other human challenge models include the use of BCG as a surrogate challenge in tuberculosis vaccine trials. While the BCG vaccine protects against disseminated tuberculosis, especially in young children who are most at risk, its ability to protect from pulmonary disease varies across the world.86 Current studies in mice, non-human primates and humans are focussed on increasing the immune protection offered by BCG, for example by using prime-boost strategies.86,87 BCG effectiveness can also be achieved by altering the vaccination route, with mucosal and intravenous routes offering enhanced protection 2018 The Authors. Immunology Published by John Wiley & Sons Ltd., Immunology, 154, 574–581 579 J. I. Gray et al. 20 Watson AM, Lam LK, Klimstra WB, Ryman KD. 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https://openalex.org/W3184198488	https://www.degruyter.com/document/doi/10.1515/almed-2021-0018/pdf	English	null	Identification of a new mutation in the human xanthine dehydrogenase responsible for xanthinuria type I	Advances in laboratory medicine	2,021	cc-by	2,879	Abstract Objectives: Hereditary xanthinuria is a rare, autosomal and recessive disorder characterized by severe hypour- icemia and increased xanthine excretion, caused by a deficiency of xanthine dehydrogenase/oxidase (XDH/XO, EC: 1.17.1.4/1.17.3.2) in type I, or by a deficiency of XDH/XO and aldehyde oxidase (AOX, EC: 1.2.3.1) in type II. Hereditary xanthinuria (HX) is a rare disorder of purine catabolism caused by an inherited xanthine dehydroge- nase/oxidase (XDH/XO, EC: 1.17.1.4/1.17.3.2) deficiency, first described in 1954 by Dent and Philpot [1]. It is characterized by very low or undetectable levels of uric acid in blood and urine, with a normal or low fractional excretion of urate together with elevated excretion of xanthine and hypoxan- thine in urine. Although most patients are asymptomatic, possible clinical symptoms include urolithiasis due to increased excretion of xanthine and myositis for xanthine deposition. No speciﬁc treatment is available, but a low purine diet and hydration must be recommended to the patient to prevent further complications. Methods: We describe a novel point mutation in the XDH gene in homozygosis found in a patient with very low serum and urine levels of uric acid, together with xanthi- nuria. He was asymptomatic but renal calculi were discovered during imaging. Results: Additional cases were found in his family and dietary recommendations were made in order to prevent further complications. Xanthinuria is an autosomal and recessive disorder that consists of three subgroups. Type I (OMIM 278300) is caused by a human XDH/XO deficiency, an enzyme which catalyzes the hypoxanthine oxidation to xanthine, and the formation of uric acid from xanthine, due to mutations in the XDH/XO gene localized on chromosome 2p23 [2–6] (Figure 1). Addi- tionally, mutations in the molybdenum cofactor sulfurase gene (MOCOS, EC: 2.8), mapped to chromosome 18q12.2, raise xanthinuria type II (OMIM 603592), developing both XDH/XO and aldehyde oxidase (AOX) deﬁciency. Both types I and II have a similar phenotype, with patients generally asymp- tomatic, and are referred to as classical HX [7]. Nevertheless, in the xanthinuria type III, there is a very different phenotype, associated with progressive neurological damage due to failure of molybdenum cofactor biosynthesis pathway that causes loss of enzymatic activity for XDH/XO, AOX, and sulﬁte oxidase [8]. The vast majority of these patients suffer a premature death and the ones that survive present convul- sions, abnormal muscle tone, developmental delay, and crystalline lens luxation. Adv Lab Med 2021; 2(4): 567–570 Identiﬁcation of a new mutation in the human xanthine dehydrogenase responsible for xanthinuria type I Keywords: hypouricemia; xanthine dehydrogenase gene; xanthinuria. https://doi.org/10.1515/almed-2021-0018 Received February 11, 2021; accepted June 17, 2021; published online July 21, 2021 Open Access. © 2021 Cristina Collazo Abal et al., published by De Gruyter. This work is licensed under the Creative Commons Attribution 4.0 International License. Genetic analysis – Next-Generation Sequencing (NSG) The NSG assay was performed using the SureSelectXT Custom kit to capture the coding and intronic flanking regions of the XDH and MOCOS genes. Barcoded samples were pooled, and captured libraries were sequenced on an Ion Proton System using the Ion PI HiQ Sequencing kit and the Ion PI Chip (ThermoFisher Scientific). Se- quences were aligned against the human genome sequence (build GRCh37/hg19) with a Torrent Mapping Alignment program. After sequence mapping, the variants were collected and annotated using the following software packages: Torrent Variant Caller (TVC 5.2–25), GATK v3.7-0, Picard 2.9.0-1-SNAPSHOT, BEDtools v2.26.0, SAMtools 1.4, ExomeDepth 1.1.10. Patient A 65-year-old man was found to have severe hypouricemia in routine analysis, present for at least 4 years. Physical ﬁndings were height, 167 cm; body weight, 73.5 kg, body mass index, 26.4 kg/m2; blood pressure, 138/90 mmHg; and pulse rate, 90 beats per minute. Physical examination showed no abnormal ﬁndings and his med- ical history included revascularization due to chronic ischemic heart disease and a bilateral inguinal herniography. His parents were not consanguineous and there was no history of urinary calculi in his family. His usual medication was bisoprolol, ator- vastatin/ezetimibe, and pantoprazole. He had never taken any antihyperuricemic agent. Determination of compounds in serum and urine On his laboratory data, the serum levels of uric acid were less than the sensitivity of the technique (<5.95 μmol/L), with a normal renal function (creatinine clearance >90 mL/min/1.73 m2). The urine analysis conﬁrmed a low excretion of purines, including uric acid (53.6 μmol/ 24 h). Serum levels of uric acid were less than 5.95 μmol/L (reference interval: 142.75–356.88 μmol/L). The urinary excretion of uric acid was 12.15 μmol/mmol creatinine (reference interval: 615.34–5,712.26 μmol/mmol creati- nine). The daily urinary excretion of xanthine and hypo- xanthine were 108.35 μmol/mmol creatinine (reference A renal echography revealed both kidneys with normal size, morphology, and echostructure. In the right kidney, two hypoechoic images (7 and 9 mm) were identiﬁed as suggestive of renal lithiasis without repercussion over the excretory system. Figure 2: Family tree. His first-degree relatives were tested, finding that at least two of his siblings had low levels of uric acid (Figure 2). One of them agreed to have his 24 h urine tested, obtaining similar xanthine and uric acid levels (Table 1). 568 AOX XDH/XO XDH/XO Hypoxanthine Xanthine Uric acid MoCoS Figure 1: Uric acid metabolism. /XO Uric acid AOX XDH/XO XDH/XO Hypoxanthine Xanthine MoCoS Xanthine Hypoxanthine Uric acid Uric acid MoCoS Figure 1: Uric acid metabolism. performance liquid chromatography (UPLC) at the Purine Research Laboratory (St Thomas’ Hospital NHS Foundation Trust, London, UK). Different mutations described during the last decade associated with xanthinuria type I and II have been reviewed by Ichida et al. [3], and other mutations have been described recently [5, 9, 10]. We report below a new muta- tion of XDH/XO associated with a case of xanthinuria type I. performance liquid chromatography (UPLC) at the Purine Research Laboratory (St Thomas’ Hospital NHS Foundation Trust, London, UK). Abstract Conclusions: Hereditary xanthinuria is an underdiagnosed pathology, often found in a routine analysis that shows hypouricemia. It is important for Laboratory Medicine to acknowledge how to guide clinicians in the diagnosis. Corresponding author: Cristina Collazo Abal, Clinical Analysis Department, University Hospital of Vigo, Vigo, Spain, E-mail: cristinacollazo@gmail.com. https://orcid.org/0000-0003- 2774-570X Susana Romero Santos and Carmen González Mao, Clinical Analysis Department, University Hospital of Vigo, Vigo, Spain. https:// orcid.org/0000-0001-8666-4032 (S. Romero Santos) Emilio C. Pazos Lago, Internal Medicine Department, University Hospital of Vigo, Vigo, Spain Francisco Barros Angueira, Galician Public Foundation for Genomic Medicine, University Hospital of Santiago de Compostela, Santiago de Compostela, Spain Daisy Castiñeiras Ramos, Laboratory of Metabolic Pathologies, University Hospital of Santiago de Compostela Santiago de Corresponding author: Cristina Collazo Abal, Clinical Analysis Department, University Hospital of Vigo, Vigo, Spain, E-mail: cristinacollazo@gmail.com. https://orcid.org/0000-0003- 2774-570X Daisy Castiñeiras Ramos, Laboratory of Metabolic Pathologies, University Hospital of Santiago de Compostela, Santiago de Compostela, Spain 568 Collazo Abal et al.: New mutation responsible for xanthinuria type I 568 Collazo Abal et al.: New mutation responsible for xanthinuria type I Table : Purine and pyrimidine results obtained in h urine. Purines and pyrimidines Reference interval, µmol/mmol creatinine Patient Brother Uracile .–. . . Thymine .–. . . Hypoxanthine .–. . . Xanthine .–. . . Uric acid .–,. . . Uridine .–. . . Deoxyadenosine .–. . . Adenosine .–. . . Deoxyguanosine .–. . . Inosine .–. . . Guanosine .–. . . Deoxyuridine .–. . . Thymidine .–. . . Table : Purine and pyrimidine results obtained in h urine. underdiagnosed pathology, often found in a routine anal- ysis that shows hypouricemia. The diagnosis of HX is made by extremely low serum and urinary uric acid together with high excretion of xanthine, once other causes are discarded. The differential diagnosis of hypouricemia is based on the proportion of uric acid excre- tion [18]. On the one hand, the possible causes of a patient having hypouricemia and elevated uric acid excretion include treatment with salicylates, intravenous contrast dye, parental nutrition, neoplasia, Wilson’s disease, syndrome of inappropriate antidiuretic hormone, Fanconi’s syndrome, cystinosis, myeloma, heavy metals, hereditary renal hypo- uricemia, and diabetes mellitus. All these were dismissed in our patient as it had a very low uric acid excretion [19, 20]. On the other hand, low levels of serum uric acid together with a low excretion has been described in neoplasms, severe hep- atopathies, treatment with XO inhibitors, and xanthinuria. The patient was asymptomatic with normal hepatic enzymes values, and he referred never had been treated with hypo- uricemic drugs. This information guided the diagnosis to a new case of xanthinuria. interval: 0.02–103.35 μmol/mmol creatinine) and 14.44 μmol/mmol creatinine (reference interval: 7.94– 92.95 μmol/mmol creatinine), respectively. Urinary levels of 2PY were of 15.9 μmol/mmol creati- nine in a second sample but it was not possible to measure 4PY, possibly because of dilution of the second urinary sample obtained (1.7 mmol creatinine/L). Secondly, an allopurinol loading test has been tradi- tionally used to determine the type of HX [21]. In healthy volunteers, oxypurinol is excreted in urine after allopurinol administrationasitismetabolizedbybothXDH/XOandAOX. On the contrary, in patients with xanthinuria type II, there is no detection of oxypurinol in urine or in serum as there is no activity for none of them. By contrast, in cases with xanthi- nuria type I, oxypurinol can be detected as there is still ac- tivity for AOX, although there is not for XDH/XO. Finally, conﬁrmation of HX can be supported by measuring XDH/XO activity in the duodenal mucosa. Very low levels of XDH ac- tivity in the duodenal mucosa would conﬁrm the diagnosis of xanthinuria. This option was ruled out considering the invasive nature of the procedure. Genetic analysis – Next-Generation Sequencing The patient was found to be homozygous for a point mu- tation of G to C in the −1 position of the intron 23 of the XDH gene (NM_000379.3(XDH):c.2545-1G>C). This is a new point mutation in the human XDH gene considered as pathogenic using American College of Medical Genetics and Genomics (ACMG) criteria [12]. This mutation present in an intron could affect the splicing process, modifying the RNA transcription, giving, as a result, an XDH deﬁcit and our patient’s phenotype. We did not ﬁnd any other muta- tion in this gene nor in the MOCOS gene, which excluded a xanthinuria type II diagnosis. It has recently been described as a three-step noninvasive diagnostic algorithm [10]. The ﬁrst step of diagnosis of HX is based on low levels of uric acid determined in serum and urine and a high concentration of xanthine in urine. The second step consists of typing HX using urinary metabolomics. It has been described that AOX participates in the catabolism of N1- -methylnicotinamide and that the ﬁnal metabolites 2PY and 4PY could be used as biomarkers to distinguish HX type I from type II. Patients with HX type II have low levels of 2PY and 4PY as there is no AOX activity [22]. The ﬁnal step is the conﬁr- mation of a mutation in the XDH/XO or MOCOS gene. Determination of compounds in serum and urine Serum and urinary uric acid levels were quantified by a uricase method (Advia 2400; Siemens Medical Solutions Diagnostics, Los Ángeles, CA, USA) and conﬁrmed by a urate oxidase method (Dimension EXL; Siemens Medical Solutions Diagnostics, Los Ángeles, CA, USA). Urinary levels of purines and pyrimidines were measured in a 24 h urine dried sample by tandem mass spectrometry (API 4000 Sciex Applied Biosystems) [11]. Urinary levels of N1-methyl-2-pyridone-5-carboxamide (2PY) and N1-methyl-4-pyridone-5-carboxamide (4PY) were measured by ultra Figure 2: Family tree Figure 2: Family tree. Collazo Abal et al.: New mutation responsible for xanthinuria type I 569 References 16. Pereg Macazaga V, García Fernández H, Regúlez M, Elorza Olabegoya JR, Beitia Martin JJ, Vázquez García JA. Xantinuria familiar, Dos nuevos casos. Med Clin 1984;82:364–5. 1. Dent CE, Philpot GR. Xanthinuria, an inborn error (or deviation) of metabolism. Lancet 1954;266:182–5. 1. Dent CE, Philpot GR. Xanthinuria, an inborn error (or deviation) of metabolism. Lancet 1954;266:182–5. 17. Mateos FA, Puig JG, Jiménez ML, Fox IH. Hereditary xanthinuria. Evidence for enhanced hypoxanthine salvage. J Clin Invest 1987; 79:847–52. 2. Ichida K, Amaya Y, Kamatani N, Nishino T, Hosoya T, Sakai O. Identiﬁcation of two mutations in human xanthine dehydrogenase gene responsible for classical type I xanthinuria. J Clin Invest 1997; 99:2391–7. 18. Esparza Martín N, García Nieto V. Hipouricemia y manejo renal del ácido úrico. Nefrologia 2011;31:44–50. 19. Shichiri M, Iwamoto H, Shiigai T. Diabetic renal hypouricemia. Arch Intern Med 1987;147:225–8. 3. Ichida K, Amaya Y, Okamoto K, Nishino T. Mutations associated with functional disorder of xanthine oxidoreductase and hereditary xanthinuria in humans. Int J Mol Sci 2012;13:15475–95. 20. Magoula I, Tsapas G, Paletas K, Mavromatidis K. Insulin-dependent diabetes and renal hypouricemia. Nephron 1991;59:21–6. 4. Diss M, Ranchin B, Broly F, Pottier N, Cochat P. Xanthinurie héréditaire de type 1: à propos de trois cas. Arch Pediatr 2015;22: 1288–91. 4. Diss M, Ranchin B, Broly F, Pottier N, Cochat P. Xanthinurie héréditaire de type 1: à propos de trois cas. Arch Pediatr 2015;22: 1288–91. 21. Ichida K, Yoshida M, Sakuma R, Hosoya T. Two siblings with classical xanthinuria type 1: signiﬁcance of allopurinol loading test. Intern Med 1998;37:77–82. 5. Iguchi A, Sato T, Yamazaki M, Tasaki K, Suzuki Y, Iino N, et al. A case of xanthinuria type I with a novel mutation in xanthine dehydrogenase. CEN Case Rep 2016;5:158–62. 5. Iguchi A, Sato T, Yamazaki M, Tasaki K, Suzuki Y, Iino N, et al. A case of xanthinuria type I with a novel mutation in xanthine dehydrogenase. CEN Case Rep 2016;5:158–62. 22. Peretz H, Watson DG, Blackburn G, Zhang T, Lagziel A, Shtauber-Naamati M, et al. Urine metabolomics reveals novel physiologic functions of human aldehyde oxidase and provides biomarkers for typing xanthinuria. Metabolomics 2012;8:951–9. 6. Antonio Orts Costa J, Zúñiga Cabrera Á, Ferrando Monleón S. Litiasis purínicas infrecuentes: déﬁcit de adenina fosforribosiltransferasa y xantinuria hereditaria. Med Clin 2002;119:508–15. 7. Ichida K, Matsumura T, Sakuma R, Hosoya T, Nishino T. Article Note: A translation of this article can be found here: https:// doi.org/10.1515/almed-2021-0059. Collazo Abal et al.: New mutation responsible for xanthinuria type I Furthermore,hypouricemiadetectedintwoofhissiblings supports the hereditary component of his condition, although they refused to confirm their results by molecular genetics. Taking all together, we could make a premature diagnosis in this patient, preventing renal complications from the renal calculi he already had and providing him, and his relatives, appropriate diet instructions and adequate monitoring. 8. Pérez-González A, Gómez-Peralta JI, Garza-Ortiz A, Barba-Behrens N. Importancia del molibdeno en los sistemas biológicos y su papel en enzimas mononucleares como parte del cofactor Moco. Educ Quím 2012:23:23–32. 9. Zhou Y, Zhang X, Ding R, Li Z, Hong Q, Wang Y, et al. Using next- generation sequencing to identify a mutation in human MCSU that is responsible for type II xanthinuria. Cell Physiol Biochem 2015;35:2412–21. 10. Mraz M, Hurba O, Bartl J, Dolezel Z, Marinaki A, Fairbanks L, et al. Modern diagnostic approach to hereditary xanthinuria. Urolithiasis 2015;43:61–7. Discussion The annual estimated incidence of HX is from 1:6,000 to 1:69,000, with over 150 patients diagnosed [13]. Just a few cases have been described in Spain where there should be an annual incidence superior to 600 cases with the actual demographic data [14–17]. This only proves that it is an In our case, the hypouricemia and low excretion of uric acid were verified by two different methods and we demonstrated the excretion of 2PY, which supports the diagnosis of HX type I. Finally, this diagnosis was confirmed by the homozygous mutation described above. 570 Research funding: None declared. 13. Orphanet: hereditary xantinuria hereditaria. Available from: https://www.orpha.net/consor/cgi-bin/Disease_Search.php? lng=EN&data_id=704&Disease_Disease_Search_disease Group=hereditary-xanthinuria&Disease_Disease_Search_ diseaseType=Pat&Disease(s)/group%20of% 20diseases=Hereditary-xanthinuria&title=Hereditary% 20xanthinuria&search=Disease_Search_Simple (Accessed 3 Mar 2021). Author contributions: CCA is the main responsible for the study conception, design, intellectual content and drafting of this paper. All authors have accepted responsibility for the entirecontentofthismanuscriptandapproveditssubmission. Competing interests: Authors state no conﬂict of interest. Informed consent: Informed consent was obtained from all individuals included in this study. Author contributions: CCA is the main responsible for the study conception, design, intellectual content and drafting of this paper. All authors have accepted responsibility for the entirecontentofthismanuscriptandapproveditssubmission. Competing interests: Authors state no conﬂict of interest. Informed consent: Informed consent was obtained from all individuals included in this study. 14. Cifuentes Delatte L, Castro-Mendoza H. Xantinuria familiar. Rev Clínica Española 1967;107:244–56. Ethical approval: The local Institutional Review Board deemed the study exempt from review. Ethical approval: The local Institutional Review Board deemed the study exempt from review. 15. Rapado A, Castro Mendoza HJ, Castilli JM, Frutos LCC M. Xanthinuria as a cause of hypouricaemia in liver disease. Br Med J 1975;2:560. Conclusions 11. Rebollido M, Cocho JA, Ramos DE, Bóveda MD, Fraga JM. Analysis of amino acids, acylcarnitines, acylglycines and organic acids in dried ﬁlter paper urine samples by Tandem Mass Spectrometry. Quim Clin 2006;25:64–74. We report here a case of xanthinuria type I due to the ho- mozygous mutation NM_000379.3(XDH):c2545-1G>C. This is a new point mutation that is suggested to be responsible for the patient’s phenotype, although additional xanthi- nuria cases are needed to confirm this association. 12. Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, et al. ACMG Laboratory Quality Assurance Committee. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med 2015;17:405–24. References Mutation of human molybdenum cofactor sulfurase gene is responsible for classical xanthinuria type II. Biochem Biophys Res Commun 2001; 282:1194–200.
https://openalex.org/W2611819586	https://repository.ubn.ru.nl//bitstream/handle/2066/200256/200256.pdf	English	null	Monitoring of somatic parameters at outpatient departments for mood and anxiety disorders	PloS one	2,018	cc-by	7,243	Monitoring of somatic parameters at outpatient departments for mood and anxiety disorders. Simoons, M.; Mulder, H.; Doornbos, D.; Schoevers, R.A.; Roon, E.N. van; Ruhé, H.G. 2018, Article / Letter to editor (PLoS One, 13, 8, (2018), article e0200520) Doi link to publisher: https://doi.org/10.1371/journal.pone.0200520 Version of the following full text: Publisher’s version Downloaded from: http://hdl.handle.net/2066/200256 Download date: 2024-10-24 Monitoring of somatic parameters at outpatient departments for mood and anxiety disorders. Simoons, M.; Mulder, H.; Doornbos, D.; Schoevers, R.A.; Roon, E.N. van; Ruhé, H.G. 2018, Article / Letter to editor (PLoS One, 13, 8, (2018), article e0200520) Doi link to publisher: https://doi.org/10.1371/journal.pone.0200520 Monitoring of somatic parameters at outpatient departments for mood and anxiety disorders. Simoons, M.; Mulder, H.; Doornbos, D.; Schoevers, R.A.; Roon, E.N. van; Ruhé, H.G. 2018, Article / Letter to editor (PLoS One, 13, 8, (2018), article e0200520) Doi link to publisher: https://doi.org/10.1371/journal.pone.0200520 Note: To cite this publication please use the final published version (if applicable). RESEARCH ARTICLE Introduction Somatic complications account for the majority of the 13–30 years shortened life expectancy in psychiatric patients compared to the general population. The study aim was to assess to which extent patients visiting outpatient departments for mood and anxiety disorders were monitored for relevant somatic comorbidities and (adverse) effects of psychotropic drugs– more specifically a) metabolic parameters, b) lithium safety and c) ECGs—during their treatment. Editor: Sinan Guloksuz, Department of Psychiatry and Neuropsychology, Maastricht University Medical Center, NETHERLANDS Editor: Sinan Guloksuz, Department of Psychiatry and Neuropsychology, Maastricht University Medical Center, NETHERLANDS Received: May 3, 2017 Accepted: June 28, 2018 Published: August 21, 2018 Editor: Sinan Guloksuz, Department of Psychiatry and Neuropsychology, Maastricht University Medical Center, NETHERLANDS Received: May 3, 2017 Accepted: June 28, 2018 Published: August 21, 2018 Editor: Sinan Guloksuz, Department of Psychiatry and Neuropsychology, Maastricht University Medical Center, NETHERLANDS Received: May 3, 2017 Accepted: June 28, 2018 Published: August 21, 2018 OPEN ACCESS Citation: Simoons M, Mulder H, Doornbos B, Schoevers RA, van Roon EN, Ruhe´ HG (2018) Monitoring of somatic parameters at outpatient departments for mood and anxiety disorders. PLoS ONE 13(8): e0200520. https://doi.org/10.1371/ journal.pone.0200520 Mirjam Simoons1,2,3, Hans Mulder1,4, Bennard Doornbos4, Robert A. Schoevers2, Eric N. van Roon3,5‡, Henricus G. Ruhe´2,6‡ 1 Department of Clinical Pharmacy, Wilhelmina Hospital Assen, Assen, The Netherlands, 2 Department of Psychiatry, Interdisciplinary Centre for Psychopathology and Emotion regulation, University of Groningen, University Medical Centre Groningen, Groningen, The Netherlands, 3 Department of Pharmacotherapy, -Epidemiology & -Economics, Department of Pharmacy, University of Groningen, Groningen, The Netherlands, 4 Mental Health Services Drenthe, Assen, The Netherlands, 5 Department of Clinical Pharmacy and Clinical Pharmacology, Medical Centre Leeuwarden, Leeuwarden, The Netherlands, 6 Department of Psychiatry, Warneford Hospital, University of Oxford, Oxford, United Kingdom a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 ‡ These authors are shared senior authors on this work. hans.mulder@wza.nl Methods We performed a retrospective clinical records review and cross-sectional analysis to assess the extent of somatic monitoring at four outpatient departments for mood and anxiety disor- ders in The Netherlands. We consecutively recruited adult patients visiting a participating outpatient department between March and November 2014. The primary outcome was per- centage of patients without monitoring measurements. Secondary outcomes were number of measurements per parameter per patient per year and time from start of treatment to first measurement. Copyright: © 2018 Simoons et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: Data are restricted as study participants did not provide consent for public data sharing. Interested, qualified researchers may request the de-identified data set by contacting Jurriaan Brouwer, MSc (jurriaan. brouwer@wza.nl) or the corresponding author. Introduction Patients with a severe mental illness (SMI), including patients with bipolar disorders and major depressive disorder, have a 13–30 year shorter life expectancy compared to the general population [1]. The majority (about 60%) of this excess mortality can be explained by somatic co-morbidity like respiratory, cardiovascular, nutritional and/or metabolic diseases [1–4]. Sev- eral factors may contribute to this increased risk of somatic morbidity and mortality, such as an unhealthy lifestyle and disparities in health care access that are associated with mental ill- ness [1,5]. In addition, the use of psychotropic drugs may cause and/or increase the vulnerabil- ity of psychiatric patients to somatic complications due to adverse effects [1,6]. In order to detect somatic complications and psychotropic drug-induced adverse effects, several guidelines and consensus documents have suggested to monitor essential somatic parameters as part of routine clinical practice in among others patients with schizophrenia, bipolar disorder and major depressive disorder [7–10]. Similarly, guidelines have been pub- lished for somatic monitoring during the use of specific classes of psychotropic drugs [7–11]. Next to the recognition that serum lithium levels, renal function and thyroid function should be monitored during lithium therapy, more recently metabolic monitoring during antipsy- chotic therapy has been advocated [7,9,11]. For patients treated for major depressive disorder only recently the first consensus document on somatic monitoring has been published in which a number of baseline and antidepressant-specific follow-up tests are recommended, in part depending on patient vulnerability characteristics [8,12]. Indeed, for antidepressants debate exists, e.g. regarding the necessity and appropriate frequency of monitoring of the elec- trocardiogram (ECG), notwithstanding a recent FDA warning [13,14]. In contrast to the expected clinical relevance of somatic monitoring of patients with SMI and the availability of the above-mentioned guidelines, several studies have shown poor adher- ence to these guidelines. For example, monitoring of serum lithium level, renal function and thyroid function in patients with bipolar disorder using lithium in the United Kingdom showed only 30–55% compliance to the available guideline [15]. A meta-analysis of 39 studies on metabolic screening in patients with predominantly schizophrenia or related disorders using antipsychotics, showed that routine baseline metabolic screening before start of pharma- cotherapy was suboptimal [16]. Given the high prevalence of somatic co-morbidities (e.g. met- abolic syndrome, pooled prevalence of 32.6% in a large cohort of SMI patients (N = 52,678) [17]), suboptimal monitoring might put patients at considerable risk for iatrogenic harm (i.e. Discussion Somatic monitoring in outpatients with mood and anxiety disorders is not routine clinical practice. Monitoring practices need to be improved to prevent psychiatric outpatients from undetected somatic complications. Competing interests: The authors have declared that no competing interests exist. Somatic monitoring practices at outpatient departments for psychiatry outpatient treatment for patients with monitoring measurements was 0.31 (range 0.0–12.9). The median time to first monitoring measurement per parameter for patients with monitoring measurements was 3.8 months (range 0.0–50.7). outpatient treatment for patients with monitoring measurements was 0.31 (range 0.0–12.9). The median time to first monitoring measurement per parameter for patients with monitoring measurements was 3.8 months (range 0.0–50.7). www.zonmw.nl/en/research-and-results/ fundamental-research/programmas/programme- detail/veni/), grant number #016.126.059. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Results We included 324 outpatients, of whom 60.2% were female. Most patients were treated for depressive disorders (39.8%), anxiety disorders (16.7%) or bipolar or related disorders (11.7%) and 198 patients (61.1%) used at least one psychotropic drug. For 186 patients (57.4%), no monitoring records were recorded (median treatment period 7.3 months, range 0–55.6). The median number of measurements per parameter per year since the start of Funding: The author(s) received no specific funding for this work, but HGR is supported by a Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO)/ZonMW VENI-Grant (https:// 1 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0200520 August 21, 2018 PLOS ONE \| https://doi.org/10.1371/journal.pone.0200520 August 21, 2018 Design and setting We performed a retrospective clinical records review and cross-sectional analysis to assess to which extent somatic monitoring had been performed in outpatients with mood and anxiety disorders. The study was conducted at four different outpatient departments for mood and anxiety disorders in the Northern part of the Netherlands. Three departments were part of two large secondary mental health care institutions and one department was part of an academic hospital. One of the three community departments partly used paper medical records for regis- tration of somatic monitoring measurements, the others used electronic medical records. Study population We included patients if they were 18 years or older and had visited the participating outpatient department for the first time after January 1st, 2010 (source population). We applied the latter inclusion criterion to ascertain that the observation period for every participant started after the introduction of several guidelines for somatic monitoring of psychiatric patients (among others antipsychotic-induced metabolic disturbances and lithium-induced side effects) [9–11,18]. We recruited patients consecutively when they visited the participating outpatient depart- ments between March and November 2014. We provided complete verbal and written infor- mation about the study, and obtained written informed consent for the use of their medication data from the community pharmacy and the use of their medical (psychiatric) history, labora- tory results and results of other measurements from the mental health care institution for the research purposes of this study. When we would doubt whether the patient understood the information or we would consider the patient impaired to consent (e.g. low IQ), we a priori would have abstained from including the patient. However for none of the patients there was any indication of impairment to consent. The independent medical ethics committee in Leeu- warden, the Netherlands (rTPO Leeuwarden; RTPO 918c), waived formal review and approval of the study protocol since participants were not subject to procedures, nor were they required to follow rules of behaviour for this study. Introduction harm resulting from treatment by a health care professional), regardless of the specific psychi- atric diagnosis. Metabolic disturbances and other somatic complications are not limited to patients with schizophrenia or patients using antipsychotics. Mood disorders are increasingly treated with PLOS ONE \| https://doi.org/10.1371/journal.pone.0200520 August 21, 2018 2 / 13 Somatic monitoring practices at outpatient departments for psychiatry combinations of lithium, antipsychotics, mood stabilizers and antidepressants. Therefore, these patients are at risk for somatic complications as well. In this study, we investigated to which extent patients visiting outpatient departments for mood and anxiety disorders were monitored for relevant somatic comorbidities and (adverse) effects of psychotropic drugs– more specifically a) metabolic parameters, b) lithium safety and c) ECGs—during their treatment. PLOS ONE \| https://doi.org/10.1371/journal.pone.0200520 August 21, 2018 Outcome measures We defined the observation period as the period between the start of treatment by the primary care giver (recorded in the medical record) and the inclusion (index) date. Our primary outcome was the percentage of patients without relevant somatic monitoring measurements in their medical records during their outpatient treatment. Our secondary outcomes were the number of measurements per somatic monitoring parameter per patient per year, as recorded in the medical records during the observation period for: I. all monitoring parameters together and, separately, II. metabolic monitoring parameters and the metabolic syndrome in the total study population, III. metabolic monitor- ing parameters and the metabolic syndrome in participants that used atypical antipsychotics, IV. lithium serum concentration, thyroid stimulating hormone (TSH) and kidney function in PLOS ONE \| https://doi.org/10.1371/journal.pone.0200520 August 21, 2018 3 / 13 Somatic monitoring practices at outpatient departments for psychiatry patients that used lithium, and V. ECG in the total study population. Another secondary out- come was the time from the start of treatment to the first monitoring measurement during the observation period. Assessment of study parameters We defined an somatic monitoring parameter as any parameter that can be measured as part of a physical examination (e.g. blood pressure, weight or ECG) or laboratory measurements (e.g. glucose- or creatinine concentration in blood or urine or a full blood examination). We checked for records of monitoring measurements in the relevant sections of the patient’s medi- cal records. All records on somatic monitoring measurements that were entered in the medical records were considered; this could be data generated by health care providers from the mental health care institution itself or communicated by other health care providers involved with the patient, as long as these measurements were retrievable from the medical records–either auto- matically from for example a laboratory system, or manually entered in the designated fields. According to the National Cholesterol Education Program Adult Treatment Panel III criteria, we considered the following criteria for assessment of the metabolic syndrome: waist circum- ference, triglycerides, high density lipoprotein (HDL), blood pressure, and glucose [19]. Medication records for each patient were retrieved from the community pharmacy for the observation period. We defined the use of a psychotropic drug as ‘a patient’s prescriptions for that psychotropic drug covering at least 80% of the observation period according to the com- munity pharmacy records’. We specifically looked at the use of antidepressants, antipsychotics and mood stabilizers (i.e. lithium, sodium valproate, lamotrigine, carbamazepine, topiramate, and levetiracetam). These drug classes were chosen because of the availability of guidelines or consensus documents for somatic monitoring of patients treated with these drugs or for the associated psychiatric diseases (e.g. antipsychotics [11], major depressive disorder [8], bipolar disorder [9], schizophrenia [10]). Statistical analysis We performed descriptive and statistical analyses using Excel 2013 (Microsoft, Redmond, Washington, USA) and IBM SPSS (version 20 for Windows; IBM Corp., Armonk, New York, USA). We report medians when distributions are non-normally distributed. For comparison of differences in dichotomous and categorical variables, we used Chi square tests and for com- parison of differences in continuous variables we used t-tests. Differences were considered sta- tistically significant when p<0.05. For comparison of categorical and continuous variables between academic and community settings or between patients with and without monitoring measurements, we used χ2 tests or Fisher’s exact tests as appropriate and t-tests, respectively. We report medians (range) when distributions are not distributed normally. We investigated the registration of monitoring parameters (yes/no) and the number of monitoring measurements during the observation period for differences per setting (both the four outpatient departments separately and the aca- demic/community departments) in univariate logistic regression models and univariate linear regression models. In all analyses, we used a p<0.05 significance level. Participants During the study period between March 2014 and November 2014, we consecutively asked 460 eligible patients to participate in the study at each of the four locations. Of these, 335 4 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0200520 August 21, 2018 Somatic monitoring practices at outpatient departments for psychiatry Fig 1. Flow diagram of patient inclusion. https://doi.org/10.1371/journal.pone.0200520.g001 Fig 1. Flow diagram of patient inclusion. https://doi.org/10.1371/journal.pone.0200520.g001 https://doi.org/10.1371/journal.pone.0200520.g001 patients gave written informed consent. In Fig 1 the flow diagram of patient inclusion is shown. The consent rate did not differ statistically between the four centres (p = 0.543). Eleven patients had to be excluded for subsequent analyses: two because they withdrew their informed consent in second instance and nine because their medical records were unavailable. patients gave written informed consent. In Fig 1 the flow diagram of patient inclusion is shown. The consent rate did not differ statistically between the four centres (p = 0.543). Eleven patients had to be excluded for subsequent analyses: two because they withdrew their informed consent in second instance and nine because their medical records were unavailable. Table 1 summarizes the characteristics of the total study population (n = 324) and of the patients treated in the academic and community settings, as obtained from the clinical records from the mental health care outpatient department and the medication records from the com- munity pharmacy. Of all participants, 60.2% were female, as can be expected in mood and anx- iety disorders. Most patients had low or medium educational levels (76.6%) and had been treated for depressive disorders (39.8%), anxiety disorders (16.7%) or bipolar or related disor- ders (11.7%). During their observation period, 198 subjects (61.1%) used at least one psycho- tropic drug during at least 80% of the observation period. Somatic monitoring practices at outpatient departments for psychiatry Table 1. Characteristics of the total study population and the populations from academic and community settings. Characteristic Valuea,b Total (n = 324) Academic setting (n = 54) Community settings (n = 270) Sex Female 195 (60.2%) 34 (63.0%) 161 (59.6%) Age (years) 43.6±12.4 40.8±13.8 44.2±12.0 Educational level Low 88 (27.2%) 7 (13.0%) 81 (30.0%) Medium 160 (49.4%) 24 (44.4%) 136 (50.4%) High 74 (22.8%) 22 (40.7%) 52 (19.3%) Unknown 2 (0.6%) 1 (1.9%) 1 (0.4%) Duration of outpatient treatment (median (range); months) 7.3 (0.0–55.6) 16.3 (2.0–55.0) 6.3 (0.0–55.6) Primary psychiatric diagnosis (DSM V diagnostic criteria)c Bipolar or related disorder 38 (11.7%) 9 (16.7%) 29 (10.7%) Depressive disorder 129 (39.8%) 16 (29.6%) 113 (41.9%) Anxiety disorder 54 (16.7%) 10 (18.5%) 44 (16.3%) Other psychiatric disorder 71 (21.9%) 14 (25.9%) 57 (21.1%) Not yet diagnosed 30 (9.3%) 5 (9.3%) 25 (9.3%) Unknown 2 (0.6%) 0 (0.0%) 2 (0.7%) Patients using a psychotropic drug at any stage during observation period 198 (61.1%) 26 (48.1%) 172 (63.7%) Of whom used: antidepressants 156 (78.4%) 20 (76.9%) 136 (78.6%) classic antipsychotics 6 (3.0%) 0 (0.0%) 6 (3.5%) atypical antipsychotics 42 (21.1%) 8 (30.8%) 34 (19.7%) lithium 42 (21.1%) 7 (26.9%) 35 (20.2%) non-lithium mood stabilizers 14 (7.1%) 1 (3.8%) 13 (7.6%) combinations of 2 psychotropic drugs 49 (24.6%) 7 (26.9%) 42 (24.3%) a Data are given as number (percentage) or mean ± standard deviation unless stated otherwise. b p<0.05, p0.01, p0.001 for academic vs. community settings. c DSM Diagnostic and Statistical Manual of Mental Disorders. a Data are given as number (percentage) or mean ± standard deviation unless stated otherwise. a Data are given as number (percentage) or mean ± standard deviation unless stated otherwise. b p<0 05 p0 01 p0 001 for academic vs community settings a Data are given as number (percentage) or mean ± standard deviation unless stated otherwise. b p<0.05, p0.01, p0.001 for academic vs. community settings. c DSM Diagnostic and Statistical Manual of Mental Disorders. p<0.001). These differences remained significant when we corrected for the longer observa- tion period in the academic setting versus the community settings in multivariate logistic regression models (all p0.038; see Table 1). Patients for whom monitoring measurements were reported (n = 138; 42.6%), had been in treatment for a significantly longer period on the index date compared to patients without monitoring measurement: 13.9 (range 0.66–55.0) vs. 4.9 (range 0.0–55.6) months (p<0.001). There was no difference in registration of monitoring measurements between patients with and without psychotropic drugs (46.5% vs. 36.5%; p = 0.077). However, for 106 of 198 (53.5%) patients who had used at least one psychotropic drug during the observation period, no moni- toring data were recorded. For patients using antidepressants, 62.8% were without monitoring measurements, which was 16.7% for patients using classic antipsychotics, 38.1% for atypical antipsychotics, 9.5% for lithium and 21.4% for other mood-stabilizers. Primary outcome: Registration of monitoring information For 186 of 324 patients (57.4%), we identified no monitoring measurements in their medical records during the median observation period of 7.3 months (range 0.0–55.6). In our logistic regression models, the lack of registration of monitoring measurements at the three commu- nity outpatient departments (62.5%, 67.8% and 58.7%) did not differ statistically between departments (p = 0.446), but was statistically different from the academic outpatient depart- ment (29.6%) both for each community department separately and grouped together (all PLOS ONE \| https://doi.org/10.1371/journal.pone.0200520 August 21, 2018 5 / 13 https://doi.org/10.1371/journal.pone.0200520.t001 Discussion This naturalistic, non-intervention study shows that monitoring of somatic parameters at out- patient departments for mood and anxiety disorders is not implemented in daily clinical prac- tice. Compared to the total study sample, a slightly higher number of metabolic measurements was found in the subsample using atypical antipsychotics. Lithium monitoring rates in the sub- population using lithium were considerably better. However, for 57.4% of all patients, there were no records of monitoring measurements. Moreover, the median time to the first mea- surement was almost four months after the initiation of outpatient treatment, indicating that a somatic screening at the first appointment is not part of routine clinical practice. Improvement of these monitoring practices provides an opportunity to ensure that somatic complications and adverse drug effects are detected and treated in order to reduce the risk of iatrogenic harm. Secondary outcomes: Monitoring frequency and timing of monitoring The median number of monitoring measurements per monitoring parameter per patient with monitoring measurements per year was 0.31 (range 0.0–12.9). Our linear regression analyses showed that this number was not different between the four outpatient departments nor between the academic and community settings (all p>0.254). 6 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0200520 August 21, 2018 Somatic monitoring practices at outpatient departments for psychiatry Fig 2 shows the frequency of monitoring for metabolic parameters and the metabolic syn- drome in the total study population (panel A) and in participants that used atypical antipsy- chotics (n = 42; panel B). The percentage of patients without metabolic measurements was 66.4% for all patients and 38.1% for patients using atypical antipsychotics. Patients who used atypical antipsychotic drugs, were monitored more often than the total population for all met- abolic parameters. In addition, Fig 2C displays the frequency of monitoring of the lithium serum concentration, TSH and kidney function in patients who used lithium (n = 42). Less than two measurements of lithium serum concentration, thyroid and kidney function per year (minimum in the Dutch guideline for Bipolar Disorders) were performed in 16.7%, 47.6% and 28.6% of patients using lithium, respectively (median treatment duration 15.1 months (range 1.0–49.1). Measurement of at least one ECG was performed in ten patients (3.1%; range 1–6 measurements), with an average observation period of 26.1 months (standard deviation 12.7). Finally, the median time to the first monitoring measurement per parameter for patients with monitoring measurements since the start of treatment was 3.8 months (range 0.0–50.7). PLOS ONE \| https://doi.org/10.1371/journal.pone.0200520 August 21, 2018 PLOS ONE \| https://doi.org/10.1371/journal.pone.0200520 August 21, 2018 Somatic monitoring in psychiatry Our results are in agreement with several studies on monitoring in psychiatric outpatients. For example, in a large benchmarking audit from The United Kingdom in lithium-treated patients, no weight had been recorded in 72% of patients, no tests had been performed on kidney and thyroid function in 19%, and 18%, respectively, and no lithium serum concentration had been taken in 9% [15]. A recent study in underserved adults with SMI taking antipsychotic drugs showed that in only 30.1% of patients adequate diabetes-specific screening (glucose serum test or HbA1C test) was performed [20]. With 90–100% screening for hypertension, diabetes and dyslipidaemia in HIV-patients compared to 40–70% in psychiatric outpatients taking antipsy- chotics, somatic monitoring frequencies were found to be considerably higher in other fields of outpatient medicine, suggesting a particular problem of poor monitoring in psychiatry [21]. Considering the similarity between these reports and our study, we believe our results are gen- eralizable to other parts of The Netherlands and Europe, although replication is warranted. Somatic monitoring is important in patients with mood and anxiety disorders because these patients are at risk for cardiovascular, nutritional and metabolic comorbidities, irrespec- tive of the use of psychotropic drugs [1,8,22]. Somatic complications account for the majority of the excess mortality which translates to a 13–30 year shorter life expectancy in the SMI pop- ulation compared to the general population [1]. The use of psychotropic drugs like antipsy- chotics or mood stabilizers but also specific antidepressants further increases the risk of somatic complications as a result of, among others, drug-induced disturbances in metabolic PLOS ONE \| https://doi.org/10.1371/journal.pone.0200520 August 21, 2018 7 / 13 Somatic monitoring practices at outpatient departments for psychiatry Fig 2. Metabolic monitoring and lithium monitoring in total and subsets of study population. Monitoring frequencies of metabolic parameters in (A) the total study population (n = 324, of whom 61.1% used psychotropic drugs) and (B) in patients using atypical antipsychotics (n = 42) and (C) lithium serum concentration, thyroid function and kidney function in patients using lithium (n = 42). Diagnostic criteria for the metabolic syndrome were: registration of measures of waist circumference, triglycerides, High Density Lipoprotein (HDL), blood pressure, and glucose, according to the National Cholesterol Education Program Adult Treatment Panel III criteria [19]. https://doi.org/10.1371/journal.pone.0200520.g002 Fig 2. Metabolic monitoring and lithium monitoring in total and subsets of study population. Somatic monitoring in psychiatry Monitoring frequencies of metabolic parameters in (A) the total study population (n = 324, of whom 61.1% used psychotropic drugs) and (B) in patients using atypical antipsychotics (n = 42) and (C) lithium serum concentration, thyroid function and kidney function in patients using lithium (n = 42). Diagnostic criteria for the metabolic syndrome were: registration of measures of waist circumference, triglycerides, High Density Lipoprotein (HDL), blood pressure, and glucose, according to the National Cholesterol Education Program Adult Treatment Panel III criteria [19]. https://doi.org/10.1371/journal.pone.0200520.g002 Fig 2. Metabolic monitoring and lithium monitoring in total and subsets of study population. Monitoring frequencies of metabolic parameters in (A) the total study population (n = 324, of whom 61.1% used psychotropic drugs) and (B) in patients using atypical antipsychotics (n = 42) and (C) lithium serum concentration, thyroid function and kidney function in patients using lithium (n = 42). Diagnostic criteria for the metabolic syndrome were: registration of measures of waist circumference, triglycerides, High Density Lipoprotein (HDL), blood pressure, and glucose, according to the National Cholesterol Education Program Adult Treatment Panel III criteria [19]. https://doi.org/10.1371/journal.pone.0200520.g002 8 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0200520 August 21, 2018 Somatic monitoring practices at outpatient departments for psychiatry parameters, liver function and ECG [6,8,23,24]. Although the effectiveness of somatic moni- toring on treatment outcomes has not been well established [25], it seems logical to monitor these known somatic complications of mood and anxiety disorders and adverse effects of psy- chotropic drugs, especially in patients with a high prevalence or a high risk of morbidity or mortality [26,27]. Importantly, this low rate of monitoring is in contrast with the SMI patients’ perceived need to have their somatic health screened and monitored, as their ability to survey their own physical health interest is reduced [28]. Lithium, metabolic and ECG monitoring Lithium monitoring was considerably better than metabolic monitoring, although for both sets of parameters improvement could still be achieved. For patients using antipsychotics or lithium, national and international consensus documents and guidelines on monitoring are available [7,11]. A possible explanation is that psychiatrists in the field of mood and anxiety disorders might not be aware of monitoring guidelines for antipsychotic use, especially when dosages of antipsychotics are low and duration of use is relatively short, or that the necessity of lithium monitoring has been more longstanding acknowledged in consensus documents or guidelines compared to metabolic monitoring during antipsychotic use. Interestingly, ECG measurements were performed in only 10 out of 324 patients. However, we cannot value this finding in terms of guideline concordance or clinical necessity, as there is no clear or uniform set of criteria for measuring the ECG and we could logistically not verify many of the various risk factors in our patient population. Baseline somatic screening An interesting finding of this study is that the first monitoring measurement was performed approximately four months after the start of treatment. This median four month delay suggests that a baseline somatic screening is not part of daily routine. Such a baseline measurement before starting any treatment is important to (1) clarify the (type of) psychiatric disorder and assist in (drug) treatment selection (2) assess the effect of co-morbidities on the psychiatric diagnosis (3) determine risk factors that may increase susceptibility to adverse effects, and (4) establish intra-individual references for any (aberrant) test result of monitoring thereafter [8]. Without a baseline measurement, the treating psychiatrist cannot determine whether aberrant measurements during treatment are the result of (drug) treatment. Reasons for not performing a baseline screening may include limited access to laboratory facilities for physicians and/or patients, insufficiently experienced or educated mental health care professionals or seemingly good somatic health (in young patients). These factors may also play a role in the lack of somatic monitoring in general. An important issue that has to be investigated further is which patients have to be measured for which parameter and how frequently, which might depend on an a-priori risk-profile instead of a one-size fits all approach. A minimum set of monitoring recommendations should be established for groups of patients depending on relevant risk factors that is cost-effective and is readily applicable in routine clinical practice. It is essential to find the right balance between costs and yield of relevant aberrances, thereby creating a set of parameters that gives monitoring information to support the clinician in making treatment decisions without pro- ducing many test results that are not hypothesis-driven or based on a specific increased risk. Implementation of somatic monitoring In previous research, the introduction of new guidelines, consensus statements, or (national) quality improvement programs alone appeared to be minimally effective in improving PLOS ONE \| https://doi.org/10.1371/journal.pone.0200520 August 21, 2018 9 / 13 Somatic monitoring practices at outpatient departments for psychiatry monitoring rates [16,29–31]. Therefore, to our opinion, the implementation of a more struc- tured monitoring program is warranted in which somatic monitoring is ensured as part of routine clinical care [32]—ideally in close collaboration between physical and mental health professionals. The differences found between academic and community mental health provid- ers suggest that collaboration between centres may also be beneficial to enhance broader implementation. A computer reminder system has been shown to be effective in supporting laboratory monitoring of psychiatric outpatients using antipsychotics [33]. In the northern part of The Netherlands, we developed the innovative care path ‘Monitoring Outcomes of Psy- chiatric Pharmacotherapy (MOPHAR)’, which is currently actively implemented as a restruc- tured routine practice for outpatient care teams. In this program, somatic monitoring of psychiatric outpatients is incorporated in routine clinical practice at the outpatient depart- ment. Primary objective of this program is to prevent, monitor and treat somatic co-morbidi- ties and adverse effects of psychotropic drugs. A nurse conducts and coordinates general somatic screenings with each patient at the first appointment and yearly thereafter. In addi- tion, recommended monitoring (among others of therapeutic effect and somatic adverse effects of psychotropic drugs) is performed according to pre-specified protocols per drug used as determined by regular medication reconciliation [34]. Mental health care providers have immediate access to this up-to-date information and minimal burden to pursue these proto- cols. This program will be further investigated for effectiveness. PLOS ONE \| https://doi.org/10.1371/journal.pone.0200520 August 21, 2018 Strengths and limitations Strengths of this study are the large population and the conduct of assessments at four loca- tions. However, a few limitations need to be considered when interpreting our results. Firstly, as we performed a retrospective review of the medical records, our results depend on their accuracy and completeness. We did not obtain additional information from health care pro- viders or patients. Therefore, we cannot exclude that monitoring of somatic parameters was done by others, for example a general practitioner, when such external results were not accessi- ble through the patient’s medical records at the outpatient department. Nevertheless, we inten- tionally designed the study to include only monitoring measurements recorded in the medical records at the outpatient departments, as this information is used for treatment decisions by the mental health care providers. Secondly, we did not systematically ascertain whether moni- toring measurements were recorded before the start of treatment at the outpatient department. To address this limitation, we performed an exploratory search for laboratory data including general practitioners’ data in 50 patients in the three months before the start of their treatment. We specifically looked at blood parameters (hemoglobin, hematocrit and full blood count), electrolytes (sodium, potassium and calcium), kidney function, blood lipids (cholesterol, tri- glycerides, HDL and low density lipoprotein (LDL)), glucose of hemoglobin A1C (HbA1C) and thyroid function. For one patient (2.0%), all these parameters were recorded, while for 31 patients (62.0%) none of these parameters were measured. This indicates that a baseline screening prior to start of outpatient treatment, for example performed by a general practi- tioner, is not routinely performed in clinical practice either. Thirdly, as mentioned before, antipsychotics in patients with mood and anxiety disorders may be used for a short period at a low dose (e.g. quetiapine 50 mg q.d.). In these patients, monitoring may have been considered irrelevant. However, only 7 out of 42 patients (16.7%) using atypical antipsychotics used low dosages (100 mg/day) quetiapine for less than one month (next to use of another psychotro- pic drug for more than 80% of the observation period in 5 patients), indicating that this alone cannot explain the relatively low monitoring frequencies in these patients. Strengths and limitations Furthermore, there is evidence that metabolic adverse effects of antipsychotics also occur during short-term use PLOS ONE \| https://doi.org/10.1371/journal.pone.0200520 August 21, 2018 10 / 13 Somatic monitoring practices at outpatient departments for psychiatry and at low dosages, thus requiring monitoring anyhow [35,36]. Finally, as we could logistically not verify somatic diseases in a valid way in these patients, we are unable to explore any associ- ations of monitoring frequency with somatic comorbidities. We did not explore any associa- tions with the psychiatric illnesses either (Table 1). However, our population had been referred to the outpatient clinic for worse than minor psychological complaints, warranting somatic monitoring at least once a year regardless of the specific psychiatric diagnosis or additional somatic comorbidities. Acknowledgments The authors would like to thank Milou Vreeman, PharmD, and Herman Veenhof, PharmD, for their assistance with data collection. The authors would like to thank Milou Vreeman, PharmD, and Herman Veenhof, PharmD, for their assistance with data collection. Conclusions and suggestions for future research In conclusion, this study shows that monitoring of somatic parameters before and during treatment in psychiatric outpatients with mood and anxiety disorders is not performed rou- tinely in clinical practice. Although risks vary between different drugs and patients, the low frequencies of systematic monitoring might increase the risk of undetected somatic complica- tions of the psychiatric illness or due to adverse effects of psychotropic drugs in these patients. The results of this study will hopefully increase the awareness of mental health care providers that better monitoring of psychiatric treatments poses an opportunity for improving the care for their vulnerable patients. Future studies should address whether special monitoring pro- grams, with routine medication reconciliation and monitoring of treatment effects, somatic complications and adverse drug effects can improve monitoring frequencies at outpatient departments for psychiatric patients. These studies should also address the questions whether such monitoring programs indeed reduce the risk of prospective somatic complications in psy- chiatric patients and how these programs optimize the benefits of improved quality of life at the lowest cost of (too many) examinations and check-ups. References 1. De Hert M, Correll CU, Bobes J, Cetkovich-Bakmas M, Cohen D, Asai I, et al. Physical illness in patients with severe mental disorders. I. 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Schoevers, Eric N. van Roon, Henricus G. Ruhe´. Conceptualization: Mirjam Simoons, Hans Mulder, Bennard Doornbos, Robert A. Schoevers, Eric N. van Roon, Henricus G. Ruhe´. Data curation: Mirjam Simoons. Data curation: Mirjam Simoons. Formal analysis: Mirjam Simoons. Funding acquisition: Hans Mulder, Robert A. Schoevers, Eric N. van Roon, Henricus G. Ruhe´. Investigation: Mirjam Simoons. Investigation: Mirjam Simoons. Methodology: Mirjam Simoons, Hans Mulder, Eric N. van Roon, Henricus G. Ruhe´. Project administration: Mirjam Simoons, Hans Mulder, Eric N. van Roon, Henricus G. Ruhe´. Resources: Mirjam Simoons, Hans Mulder, Robert A. Schoevers, Eric N. van Roon, Henricus G. Ruhe´. Supervision: Mirjam Simoons, Hans Mulder, Bennard Doornbos, Robert A. Schoevers, Eric N. van Roon, Henricus G. Ruhe´. Visualization: Mirjam Simoons. 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https://openalex.org/W3057038768	https://cmjournal.biomedcentral.com/track/pdf/10.1186/s13020-020-00367-1	English	null	The scientific elucidation of daodi medicinal materials	Chinese medicine	2,020	cc-by	8,640	© The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://crea- tivecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdo- main/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Liu et al. Chin Med (2020) 15:86 https://doi.org/10.1186/s13020-020-00367-1 Liu et al. Chin Med (2020) 15:86 https://doi.org/10.1186/s13020-020-00367-1 Chinese Medicine Chinese Medicine Open Access The scientific elucidation of daodi medicinal materials Xindan Liu1 , Ying Zhang1, Menghua Wu1, Zhiguo Ma1, Zihan Huang1, Fang Tian1, Sihan Dong2, Simin Luo2, Yu Zhou2, Jinju Zhang2, Nanxin Li2, Xiaofang He2 and Hui Cao1 Abstract Daodi medicinal materials (DMMs), with unique characteristics and specific ecological growing environments, are rec- ognized as high-quality medicinal products of Chinese medicinal materials (CMMs). The quality evaluation of CMMs is fundamental for standardization. The concept and application of DMMs have a long history as described in records in ancient books and rooted in practice and experience over generations. DMM is the specific term for pure, superior medicinal herbs with the following characteristics: optimum harvest season (reflecting the appropriate develop- mental stage of the plant), scrupulous processing, traditional preparation technology, etc. As DMM and high-quality medicinal products are traditionally thought to be closely related, modern scientific studies that confirm the associa- tion of these products are described. This article aims to clarify the scientific elucidation of DMMs. Keywords: Daodi medicinal materials, Chinese medicinal materials, Ecological environment, Chemical components, Pharmacological functions Background was formally listed under each medicinal herb heading, specifying daodi production regions. It has been sug- gested that the quality of CMMs is highly correlated with their geographical origins. In ancient times, the identi- fication of DMMs was commonly carried out based on the characteristics of the superficies, and this approach depended to a certain extent on empirical experiences and assumptions. Currently, modern scientific analyti- cal techniques may be applied to confirm the validity of associations between high-quality medicinal products and DMM to ensure their utility, clarifying the scientific understanding of daodi medicinal materials. In recent years, as the use of Chinese medicinal materi- als (CMMs) has increased, the international attention paid to the safety, stability and efficacy of CMMs has increased. Some authentic and superior CMMs that are grown in specific regions and widely recognized as hav- ing better therapeutic effects are called daodi medicinal materials (DMMs) [1]. DMMs, based on the theory, ori- gin, processing, and prominent curative effect of CMMs, are the essence of Chinese cultural heritage [2]. DMMs were first recorded in Zhen Zhu Nang Yao Xing Fu (Pre- cious Drus in Rhyme), a book written 700 years ago. The term “daodi medicinal material” is widely found in Ben Cao Pin Hui Jing Yao (Essentials of Materia Medica Dis- tinctions), a book compiled by the Imperial Hospital dur- ing the Ming Dynasty (1368–1644 A.D.), in which 268 medicinal herbals are listed. The entry “original source” The word “dao” (in “daodi”) is an ancient Chinese unit of measurement used to divide administrative districts, and this term can be retraced to the Eastern Han Dynasty (25–220 A.D.) as described in Hou Han Shu (Book of Later Han, 432–445 A.D.) [3]. In the Tang Dynasty (618– 907 A.D.), the nation was divided into 10 “dao” accord- ing to landscapes in the Zhenguan Period, and then the number was increased to 15 “dao” in the Kaiyuan Period. Currently, “dao” is conceptually similar to the modern organizational system of provinces. The word “di” (in Correspondence: zhangying@jnu.edu.cn; kovhuicao@aliyun.com 1 Research Center for Traditional Chinese Medicine of Lingnan (Southern China), Jinan University, Guangzhou 510632, China Full list of author information is available at the end of the article Correspondence: zhangying@jnu.edu.cn; kovhuicao@aliyun.com 1 Research Center for Traditional Chinese Medicine of Lingnan (Southern China), Jinan University, Guangzhou 510632, China Full list of author information is available at the end of the article Correspondence: zhangying@jnu.edu.cn; kovhuicao@aliyun.com 1 Research Center for Traditional Chinese Medicine of Lingnan (Southern China), Jinan University, Guangzhou 510632, China Full list of author information is available at the end of the article Liu et al. Chin Med (2020) 15:86 Page 2 of 11 Daodi medicinal materials are the essence of Chinese cultural heritage Daodi medicinal materials are the essence of Chinese cultural heritage “daodi”) refers to regions and geography. Nowadays, “daodi medicinal materials” refers to the distinctively higher quality of the medicinal materials that grow in a certain area. Over the long course of clinical selection, by discarding the inferior and retaining the superior, DMMs have been acquired. DMMs represent the influence of populations, together with optimum harvest season, habitat and grow- ing conditions such as sunshine, soil, and water. Gener- ally, the identification of DMMs is a useful tool for the quality evaluation of CMMs. The establishment of DMMs is related to resources, agricultural technology and CMM development. In ancient China, due to agriculture production, rich expe- rience along with advanced technology in growing and processing medicinal herbals was accumulated, which resulted in the exchange of resources around the world [4]. In addition, the vast territory of China, including plains, hills, mountains, lakes, rivers and seas with differ- ent climates, sunshine, soils and ecological environments, provides favorable conditions for the growth of medici- nal herbs. Through meticulous selection over the course of production and continuous clinical tests, DMMs have been proven to be definitely curative in medical treat- ments and therefore have been handed down from gen- eration to generation [5].f Populationsf p Different growing environments may greatly influence the quality and chemical components of closely related populations. Modern experimental research, especially molecular biological identification, has validated that the different production areas of CMMs are closely related to the quality and DNA sequence divergence of CMMs [6–8]. For example, as the dominant constituents, patch- oulol and pogostone are the basis for the anti-inflam- matory activity of Pogostemon cablin (Blanco) Benth. [9]. And it has been found that pogostone also exhibits potent anti-fungal [9], antiapoptotic [10], antioxidant [11], and immunosuppressive [12] properties. In our previous study, P. cablin produced in Shipai (in Guang- zhou city, Guangdong province, SP) and Gaoyao (in Zhaoqing city, Guangdong province, GY) differed from P. cablin cultivated in Hainan province (HN) and Zhan- jiang city (in Guangdong province, ZJ) not only in the total amount of volatile oil but also in genotype [13–15]. According to the composition of the volatile oil, P. cab- lin is divided into two chemotypes: SP and GY cultivars belong to the pogostone-type, while the HN and ZJ cul- tivars belong to the patchoulol-type (Fig. 1). Moreover, we have demonstrated that the sequence divergence of both the matK and 18S rRNA genes among 6 samples of P. cablin from different locations was well correlated with the regions of cultivation and intraspecific essential oil chemotypes (Fig. 2) [16]. The same is true for other medicinal herbs. The major pharmacological compo- nents in Cnidium monnieri (L.) Cuss. are coumarins DMM can be very effective if proper consideration is given to the characteristics of the original sources, growth and seasonal changes of the material; however, the same medicinal herb grown in different areas does not have the same effectiveness even if they are the same plant. In China, traditional Chinese medicine doctors usually select a superior populations or variety to pre- scribe based on geographical features. In addition, dif- ferent harvest times or processing methods can greatly influence the quality of CMMs in terms of chemical com- ponents and pharmacological functions. Thus, DMM, as an authentic and superior medicinal product, needs to be validated by modern analytical methods for each CMM. In this paper, the scientific elucidation of DMMs will be introduced, as well as a putative production chain for processing traditional, experience-based CMMs. Fig. 1 Structures of the pogostone and patchoulol in Pogostemon cablin Fig. 1 Structures of the pogostone and patchoulol in Pogostemon cablin Fig. Populationsf 1 Structures of the pogostone and patchoulol in Pogostemon cablin Page 3 of 11 Liu et al. Chin Med (2020) 15:86 e oil composition in the leaves (a), comparison of the variable sites in the matK sequence (b), comparison of the variable sites in the uence (c) and cluster trees of both the matK and 18S rRNA gene sequences (d) of Pogostemon cablin (Blanco) Benth. from 6 different top number indicates the nucleotide position upstream of the matK (b) and the 18S rRNA (c) sequence, an asterisk () indicates the de as the P1 sequence, and a hyphen (—) indicates an alignment gap (c) Fig. 2 Volatile oil composition in the leaves (a), comparison of the variable sites in the matK sequence (b), comparison of the variable sites in the 18S rRNA sequence (c) and cluster trees of both the matK and 18S rRNA gene sequences (d) of Pogostemon cablin (Blanco) Benth. from 6 different locations. The top number indicates the nucleotide position upstream of the matK (b) and the 18S rRNA (c) sequence, an asterisk () indicates the same nucleotide as the P1 sequence, and a hyphen (—) indicates an alignment gap (c) Fig. 2 Volatile oil composition in the leaves (a), comparison of the variable sites in the matK sequence (b), comparison of the variable sites in the 18S rRNA sequence (c) and cluster trees of both the matK and 18S rRNA gene sequences (d) of Pogostemon cablin (Blanco) Benth. from 6 different locations. The top number indicates the nucleotide position upstream of the matK (b) and the 18S rRNA (c) sequence, an asterisk () indicates the same nucleotide as the P1 sequence, and a hyphen (—) indicates an alignment gap (c) Liu et al. Chin Med (2020) 15:86 Page 4 of 11 et al. [25], Chaenomeles speciosa (Sweet) Nakai pro- duced the highest quality yields when harvested in early July. In the case of Desmodium styracifolium (Osb.) Merr., the best collection season was early October, when the highest concentrations of polysaccharides, flavonoids and schaftoside were measured [26]. [17]. Similarly, according to the coumarin chemotypes, C. monnieri has been classified into three chemotypes, the osthol-linear furanocoumarins-type (chemotype I), principally cultivated in regions of Jiangsu and Hunan provinces; the angular furanocoumarins-type (chemo- type II), mainly produced in Heilongjiang province; and the transition-type (chemotype III), largely came from Henan and Hebei regions [18]. Populationsf It was found that there were 12 variable sites in the matK gene sequence of C. monnieri from different populations. A phylogenetic tree constructed by the neighbor-joining (NJ) method showed that the phylogenetic relationship of 6 C. monnieri cul- tivars was well correlated with their geographical distri- bution and intraspecific coumarin chemotypes of (Fig. 3) [19]. Likewise, Laboratory research showed a clear cor- relation between the rDNA ITS sequence and the phe- notype of Dendrobium officinale Kimura et Migo from different populations [20]. The phylogenetic relationship predicted by the 5S rRNA spacer region data correlated well with the essential oil chemotype of Acorus calamus L. collected from various locations [21]. However, the dif- ference in the alkaloid content of Fritillaria thunbergii Miq. from various habitats did not result from variation in the 5S rRNA sequence but from the microenviron- ment [22]. It’s reported that 5S rRNA is highly-conserved arcoss all species [23], thus, different environments did not produce changes in 5S rRNA of different F. thun- bergii populations, but produced differences in second- ary metabolites. In these cases, it can be concluded that genetic diversity existed among different populations is relevant to the cultivation regions except some highly- conserved DNA markers. Sunshine S h Sunshine is an important factor for the formation of DMMs. For example, the content of volatile compo- nents in Houttuynia cordata Thunb. was closely related to light intensity: monoterpenoids and nonterpenoids were positively and negatively associated with light intensity, respectively [27]. In Viola yedoensis Makino, the content of flavonoids and coumarins was positively correlated with light intensity [28]. Although it has been demonstrated that higher concentrations of total patchoulol corresponded to lower light intensity in P. cablin, there was no statistically significant correlation between patchoulol content and shade [29]. In recent years, circadian clocks that temporally organize many aspects of growth and metabolism have even been found in numerous plant species [30–32]. For example, in Antirrhinum majus L., monoterpene synthase mRNA levels and corresponding monoterpene emission, which followed diurnal rhythms, were controlled by a circa- dian clock [33]. A similar daily fluctuation was found in the endogenous level of geranyl acetate and in the expression of its biosynthetic gene, alcohol acetyl trans- ferase in Rosa rugosa Thunb. [31]. Harvest time Soil is also important for the identification of DMMs and for the evaluation of quality. Modern pharmacologic studies have proven that the specific composition of soil has a large influence on the quality and quantity of the chemicals in medicinal herbs. For example, soil available iron (Fe) could promote the accumulation of flavonoids, while soil available manganese (Mn), total potassium (K), and available K had an inhibitory effect on flavonoid content in Spatholobus suberectus Dunn [34]. In Citrus grandis ‘Tomentosa’, the content of soil available copper (Cu), zinc (Zn), Mn, boron (B), and molybdenum (Mo) was positively associated with flavonoid concentrations and naringin concentrations [35]. There was a positive correlation between soil total nitrogen (N), available K and emodin in Polygonum cuspidatum Sieb. et Zucc. [36]. Soil Mn was a favorable factor for accumulating schisan- therin A in Schisandra sphenanthera Rehd. et Wils., as a significant correlation was also found between these fac- tors [37]. Soil is also important for the identification of DMMs and for the evaluation of quality. Modern pharmacologic studies have proven that the specific composition of soil has a large influence on the quality and quantity of the chemicals in medicinal herbs. For example, soil available iron (Fe) could promote the accumulation of flavonoids, while soil available manganese (Mn), total potassium (K), and available K had an inhibitory effect on flavonoid content in Spatholobus suberectus Dunn [34]. In Citrus grandis ‘Tomentosa’, the content of soil available copper (Cu), zinc (Zn), Mn, boron (B), and molybdenum (Mo) was positively associated with flavonoid concentrations and naringin concentrations [35]. There was a positive correlation between soil total nitrogen (N), available K and emodin in Polygonum cuspidatum Sieb. et Zucc. [36]. Soil Mn was a favorable factor for accumulating schisan- therin A in Schisandra sphenanthera Rehd. et Wils., as a significant correlation was also found between these fac- tors [37]. CMMs harvested during different growing periods contain different plant metabolites. For instance, the content of essential oil increased as the age of P. cablin increased: patchoulol, α-bulnesene and other sesquiter- penes accumulated to high concentrations at 210 days after maturation [24]. According to the report from Jin Fig. 3 Phylogenetic tree of the matK gene sequence (E) of Cnidium monnieri (L.) Cuss. from six locations (C1–C6) Fig. 3 Phylogenetic tree of the matK gene sequence (E) of Cnidium monnieri (L.) Cuss. from six locations (C1–C6) Fig. Traditional descriptions of daodi medicinal materials by famous physicians in ancient China Traditional descriptions of daodi medicinal materials by famous physicians in ancient China In the use of CMMs, a large emphasis has been placed on the identification of DMMs since ancient times. As recorded in Shen Nong Ben Cao Jing (The Divine Shen- nong’s Classic of Materia Medica, 25–220 A.D.) [51], “each medicinal material has laws for its production region, authenticity, and freshness.” In various chapters of that book, locations in ancient kingdoms and regions, such as mountain valley, river valley or marshes, were mentioned for the first time as medicinal herbs sources. This record indicates that different CMMs come from certain specific areas. In his immense book Ben Cao Jing Ji Zhu (Collection of Commentaries on the Classic of the Materia Medica, 480–498 A.D.) [52], Tao Hongjing, a well-known physician in the Northern and Southern Dynasties (420–589 A.D.), used such terms “good”, “quite good”, “fairly good”, “excellent” and “best” to describe the effects of over 40 medicinal herbals commonly used in medical treatment. Moreover, correlations between the sources, developmental stages and efficacy of these medicinal herbs were described. Sun Simiao, a famous physician and pharmacologist of the Tang Dynasty (618–907 A.D.), stated the following in his book Qian Jin Yi Fang (Formulas Worth a Thousand Gold Pieces, 682 A.D.) [53]: “Medicinal herbs used by ancient phy- sicians were always from designated original sources, which accounted for their great effectiveness in medi- cal treatment”. In that book, he comprehensively sorted 519 DMMs and systematically stipulated 133 regions of production. According to Kou Zongshi, a famous physi- cian of the Song Dynasty (960–1279 A.D.), in his book Ben Cao Yan Yi (Extension of the Materia Medica, 1116 A.D.), “in prescribing medicinal herbs, care should always be taken to select those from proper sources to ensure their effectiveness”, greatly emphasizing the designated original sources of medicinal herbs [54]. During the Jin and Yuan Dynasties (1115–1368 A.D.), the text Yong Yao Fa Xiang (Medication Method, 1249 A.D.) also suggested that one could achieve excellent treatment results only by using DMMs with proper production regions and harvest time. Then, the Ming Dynasty (1368-1644 A.D.) docu- ment Ben Cao Meng Quan (Materia Medica Companion, 1565 A.D.) stated that “the effect will be definitely dif- ferent if medicinal materials are produced in a different environment” [55]. The record in Yi Xue Yuan Liu Shi Harvest time 3 Phylogenetic tree of the matK gene sequence (E) of Cnidium monnieri (L.) Cuss. from six locations (C1–C6) Fig. 3 Phylogenetic tree of the matK gene sequence (E) of Cnidium monnieri (L.) Cuss. from six locations (C1–C6) Liu et al. Chin Med (2020) 15:86 Page 5 of 11 Liu et al. Chin Med (2020) 15:86 Water components and predict the large-scale distribution of target medicinal herbs such as P. cablin [44], Artemi- sia annua L. [45], Polygonum multiflorum Thunb. [46], Morinda officinalis How [47], Aquilaria sinensis (Lour.) Gilg [48], Rheum tanguticum Maxim. ex Balf. [49], Amo- mum villosum Lour. [50], etc. components and predict the large-scale distribution of target medicinal herbs such as P. cablin [44], Artemi- sia annua L. [45], Polygonum multiflorum Thunb. [46], Morinda officinalis How [47], Aquilaria sinensis (Lour.) Gilg [48], Rheum tanguticum Maxim. ex Balf. [49], Amo- mum villosum Lour. [50], etc. Another matter regarding DMMs that warrants atten- tion is water. As shown in Aconitum carmichaelii Debx., the heavy metals cadmium (Cd), arsenic (As), mercury (Hg), and lead (Pb) concentrations in it were positively associated with the water in Fujiang River (p < 0.05) [38]. This result indicates that the quality of CMMs can also be affected by water. To meet the growing demand of DMMs, additional detailed studies should be undertaken in this field. Comprehensive ecological factors For example, the ancient Chinese medicine book Xin Xiu Ben Cao (Newly Revised Materia Medica, 659 A.D.) [57] said that “Fraxinus chinensis Roxb., which can change the color of water to a fluorescent color after soaking, is thought to be superior in quality” (Fig. 5). Currently, scientific evidence supporting the rational for such description is available. F. chinensis produced from Shaanxi province has a higher content of aesculin and aesculetin than that produced in Sichuan province and Liaoning province, and its stronger fluorescence reac- tion is consistent with the description written in ancient times [58, 59]. The same observation is true for Astra- galus membranaceus (Fisch.) Bge. This herb is principally cultivated in a region in Shanxi province, and the cultivar produced in this region contains more astragaloside than do cultivars produced in Shandong, Inner Mongolia, Hebei and Jilin provinces [60]. It is generally recognized that Rehmannia glutinosa Libosch. cultivated in Henan province is of particularly high quality. Modern experi- mental studies have demonstrated that higher levels of the active constituent catalpol content are present in R. glutinosa grown in this region than in cultivars grown in areas of Xianyang (in Shaanxi province) and Dali (in Shaanxi province) [61]. Similarly, A. villosum cultivated in Yangchun (in Guangdong province) is believed to be (Origins of Medicine, 1767 A.D.) attached similar impor- tance to the use of DMMs collected from certain original sources [56]. All of this historical literature showed that the use of DMMs has been a practice since ancient times. The scientific elucidation of daodi medicinal materials DMMs are the subset of CMMs that meet the high- est quality criteria. DMMs are not only associated with specific geographic regions (Fig. 4) but also linked to the chemical components and pharmacological function of CMMs. Comprehensive ecological factors Comprehensive research on the relationship between different ecological environment factors and the qual- ity of CMMs has also been recorded. For example, in Scutellaria baicalensis Georgi, most of the chemical constituents were negatively correlated with latitude and positively correlated with temperature. Generally, the contents of 21 chemical constituents were higher at low latitudes than at high latitudes. By gradual regres- sion analysis, it was found that the content of baicalin in S. baicalensis was negatively correlated with latitude. Similarly, the content of inorganic elements in soil was excessively high (magnesium (Mg) and calcium (Ca) excluded), which had a negative effect on the accumula- tion of chemical constituents in S. baicalensis [39, 40]. Taking the well-known antioxidant herb Panax ginseng C. A. Mey. as another example, low temperature was a favorable factor for the accumulation of ginsenosides, as a negative correlation was found between temperature and ginsenoside contents within a certain temperature range, while the levels of soil available B, effective Fe and available N were positively correlated to ginsenoside con- tents [41]. In recent years, due to overexploitation, the destruction of the ecological environment and the lack of proper cultivation practices, the geographical distri- butions of most DMMs may undergo large changes. For example, although the Changzhi region of Shanxi prov- ince and provinces of north-east China was P. ginseng’s original production center, the present production center of it is in Xiaoxinganling region (in Heilongjiang prov- ince) and Changbaishan region (in Jilin province) [42]. Similarly, Panax notoginseng (Burk.) F. H. Chen histori- cally came from the Tianzhou region (in Guangxi prov- ince), but now the dominant medicinal material comes from Wenshan region (in Yunnan province) [43]. In these cases, predicting the geographical distribution of CMMs is important for resource conservation and regional man- agement. Therefore, a geographic information system based on a computer program (TCMGIS) was developed to predict the distribution of CMMs. By integrating geo- graphic location, climate and soil type databases, TCM- GIS was able to determine the impacts of environmental Liu et al. Chin Med (2020) 15:86 Liu et al. Chin Med (2020) 15:86 Page 6 of 11 Page 6 of 11 a certain production region are often of high quality (Table 1). CMM Chinese medicinal material Daodi medicinal materials are the basis of the medicinal industry and clinical practice y p CMMs are the materials processed into decoction ingre- dients or used to produce proprietary drugs. The iden- tification of DMMs is important in quality evaluation and disease treatment. In addition to the optimum har- vest season, the processing and standard prescription of CMMs produce the unique characteristics of DMMs, and a plausible production chain of CMMs is hypothe- sized in Fig. 6. The production chain of CMMs is based on the content we described above, including popula- tions, designated growing regions (daodi), and harvest season, as well as the extensive quality control knowledge accumulated for CMMs by Liu et al. [77–80]. Compared to previous production chain of CMMs [77–80], factors such as populations, designated growing regions and Chemical components Chin Med (2020) 15:86 Page 8 of 11 Page 8 of 11 superior in quality. The effective medicinal elements in A. villosum is bornyl acetate. Modern experimental research has validated that Yangchun-cultivated A. villosum has the highest effective component content among different populations [62–64]. In other example, P. cablin culti- vated in Shipai (in Guangzhou city, Guangdong province) is of particularly high quality. It produced higher levels of the active constituent pogostone than that cultivated in Gaoyao (in Zhaoqing city, Guangdong province), Leizhou (in Zhanjiang city, Guangdong province), Wuchuan (in Zhanjiang city, Guangdong province) and Hainan prov- ince [65–70]. Interestingly, the same is true for toxicol- ogy. A. carmichaelii produced from plantation sites at Jiangyou county of Sichuan province is believed to be superior in quality. The proportions of the major bioac- tive constituents monoester alkaloids to toxic constitu- ents diester alkaloids amount among 5 samples of A. carmichaelii from different localities were well correla- tive with their regions of cultivation. The highest propor- tion occurred in cultivar Jiangyou (in Sichuan province), followed by cultivars Hanzhong (in Shaaxi province), Butuo (in Sichuan province), Weishan (in Yunnan prov- ince), and Anxian (in Sichuan province) [71]. in quality. Accordingly, the hepatoprotective effect is the best for the Huoshan cultivar, second for the Yunnan cul- tivar, and last for the other region cultivars [73]. P. cablin has been classified into two chemotypes, the patchoulol- type, including cultivars HN and ZJ, and the pogostone- type, including cultivars SP and GY. Accordingly, this cultivars produced in GY are more potent than those from ZJ in terms of promoting digestion [74] and anti- bacterial [75] effects. In addition, the toxicity of DMMs is often less potent than that of non-DMMs. For instance, P. multiflorum is principally cultivated in region Deqing county of Guangdong province, which has the largest output and the longest history of medicinal use. Accord- ingly, cultivar Deqing showed less potent cytotoxicity than cultivar Chongqing in HepG2 and LO2 cells [76]. These studies show considerable promise for explaining the scientific mechanism of DMM superiority. Chemical components As we described above, ecological environments such as topography, sunshine, soil, and water directly influence the secondary metabolites (many of which are bioactive components) in medicinal herbs. The many names of DMMs reflect the connotations of production regions; for instance, “qin pi” (Fraxinus chinensis Roxb.), “fen qi” (Astragalus membranaceus (Fisch.) Bge.), “huai di huang” (Rehmannia glutinosa Libosch.) and “ba dou” (Croton tig- lium L.), where “qin”, “fen”, “huai”, and “ba” refer to the names of regions used over the course of ancient West- ern Zhou Dynasty (1046–771 B.C.) [1]. Modern experi- mental research has validated that DMMs growing in Fig. 4 The distribution of some daodi medicinal materials in China Fig. 4 The distribution of some daodi medicinal materials in China Fig. 4 The distribution of some daodi medicinal materials in China Page 7 of 11 Page 7 of 11 Liu et al. Chin Med (2020) 15:86 Table 1 The contents (%) of active constituents in different Chinese medicinal materials in daodi production region and non-daodi production regions No. CMM Active constituents Content (%) References Daodi production region Non-daodi production region 1 Fraxinus chinensis Roxb. Aesculin, aescu- letin Shaanxi: aesculin (4.37%), aescu- letin (1.92%) Sichuan: aesculin (1.10%), aesculetin (0.21%) Liaoning: aesculin (2.21%), aescu- letin (0.19%) [58, 59] 2 Astragalus membranaceus (Fisch.) Bge. Astragaloside Shanxi: 0.37% Shandong: 0.12% Inner Mongolia: 0.11% Hebei: 0.29% Jilin: 0.32% [60] 3 Rehmannia gluti- nosa Libosch. Catalpol Henan: 0.76% Xianyang, Shaanxi: 0.34% Dali, Shaanxi: 0.33% [61] 4 Amomum vil- losum Lour. Bornyl acetate Yangchun, Guangdong: 65.82% Gaozhou, Guang- dong: 55.95% Guangxi: 61.75% Yunnan: 50.92% Burma: 60.71% [62–64] 5 Pogostemon cablin (Blanco) Benth. Pogostone Shipai, Guang- zhou, Guang- dong: 68.43% Gaoyao, Zhao- qing, Guang- dong: 26.15% Leizhou, Zhanji- ang, Guang- dong: 4.78% Wuchuan, Zhanjiang, Guangdong: 5.20% Hainan: 8.97% [65–70] Table 1 The contents (%) of active constituents in different Chinese and non-daodi production regions Fig. 5 The herbal classic Xin Xiu Ben Cao (Newly Revised Materia Medica) describes the metachromatism-based quality evaluation of “qin pi” (Fraxinus chinensis Roxb.) escribes the metachromatism-based quality evaluation of “qin pi” Fig. 5 The herbal classic Xin Xiu Ben Cao (Newly Revised Materia Medica) describes the metachromatism-based quality evaluation of “qin pi” (Fraxinus chinensis Roxb.) Fig. 5 The herbal classic Xin Xiu Ben Cao (Newly Revised Materia Medica) describes the metachromatism-based quality evaluation of “qin pi” (Fraxinus chinensis Roxb ) Liu et al. DMMs: daodi medicinal materials; CMMs: Chinese medicinal materials; SP: Shipai (in Guangzhou City, Guangdong province); GY: Gaoyao (in Zhaoqing City, Guangdong province); HN: Hainan province; ZJ: Zhanjiang City (in Guang- dong province); NJ: neighbor-joining; Fe: iron; Mn: manganese; K: potassium; Cu: copper; Zn: zinc; B: boron; Mo: molybdenum; N: nitrogen; Cd: cadmium; As: arsenic; Hg: mercury; Pb: lead; Mg: magnesium; Ca: calcium; TCMGIS: geo- graphic information system based on a computer program. Acknowledgements Not applicable. 14. Zhang Y, Chen Y, Zhang JC, Yang MS, Cao H, Xiao PG. Correlation between ITS genotype and geographical distribution of Pogostemon cablin. Acta Pharm Sin. 2007;42(1):93–7. 14. Zhang Y, Chen Y, Zhang JC, Yang MS, Cao H, Xiao PG. Correlation between ITS genotype and geographical distribution of Pogostemon cablin. Acta Pharm Sin. 2007;42(1):93–7. Conclusions Received: 12 May 2020 Accepted: 13 August 2020 Received: 12 May 2020 Accepted: 13 August 2020 DMMs have long maintained, currently have, and will continue to maintain a good reputation on the basis of their excellent curative effects. In our review, DMM is the specific term for pure, superior medicinal herbs with the following characteristics: optimum harvest season (reflecting the appropriate developmental stage of the plant), scrupulous processing, traditional preparation technology, etc. Historical literature, modern phyto- chemical and pharmacological methods have provided additional scientific data and a theoretical basis to vali- date the mechanisms of DMMs. In addition, every pro- cedure in the production chain of CMMs should be standardized to guarantee the prominent curative effect of medicinal materials. Effectively establishing a correla- tion among the active components, clinical efficacy and identity of DMMs is an important aspect in the quality evaluation of CMMs. The core scientific elucidation of DMMs should be continuously carried out, and multidis- ciplinary measures should be adopted to explore scien- tific and practical methodologies for the further research of DMMs. Author details 1 Author details 1 Research Center for Traditional Chinese Medicine of Lingnan (Southern China), Jinan University, Guangzhou 510632, China. 2 College of Pharmacy, Jinan University, Guangzhou 510632, China. 1 Research Center for Traditional Chinese Medicine of Lingnan (Southern China), Jinan University, Guangzhou 510632, China. 2 College of Pharmacy, Jinan University, Guangzhou 510632, China. Competing interests The authors declare that they have no competing interests. Funding Thi k This work was supported by the 6th National Academic Experience Inherit- ance Program of Famous Chinese Medicine Experts (Prof. Hui Cao) (No. 176-2017-XMZC-0166-01). Pharmacological functions Pharmacological functions are actually the outside mani- festations of CMMs. In the case of DMMs, the conditions in a certain region are thought to confer clinical supe- riority, and for this reason, DMMs are considered the most efficacious among CMMs [72]. Scientific evidence supporting the alleged clinical superiority of DMMs is the subject of ongoing research. For example, Dendro- bium huoshanense C. Z. Tang et S. J. Cheng produced in Huoshan (in Anhui province) is considered to be superior Fig. 6 A putative production chain for CMMs Fig. 6 A putative production chain for CMMs Liu et al. Chin Med (2020) 15:86 Page 9 of 11 Page 9 of 11 harvest time directly influenced the quality of CMMs were emphasized. 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https://openalex.org/W4303183654	https://www.frontiersin.org/articles/10.3389/fenrg.2022.996556/pdf	English	null	Quadratic regression estimation of hybridized nanoliquid flow using Galerkin finite element technique considering shape of nano solid particles	Frontiers in energy research	2,022	cc-by	12,175	Quadratic regression estimation of hybridized nanoliquid ﬂow using Galerkin ﬁnite element technique considering shape of nano solid particles OPEN ACCESS EDITED BY Adnan Abbasi, Mohi-ud-Din Islamic University, Pakistan REVIEWED BY Muhammad Hafeez, Gdansk University of Technology, Poland Ali Akgül, Siirt University, Turkey CORRESPONDENCE Wasim Jamshed, wasiktk@hotmail.com SPECIALTY SECTION This article was submitted to Process and Energy Systems Engineering, a section of the journal Frontiers in Energy Research RECEIVED 17 July 2022 ACCEPTED 08 August 2022 PUBLISHED 07 October 2022 Mustafa Mutiur Rahman1, Wasim Jamshed2, Suriya Uma Devi. S3, Rabha W. Ibrahim4, Amjad Ali Pasha5, Basma Souayeh6, Rabia Safdar7, Mohamed R. Eid8,9, Syed M. Hussain 10 and El Sayed M. Tag El Din11 Mustafa Mutiur Rahman1, Wasim Jamshed2, Suriya Uma Devi. S3, Rabha W. Ibrahim4, Amjad Ali Pasha5, Basma Souayeh6, Rabia Safdar7, Mohamed R. Eid8,9, Syed M. Hussain 10 and El Sayed M. Tag El Din11 1Department of Mechanical and Mechatronics Engineering, University of Waterloo, Waterloo, ON, Canada, 2Department of Mathematics, Capital University of Science and Technology (CUST), Islamabad, Pakistan, 3Department of Mathematics, KPR Institute of Engineering and Technology, Coimbatore, India, 4Mathematics Research Center, Department of Mathematics, Near East University, Mersin, Turkey, 5Aerospace Engineering Department, King Abdulaziz University, Jeddah, Saudi Arabia, 6Department of Physics, King Faisal University, College of Science, Al-Ahsa, Saudi Arabia, 7Department of Mathematics, Lahore College Women University, Lahore, Pakistan, 8Department of Mathematics, Faculty of Science, New Valley University, Al-Kharga, Al-Wadi Al-Gadid, Egypt, 9Department of Mathematics, Faculty of Science, Northern Border University, Arar, Saudi Arabia, 10Department of Mathematics, Faculty of Science, Islamic University of Madinah, Medina, Saudi Arabia, 11Electrical Engineering, Faculty of Engineering and Technology, Future University in Egypt, New Cairo, Egypt Rahman MM, Jamshed W, Devi. S SU, Ibrahim RW, Pasha AA, Souayeh B, Safdar R, Eid MR, Hussain SM and Tag El Din ESM (2022), Quadratic regression estimation of hybridized nanoliquid ﬂow using Galerkin ﬁnite element technique considering shape of nano solid particles. Because of its multivariate particle suspension approach, the developing class of ﬂuid has a better level of stability as well as increased heat transfer. In this regard, hybrid nanoﬂuid outperforms ordinary ﬂuid and even well-known nanoﬂuid. In a slick environment, we investigate its ﬂuidity and heat transfer qualities. Nano-leveled particle morphologies, porousness materials, variable thermal conductivity, slippage velocity, and thermal radiative effects are all being studied. The Galerkin ﬁnite element method is a numerical methodology for numerically solving the governing equations (G-FEM). TYPE Original Research PUBLISHED 07 October 2022 DOI 10.3389/fenrg.2022.996556 TYPE Original Research PUBLISHED 07 October 2022 DOI 10.3389/fenrg.2022.996556 TYPE Original Research PUBLISHED 07 October 2022 DOI 10.3389/fenrg.2022.996556 Introduction and HT of a stretching/shrinking slip by the virtue of MBNM. Akram et al. (Akram et al., 2022) analyzed the electroosmotic movement of silver-water HNF controlled by using two altered methods for NF including the MBNM. Areekara et al. (Areekara et al., 2022) suggested a study on NF movement with asymmetrical heat foundation and representative boundary conditions with the application by MBNM. Different simulations, including the regulator rule scheme, Carreau’s scheme, Cross’ scheme, and Ellis’s scheme, are offered to shed light on the behavior of HNFs, however, few researchers have investigated the Williamson liquid scheme (WLS). Williamson (1929) thought about the ﬂow of hybrid nanoﬂuids (HNFs) such as (pseudo-plastic liquids), proposed an equation system to represent the ﬂow of HNFs, and then empirically veriﬁed the results. In an advanced gravitational investigation, researchers proposed that an echoing level of a WLS should movement concluded an inspired superﬁcial. A real ﬂuid has both the lowest and highest operational viscosities that relate to its molecular structure. The WLS measures together the lowest and highest thicknesses. During the attendance of revolution, Said et al. (Said et al., 2021) planned a 3D-class of HNF to additional upsurge the heat transfer (HT) rate completed by widening slip. Mandal et al. (Mandal et al., 2022) exploited an artiﬁcial neural network to form investigational statistics. Saha et al. (Saha et al., 2022) described an investigation of HT and rheological possessions of HNFs for refrigeration presentations. Survey studies by Al-Chlaihawi et al. (Al-Chlaihawi et al., 2022), Kursus et al. (Kursus et al., 2022), Xiong et al. (Xiong et al., 2021), and Muneeshwaran et al. (Muneeshwaran et al., 2021) respectively, can be located in this direction, while Dubey et al. (Dubey and Sharma, 2022) offered a short survey in HNF on mechanics revisions. Syed and Jamshed (Hussain and Jamshed, 2021) considered the movement of MHD tangent HNF via a strained slip’s boundary layer. In accumulation, the demonstration of the extended HT of tangent hyperbolic ﬂuids crossways a nonlinearly ﬂuctuating slide containing HNFs was tested by Qureshi (Qureshi, 2022), Jamshed et al. (Jamshed et al., 2021a), and Parvin et al. (Parvin et al., 2021). Nanoﬂuids own the characteristics of the non-Newtonian ﬂuid, together with the viscoelastic properties. Extended experimental work research is required to develop nanoﬂuid viscosity models for use in simulation studies (Wang and Mujumdar, 2008; Bilgili et al., 2021). Introduction Therefore, the Powell- Eyring ﬂuid is considered in the current model, together with the signiﬁcance of non-Newtonian ﬂuid properties. This type of ﬂuid is proposed by Powell and Eyring (Hayat and Nadeem, 2018) in 1944. Moreover, Powell-Eyring ﬂuid is one type of visco- elastic ﬂuid. Eyring–Powell ﬂuid model implements a higher- level complicated mathematical framework, but it is found to be the greater model over previous viscoelastic ﬂuid models. This model is founded on the kinetic theories of liquids, not on empirical expressions. In addition, Eyring–Powell ﬂuid model has Newtonian properties at low and great shear stress. Examples of Powell-Eyring ﬂuids are polymer melts and solids suspended in non-Newtonian liquids. The signiﬁcant implementations of Powell-Eyring ﬂuid have been observed in engineering, manufacturing, and industrial areas such as polymers, pulp, plasma, and other biological technology. However, several researchers have investigated the properties of non-Newtonian Powell-Eyring nanoﬂuid (Hayat et al., 2015; Malik et al., 2015; Hayat et al., 2017). Aziz and Afy (El-Aziz and Aﬁfy, 2019) chose the shooting technique, together with the Buongiorno nanoﬂuid model to obtain the Casson nanoﬂuid’s numerical solution over a stretching sheet. At the initial stages of ﬂow (primary and secondary ﬂow), they concluded that the Hall parameter upsurges in the convective rate of heat and mass transfer, together with the drag coefﬁcient. Moreover, the nanoparticle volume concentration parameter increases for increasing velocity slip values. Consequently, the Sherwood number is reduced. Subsequently, the inﬂuences on the magnet ﬁeld and Soret- Dufour have been reportedly on non-rotational Newton’s Oldroyd-B nanoﬂuid stream bounded by the stretched sheet (Ali et al., 2021). This model is also being restricted under the modiﬁcation of Fourier’s law. For the step of numerical ﬁndings, the Galerkin-Finite element system was developed. Puneeth et al. (Shankaralingappa et al., 2021) measured 3D- assorted convection movement of HNFs of a non-linear widening surface using a modiﬁed Buongiorno’s nanoﬂuids model (MBNM). Rana et al. (Rana et al., 2021) presented a study of HNF movement past a perpendicular platter with nanoparticle aggregation kinematics, the current slide, and important buoyancy force possessions utilizing MBNM. Mahanthesh et al. (Mahanthesh et al., 2021) estimated the HT optimization of HNFs with the help of MBNM. Owhaib and Al-Kouz (Owhaib and Al-Kouz, 2022) and Owhaib et al. (Owhaib et al., 2021) employed the concept of MBNM in 3D systems of movement and HT of bi-directional overextended HNF ﬁlm showing an exponential heat generation. Quadratic regression estimation of hybridized nanoliquid ﬂow using Galerkin ﬁnite element technique considering shape of nano solid particles For this analysis, a Powell-Eyring hybrid nanoﬂuid (PEHNF) ﬂowing via a permeable stretchable surface is used, which comprises two types of nanoparticles (NP), copper (Cu), and titanium alloy (Ti6Al4V) dispersed in sodium alginate (C6H9NaO7). The heat transfer ratio of PEHNF (Ti6Al4V-Cu/C6H9NaO7) remained much greater than that of conventional nanoﬂuids (Cu-C6H9NaO7), with a range of 43%–54%. When lamina particles are present, the thermal conductivity of the boundary layer increases dramatically, while spherical nanoparticles have the lowest thermal conductivity. As nanoparticles are added under their fractional sizes, radiative heat conductance, and ﬂexible heat conductance, the system’s entropy increases. The ﬂow system’s ability to transport mass decreases when molecule diffusivity decreases dramatically. This is theoretically related to a rise in Schmidt number against molecular diffusivity. Front. Energy Res. 10:996556. doi: 10.3389/fenrg.2022.996556 Front. Energy Res. 10:996556. doi: 10.3389/fenrg.2022.996556 © 2022 Rahman, Jamshed, Devi. S, Ibrahim, Pasha, Souayeh, Safdar, Eid, Hussain and Tag El Din. This is an open- access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Frontiers in Energy Research 01 frontiersin.org Rahman et al. 10.3389/fenrg.2022.996556 KEYWORDS KEYWORDS Powell-Eyring hybridized nanoﬂuid, modiﬁed Buongiorno’s structure, shape factor, irreversibility analysis, Galerkin ﬁnite element technique KEYWORDS Powell-Eyring hybridized nanoﬂuid, modiﬁed Buongiorno’s structure, shape factor, irreversibility analysis, Galerkin ﬁnite element technique Powell-Eyring hybridized nanoﬂuid, modiﬁed Buongiorno’s structure, shape factor, irreversibility analysis, Galerkin ﬁnite element technique Introduction presented the latest updating that involves the traditional nanoﬂuids with the features of heat and mass transmission in a different physical situation. A statistical technique called quadratic regression estimation (QRE) is considered to identify the parabola equation that ﬁnest ﬁts a given collection of data. Finding the equation of the conventional line that most closely ﬁts a collection of information is the goal of this sort of regression, which is an extension of modest linear regression. Jamei et al. (Jamei et al., 2022) estimated the thickness of HNFs for current energy using the QRE. Nandi et al. (Nandi et al., 2022a; Nandi et al., 2022b) suggested different investigations on HNFs based on QRE. Bhattacharyya et al. (Bhattacharyya et al., 2022) introduced a numerical and statistical method to capture the movement characteristics of HNFs containing copper and grapheme HNs utilizing QRE. Kumbhakar and Nandi (Kumbhakar and Nandi, 2022) presented an unsteady MHD radiative-dissipative movement of HNFs of a widening sheet with slide and convective conditions employing QRE. Said et al. (Said et al., 2022) considered the application of the original outline by collaborative boosted QRE of HNFs. Chen et al. (Chen et al., 2022a; Chen et al., 2022b) gave a long approximation of the physical properties of HNFs. By focusing on the ﬂowing rapidity of a Powell-Eyring HNFs as well as thermal transmission with changing heat and current conductance ﬂowing through a stretched permeable material, this work intends to bridge a gap in the previous survey and ﬁll a knowledge gap. The ﬂow of nanoliquid was geometrically modeled using a single-phase nanoliquid. The foundation liquid in the investigation of copper (Cu) and titanium alloy (Ti6Al4V) hybrid nanoparticles is sodium alginate (C6H9NaO7). The regulatory equations of the Powell-Eyring hybridization nanoliquid are transformed into ordinary differential equations (ODEs). The inﬂuences of porous parameters, thermal radiative ﬂuxing, and variable thermal conductance are considered in the examination. Then, the effects of the slippage velocity and nanoparticle shape factors are probed in ﬂowing and entropy aspects. The obtained ODEs are solved numerically using the Galerkin ﬁnite element technique and the necessary prevailing parametric parameters. Numerical results are shown graphically, and comments are built upon. In-depth research has been done on the possessions of particle morphologies, the convective slide boundary condition, the thermal energy movement, and the slippery velocity. Introduction Hussain et al. (Hussain et al., 2022a) characterized a biochemical response and current of HNFs ﬂow- through solar gatherer as potential solar energy applying the idea of MBNM. Roşca et al. (Roşca et al., 2021) engaged the movement Porous media models (PMMs), frequently referred to as porous materials, have pores (vacuums). The thin part of the material is referred to as the “matrix” or “frame.” Typically, a ﬂuid is injected into the pores (ﬂuid or fume). Though the material that makes up the frame is regularly hard, structures Frontiers in Energy Research 02 frontiersin.org 10.3389/fenrg.2022.996556 Rahman et al. Rahman et al. interior temperature compeers. For more details see Refs (Akgül et al., 2022; Attia et al., 2022; Ahmad et al., 2022b; Bilal et al., 2022; Qureshi et al., 2022; Safdar et al., 2022). like foams might proﬁt from the idea of PMM. To apply solar heat, Jamshed et al. (Jamshed et al., 2021b) employed PMM in solar aircraft combining tangent HNFs. Using the PMM in HNFs, Shahzad et al. (Shahzad et al., 2021) developed a comparative mathematical study of HT. Numerical conduct of a 2D-Magneto double-diffusive convection ﬂow of HNF over PMM was provided by Parvin et al. (Parvin et al., 2021). Faisal et al. (Shahzad et al., 2022a) reported that using HNFs rather than PMM increased the thermal efﬁcacy of solar water pumps. Banerjee and Paul (Banerjee and Paul, 2021) examined the most recent research and advancements concerning PMM combustion applications. For pebble-bed devices, Zou et al. (Zou et al., 2022) designed an explicit system of stone heat in the PMM. PMM substantiation using stress drip dimensions was suggested by Lee et al. (Lee et al., 2022). On a constructed soaking soil pile model, Cui et al. (Cui et al., 2021) investigated a numerical analysis of the solution for longitudinal quivering of a ﬂuctuating pile based on PMM. A machine learning approach was taken by Alizadeh et al. (Alizadeh et al., 2021) to calculate transference and thermodynamic processes in metaphysical systems HT in HNFs movement in PMM. A non-homogenous HNF was proposed by Rashed et al. (Rashed et al., 2021) for 3D convective movement in enclosures with assorted PMM (AdnanKhan et al., 2021; Adnan and Ashraf, 2022a; Khan et al., 2022a; Alharbi et al., 2022; Ashraf et al., 2022a; Ashraf et al., 2022b; Adnan and Ashraf, 2022b; Khan et al., 2022b; Khan et al., 2022c; Murtaza et al., 2022). Introduction The rate of HT through a component thickness of a material per unit area per temperature variation is known as the variable thermal conductivity (VTC) of that material. Alternatively said, the VTC is inversely proportional to the temperature capacity. Gbadeyan et al. (Olabode et al., 2021) studied the effect of VTC and thickness on Casson NF movement with convective warming and velocity slide. Mabood et al. (Mabood et al., 2021) impacted the Stefan blowing and mass convention on the movement of HNF of VTC in a revolving disk. Abouelregal et al. (Abouelregal et al., 2021) checked the thermo-viscoelastic fractional model of revolving HNFs with VTC owing to mechanical and current loads. Swain et al. (Swain et al., 2021) utilized the HT and stagnation- point movement of inﬂuenced HNFs with VTC. Also, Ahmed et al. (Ahmed et al., 2021) considered the HT of MHD movement of HNFs via an exponential penetrable widening arched surface with VTC. Mahdy et al. (Mahdy et al., 2021) employed the VTC and hyperbolic two-temperature philosophy throughout the magneto- photothermal model of semiconductors induced by laser pulses. Hobiny and Ibrahim (Hobiny and Abbas, 2022) analyzed the impacts of VTC in a semiconducting medium utilizing the ﬁnite element technique. Ahmad et al. (Ahmad et al., 2022a) studied the unsteady 3D-bio convective movement of HNFs by an exponentially widening sheet with VTC and chemical reaction. Din et al. (Din et al., 2022) assumed the entropy generation from convective released moving exponential porous ﬁns with VTC and Flow examination 1) Unsteady two-dimensional laminar ﬂowing. the suitable boundary conditions are (Aziz et al., 2021): 2) Boundary-layer guesstimate. v1(x, 0) Uw + Nαzv1 zy, v2(x, 0) Vα, −kαzΘ zy hα(Θw −Θ)C C* w, (7) v1 →0, Θ →Θ∞, C* →C* ∞as y →∞. (8) 3) Modiﬁed Buongiorno’s structure. 4) Non-Newtonian PEHNFs. 5) Different effects i.e., porous medium, variable thermal conductivity, radiative ﬂowing, and nanomolecules shaped inﬂuence. 5) Different effects i.e., porous medium, variable thermal conductivity, radiative ﬂowing, and nanomolecules shaped inﬂuence. (8) Where a ﬂow speed is of the structure v⃖ [v1(x, y, t), v2(x, y, t), 0]. Time is denoted by t, Θ signiﬁes a ﬂuid temperature. The penetrability of an expanding plate is symbolized by Vα. Nα is the slip length. The porousness of NF is characterized by k. The additional parameters like thermal conductivity of nanosolid and heat transmission factor are represented by k0 and hf, respectively. 6) Penetrable expanding surface. 6) Penetrable expanding surface. 7) Slip and convective boundary constraints. The exact formulary of the stress tensor of ﬂuid follows the Powell-Eyring relationship is provided as (Aziz et al., 2021): τij μhnfzui zxj + 1 ~ζ sinh−1 1 ϱ* zui zxj . (2) (2) The mongrelized nanoliquid is combined principally with Cu nano molecules in machine grease standard liquid at a constant fractional size (ϕCu) and it is put at 0.09 all about the study. Ti6Al4V NP were put together into HNFs consuming attentiveness scope (ϕTA). Here, μhnf is the mechanical viscosity of PEHNF, and ~ζ and ϱ* are matter constants. The movement geometric is illuminated in Figure 1. The combination of nanomolecules in the baseﬂuid runs to a difference in the characteristics thermophysically. Table 1 summarises the relevant parameters for PENF (Reddy et al., 2014; Din et al., 2022). Frontiers in Energy Research Flow examination Analysis of movement shows how a superﬁcial moves horizontally at an accelerating rate. Uw(x, t) ex 1 −Ωt, (1) (1) where e is the preliminary increasing amount. Solitary slip heat is Θw(x, t) Θ∞+ ex 1−Ωt and based on the suitability, pretend to be frontiersin.org Frontiers in Energy Research 03 Rahman et al. 10.3389/fenrg.2022.996556 TABLE 1 Thermo-physical properties of PENF. TABLE 1 Thermo-physical properties of PENF. FIGURE 1 Flow model illustration. zv1 zx + zv2 zy 0, (3) zv1 zt + v1 zv1 zx + v2 zv1 zy ⎛ ⎜ ⎜ ⎜ ⎝]hnf + 1 ρhnf~ζϱ ⎞⎟⎟⎟⎠z2v1 zy2 − 1 2~βϱ3ρhnf zv1 zy 2z2v1 zy2 − μhnf ρhnfkv1, (4) zΘ zt + v1 zΘ zx + v2 zΘ zy 1 ρCpκhnf z zy κhnf (Θ) zΘ zy − 1 ρCphnf zqr zy , (5) v1 zC* zx + v2 zC* zy DB z2C* zy2 + DT T∞ z2Θ zy2 −k1 C* −C* ∞. (6) th it bl b d diti (A i t l 2021) TABLE 1 Thermo physical properties of PENF. Aspect Nanoliquid Viscid (μ) μnf μf(1 −ϕ)−2.5 Density (ρ) ρnf (1 −ϕ)ρf −ϕρs Heat capacity (ρCp) (ρCp)nf (1 −ϕ)(ρCp)f + ϕ(ρCp)s Thermal conductivity (κ) κnf κf [(κs+(m−1)κf)−(m−1)ϕ(κf−κs) (κs+(m−1)κf)+ϕ(κf−κs) ] Aspect Nanoliquid Viscid (μ) μnf μf(1 −ϕ)−2.5 Density (ρ) ρnf (1 −ϕ)ρf −ϕρs Heat capacity (ρCp) (ρCp)nf (1 −ϕ)(ρCp)f + ϕ(ρCp)s Thermal conductivity (κ) κnf κf [(κs+(m−1)κf)−(m−1)ϕ(κf−κs) (κs+(m−1)κf)+ϕ(κf−κs) ] FIGURE 1 Flow model illustration. zv1 zx + zv2 zy 0, (3) zv1 zt + v1 zv1 zx + v2 zv1 zy ⎛ ⎜ ⎜ ⎜ ⎝]hnf + 1 ρhnf~ζϱ* ⎞⎟⎟⎟⎠z2v1 zy2 − 1 2~βϱ3ρhnf zv1 zy 2z2v1 zy2 − μhnf ρhnfkv1, (4) zΘ zt + v1 zΘ zx + v2 zΘ zy 1 ρCpκhnf z zy κhnf (Θ) zΘ zy − 1 ρCphnf zqr zy , (5) v1 zC* zx + v2 zC* zy DB z2C* zy2 + DT T∞ z2Θ zy2 −k1 C* −C* ∞. (6) (3) stable at x 0, e, Θw and Θ∞let the current difference ratio, surface heat, and free-streaming heat, consistently. (5) Under the following hypotheses and constraints, the theoretical framework is selected: v1 zC zx + v2 zC* zy DB z2C* zy2 + DT T∞ z2Θ zy2 −k1 C* −C* ∞. (6) 1) Unsteady two-dimensional laminar ﬂowing. Framed model Classic formulas (Aziz et al., 2021) of a viscidness P-EHNF beside with entropy assembly beﬁttingly adapted underneath normal boundary-layer approximations via a penetrable substantial, porous medium, variable thermal conductivity, and radiative ﬂowing are ϕ is the nanomolecules fractional volume factor. μf, ρf, (Cp)f, σf and kf are dynamical viscidness, density, efﬁcient heat capacitance, and the electrical and thermal conductance of the base ﬂuid, correspondingly. The further attributes ρs, (Cp)s, Frontiers in Energy Research 04 frontiersin.org Rahman et al. 10.3389/fenrg.2022.996556 TABLE 2 Thermophysical properties of PEHNF. TABLE 2 Thermophysical properties of PEHNF. Aspect Hybrid nanoliquid Viscid (μ) μhnf μf(1 −ϕCu)−2.5(1 −ϕTA)−2.5 consistency (ρ) ρhnf (1 −ϕTA){(1 −ϕCu)ρf + ϕCuρp1}] + ϕTAρp2 Heat capacity (ρCp) (ρCp)hnf [(1 −ϕTA){(1 −ϕCu)(ρCp)f + ϕCu(ρCp)p1}] +ϕz(ρCp)p2 Thermal conductivity (κ) κhnf κnf [(κp2+(m−1)κgf)−(m−1)ϕTA(κnf−κp2) (κp2+(m−1)κnf)+ϕTA(κnf−κp2) ], κnf κf [ (κp1+(m−1)κf)−(m−1)ϕCu(κf−κp1) (κp1+(m−1)κf)+ϕCu(κf−κp1) ] FIGURE 2 Standards of the practical shaped element for diverse particle shaped. The material possessions of the crucial sodium alginate (C6H9NaO7) and the various NP exploited in this research are indicated in Figure 3 (Xu and Chen, 2017; Makinde et al., 2018; Jamshed et al., 2021d). Aspect Hybrid nanoliquid Viscid (μ) μhnf μf(1 −ϕCu)−2.5(1 −ϕTA)−2.5 consistency (ρ) ρhnf (1 −ϕTA){(1 −ϕCu)ρf + ϕCuρp1}] + ϕTAρp2 Heat capacity (ρCp) (ρCp)hnf [(1 −ϕTA){(1 −ϕCu)(ρCp)f + ϕCu(ρCp)p1}] +ϕz(ρCp)p2 Thermal conductivity (κ) κhnf κnf [(κp2+(m−1)κgf)−(m−1)ϕTA(κnf−κp2) (κp2+(m−1)κnf)+ϕTA(κnf−κp2) ], κnf κf [ (κp1+(m−1)κf)−(m−1)ϕCu(κf−κp1) (κp1+(m−1)κf)+ϕCu(κf−κp1) ] FIGURE 2 Standards of the practical shaped element for diverse particle shaped. The material possessions of the crucial sodium alginate (C6H9NaO7) and the various NP exploited in this research are indicated in Figure 3 (Xu and Chen, 2017; Makinde et al., 2018; Jamshed et al., 2021d). σf and ks are the density, active heat capacitance, and the electrical and thermal conductance of nanomolecules, correspondingly. The physical properties of PEHNF are deﬁned in Table 2 (Devi and Devi, 2016). FIGURE 3 Substance effects of C6H9NaO7 and NPs at 293 K. Herein, μhnf, ρhnf, ρ(Cp)hnf and κhnf indicates the dynamic viscous, density, speciﬁc temperature capacitor, and current conductivity of HNF. ϕ is a fractional size factor and ϕhnf ϕCu + ϕTA is the size parameter of solid- nanoparticle combination. μf, ρf, (Cp)f and κf are dynamic viscosity, density, speciﬁc heat capacity, and the thermal conducting of the ordinary ﬂuid. Frontiers in Energy Research Drag force and nusselt quantity The drag force Cf together with a Nusselt amount Nux are the physical amounts of importance that dominate the light and can be conﬁrmed as (Aziz et al., 2021) v1 zψ zy, v2 −zψ zx. (10a) (10a) and similarity transformations are and similarity transformations are Cf τw ρfU2 w , Nux xqw kf%Θw −Θ∞& (19) (19) δx, y e ]f(1 −Ωt) y, ψx, y ]fe (1 −Ωt) xf(δ), θ(δ) (Θ −Θ∞)(Θw −Θ∞)−1hδ(Θ −Θ∞)(Θw −Θ∞). where τw and qw correspond to the heating ﬂux revealed by (11) (11) τw ⎛ ⎝⎛ ⎝μhnf + 1 ~ζϱ* ⎞⎠zv1 zy − 1 6~ζϱ3 zv1 zy 3 ⎞⎠ y0 , qw −khnf⎛ ⎜ ⎝1 + 16 3 σT3 ∞ κ]f ρCpf ⎞⎟⎠zΘ zy y0 . ( into Eqs. 3–6. We get 1 ϕx1ϕx2 + β1 ϕx1 f‴ + ff″ −f′ 2 −ξf′ + δ 2f″ −β1β2 ϕx2 f″ 2f‴ 1 ϕx1ϕx2 + β1 ϕx1 f‴ + ff″ −f′ 2 −ξf′ + δ 2f″ −β1β2 ϕx2 f″ 2f‴ −Γf′ 0, (20) Employing the dimensionless makeovers (11), one acquires (12) (12) θ″1 + γθ + 1 ϕx4 PrNr + γθ′ 2 + Pr ϕx3 ϕx4 fθ′ −f′θ −ξθ + δ 2θ′ 0. (13) h″ + PrScfh′ + Nt Nb θ″ −Sc 8h 0. (14) CfRex 1 2 1 ϕx1ϕx2 + β1f″(0) −β1β2 3 f″(0) 3 , NuxRe−1 2 x −khnf kf (1 + Nr)θ′(0). f (21) (21) with with Where Nux signiﬁes Nusselt quantity and Cf specify the skin resistance. Rex uwx ]f signiﬁes Rex uwx ]f signiﬁes local Reynolds amount based on uw(x). f(0) S, f′(0) 1 + εf″(0), θ′(0) −Bα(1 −θ(0)) f′(δ) →0, f″(δ) →0, θ(δ) →0, h (δ) →0 as δ →∞. (15) where ϕxi ′s; 1 ≤i ≤4 in Eqs. 12, 13 establishes the thermophysical characteristics of the Powell-Eyring nanoﬂuid. Framed model ρp1, ρp2, (Cp)p1, (Cp)p2, κp1 and κp2 are the density constancy, precise heat competence, and current conducting of the solid- nanoparticle. Heat conditional of heat conducting for hybrid nanoliquid blend is followed as (Jamshed et al., 2021c): PEHNF radiative ﬂow only travels a little space resulting in the thickener of NF. Because this is happening, Rosseland’s guesstimate for radiative ﬂuxing (Shahzad et al., 2022b), is used in Eq. 5 and it is given κhnf (Θ) khnf1 + γ(Θ −Θ∞)(Θw −Θ∞)−1 (9) (9) The signiﬁcance of several nanomolecules shaped is identiﬁed as the nanomolecules shaped inﬂuence. Figure 2 gives the values of the practical shaped component for different element shapes are obtained as (Akgül et al., 2022): Figure 3 demonstrates the themophysical values of the used materials of HNF. qr −4σ* 3k* zΘ4 zy , (10) (10) Where σ* is Stefan-Boltzmann amount and k* is the absorbing factor. Where σ* is Stefan-Boltzmann amount and k* is the absorbing factor. frontiersin.org frontiersin.org Frontiers in Energy Research 05 Rahman et al. 10.3389/fenrg.2022.996556 Model solution similar solutions for the suggested problematic computational results are processed for local similar considerations. similar solutions for the suggested problematic computational results are processed for local similar considerations. Boundary-value problem (BVP) formulas (3–6) are converted in the deﬁnition of the similarity procedure that converts the regulating PDEs to ODEs. Advancing streaming function ψ as the next Frontiers in Energy Research Numerical implementation: Galerkin ﬁnite element method where ϕxi ′s; 1 ≤i ≤4 in Eqs. 12, 13 establishes the thermophysical characteristics of the Powell-Eyring nanoﬂuid. ϕx1 (1 −ϕCu)2.5(1 −ϕTA)2.5, ϕx2 (1 −ϕTA)1 −ϕCu + ϕ1 ρp1ρf + ϕTA ρp2 ρf , (16) ϕx3 1 −ϕTA ⎧ ⎪ ⎨ ⎪ ⎩1 −ϕCu + ϕCu ρCpp1 ρCpf ⎫ ⎪ ⎬ ⎪ ⎭+ ϕTA ρCpp2 ρCpf , (17) ϕx4 ⎡⎢⎣ κp2 + (m −1)κnf −(m −1)ϕTiO2 κnf −κp2 κp2 + (m −1)κnf + ϕTiO2 κnf −κp2 ⎤⎥⎦ ⎡⎢⎣ κp1 + (m −1)κf + ϕCu κf −κp1 κp1 + (m −1)κf −(m −1)ϕCu κf −κp1 ⎤⎥⎦. (18) The relevant constraints of the present system were studied numerically using the ﬁnite element technique. The ﬁnite element approach is based on the partitioning of the desired domain into elements (ﬁnite). The FES (ﬁnite element scheme) is covered in this section. Figure 4 depicts the ﬂow chart of the ﬁnite element method. This method has been employed in numerous computational ﬂuid dynamics (CFD) problems; the assistances of employing this methodology are discussed further below. II- A Galerkin ﬁnite element manner (G-FEM) is utilized to determine the solutions of highly elliptic equations (Brewster, 1992) (non- linear). Using a ﬁnite element technique, the domain of the current exemplary is broken into small parts. G-FEM is used in a variety of applications, including electrical systems, solid mechanics, chemical processes, and ﬂuid-related challenges. The phases of the G-FEM strategy are as next: (16) (17) (18) Meticulous authentication is done on Eq. 3. Notation ′ is used for the demonstration of derivatives concerning δ. The parametric values were deﬁned in Table 3. It is noticed a variety of factors depend on the similarity variable ″Ω″ and unsteadiness. Consequently, to acquire non- Phase-I. Weak form is derived from strong form (mentioned ODEs), and residuals are computed. frontiersin.org 06 Rahman et al. 10.3389/fenrg.2022.996556 TABLE 3 Explanation of the entrenched control constraints. TABLE 3 Explanation of the entrenched control constraints. Numerical implementation: Galerkin ﬁnite element method Symbol Name Formula Default value β1 Non-Newtonian Powell-Eyring-I β1 1 μf ~ζϱ* 0.1 β2 Non-Newtonian Powell-Eyring-II β2 U3 w 2ϱ2 ]fx 0.1 Γ Porous media Γ ]f(1−Ωt) ek 0.1 Pr Prandtl number Pr ]f αf 6.5 ϕ Volume fraction - 0.18 m Shape factor - 3 S Suction/injection parameter S −Vα 1 ]f e ( 0.4 Nr Thermal radiation parameter Nr 5.33 σΘ3 ∞ κ]f(ρCp)f 0.3 Bα Biot number Bα hα kα ]f(1−Ωt) e ( 0.2 Sc Schmidt number Sc v DB 0.3 Nb Brownian motion Nb τDB(C w−C* ∞) ] 0.1 Nt Thermophoresis parameter Nt τDT(EEw−EE∞) ]EE∞ 0.3 ε Velocity slip ε e ]f(1−Ωt) ( Nα 0.3 Formula to achieve energy irreversibility and reduce waste. Design is prearranged about entropy generation in NFs (Hussain, 2022) Phase -II. Shape functions are linearly taken, and G-FEM is used to generate a weak form. Phase -III. The assembly method is used to build stiffness elements, and a global stiffness matrix is created. EG khnf Θ2 ∞ ⎧ ⎪ ⎨ ⎪ ⎩zΘ zy 2 + 16 3 σΘ3 ∞ κ]f ρCpf zΘ zy 2⎫ ⎪ ⎬ ⎪ ⎭+ μhnf Θ∞ zv1 zy 2 + μhnfv2 1 kΘ∞ (21) Phase -IV. Using the Picard linearizing technique, an algebraic structure (non-linear equalities) is produced. (21) Phase -V. Employing the next halting conditions, algebraic equations are simulated using 10−5 (computational tolerance). The irreversibility of thermal transport is represented by the leading term in the preceding equation, while the frictional and porous media effects are represented by the following terms. The nondimensional entropy establishment is prearranged by NG [(Jamshed and Nisar, 2021; Jamshed et al., 2022a)]. ''''''' δi+1 −δi δi ''''''' < 10−5. (23) (23) Additionally, the Galerkin restricted constituent technique’s watercourse summary is represented in Figure 4. NG Θ2 ∞e2EG kf(Θw −Θ∞)2. (22) (22) Code authentication Eq. 11 is utilized to obtain a dimensionless equation regarding entropy formation as follows, On the one hand, the validity of the computational technique was tested by comparing the current method’s performance to the available data on heat transfer rate in Refs. (Hussain et al., 2022b; Bouslimi et al., 2022). Table 3 demonstrates the consistent comparison found across the investigations. The current study’s provided results, on the other hand, are quite accurate. NG Reϕ4(1 + Nr)θ′ 2 + 1 ϕx1 Br Λ f″ 2 + Γf′ 2 , (23) (23) Here, Reand Brindicate Reynolds and Brinkmann quantities. Here, Reand Brindicate Reynolds and Brinkmann quantities. Λ signs nondimensional current gradient. Here, Reand Brindicate Reynolds and Brinkmann quantities. Λ signs nondimensional current gradient. Λ signs nondimensional current gradient. Quadratic regression assessment of frictional force and thermal gradients of the surface A crucial aspect that interests scientists and researchers is the reduction of energy resource waste. Therefore, these necessary outcomes for researchers are gained by enhancing the functionality of earlier systems. Systems’ entropy creation is examined in order Quadratic regression analysis (QRA) is the statistical procedure that was used to test the elements that inﬂuence the ﬂow in the Frontiers in Energy Research 07 frontiersin.org Rahman et al. 10.3389/fenrg.2022.996556 Cfx(est) Cfx + h1S + h1ε + h3S2 + h4ε2 + h5Sε, (24) (24) FIGURE 4 G-FEM ﬂow diagram. Likewise, towards the Quadratic regression assessment of Nusselt quantity Nux, time-dependent variant ξ and radiative variable Nr were tested under 100 consistent values were presented as Nux(est) Nux + p1ξ + p1Nr + p3ξ2 + p4(Nr)2 + p5ξNr, (25) with h1, h2, h3, h4, h5 and are the factors of QRA to guide for the reduced Cfx and Nux, congruently. Nux(est) Nux + p1ξ + p1Nr + p3ξ2 + p4(Nr)2 + p5ξNr, (25) with h1, h2, h3, h4, h5 and are the factors of QRA to guide for the reduced Cfx and Nux, congruently. Tables 4, 5 illustrate correspondences between the frictional factor (Cfx) and the Nusselt quantity (Nux) under signiﬁcant constraints for β1 = 0.1, β2, = 0.3, , ξ 0.2, Γ = 0.1, ϕ = 0.18, ϕCu = 0.09, ε = 0.3, γ = 0.2, Nr = 0.3, Bα = 0.3, S = 0.1, m 3, Pr = 6.5, Re = 5 and Br = 5. The ideal relative error limits σ1 was deduced by the relation σ1 \|Cfx(est) −Cfx\|/Cfx, similarly, σ2 \|Nux(est) −Nux\|/Nux is employed for the relative error limits σ2. It can be evident that for the factors S or Nr, both the frictional factor (Cfx) and the Nusselt quantity (Nux) tends to reduce the higher values of inﬂuencing factors. Variations in the velocity slip clarify the dominance of speed slippage ε over the suction factor S in the shear stress manipulations. Because of the thermal outcome, the thermal slippage constrain plays a vital role in the heat transference rate. The reduced frictionless force mechanisms hold an upper hand over the QRA technique with fast and better convergence when the optimal regression estimate was introduced to the process and the percentage difference tends to be nearly zero. G-FEM ﬂow diagram. Outcomes and review The reason for this illustration is that when a large temperature gradient occurs at the surface, more entropy is produced, causing higher oscillations in nanoparticle mobility. being induced by inﬁnite ﬂuid viscosity. As a result, yield stress restricts the ﬂow, resulting in a velocity decrement towards the inﬁnite ﬂuid stream. The heat propagation in the Powell- Eyring ﬂuid heat propagation was boosted, as shown in Figure 5B. This effect is caused by the support of the stretching surface in overcoming the material yield stress dominance. In addition, temperature-dependent control ﬂuid viscosity causes the temperature distribution to rise at various levels. As the Powell-Eyring effect is augmented, the molecular bond is disrupted, and the particles are allowed to move freely. As a result, Powell-Eyring ‘s term has a minimal rising effect Figure 5B. In Figure 5C, the entropy variation is plotted versus the Powell-Eyring term. Under the increment of β2, the curves show different patterns. It illustrates an augmentation near the stretching wall, while a modest reduction is detected at a distance from it. The reason for this illustration is that when a large temperature gradient occurs at the surface, more entropy is produced, causing higher oscillations in nanoparticle mobility. TABLE 5 Frictional factor (Cfx) and ideal relative error bound (σ1) for various values of suction (S) and speed slippage constraint (ε). S Cfx h1 h2 h3 h4 h5 σ1 0.5 -1.3257 -0.8022 1.6029 0.0250 -1.3232 0.9033 0.0196 1.5 -1.4202 -0.7394 1.7238 0.0421 -1.4197 0.7956 0.0185 2.5 -1.7149 -0.6527 1.9069 0.0829 -1.5819 0.6210 0.0173 3.5 -1.9015 -0.4951 2.2036 0.1901 -1.7452 0.3502 0.0104 Outcomes and review system one at a time. Technically, this process works with the single key aspect and tends to explore its signiﬁcance over the ﬂow by keeping the other constraints as constants. In this section, the features of frictional force and Nusselt quantity are examined under Quadratic regression analysis (QRA). Results of the numerical procedure adapted for the parametrical studies were showcased and discussed in this section. Inﬂuence constraints like β1, β2, ξ, Γ, ϕ, ε, Nr, Bα, S, γ, Re and Br were worked over the crucial aspects of ﬂow, thermal, entropy, and concentration dispersion in the system. Plots from 6(a)-11(b) for C6H9NaO7 traditional PENF and Ti6Al4V-Cu/C6H9NaO7 PEHNF tend to visually illustrate the outcomes and signiﬁcant impact of such parameters. Regarding the frictional factor fx , after testing with 100 combinations of suction (S) and speed slippage constrain (ε) between 0.2 and 1.1, it was noted that those constraints tend to resist frictional factors. QRA for the predicted Cfx owed to pressure inﬂuence S and speed slippage ε variation is delivered by TABLE 4 Comparing of −θ9(0) with Pr when ξ 0, ϕ 0, ϕhnf 0, γ 0, ε 0, Nr 0, S 0 and Bα →∞. Pr Bouslimi et al. (Hussain et al., 2022b) Hussain (Bouslimi et al., 2022) Present Results 72 × 10−2 0.80876181 0.80876181 0.80878120 1×100 1.00000000 1.00000000 1.00000000 3×100 1.92357420 1.92357420 1.92357114 7×100 3.07314651 3.07314651 3.07335681 10×100 3.72055429 3.72055429 3.72055845 Frontiers in Energy Research 08 10.3389/fenrg.2022.996556 Rahman et al. being induced by inﬁnite ﬂuid viscosity. As a result, yield stress restricts the ﬂow, resulting in a velocity decrement towards the inﬁnite ﬂuid stream. The heat propagation in the Powell- Eyring ﬂuid heat propagation was boosted, as shown in Figure 5B. This effect is caused by the support of the stretching surface in overcoming the material yield stress dominance. In addition, temperature-dependent control ﬂuid viscosity causes the temperature distribution to rise at various levels. As the Powell-Eyring effect is augmented, the molecular bond is disrupted, and the particles are allowed to move freely. As a result, Powell-Eyring ‘s term has a minimal rising effect Figure 5B. In Figure 5C, the entropy variation is plotted versus the Powell-Eyring term. Under the increment of β2, the curves show different patterns. It illustrates an augmentation near the stretching wall, while a modest reduction is detected at a distance from it. Impact of penetrable material factor (Γ) the proﬁle of temperature and entropy have been analyzed and investigated in Figure 7A,B. In Figure 7A, the inﬂuence of the increment in nanoparticle shape parameter (m) on the proﬁle of temperature has been depicted, observations show that the temperature increment because of the ascent in shape factors. Physically the increment in thermal conductivity and thermal boundary-layer thickener are the main causes of such an outcome. Additionally compared to a sphere, hexahedron, tetrahedron, and column shape nanoparticles, lamina has engendered more enhancement in the temperature (Pasha et al., 2022). Besides, the function of temperature for lamina shape nanoparticles has been continuously more than that of a sphere, hexahedron, tetrahedron, column shape nanoparticles, the temperature range in PEHNF (Ti6Al4V-Cu/C6H9NaO7) case is higher than that of PENF (Cu-C6H9NaO7) case. The impact of increasing the nanoparticle shape parameter (m) on the proﬁle of entropy has been presented in Figure 7B, and measurements demonstrate that entropy increases as shape factors increase. It is worth noting that the PEHNF (Ti6Al4V-Cu/ C6H9NaO7) nanoﬂuid phase had a higher initial entropy than the PENF (Cu-C6H9NaO7) nanoﬂuid phase. Later, when the shape factor is separated from the stream by a sufﬁcient distance, it ﬁercely behaves in the other direction and inﬂuences the entropy rate in the stretching porous device, as observed. The graphical results of permeability parameter (Γ) against the ﬂow, thermal, and entropy distribution of PENF (Cu-C6H9NaO7) and PEHNF (Ti6Al4V-Cu/C6H9NaO7) are presented in Figure 6A–C. Figure 6A shows the descending behavior of Powell-Eyring ‘s non-liquid ﬂow curve through the porous medium, resulting in a plate surface while velocity is dragged. In fact, as the porosity effect enhances, the ﬂow pores increase signiﬁcantly, resulting in fewer nanoparticle collisions and lower heat generation. Viscous force controls buoyancy, thereby slowing the ﬂow rate. The opposite effect is portrayed in Figure 6B. As shown in Figure 6B, increasing the porosity effect enhances the ﬂow temperature. Figure 6C portrayed the entropy generated NG against (Γ). This shows an increment near the surface, whereas a slight reduction is observed away from the surface. This result is caused by the large temperature difference near the surface, which causes more entropy to be produced. In industrial applications, the contribution of the porous medium permeability is to control the spin coating ﬂow properties. Greater permeability, which can be depicted as bigger pore spaces provide better nanoparticle percolation. Impact of penetrable material factor (Γ) Besides, this effect (higher mobility) relates to reduced friction at the sheet surface. Impact of powell-eyring parameter (β2) The illustration of the non-Newtonian Powell-Eyring ﬂuid term (β2) on the model proﬁles (ﬂow velocity, temperature, and entropy) against a rising stream term (β2) were depicted in Figures 5A,B,C. The velocity proﬁle (Figure 5A) is decreased as the material term increases due to the material term shear FIGURE 5 (A) f′(δ), (B) θ(δ), and (C) NG with diverse β2 values. FIGURE 5 (A) f′(δ), (B) θ(δ), and (C) NG with diverse β2 values. FIGURE 5 (A) f′(δ), (B) θ(δ), and (C) NG with diverse β2 values. FIGURE 5 (A) f′(δ), (B) θ(δ), and (C) NG with diverse β2 values. 09 09 Frontiers in Energy Research frontiersin.org Rahman et al. 10.3389/fenrg.2022.996556 FIGURE 6 (A) f′(δ), (B) θ(δ), and (C) NG with diverse Γ values. FIGURE 7 (A) θ(δ) and (B) NG with diverse m values. Rahman et al. 10.3389/fenrg.2022.996556 FIGURE 6 (A) f′(δ), (B) θ(δ), and (C) NG with diverse Γ values. FIGURE 6 (A) f′(δ), (B) θ(δ), and (C) NG with diverse Γ values. FIGURE 6 (A) f′(δ), (B) θ(δ), and (C) NG with diverse Γ values. FIGURE 6 (A) f′(δ), (B) θ(δ), and (C) NG with diverse Γ values. FIGURE 7 (A) θ(δ) and (B) NG with diverse m values. FIGURE 7 (A) θ(δ) and (B) NG with diverse m values. 10 Frontiers in Energy Research frontiersin.org Rahman et al. 10.3389/fenrg.2022.996556 FIGURE 8 (A) θ(Γ) and (B) NG with diverse Bα values. FIGURE 9 (A) f′(δ), (B) θ(δ), and (C) NG with diverse ε values. FIGURE 8 (A) θ(Γ) and (B) NG with diverse Bα values. FIGURE 8 (A) θ(Γ) and (B) NG with diverse Bα values. FIGURE 9 (A) f′(δ), (B) θ(δ), and (C) NG with diverse ε values. FIGURE 9 (A) f′(δ), (B) θ(δ), and (C) NG with diverse ε values. FIGURE 9 (A) f′(δ), (B) θ(δ), and (C) NG with diverse ε values. 11 Frontiers in Energy Research frontiersin.org Rahman et al. 10.3389/fenrg.2022.996556 FIGURE 10 NG with diverse (A) Re and (B) Br values. FIGURE 10 NG with diverse (A) Re and (B) Br values. Frontiers in Energy Research Impact of velocity slip variable (ε) The impacts of velocity slip parameters (ε) on the velocity ﬁeld, temperature ﬁeld, and entropy generation are plotted in Figure 9A–C, by choosing C6H9NaO7 as the base ﬂuid. The effect of the strain parameter is reported from the PENF (Cu- C6H9NaO7) and PEHNF (Ti6Al4V-Cu/C6H9NaO7) momentum distribution (Figure 9A). This parameter is obtained from the boundary conditions of the current model. The gradual increment of the velocity slip enhances the ﬂuid viscosity, thus decreasing the ﬂuid velocity. The higher concentration of slip velocity (ε) values lowers the thermal boundary layer thickness (Figure 9B), which the diminution of the proﬁle is associated with the Williamson nanoﬂuids. At the same time, the slip velocity parameter slows the collisions with molecular diffusion. When the concentration of nanoparticles is higher in a system, the system is associated with the instantaneous possessions of thermal convection, transmission, and kinematic viscosity. Figure 9C shows the plot of entropy generated (NG) versus (ε). The plot of NG shows an effective reduction in this proﬁle because the location is far from the plate. Due to the act of the velocity slip, entropy gradually decreases. Impact of the biot number (Bα) The visualization of the temperature proﬁle against Biot numbers (Bα) for PEHNF (Ti6Al4V-Cu/C6H9NaO7) and PENF (Cu-C6H9NaO7) nanoparticles is displayed in Figure 8A. Overall, Finally, the achieved outcome of the impact of the changes in various parameters and 5 shape nanoparticles by the names of a sphere, hexahedron, tetrahedron, column, and lamina on Frontiers in Energy Research 12 frontiersin.org Rahman et al. 10.3389/fenrg.2022.996556 FIGURE 11 (A). Concentration h(δ) via Sc (B). Concentration h(δ) via 8. TABLE 6 Nusselt quantity (Nux) and ideal relative error bound (σ2) for various values of time-dependent variant (ξ) and radiative variable (Nr). γ Nux p1 p2 p3 p4 p5 σ2 0.04 -1.8017 2.1244 -0.7419 -2.0179 0.1720 -0.3247 0.0043 0.10 -1.6956 2.0156 -0.6276 -1.9856 0.1254 -0.2729 0.0054 0.16 -1.4209 1.8057 -0.4025 -1.7514 0.0982 -0.2088 0.0079 0.22 -1.2023 1.6412 -0.3253 -1.6049 0.0717 -0.1054 0.0090 Entropy changes regarding Reynolds (Re) and brinkman numbers (Br) The effect of the Reynolds number (Re) on the entropy proﬁle is shown in Figure 10A when both types of nanoﬂuids are bounded by the stretching sheet. It is noted that the higher Re boosts the level of entropy that can be generated in the ﬂuid system. Figure 10B shows the relationship between the entropy generation NG and the values of the Brinkman number (Br), showing that increasing Brinkman number (Br) enhances entropy generation. Brinkman number (Br) deﬁnes the viscous inﬂuence of ﬂuid behavior. As a consequence, high Brinkman numbers (Br) denotes that ﬂuid friction is the utmost factor of entropy generation. In both the Reynolds number and Brinkman number relationship, Cu-C6H9NaO7 nanoparticles are found to have higher entropy level, compared to Ti6Al4V-Cu/C6H9NaO7 nanoparticles. the enhancing (Bα) rises a temperature. The thermal thin state is regarding the low Biot number: The condition of uniform temperature region within the body (nano-polymer surface). Higher values of Bα indicate the thermal thick state in which non-uniform of temperature domains occur. The plot of NG against (Bα)as displayed in Figure 8B discovers that the entropy proﬁle is insensitive (only slight changes) with the increasing (Bα) at the stretching surface, compared to the position away from it. It is found that less enhancement in entropy proﬁle is observed near the stretching walls. However, a decline in entropy generation is observed with the growth of (Bα). Frontiers in Energy Research Frontiers in Energy Research 13 frontiersin.org 10.3389/fenrg.2022.996556 Rahman et al. Concentration changes regarding schmidt number (Sc) and chemical reaction parameter (8) Parametrical study on drag force (Cf) and nusselt number (Nu) TABLE 7 CfRex 1 2 and NuRex −1 2 values at Pr 6.5, β1 0.01 and m 3. Entropy changes regarding Reynolds (Re) and brinkman numbers (Br) β2 ξ Γ ϕ ϕTA ε γ Nr Bα S CfRex 1 2 Cu- C6H9NaO7 CfRex 1 2 Ti6Al4V -Cu/C6H9NaO7 NuRex −1 2 Cu- C6H9NaO7 NuRex −1 2 Ti6Al4V -Cu/C6H9NaO7 0.1 0.2 0.1 0.18 0.09 0.3 0.2 0.3 0.3 0.1 1.8921 2.1301 0.5563 1.1216 0.2 1.8562 2.0953 0.5269 1.1033 0.3 1.8134 2.0264 0.4903 1.0761 0.2 1.8921 2.1301 0.5563 1.1216 0.6 1.9309 2.1745 0.5732 1.1905 0.8 1.9647 2.2068 0.5907 1.2351 0.1 1.8921 2.1301 0.5563 1.2516 0.3 1.9163 2.1564 0.5374 1.2167 0.4 1.9357 2.1849 0.5012 1.1856 0.09 1.8089 - 0.4718 - 0.15 1.8454 - 0.5025 - 0.18 1.8921 - 0.5563 - 0.0 - 1.8089 - 0.4718 0.06 - 2.0963 - 1.1091 0.09 - 2.1301 - 1.1216 0.1 1.9728 2.2060 0.6175 1.8116 0.2 1.9359 2.1642 0.5845 1.1520 0.3 1.8921 2.1301 0.5563 1.1216 0.1 1.8921 2.1301 0.6437 1.2372 0.2 1.8921 2.1301 0.6044 1.1530 0.3 1.8921 2.1301 0.5563 1.1216 0.1 1.8921 2.1301 0.5119 1.0906 0.3 1.8921 2.1301 0.5563 1.1216 0.5 1.8921 2.1301 0.5729 1.5238 0.1 1.8921 2.1301 0.5218 1.1016 0.3 1.8921 2.1301 0.5563 1.1216 0.4 1.8921 2.1301 0.5980 1.1464 0.1 1.8921 2.1301 0.5563 1.1216 0.3 1.9127 2.1527 0.5864 1.1504 0.2 1.9432 2.1965 0.6238 1.1714 TABLE 7 CfRex 1 2 and NuRex −1 2 values at Pr 6.5, β1 0.01 and m 3. Frontiers in Energy Research Future track C6H9NaO7) nanoparticles. The Nusselt number enhances with an augmentation ξ, whereas an opposite function is remarked with the addition of β2, γ and Nr for both PEHNF (Ti6Al4V-Cu/ C6H9NaO7) and PENF (Cu-C6H9NaO7) nanoparticles (Figure 7). These outcomes can be a guideline for the industry and technology, to choose the appropriate working ﬂuid for improved productivity in the associated device or prototype Subsequently, this research work can be extended by applying the model of rotating disk ﬂow considering ferromagnetic nanoparticles (Jamshed et al., 2021e; Pasha et al., 2022). The FEM could be applied to a variety of physical and technical challenges in the future (Jamshed et al., 2022b; Hussain et al., 2022b; Hussain et al., 2022a). Acknowledgments 1) Along the far stream, the velocity ﬁeld is reduced for the upsurging Powell-Eyring ﬂuid (β2), porosity (Γ), volume fraction (ϕ), and velocity slip (ε). 1) Along the far stream, the velocity ﬁeld is reduced for the upsurging Powell-Eyring ﬂuid (β2), porosity (Γ), volume fraction (ϕ), and velocity slip (ε). Authors are grateful to the Deanship of Scientiﬁc Research, Islamic University of Madinah, Ministry of Education, KSA for supporting this research work through research project grant under Research Group Program/1/804. 2) The temperature proﬁle of Powell-Eyring ﬂuid for both cases of Cu-C6H9NaO7 (conventional nanoﬂuid) and Ti6Al4V-Cu/ C6H9NaO7 (hybrid nanoﬂuid) intensiﬁes under the increment of γ, Γ, ϕand Bα. 3) The temperature distribution is affected by most of the physical quantities, which denotes that nanoﬂuids have a high heat exchange rate. This property helps control the temperature during spin coating processes. Parametrical study on drag force (Cf) and nusselt number (Nu) The coefﬁcients in the components of the ﬂow and heat transmission are namely dragged force (Cf) and Nusselt numbers (Nux), respectively. These parameters are tabulated in Tables 5, 6 showing their numerical values. Table 7 shows that a frictional force factor has non-uniform changes for both of the nanoparticles with an increasing ε and ξ. Also, it is observed that the drag force coefﬁcient upsurges with an increment of Γ and ϕ. Besides, the reduction is remarked with rising ε for both the case of nanoparticles. Furthermore, the increasing values of Bα and Nr have no impact on drag force coefﬁcient for both PEHNF (Ti6Al4V-Cu/C6H9NaO7) and PENF (Cu- Figure 11A exhibits dispersal of concentration h(δ) towards the signiﬁcant Schmidt number (Sc) which has a vital impact on it. Technically for the higher Schmidt number (Sc), the progress in the molecular diffusion tends to get reduced which restricts the mass transference in the system which can be visualized from Figure 11A. On other hand, Figure 11B discloses the inﬂuence of chemical reaction constraints on mass diffusion. The effective chemical reaction process exerts more mass in the system which makes the mass transference process harder and this may lead to the deceleration noted in Figure 11B for the higher values of chemical reaction constraint. Frontiers in Energy Research 14 frontiersin.org Rahman et al. 10.3389/fenrg.2022.996556 Conﬂict of interest The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. 4) The entropy proﬁle against Powell-Eyring ﬂuid (β2), Porosity term (Γ), volume fraction (ϕ, ϕhnf) and Biot number (Bα) and shape factor (m) explore dual behavior. 5) The shape of nanoparticles is namely sphere, hexahedron, tetrahedron, lamina, and column. Among them, lamina has the greatest impact on the function of temperature and entropy. Concluding consequences and forthcoming course In this mathematical investigation, the heat transmission in a Powell-Eyring hybrid nanoﬂuid (PEHNF) model bounded by an expanding surface is investigated. This model is implemented in a thermal system, which is inspired by the modiﬁed Buongiorno’s NF prototype. The presence of nanoparticles such as Cu- C6H9NaO7 and Ti6Al4V-Cu/C6H9NaO7 nanoparticles are implemented in this model. The inﬂuences of porous media, thermal radiative ﬂow, and variable thermal conductivity are taken into account in the mathematical model. Furthermore, the effect of the nanoparticles’ shape factors is determined, and their impacts can be observed in thermal and entropy aspects. The numerical solutions for the current mathematical model can be achieved by following these steps: 1) Apply similarity solution to convert PDEs to ODEs, and 2) solve the ODEs with Galerkin ﬁnite element plan. The primary outcomes from this investigation are listed for the proﬁles such as velocity, temperature, and concentration, together with the coefﬁcients of drag force and Nusselt number. These outcomes are listed below: Author contributions WJ, MR and SS formulated the problem. WJ and RI solved the problem. WJ, MR, SS, RI, BS, RS, AP, ME, SH and ED computed and scrutinized the results. 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https://openalex.org/W4294537086	https://bmcpublichealth.biomedcentral.com/counter/pdf/10.1186/s12889-022-14101-z	English	null	Translation and validation of the Chinese ABCD risk questionnaire to evaluate adults’ awareness and knowledge of the risks of cardiovascular diseases	BMC public health	2,022	cc-by	6,420	Translation and validation of the Chinese ABCD risk questionnaire to evaluate adults’ awareness and knowledge of the risks of cardiovascular diseases Yan Liu1,2, Wei Yu3†, Mei Zhou3, Fang Li1, Farong Liao3, Zhengyu Dong4, Hairong Wang3, Jiaqing Chen5 and Lingling Gao2 Abstract Background: Assessment of health beliefs and risk perception is a critical means to prevent coronary heart dis- ease, but there are few such studies on assessment in the Chinese population. Given the demonstrated value and widespread use of the Attitudes and Beliefs about Cardiovascular Disease Risk Questionnaire (ABCD), this study was designed to translate it into Chinese, and to evaluate its reliability and validity in a Chinese population. Methods: The Chinese version of the ABCD was created using the Beaton translation model, which included forward and backward translation. The reliability and construct validity of the Chinese ABCD were examined in a sample of 353 adults who participated in the public welfare projects of the Chinese National Center for Cardiovascular Diseases in Guilin city, Guangxi. Exploratory factor analysis (EFA) and confirmatory factor analysis (CFA) were performed to examine the factor structure of the Chinse ABCD. The internal consistency of the questionnaire was assessed using Cronbach’s α and corrected item-total correlations. Results: We deleted item 7 in the knowledge dimension of the Chinese ABCD and added two items about smok- ing and sleep knowledge, while retaining 25 of the original items, so that it finally included 27 items. The correlations were .20–.90; the correlations between each item and the total score of the ABCD were .34–.86; and the item-level Content Validity Index (I-CVI) was .86–1.00. The results of the EFA showed that all items were close to .40, and the cumulative variance contribution rate was 63.88%. The model fit was acceptable (χ2 = 698.79, df = 243, χ2/df = 2.87, P < 0.001, SRMR = 0.06, RMSEA = 0.05, CFI = 0.96, and TLI = 0.94) according to the CFA. The Cronbach’ s α of the entire questionnaire was .86, and the α of each of dimension was .65, .90, .88, and .78. The split-half reliability of the entire the ABCD was .67, and the test-retest reliability was .97 (P < 0.05). The questionnaire had good reliability and validity and was associated with sociodemographic and health-related characteristics (smoking and Body Mass Index). †Yan Liu and Wei Yu contributed equally to this work. Liu et al. BMC Public Health (2022) 22:1671 https://doi.org/10.1186/s12889-022-14101-z © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Liu et al. BMC Public Health (2022) 22:1671 Page 2 of 8 Conclusion: The Chinese version of the ABCD has good reliability and validity, and provides a reliable assessment tool for measuring public health beliefs about the risk of cardiovascular disease, promoting the primary prevention of coronary heart disease. Keywords: Cardiovascular disease, Health beliefs, Risk perception, Translation Translation and adaptation of the Chinese ABCD p To ensure the quality of the research methodology, the questionnaire was evaluated according to the contents of the COnsensus-based Standards for the selection of health status Measurement INstruments (COSMIN) checklist [11], and the study’s report was adhered to the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) [12]. After obtaining the consent and authorization of the original author of the ABCD, a research group was established to perform a Chinese translation of it using the Beaton translation model [13, 14]. First, forward translation of the ABCD Introduction outpatient department of the Affiliated Hospital of Guilin Medical University from October 2021 to January 2022 in Guilin, Guangxi province, China. The inclusion crite- ria were: being a permanent resident of Guilin for over 6 months, age 35 years or older, and not being diagnosed with a mental or cognitive disorder. Cardiovascular diseases (CVDs) are the leading cause of death and disability in the world, mainly because of ischemic heart disease and stroke [1]. According to the latest Global Burden of Cardiovascular Diseases study, the number of patients worldwide with CVDs reached 523 million in 2019, and the morbidity due to CVDs was 330 million in China. Furthermore, the highest rates of morbidity and mortality from CVDs are in China [2], which is partly related to the increase in the elderly popu- lation of China. Because long-term, unhealthy lifestyles exacerbate the risk of CVDs in the elderly, the Chinese Guidelines on Healthy Lifestyle to Prevent Cardio-met- abolic Diseases make some recommendations to reduce risk factors, such as, to stop smoking, to eat a rational diet, and to engage in physical activity and other healthy lifestyle habits. Altering bad habits and maintaining a healthy lifestyle is important to prevent CVDs, which are affected by one’s health beliefs. It is well known that health beliefs affect one’s perceptions and health knowl- edge of behavioral risks [3, 4]. There is evidence that individuals who have health knowledge will engage in healthier behaviors to reduce the incidence of CVDs [5].hf The sample size was determined based on the general rule that the sample should contain 5–10 participants for each item to be analyzed by factor analysis. Given that the English ABCD questionnaire has 26 items, and assuming a 20% rate of invalid questionnaires, the calcu- lated sample size was 325 cases, but it was determined that the sample size should be 374 cases. The original ABCDh The ABCD is a self-assessment tool to evaluate of an individual’s health knowledge, perceived risks, and ben- efits, which was developed in 2017 by Woringer et al. [7], based on the Health Belief Model and the Tran- stheoretical Model. It consists of 26 items that measure four dimensions, including CVD knowledge, percep- tion of risks, perception of benefits, and healthy eating intentions. The knowledge dimension is measured using dichotomous response options (yes/no questions), and the other three dimensions are measured using a 4-point Likert scale, with responses ranging from 1 = “com- pletely disagree” to 4 = “completely agree.” The ABCD’s total score ranges from 18 to 80 points. The higher the score, the higher the perceived risk of preventing CVD. It is currently used to assess the perceived risk of CVD in England’s health-examination population, the Hungarian community population [8], and Dutch adults [9, 10]. Therefore, effective and reliable assessments of individ- uals’ knowledge and perceptions of risks are essential. In 2015, Liu et al. developed a Chinese version of a health- belief scale for diabetic patients about the prevention of CVDs [6], but it was not for the general population. At present, the Attitudes and Beliefs about Cardiovascular Disease Risk Questionnaire (ABCD), developed by the British National Health Service Program to measure the general population’s perceptions and knowledge of the risks of CVDs, is widely used abroad [7–9]. However, the ABCD has not been translated to Chinese and validated in a Chinese sample. Hence, this study’s aims were to translate the ABCD to Chinese and to evaluate its psy- chometric performance in a Chinese sample using clas- sical test theory. In addition, the Chinese version of the questionnaire was applied to the cognition and assess- ment of CVD risk in a population in a cardiovascular dis- ease screening program. Statistical analysesh The structural validity of the questionnaire was verified using factor analysis to analyze the data; the factor anal- ysis was conducted with the freeware statistical pack- age Jamovi (V2.25). The data were randomly divided into two groups: exploratory factor analysis (EFA) was performed on the data from one group (n = 176), and confirmatory factor analysis (CFA) was performed on the data from the other group (n = 177). The degree of fit of the CFA model was assessed by common sta- tistical parameters, including the chi-square (χ2) test, the standardized root mean residual (SRMR), the root mean square error of approximation (RMSEA), the Tucker-Lewis Index (TLI), and the Comparative Fit Index (CFI). The reliability of the questionnaire was analyzed by test-retest reliability, split-half reliability, alternate reliability, and the internal consistency coef- ficient. All other statistical computations, including bivariate Spearman’s correlations and group compari- sons were conducted using the SPSS (V25) statistical software package. After three rounds of evaluation and cultural background debugging for language habits, cultural background, content relevance, etc., the team added two items about smoking and sleep, which were based on items in the original knowledge dimension of the ABCD; the two items added to the knowledge dimension were item 9 (“People who smoke are at risk of having a heart attack or stroke”) and item 10 (Having enough sleep (7–8 hours per day) will help you lower your risk of having a heart attack or stroke”). In contrast, item 7 (“HDL refers to ‘good’ cho- lesterol, and LDL refers to ‘bad’ cholesterol”) was deleted because it appeared to be too specialized, as nearly half of the people (49.29% (n = 173) who completed the pre- test failed to respond to the item. Hence, there were finally nine items in the knowledge dimension. Because Chinese residents have different living habits than foreign resi- dents, Chinese residents found it difficult to understand terms such as gardening and moderate intensity exercise. Therefore, the relevant content of items 2, 3, 6, and 22 were interpreted. For example, the translation of “garden- ing” in item 2 was interpreted as “digging to plant vegeta- bles or flowers.” For item 3, “moderate intensity exercise” was defined as “running or activities at 60% to 70% of maximum heart rate, where maximum heart rate (times /min)=220-age.” Due to the different drinking habits of Sample and procedures Finally, the 40 patients who met the standards of admission to the study were selected to complete the Chinese version in order to evaluate its reliability and validity. After modifying the wording of the individual items of the questionnaire, the expert committee reviewed and evaluated it again, and the final Chinese version of the questionnaire was created. [Approval Number: QTLL202157]. During the evaluation process, the subjects of the study gave their informed consent and signed consent forms on site. The partici- pation of subjects was based on the principle of “pro- portional universalism” and covered vulnerable groups rather than being targeted [15]. Sociodemographic characteristics of the samples Sociodemographic characteristics of the samples A total of 374 questionnaires were distributed to adults, and all 374 of them were returned, resulting in an effec- tive recovery rate of 100%. Excluding questionnaires with missing answers and repetitive answers, 353 valid ques- tionnaires were obtained, for an effective rate of 94.39%. Two-thirds of the participants were female (63.5%, n = 353), and the mean age of the sample was somewhat over 55 years (M = 55.75; SD = 10.10), ranging from 35 to 76 years. The largest portion of the sample consisted of respondents with a college or a higher level of educa- tion (31.2%), followed by senior high-school graduates (44.7%), graduates of junior middle-school (17.3%), and participants with a primary education (6.8%). The occu- pations of the participants were mainly retirees (45.3%), technicians (24.6%), administrators (8.5%), farmers (3.1%), and others (18.4%). Sample and procedures A convenience sample of persons who attended a CVD screening program was recruited for the study from an Liu et al. BMC Public Health (2022) 22:1671 Liu et al. BMC Public Health (2022) 22:1671 Page 3 of 8 was performed independently by two experts who had experience translating medical questionnaires abroad for 2 years. A comprehensive analysis of the two trans- lations was conducted to select the most appropriate question content for the Chinese version, and version 1 was created. Second, the Chinese version was back- translated to English independently by language pro- fessionals in Sun Yat-Sen University and a doctor of cardiovascular medicine in the United States who lived and worked there for over 20 years. After discus- sion about and analysis of the two translated versions, a comprehensive translated version was created. Third, the second Chinese version (version 2) was revised based on the review and discussion of it by the mem- bers of an opinion group. Next, the field Chinese ver- sion was sent to an expert committee of who reviewed the translation methodology to make cultural adjust- ments for Chinese populations. Finally, the 40 patients who met the standards of admission to the study were selected to complete the Chinese version in order to evaluate its reliability and validity. After modifying the wording of the individual items of the questionnaire, the expert committee reviewed and evaluated it again, and the final Chinese version of the questionnaire was created. was performed independently by two experts who had experience translating medical questionnaires abroad for 2 years. A comprehensive analysis of the two trans- lations was conducted to select the most appropriate question content for the Chinese version, and version 1 was created. Second, the Chinese version was back- translated to English independently by language pro- fessionals in Sun Yat-Sen University and a doctor of cardiovascular medicine in the United States who lived and worked there for over 20 years. After discus- sion about and analysis of the two translated versions, a comprehensive translated version was created. Third, the second Chinese version (version 2) was revised based on the review and discussion of it by the mem- bers of an opinion group. Next, the field Chinese ver- sion was sent to an expert committee of who reviewed the translation methodology to make cultural adjust- ments for Chinese populations. Ethics and participant’s consenth This study has been approved by the Ethics Committee of the Affiliated Hospital of Guilin Medical University Liu et al. BMC Public Health (2022) 22:1671 Liu et al. BMC Public Health (2022) 22:1671 Liu et al. BMC Public Health (2022) 22:1671 Page 4 of 8 Chinese residents, item 6 “drinking high levels of alco- hol” was translated as “excessive drinking” (daily alcohol intake > 24 g; note: The amount of alcohol intake was calculated as alcohol content (% v/v) × drinking amount (mL)/100 × 0.8 of the bottle). Weight is usually calculated by kilogram or jin in China, whereas, it is usually based on portions in foreign countries; therefore, “five portions of fruit and vegetables” were annotated as “400 g or 8 liang.” EFA, and the cumulative variance contribution rate was 62.84%. A sufficient number of factors were determined from the Scree Plot and a parallel analysis (PA). In PA, the data can be used to generate a certain number of simulated datasets, so the factors whose eigenvalues were greater than 1.00 and higher than the threshold value extracted to obtain three factors, were compared with the original ABCD factors, and found to be the same (Fig. 1). The EFA was conducted by using the maximum variance method to evaluate the item results, which showed that all the items were close to .40, as shown in Table 1. EFA, and the cumulative variance contribution rate was 62.84%. A sufficient number of factors were determined from the Scree Plot and a parallel analysis (PA). In PA, the data can be used to generate a certain number of simulated datasets, so the factors whose eigenvalues were greater than 1.00 and higher than the threshold value extracted to obtain three factors, were compared with the original ABCD factors, and found to be the same (Fig. 1). The EFA was conducted by using the maximum variance method to evaluate the item results, which showed that all the items were close to .40, as shown in Table 1. The reliability of the questionnaire Cronbach’s α is commonly used as the internal consist- ency coefficient of a questionnaire. Our studies have shown that the Cronbach’s α of the entire questionnaire was .86, and it was .65, .90, .88, and .78 for each of the four dimensions. Split-half reliability was calculated by the odd and even grouping method. Spearman’s correla- tion was used to analyze the two halves of the data. The results showed that the correlation of the entire ques- tionnaire was .67, and the correlation of each dimen- sion was .63, .79, .78, and .62. The test-retest reliability of the questionnaire was based on the correlation between the pretest and retest data, using Pearson’s correlation The validity of the ABCD Content validity The CFA was used to test the ABCD’s structural valid- ity further by determining the degree to which it fit the EFA model. The results showed that the model fit was acceptable (χ2 = 698.79, df = 243, χ2/df = 2.87, P < 0.001; SRMR = 0.06; RMSEA = 0.05; CFI = 0.96; and TLI = 0.94) as shown in Table 2. Seven CVD experts were invited to evaluate the Content Validity Index (CVI) of the ABCD, which was assessed with the CVI at the Item level (I-CVI), the Scale-level Content Validity Index/Universal Agreement Validity Index (S-CVI/UA), and the Scale-level Content Validity Index Average (S-CVI/Ave). A 4-level scoring method was adopted, with scores ranging from 1 (irrelevant) to 4 (very relevant). The I-CVI was 3 or 4 points for each item divided by the total number of experts; the S-CVI/ UA was 3 or 4 points for all items, divided by the total number of experts; and the S-CVI/Ave was the average of the I-CVI for all items. The values of the I-CVI, S-CVI/ UA, and S-CVI/Ave were .86–1.00, .82, and .97, which indicate good content validity. Discussionh The research team adopted the Chinese ABCD and con- ducted an on-site survey of Chinese adults to verify its psychometric properties, including its content valid- ity and structural validity. Content validity refers to the accuracy of the item content to achieve the expected measurement results (I-CVI ≥ 0.78, S-CVI/UA ≥ 0.8, and S-CVI/Ave ≥ 0.9) [13]. In this study, the I-CVI was .86–1.00, the S-CVI/UA was .82, and the S-CVI/Ave was .97, indicating that the content validity of Chinese ABCD was good. Structural validity reflects the degree of inte- gration between the ABCD’s structure and the theory or framework on which it is based, which requires item loadings that are greater than .40 and a cumulative vari- ance contribution rate not less than 50%. On the whole, all the measurement items had a significance level of P < 0.001, and the standardized loadings were all greater than .70 in the EFA results of this study, indicating that there was good correspondence between the factors and the measurement items, and the aggregation validity was good. In addition, the SRMR was close to .08 and the RMSEA was below .06, as required, whereas the TLI and CFI were over .90, indicating a good fit [16]. The factor analysis results confirmed the structural validity of the coefficient, to test the repeatability of the results. Three weeks after the 40 participants who took the pre-test of the ABCD, completed a post-test of it; the test-retest Table 2 Factor loadings of the CFA 95% Confidence Interval Factor Indicator Stand. Construct validityh The sample data was suitable for factor analysis based on the Kaiser-Meyer-Olkin (KMO) measure and Bartlett’s test of sphericity. In this study, the KMO of .86 and Bar- tlett’s χ2 value of 2453.0 (P < 0.01) met the conditions for Fig. 1 Scree Plot of the EFA Liu et al. BMC Public Health (2022) 22:1671 Page 5 of 8 Table 1 Factor loadings of the EFA The “Minimum residual” extraction method was used in combination with “Varimax” rotation; the hidden loadings were below 0.3 Factor Item 1 2 3 Uniqueness Perceived Risk 6 0.89 0.19 Perceived Risk 5 0.89 0.19 Perceived Risk 4 0.86 0.24 Perceived Risk 3 0.85 0.22 Perceived Risk 2 0.84 0.27 Perceived Risk 1 0.77 0.35 Perceived Risk 8 0.76 0.39 Perceived Risk 7 0.63 0.59 Healthy Eating Intentions 1 0.76 0.33 0.40 Healthy Eating Intentions 2 0.71 0.36 Perceived Benefits 6 0.71 0.43 Perceived Benefits 7 0.67 0.35 0.39 Healthy Eating Intentions 3 0.50 0.71 Perceived Benefits 5 0.39 0.83 Perceived Benefits 1 0.88 0.14 Perceived Benefits 2 0.87 0.13 Perceived Benefits 3 0.46 0.69 0.28 Perceived Benefits 4 0.45 0.54 0.48 reliability of the questionnaire was .97 (P < 0.05). The rela- tionship of the questionnaire data with the demographic characteristics of the Chinses sample are presented in Table 3. Discussionh Estimate Lower Upper Z p Factor 1 Perceived Risk 1 0.87 0.73 0.96 14.65 < 0.001 Perceived Risk 2 0.82 0.70 0.94 13.31 < 0.001 Perceived Risk 3 0.80 0.69 0.94 12.96 < 0.001 Perceived Risk 4 0.81 0.64 0.86 13.13 < 0.001 Perceived Risk 5 0.96 0.90 1.136 17.58 < 0.001 Perceived Risk 6 0.74 0.61 0.86 11.49 < 0.001 Perceived Risk 7 0.89 0.81 1.05 15.26 < 0.001 Perceived Risk 8 0.97 0.89 1.12 17.74 < 0.001 Factor 2 Perceived Benefits 1 0.81 0.53 0.72 13.07 < 0.001 Perceived Benefits 2 0.95 0.62 0.78 16.99 < 0.001 Perceived Benefits 3 0.85 0.57 0.75 14.13 < 0.001 Perceived Benefits 4 0.78 0.54 0.75 12.37 < 0.001 Perceived Benefits 5 0.91 0.62 0.80 15.74 < 0.001 Perceived Benefits 6 0.91 0.55 0.71 15.79 < 0.001 Perceived Benefits 7 0.70 0.46 0.67 10.62 < 0.001 Factor 3 Healthy Eating Intentions 1 1.00 0.93 1.15 18.84 < 0.001 Healthy Eating Intentions 2 0.83 0.74 0.98 13.74 < 0.001 Healthy Eating Intentions 3 0.98 0.92 1.14 18.12 < 0.001 Table 2 Factor loadings of the CFA Liu et al. BMC Public Health (2022) 22:1671 Page 6 of 8 Table 3 Group comparisons of the questionnaire Body Mass Index (BMI) is a person’s weight in kilograms (or pounds) divided by the square of height in meters (or feet). Discussionh a The significance level of the mean difference is .05 Characteristics n(%) ABCD Risk Questionnaire Mean (SD) Knowledge Risk Benefits Eating Total Total 353 7.40 (1.69) 15.98 (7.63) 20.24 (4.87) 7.75 (2.78) 51.38 (11.89) Gender Female 224 (63.46) 7.39 (1.69) 16.04 (6.80) 19.80 (4.87) 7.58 (3.06) 51.69 (11.97) Male 129 (36.54) 7.41 (1.70) 15.94 (8.09) 20.50 (4.86) 7.85 (2.61) 50.86 (11.77) P 0.910 0.902 0.198 0.393 0.528 Educational level Primary degree or below 24 (6.80) 7.66 (1.49) 16.04 (7.51) 19.95 (5.13) 7.58 (2.04) 51.25 (11.62) Junior middle-school degree 61 (17.28) 7.27 (1.75) 14.81 (7.86) 20.45 (4.57) 7.91 (2,71) 50.47 (12.85) Senior high-school degree 158 (44.76) 7.18 (1.90) 14.30 (6.95) 20.20 (4.84) 7.65 (2.94) 49.34 (11.02) College degree or higher 110 (31.16) 7.73 (1.31) 19.01 (7.65) 20.25 (5.06) 7.85 (2.74) 54.86 (11.98) P 0.034a <0.001a 0.976 0.884 0.002a Employment status Retirees 160 (45.33) 7.18 (1.85) 15.89 (8.31) 20.06 (5.54) 7.73 (2.89) 50.87 (13.09) Technicians 87 (24.65) 7.36 (1.62) 16.68 (5.97) 19.82 (4.24) 7.71 (2.66) 51.59 (9.82) Administrators 30 (8.50) 8.23 (0.81) 16.03 (7.84) 21.06 (4.63) 7.80 (2.38) 53.13 (11.01) Farmers 11 (3.12) 7.72 (1.19) 14.90 (5.35) 20.45 (1.86) 8.09 (1.30) 51.18 (5.89) Others 65 (18.41) 7.56 (1.67) 15.40 (8.22) 20.84 (4.32) 7.78(3.06) 52.60 (12.62) P <0.001a 0.756 0.607 0.995 0.904 Residential location Urban 314 (88.95) 7.36 (1.76) 15.94 (7.77) 20.37 (4.79) 7.76 (2.81) 51.45 (12.22) Suburban 22 (6.23) 7.36 (1.00) 15.45 (6.38) 17.59 (6.11) 7.27 (2.88) 47.68 (8.35) Rural 17 (4.82) 8.11 (0.92) 17.23 (6.67) 21.29 (3.58) 8.23 (2.13) 54.88 (8.07) P 0.016a 0.754 0.080 0.559 0.164 Annual household income (yuan, RMB) < 50,000,00 121 (34.28) 7.33 (1.75) 15.48 (7.19) 19.55 (4.68) 7.37 (2.86) 49.74 (11.48) 50,000,00-100,000,00 131 (37.11) 7.50 (1.79) 16.01 (8.00) 20.67 (4.72) 7.77 (2.63) 51.96 (12.14) >100,000,00 101 (28.61) 7.36 (1.50) 16.52 (7.69) 20.52 (5.22) 8.18 (2.83) 52.60 (11.94) P 0.696 0.802 0.152 0.093 0.159 Smoking status Smoker 155 (43.91) 6.33 (1.92) 12.98 (8.00) 19.47 (5.09) 7.33 (3.11) 46.13 (12.35) Non-smokers 198 (56.09) 8.24 (0.81) 18.32 (6.44) 20.84 (4.61) 8.08 (2.45) 55.50 (9.73) P <0.001a <0.001a 0.009a 0.014a <0.001a BMI(kg/m2) < 18.5 38 (10.76) 6.65 (2.17) 4.81(5.50) 18.50 (6.60) 6.26 (3.53) 36.23 (11.43) 18.5–23.9 153 (43.34) 8.42 (0.64) 17.88(6.83) 20.98 (4.61) 8.16 (2.50) 55.45 (10.22) ≥24 162 (45.89) 6.61 (1.75) 16.80(6.55) 19.95 (4.51) 7.72 (2.73) 51.09 (10.50) P <0.001a <0.001a 0.033a 0.007a <0.001a questionnaire, which was consistent with the results of Martos et al. [8]. and test-retest reliability. Conclusion In summary, the English version of the ABCD question- naire was translated into Chinese in this study following strict methodological standards for translating measure- ment tools, and we added content to measure smoking- and sleep-related knowledge. After deleting two items with low response rates and high repetition rates, the Chinese version of the ABCD we created has 27 items. The reliability and validity of the ABCD was only tested with adults, so other studies are needed with younger samples. The Chinese version of ABCD maintained the content and semantic equivalence of the English version as much as possible, and the Chinese version has good reliability and validity. However, its split-half reliability is low, and the sample size should be increased in subse- quent studies. The Chinese version of ABCD provides a reliable tool for assessing the public’s health beliefs about the risk of CVDs, and it provides a self-assessment tool to enhance the public’s awareness of early prevention of CVDs. fi Split-half reliability measures the homogeneity of a scale by dividing its items into two parts and calculating the correlation between the two parts. The split-half reli- ability of a scale is very good if it is over .60, and it was better that that for our version of the ABCD. Retest reli- ability is an index to evaluate the stability of the scale. For our sample, a large number of correlations showed good stability, and the test-retest reliability of the question- naire was better than the criterion correlation of .78. Our study, which was conducted in the same hospital, yielded a test-retest reliability for the ABCD of .97, which indi- cates very high stability. As for the perception of cardiovascular disease risk, unlike the results of Martos et al. [8], the measures of smoking and Body Mass Index (BMI) were significantly correlated with risk perception, which may be related to the national cultural environment and dietary habits. In China, where tobacco consumption is the highest in the world, smoking has a great impact on people’ s health and it is a well-known risk factor for CVD. Chinese peo- ple have a dietary habit that consists of a rich food at din- ner, and not exercising after meals [18], which has lead to an increased BMI, but their awareness of the association of the risk for cardiovascular disease with a higher BMI is inadequate. Limitations Although the methodology used to translate the ques- tionnaire was reasonable, the current research has some limitations. For example, the Jamovi software we used in the study only met the requirements of first-order CFA, and it failed to modify the model. In addition, the study’s sample was obtained by convenience sampling and con- sisted mostly of urban residents who participated in the early risk screening program of the National Center for Cardiovascular Diseases. Therefore, this may have Discussionh A Cronbach’s α greater than .70 indicates that a scale’s internal consistency/internal reli- ability is acceptable, with .65–.70 indicating it is gener- ally acceptable, with.70–.08 indicating it is good, and .80–.90 indicating it is outstanding [17]. The Cronbach’s α of the knowledge dimension of the questionnaire in this Reliability refers to the degree of consistency of the results of a questionnaire across different times, investi- gators, and scenarios, and it is mainly evaluated by inter- nal consistency/internal reliability, split-half reliability, Liu et al. BMC Public Health (2022) 22:1671 Liu et al. BMC Public Health (2022) 22:1671 Page 7 of 8 resulted in self-selection bias. Further assessments of the ABCD should use other methods to provide a more bal- anced sample. study was .65, which was lower than the alpha for trans- lations of the ABCD into Dutch (α = .75) [9], and higher than its translation into Hungarian (α = .50) [8]. This low Cronbach’s α may be due to the specialized knowledge included in the original ABCD questionnaire. Therefore, when the questionnaire is translated into other national languages, it will be translated in accordance with the local language, so that respondents can easily understand it. However, the data for the knowledge items were all within the acceptable range, meaning that the items con- tributed sufficiently to the overall knowledge score. Acknowledgements Th k ll h d Thanks to all the study authors and to the National Center for Cardiovascular Disease for providing the data platform. Funding This research was supported by the Early Screening and Comprehensive Intervention Program for High-risk Groups of Cardiovascular Diseases of the National Center for Cardiovascular Diseases (Contract Number: GuiLin Center For Disease Control And Prevention[2019]32), Self-funded Scientific Research Project of Health Department of Guangxi Zhuang Autonomous Region (Con- tract Number: Z20190111), and the Ethics Committee of Affiliated Hospital of Guilin Medical University, grant number: QTLL202157. Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s12889-022-14101-z. Additional file 1. Additional file 1. Additional file 1. Conclusion The results of this study showed that people with a high level of education had greater awareness of cardiovascular risks, suggesting that we need to attend to people with lower educational levels in health education in the future. Later studies should pay more attention to these associations and provide targeted individualized education. Authors’ contributions Liu Yan and Yu Wei conceived and designed this study. Liu Yan, Gao Lingling, Dong Zhengyu, Yu Wei, and Chen Jiaqing conducted the statistical analysis and interpreted the survey results, and contributed to the preparation of the questionnaire and the literature review. Liao Farong, Li Fang, Wang Hairong, and Zhou Mei supported the statistical analysis and survey methods. After Liu Yan wrote the first draft, all the authors critically reviewed it, and the final manuscript was read and approved by all the authors. References 1. World Health Organization. Cardiovascular diseases (CVDs). 2021. https:// www.who.int/news-room/fact-sheets/detail/cardiovascular-diseases- (cvds). Accessed 11 June 2021. 1. World Health Organization. Cardiovascular diseases (CVDs). 2021. https:// www.who.int/news-room/fact-sheets/detail/cardiovascular-diseases- (cvds). Accessed 11 June 2021. Declarations lifestyle and socioeconomic disparities among adults in vulnerable communities of Belgium and England. BMC Public Health. 2022;22(1):197. https://doi.org/10.1186/s12889-022-12608-z. lifestyle and socioeconomic disparities among adults in vulnerable communities of Belgium and England. BMC Public Health. 2022;22(1):197. https://doi.org/10.1186/s12889-022-12608-z. Author details 1 14. Zang YL, Liu W, Lou FL. Translation technology of nursing measuring instrument. Chinese J Nurs Edu. 2009;6(6):275–7. [in Chinese]. 1 Nursing Department, Affiliated Hospital of Guilin Medical University, Guangxi, China. 2 School of Nursing, Sun Yat-sen University, Guangzhou, China. 3 Car- diovascular Medicine Ward, Affiliated Hospital of Guilin Medical University, Guangxi, China. 4 Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China. 5 Rhode Island Hospital, affiliated with the Warren Alpert Medical School of Brown University, Providence RI, USA. 15. Marmot M, Bell R. Fair society, healthy lives. Public Health. 2012;126:S4– S10. https://doi.org/10.1016/j.puhe.2012.05.014. 16. Shi JZ, Mo XK, Sun ZQ. Content validity index in scale development. J Cent South Univ (Med Sci). 2012;2(37):152–5. [in Chinese]. 17. Liu K, Zhang MF, Zhang LF, Po XQ. How to test the reliability of measure- ment tools in nursing research. J Nurs Train. 2015;30(13):1201–1203. [in Chinese]. Received: 17 May 2022 Accepted: 29 August 2022 18. Wei XQ, Yu DM, Ju LH, Cheng X, Zhao LY. Analysis of the correlation between eating away from home and BMI in adults 18 years and older in China: data from the CNNHS 2015. Nutrients. 2021;14(1):146. https://doi. org/10.3390/nu14010146. Consent for publication Not applicable. Not applicable. 13. Beaton DE, Bombardier C, Guillemin F, Ferraz MB. Guidelines for the process of cross-cultural adaptation of self-report measures. Spine (Phila Pa 1976). 2000;25(24):3186–91. https://doi.org/10.1097/00007632-20001 2150-00014. Availability of data and materials The datasets generated and analyzed during the current study are not pub- licly available due to the requirements of the National Cardiovascular Center of China for permitting access to foreign researchers, but they are available from the corresponding author upon a reasonable request. Liu et al. BMC Public Health (2022) 22:1671 Page 8 of 8 Liu et al. BMC Public Health (202 Ethics approval and consent to participate 11. Mokkink LB, Terwee CB, Patrick DL, Alonso J, Stratford PW, Knol DL, et al. The COSMIN checklist for assessing the methodological quality of studieson measurement properties of health status measurement instruments:an international Delphi study. Qual Life Res. 2010;19(4):539– 49. https://doi.org/10.1007/s11136-010-9606-8. This study has been approved by the Ethics Committee of the Affiliated Hospital of Guilin Medical University [Approval Number: QTLL202157]. During the evaluation process, the subjects of the study gave their informed consent and signed consent forms on site. All procedures methods were conducted in accordance with relevant guidelines and regulations of the Declaration of Helsinki. 12. Von Elm E, Altman DG, Egger M, PocockSJ GPC. Vandenbroucke JP.STROBE initiative. The strengthening the reporting of observational studies in epidemiology (STROBE) statement: guidelines for reporting observational studies. Int J Surg. 2014;12(12):1495–9 https://doi.org/10. 1016/j.ijsu.2014.07.013. Competing interests The authors have no conficts of interest to disclose. Publisher’s Note S i N i Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. 2. Roth GA, Mensah GA, Johnson CO, Addolorato G, Ammirati E, Bad- dour LM, et al. Global burden of cardiovascular diseases and risk factors, 1990-2019: update from the GBD 2019 study. J Am Coll Cardiol. 2020;76(25):2982–3021. 3. Chinese Preventive Medicine Association, Branch of Heart Disease Pre- vention and Control, Chinese Preventive Medicine Association, Chinese Diabetes Society, Branch of Stroke Prevention and Control, Chinese Preventive Medicine Association, Chinese Health Management Society, Branch of Non-communicable Chronic Disease Prevention and Control, Chinese Preventive Medicine Association, Branch of Hypertension, et al. Chinese guideline on healthy lifestyle to prevent cardiometabolic diseases. Chinese Circ J. 2020;35(3):209–9. [in Chinese]. 4 H l EL D i f h l h b li f d b h i L P h l i l 3. Chinese Preventive Medicine Association, Branch of Heart Disease Pre- vention and Control, Chinese Preventive Medicine Association, Chinese Diabetes Society, Branch of Stroke Prevention and Control, Chinese Preventive Medicine Association, Chinese Health Management Society, Branch of Non-communicable Chronic Disease Prevention and Control, Chinese Preventive Medicine Association, Branch of Hypertension, et al. Chinese guideline on healthy lifestyle to prevent cardiometabolic diseases. Chinese Circ J. 2020;35(3):209–9. [in Chinese]. 4 Hartley EL Determinants of health beliefs and behavior L Psychological 4. Hartley EL. Determinants of health beliefs and behavior. L. Psychological determinants. Am J Public Health Nations Health. 1961;51:1541–7. 5. Lim BC, Kueh YC, Arifin WN, Ng KH. Modelling knowledge, health beliefs, and health-promoting behaviours related to cardiovascular disease prevention among Malaysian university students. PLoS One. 2021;4(16):e0250627. https://doi.org/10.1371/journal.pone.0250627. 6. Liu YJ, Wang M, Dong XF, Xu QL. Reliability and vaIidity of the Chinese version health beIiefs related to cardiovascular disease scaIe. J Nurs Sci. 2015;30(15):23–5 [in Chinese]. 7. Woringer M, Nielsen JJ, Zibarras L, Evason J, Kassianos AP, Harris M, et al. Development of a questionnaire to evaluate patients’ awareness of car- diovascular disease risk in England’s National Health Service Health Check preventive cardiovascular programme. BMJ Open. 2017;7(9):e014413. https://doi.org/10.1136/bmjopen-2016-014413. • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. 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Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: 8. Martos T, Csabai M, Bagyura Z, Ocsovszky Z, Rafael B, Sallay V, et al. Cardiovascular disease risk perception in a Hungarian community sample: psychometric evaluation of the ABCD risk perception question- naire. BMJ Open. 2020;10(7):e036028. https://doi.org/10.1136/bmjop en-2019-036028. 8. Martos T, Csabai M, Bagyura Z, Ocsovszky Z, Rafael B, Sallay V, et al. Cardiovascular disease risk perception in a Hungarian community sample: psychometric evaluation of the ABCD risk perception question- naire. BMJ Open. 2020;10(7):e036028. https://doi.org/10.1136/bmjop en-2019-036028. 9. Hassen HY, Aerts N, Demarest S, Manzar MD, Abrams S, Bastiaens H. Vali- dation of the Dutch-Flemish translated ABCD questionnaire to measure cardiovascular diseases knowledge and risk perception among adults. Sci Rep. 2021;11(1):8952. https://doi.org/10.1038/s41598-021-88456-5. 10. Hassen HY, Bowyer M, Gibson L, Abrams S, Bastiaens H. Level of cardiovas- cular disease knowledge, risk perception and intention towards healthy
https://openalex.org/W1898800845	https://portal.research.lu.se/files/3461767/8839552	English	null	Birth position and obstetric anal sphincter injury: a population-based study of 113 000 spontaneous births	BMC pregnancy and childbirth	2,015	cc-by	6,534	Citation for published version (APA): Elvander, C., Ahlberg, M., Thies-Lagergren, L., Cnattingius, S., & Stephansson, O. (2015). Birth position and obstetric anal sphincter injury: a population-based study of 113 000 spontaneous births. BMC Pregnancy and Childbirth, 15, Article 252. https://doi.org/10.1186/s12884-015-0689-7 Birth position and obstetric anal sphincter injury: a population-based study of 113 000 spontaneous births. Birth position and obstetric anal sphincter injury: a population-based study of 113 000 spontaneous births. Elvander, Charlotte; Ahlberg, Mia; Thies-Lagergren, Li; Cnattingius, Sven; Stephansson, Olof Elvander, Charlotte; Ahlberg, Mia; Thies-Lagergren, Li; Cnattingius, Sven; Step Published in: BMC Pregnancy and Childbirth Published in: BMC Pregnancy and Childbirth DOI: 10.1186/s12884-015-0689-7 Link to publication Citation for published version (APA): Elvander, C., Ahlberg, M., Thies-Lagergren, L., Cnattingius, S., & Stephansson, O. (2015). Birth position and obstetric anal sphincter injury: a population-based study of 113 000 spontaneous births. BMC Pregnancy and Childbirth, 15, Article 252. https://doi.org/10.1186/s12884-015-0689-7 Citation for published version (APA): Elvander, C., Ahlberg, M., Thies-Lagergren, L., Cnattingius, S., & Stephansson, O. (2015). Birth position and obstetric anal sphincter injury: a population-based study of 113 000 spontaneous births. BMC Pregnancy and Childbirth, 15, Article 252. https://doi.org/10.1186/s12884-015-0689-7 Total number of authors: 5 5 RESEARCH ARTICLE Open Access © 2015 Elvander et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. 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Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. LUND UNIVERSITY PO Box 117 221 00 Lund +46 46-222 00 00 Elvander et al. BMC Pregnancy and Childbirth (2015) 15:252 DOI 10.1186/s12884-015-0689-7 Abstract Background: The association between birth position and obstetric anal sphincter injury (OASIS) in spontaneous vaginal deliveries is unclear. Methods: The study was based on the Stockholm-Gotland Obstetric Database (Sweden) from Jan 1st 2008 to Oct 22nd 2014 and included 113 279 singleton spontaneous vaginal births with no episiotomy. We studied risk of OASIS with respect to the following birth positions: a) sitting, b) lithotomy, c) lateral, d) standing on knees, e) birth seat, f) supine, g) squatting, h) standing and i) all fours. All analyses were stratified for parity. General linear models were used to calculate risk ratios (RR) adjusted for maternal, pregnancy and fetal characteristics. Results: The rates of OASIS among nulliparous women, parous women and women undergoing vaginal birth after a caesarean (VBAC) were 5.7 %, 1.3 % and 10.6 %, respectively. The rates varied by birth position: from 3.7 to 7.1 % in nulliparous women, 0.6 % to 2.6 % in parous women and 5.6 % to 18.2 % in women undergoing VBAC. Regardless of parity, the lowest rates were found among women giving birth in standing position and the highest rates among women birthing in the lithotomy position. Compared with sitting position, the lithotomy position involved an increased risk of OASIS among nulliparous (adjusted RR 1.17, 95 % CI 1.06-1.29) and parous women (adjusted RR 1.66, 95 % CI 1.35-2.05). Birth seat and squatting position involved an increased risk of OASIS among parous women (adjusted RR [95 % CI] 1.36 [1.03-1.80] and 2.16 [1.15-4.07], respectively). Independent risk factors for OASIS were maternal age, head circumference ≥35 cm, birth weight ≥4000 g, length of gestation ≥40 weeks, prolonged second stage of labour, non-occiput anterior presentation and oxytocin augmentation. Conclusions: Compared with sitting position, lateral position has a slightly protective effect in nulliparous women whilst an increased risk is noted among women in the lithotomy position, irrespective of parity. Squatting and birth seat position involve an increase in risk among parous women. Keywords: OASIS, Anal sphincter tear, Upright delivery, Perineal tear Birth position and obstetric anal sphincter injury: a population-based study of 113 000 spontaneous births Charlotte Elvander1, Mia Ahlberg1, Li Thies-Lagergren2, Sven Cnattingius1 and Olof Stephansso harlotte Elvander1, Mia Ahlberg1, Li Thies-Lagergren2, Sven Cnattingius1 and Olof Stephansson1 * Correspondence: li.thies-lagergren@med.lu.se 2The Department of Health Sciences, Faculty of Medicine, Lund University, Box 117, 221 00 Lund, Sweden Full list of author information is available at the end of the article Background recumbent/sitting or lithotomy position [4–7]. It is claimed that these positions enable the attending mid- wife or obstetrician to monitor the fetus and facilitate a hands-on approach to perineal management to lower the risk of obstetric anal sphincter injuries (OASIS) [8]. For centuries, pros and cons about different birth posi- tions have been debated. For upright labour, several physiological advantages have been hypothesized and measured, such as effects of gravity, stronger uterine contractions, maternal satisfaction and feeling of con- trol [1–3]. However, today the majority of women in the Western societies deliver in a dorsal, semi- Obstetric anal sphincter injuries are related to long term maternal complications, such as anal incontinence [9–11], sexual dysfunction [12], pain [13] and a reduced quality of life [14–16]. The rates of OASIS have increased in Sweden and in many other high-income countries over the last decades [17–20]. It has been unclear whether such trends reflect differences in * Correspondence: li.thies-lagergren@med.lu.se 2The Department of Health Sciences, Faculty of Medicine, Lund University, Box 117, 221 00 Lund, Sweden Full list of author information is available at the end of the article Elvander et al. BMC Pregnancy and Childbirth (2015) 15:252 Page 2 of 9 study cohort consisted of 113 279 live singleton term non-instrumental births. populations, differences in diagnosis and registration, or differences in management of delivery between and within countries [19, 21–23]. Previously reported non-modifiable risk factors for OASIS are primiparity [22, 24], previous caesarean de- livery [22, 25], high birth weight [17, 22, 24, 26–29], oc- ciput posterior position [26, 17–30, 31], prolonged second stage of labour [28, 29, 30] and increasing gesta- tional age [24, 30]. Studies disagree on whether maternal age influences risk of OASIS [5, 22, 17]. Modifiable risk factors include instrumental delivery [20, 25, 28, 17–31, 32] augmentation [26] and midline episiotomy [32]. In- consistent findings have also been reported for the as- sociation between birth position and OASIS [4, 5, 33, 34] Further, considering women’s right to make choices concerning birth position, it is also important to exam- ine associations between birth position and risk of OASIS. Exposures h d The midwife reported birth position in the medical record system within the next hours after birth. The recording system allowed the midwife to choose be- tween seven different positions which (translated from Swedish) were; sitting, lithotomy, lateral, standing on knees, supine squatting, and standing. If another birth position was used it was documented using free text under the heading of “Other:…”. All births on the birth seat and in all fours position were collected from this heading. Remaining births in free text were sorted into appropriate birth positions if possible. The residual 166 births that were not understandable were collapsed with 583 births that did not have any infor- mation on birth position into the birth position cat- egory “unknown”. There were no specific criteria for each position; the midwife freely decided which head- ing she/he judged to best describe the birth position used by the woman. The collected data does not re- veal level of elevation of the head of the bed. In the present investigation, we used the following categories of birth position: a) sitting, b) lithotomy, c) lateral, d) standing on knees, e) birth seat, f) supine, g) squat- ting, h) standing or i) all fours. The most frequently used birth position was sitting, which was used as reference category. The aim of this study was to investigate the associ- ation between birth position and occurrence of OASIS in spontaneous vaginal deliveries using a large population- based cohort in Sweden. Covariates Selection of potential confounding factors were based on biological plausibility and on results from previous studies. From the partograph and birth records we obtained information on maternal age, induction of labor, use of epidural analgesia (EDA), oxytocin aug- mentation, duration of second stage, head circumfer- ence, presentation, birth weight, and hospital delivery unit. Data on maternal weight and height for calcula- tion of body mass index (BMI) was retrieved from Outcome measurements Th The primary outcome was obstetric anal sphincter in- jury (OASIS). Cases were defined by having both a Diagnosis Related Groups (DRG) code (i.e. a surgical code for suturing of OASIS) in combination with a definition by the midwife or the obstetrician by a checkbox in the obstetric electronic case notes and/or International Classification of Diseases, tenth revision (ICD-10) code for sphincter injury. The DRG code used was MBC33 and the following ICD-10 codes: O702, O702C, O702D, O702X (grade III which in- volve the anal sphincter complex) and O703 (grade IV, which extend to the rectal mucosa). In Sweden, midwives are the primary caregivers during normal labour and birth. However, if compli- cations occur, the midwife will notify an obstetrician. Swedish midwives diagnose and suture first and sec- ond degree perineal lacerations. If a midwife is un- sure of the degree of the laceration or suspects a larger laceration (third or fourth degree), an obstetri- cian will be called upon for diagnosis and repair of the trauma. Methods Data on mother, delivery and infant characteristics were obtained from the population-based Stockholm- Gotland Obstetric Database, based on the medical record system used for all maternity, delivery and postnatal care units in the region. Data from the medical record system is forwarded daily to the data- base, which contains information from 2008 and on- wards. The database includes prospectively recorded standardized information from antenatal care, delivery (with partograph data) and the postpartum period for both mother and infant. Statistical analyses Nulliparous and parous women were analysed separ- ately. Furthermore, women with vaginal birth after a primary caesarean section (VBAC) have shown to be at higher risk for OASIS than both nulliparous and other parous women [34]. Women with VBAC (n = 2 828) were therefore included in a separate group. Character- istics of the nulliparous women, parous women and women undergoing VBAC in relation to birth position were estimated and presented along with prevalence rates of OASIS in descriptive Tables 1 and 2. Due to low numbers of OASIS in each birth position group, further analyses were not performed on the VBAC group. Table 1 shows maternal, neonatal and obstetrical char- acteristics of the nulliparous women, parous women and women undergoing VBAC in relation to birth position. Irrespective of parity, women giving birth in the lithotomy position were characterized by high rates of induction, EDA, oxytocin augmentation, long second stages, infants with large head circumferences, high birth weights and births of infants presenting in non-occiput anterior presentations. The prevalence of OASIS among nulliparous women was 5.7 %, among parous women 1.3 % and among women undergoing VBAC 10.6 % (Table 2). The rate of OASIS varied by birth position: from 3.7 to 7.1 % in nul- liparous women, 0.6 % to 2.6 % in parous women and 5.6 % to 18.2 % in women undergoing VBAC. The low- est rates were, regardless of parity, found among women giving birth in standing position. We used general linear models to calculate risk ratios (RRs) and 95 % confidence intervals (CI) to estimate as- sociations between OASIS and the following categorical variables: maternal age (<35 or ≥35 years), maternal height (<167 or ≥167 cm), BMI (<25 or ≥25 kg/m2), head circumference (<35 or ≥35 cm), birth weight (<4000 or ≥4000 g), gestational age (<40 or ≥40 weeks), EDA (no/yes), augmentation with (synthetic) oxytocin (no/yes), duration of second stage (<90 or ≥90 min), non-occiput anterior presentation (no/yes), induction (no/yes) hospital delivery units (A, B, C, D, E, F and G) and changes over time in years. In Sweden, dystocia dur- ing second stage refers to >2 h for the descending phase alternatively >3 h if epidural anesthesia is used, and >1 h for the pushing phase. There are no clear differences made with regards to parity. Study population h d During the study period (from January 1st, 2008, through Oct 22nd, 2014), 175 522 singleton births were recorded. We excluded cesarean and vaginal instru- mental births (n = 49 422), preterm births (≤36 com- pleted weeks; n = 4 408), births that required an episiotomy (n = 4 508), births in non-cephalic presenta- tions (n = 3 749), and stillbirths (n = 156). The final Elvander et al. BMC Pregnancy and Childbirth (2015) 15:252 Elvander et al. BMC Pregnancy and Childbirth (2015) 15:252 Page 3 of 9 Page 3 of 9 2009/275-31 and No. 2012/365-32). According to the Per- sonal Data Act in Sweden, informed consent does not have to be obtained from each individual in this type of registry based research. the records of the first antenatal visit, usually in the first trimester. Hospital delivery unit was included as a potential confounder to adjust for potential cluster effects. Gestational age was determined using the fol- lowing hierarchy: a) date of embryo transfer (3.0 %), b) early second trimester ultrasound (95.2 %), c) date of last menstrual period reported at the first antenatal visit (1.8 %) and d) from a postnatal assessment (<1 %). Duration of second stage was determined using the recordings of exact times in the mother’s electronic case notes from full dilatation to delivery. This variable had 7 % missing among nulliparous women and 24 % missing among parous women. Results In total, we studied 44 942 births to nulliparous women, 65 486 births to parous women, and 2828 births to women undergoing VBAC. The most frequently used birthing position was sitting, both among nulliparous women (38.5 %), parous women (41.3 %) and women undergoing VBAC (37.3 %) (Fig. 1). There were large variations in practice of birth po- sitions amongst the seven in-hospital delivery units. For instance, the rate of the sitting position varied from 17.0 to 60.7 % and the use of birth seat from 0.3 to 33.0 % (Fig. 2). Statistical analyses Due to this quite unclear description and definition of a long second stage in Sweden we decided to use 90 min for all women. Among nulliparous women and compared with the sit- ting position, the lithotomy position was associated with a modestly increased risk of OASIS in the adjusted ana- lysis (Table 3). Only lateral position was associated with a reduced risk of OASIS in nulliparous women (adjusted RR 0.79, 95 % CI 0.68-0.92). Among parous women, the unadjusted analyses showed that lithotomy position was, compared with sit- ting position, associated with a more than two-fold in- creased risk of OASIS (Table 3). This increased risk was attenuated after adjustments. The birth seat position was associated with almost a 40 % increased risk of OASIS, and this increased risk was not attenuated after adjust- ments. Also in parous women, squatting position was not significantly associated with OASIS in the univariate analyses. In in the adjusted analysis, squatting position was associated with a more than two-fold increased risk of OASIS (Table 3). Other birth positions were not asso- ciated with risk of OASIS in parous women. The covariates were then entered into the multivari- able model, and variables that remained significant after adjustments for either nulliparous or parous women were presented. The statistical software package SPSS 20.0 (SPSS Inc., Chicago, IL, USA) was used for all data analyses. The regional ethical committee at Karolinska Institutet, Stockholm, Sweden approved the study protocol (No. Statistical analyses Table 1 Prevalence of maternal and neonatal characterstics in relation to birth position Characteristics (%) Total Sitting Lithotomy Lateral Knee Birth seat Supine Squatting Standing All fours Unknown Nulliparous women Number 44 942 17 294 11 906 5850 2660 4913 1214 342 243 235 285 % Maternal age >35 years 13 11 14 12 14 14 10 10 17 15 17 Head circumference >35 cm 55 53 58 54 53 57 51 47 53 61 53 Birthweight > 4000 g 11 10 14 10 10 11 10 10 12 14 12 Length of second stage >90 min 45 43 58 27 35 39 36 39 37 40 45 Gestational age >40 weeks 60 59 63 59 59 62 54 57 61 65 59 Induction 15 14 19 14 10 11 14 13 7 8 18 Epidural analgesia 57 57 68 53 43 51 50 56 42 53 55 Non-occiput anterior presentation 3 2 4 2 1 2 2 3 3 2 14 Oxytocin augmentation 61 60 79 52 43 50 50 54 40 51 58 Parous women Number 65 486 27 054 7410 12 631 7478 5025 3687 469 858 427 447 % Maternal age >35 years 35 32 36 36 41 40 31 34 45 41 35 Head circumference >35 cm 65 62 71 67 67 71 59 63 67 69 65 Birthweight > 4000 g 22 20 29 22 23 24 16 22 24 28 24 Length of second stage >90 min 7 5 16 5 4 7 3 9 7 8 6 Gestational age >40 weeks 57 55 61 57 59 61 52 57 59 61 56 Induction 14 14 21 14 9 9 15 11 10 11 33 Epidural analgesia 26 26 39 25 19 28 18 28 16 20 24 Non-occiput anterior presentation 4 3 7 4 2 4 3 4 2 3 20 Oxytocin augmentation 26 25 48 23 17 23 20 25 16 19 22 Women undergoing VBAC Number 2828 1054 864 387 149 225 77 21 18 16 17 % Maternal age >35 years 31 28 32 32 37 29 29 38 50 38 35 Head circumference >35 cm 63 62 67 62 68 61 54 57 71 75 53 Birthweight > 4000 g 18 19 18 18 18 17 12 19 22 50 0 Length of second stage >90 min 39 36 50 34 23 42 29 40 12 31 29 Gestational age >40 weeks 61 60 62 62 55 68 58 62 56 69 47 Induction 15 14 18 13 11 11 10 24 6 6 18 Epidural analgesia 62 60 71 58 53 59 56 67 33 69 47 Non-occiput anterior presentation 2 2 3 2 0 2 1 5 6 6 18 Oxytocin augmentation 58 55 71 49 41 51 48 57 28 63 53 Elvander et al. Statistical analyses BMC Pregnancy and Childbirth (2015) 15:252 Page 4 of 9 Elvander et al. BMC Pregnancy and Childbirth (2015) 15:252 Page 5 of 9 Page 5 of 9 Table 2 Prevalence (%) of OASIS in relation to birth position Total Sitting Lithotomy Lateral Knee Birth seat Supine Squatting Standing All fours Unknown Nulliparous women N of OASIS 2 548 931 850 250 126 279 58 15 9 13 17 OASIS (%) 5.7 5.4 7.1 4.3 4.7 5.7 4.8 4.4 3.7 5.5 6.0 3rd degree (%) 5.2 4.9 6.5 4.0 4.3 5.1 4.4 3.8 3.7 5.5 5.5 4th degree (%) 0.5 0.5 0.6 0.3 0.4 0.6 0.4 0.6 0.0 0.0 0.5 Parous women N of OASIS 859 323 194 124 70 82 37 10 5 6 8 OASIS (%) 1.3 1.2 2.6 1.0 0.9 1.6 1.0 2.1 0.6 1.4 1.8 3rd degree (%) 1.1 1.1 2.0 0.9 0.8 1.4 0.9 1.7 0.6 1.2 1.7 4th degree (%) 0.2 0.1 0.6 0.1 0.1 0.2 0.1 0.4 0.0 0.2 0.1 Women undergoing VBAC N of OASIS 301 100 106 32 16 27 13 3 1 1 2 OASIS (%) 10.6 9.5 12.3 8.3 10.7 12.0 18.2 14.3 5.6 6.3 11.8 3rd degree (%) 10.2 9.5 10.9 7.4 10.7 11.6 18.2 14.3 5.6 1.2 11.8 4th degree (%) 0.6 0.0 1.4 1.0 0.0 0.4 0.0 0.0 0.0 0.2 0.0 Table 2 Prevalence (%) of OASIS in relation to birth position In multivariate analyses of nulliparous and parous women, we also found the following factors to be associ- ated with increased risk of OASIS: large head circumfer- ence, high birth weight, prolonged second stage of labour, gestational age ≥40 weeks, and non-occiput an- terior presentation (Table 3). High maternal age, oxyto- cin augmentation and hospital delivery unit was only associated with increased risk of OASIS in nulliparous women. Epidural analgesia had a protective effect in nul- liparous women but was not significantly associated with OASIS in parous women. Maternal height, BMI and in- duction were not associated with increased risks of OASIS in the adjusted analysis (data not shown). position also implied an increased risk of OASIS in parous women. Compared with the reference category of sitting position, lateral position was associated with a slightly protective effect in nulliparous women. Statistical analyses Irrespective of parity, births in the lithotomy position had higher rates of OASIS but were more often accom- panied by other risk factors for OASIS, such as high birth weight, large head circumference, non-occiput an- terior presentation, oxytocin augmentation and a pro- longed second stage of labour. These factors partly explained the association between lithotomy position and risk of OASIS. The increased risk that remained after adjustments supports findings from a Swedish ob- servational cohort study [4]. Two additional studies have shown slightly increased risks of perineal trauma in lithot- omy position, but these studies were not powered to spe- cifically study birth position and risk of OASIS [35, 36]. Possible explanations of the association between lithotomy Discussion We found that the lithotomy position was associated with increased risk of OASIS, especially in parous women. The less frequently used birth seat and squatting Fig. 1 Rates of birthing positions (%) among nulliparous women, parous women and women undergoing VBAC, respectively Fig. 1 Rates of birthing positions (%) among nulliparous women, parous women and women undergoing VBAC, respectively Elvander et al. BMC Pregnancy and Childbirth (2015) 15:252 Page 6 of 9 Fig. 2 Rates (%) of birth positions used in relation to in-hospital birth unit (a-g) The increased rate of OASIS among women undergo- ing VBAC may be explained by fetopelvic disproportion leading to a primary caesarean section possibly predis- posing to OASIS at first vaginal delivery [25, 38]. We lacked information about indication for these women’s caesarean sections and if the birthing process in women undergoing VBAC differed from non-VBAC women. birth position and risk of OASIS may be that lithotomy position increases pressure sensations in the perineal area and decreases the woman’s ability to moderate the tempo of her own pushing efforts [35]. Further, the lithotomy position might increases the risk of OASIS by causing a greater pressure towards the sphincter during the expul- sion of the infant compared with alternate positions. The 6 % rate of OASIS among nulliparous women in our study population is reflected by the official rate re- ported by The National Board of Health and Welfare [39]. There is a significant difference in the Nordic countries in the incidence of OASIS [23]. It has been hy- pothesized that differences in delivery techniques and use of episiotomies may be one reason [23]. Although a Norweigan intervention study suggest that the obstetric practice and hands on practice makes a difference [37] another meta-analysis concluded that hands off (or poised) versus hands on showed no effect on third- and fourth-degree tears [40]. In Sweden, episiotomies are un- common with a rate of <8 % among nulliparous women, while in Norway and Finland, corresponding rates are 23 % and 65 %, respectively [37, 41]. We found a doubled risk of OASIS in squatting pos- ition among parous women. The increased risk in this position has been reported previously [4]. In contrast to others [5], we analysed birth seat position and squatting separately. Discussion Women in the squatting position lean for- ward more than women in the birth seat position who most often lean back into the arms of their partner sit- ting behind on a chair. Although an association between birth seat position and increase in second-degree tears previously has been reported [8], the association be- tween birth seat and increased risk of OASIS in parous women found in our study is a novel and important finding. It is plausible that both the squatting position and the use of birth seat may lead to a too rapid expul- satory phase in parous women. This, in combination with challenging circumstances for the midwife to hands on protect the perineum may increase the risk of OASIS. Slowing the delivery of the infants’ head and supporting the perineum are interventions that have shown to lower the risk of OASIS [37]. Strengths and limitations Major strengths of this study are the large sample size of births from all maternity and delivery units within a geo- graphically defined area, and that data were obtained Elvander et al. BMC Pregnancy and Childbirth (2015) 15:252 Page 7 of 9 Page 7 of 9 from prospectively recorded information in antenatal management techniques [40] and such information was Table 3 Unadjusted and adjusted risk ratios and 95 % CI for OASIS among nulliparous and parous Nulliparous (n = 44 942) Parous (n = 65 486) Unadjusted risk ratio Adjusted risk ratio* Unadjusted risk ratio Adjusted risk ratio* 95 % CI 95 % CI 95 % CI 95 % CI Total Sitting 1.00 1.00 1.00 1.00 Lithotomy 1.33 (1.21-1.46) 1.17 (1.06-1.29) 2.19 (1.83-2.62) 1.66 (1.35-2.05) Lateral 0.79 (0.69-0.91) 0.79 (0.68-0.92) 0.82 (0.67-1.01) 0.84 (0.66-1.06) Knee 0.88 (0.73-1.06) 0.88 (0.71-1.09) 0.78 (0.60-1.00) 0.81 (0.58-1.11) Birth seat 1.05 (0.92-1.21) 1.05 (0.90-1.22) 1.37 (1.07-1.74) 1.36 (1.03-1.80) Supine 0.89 (0.68-1.16) 0.91 (0.67-1.22) 0.84 (0.60-1.18) 0.84 (0.55-1.30) Squatting 0.81 (0.49-1.36) 0.66 (0.36-1.24) 1.79 (0.95-3.35) 2.16 (1.15-4.07) Standing 0.69 (0.36-1.33) 0.71 (0.34-1.49) 0.49 (0.20-1.18) 0.40 (0.10-1.61) All four 1.03 (0.59-1.78) 1.12 (0.62-2.05) 1.18 (0.53-2.64) 0.95 (0.30-2.96) Maternal age (years) <35 1.00 1.00 1.00 1.00 >35 1.25 (1.12-1.39) 1.21 (1.08-1.36) 1.13 (0.99-1.30) 1.12 (0.96-1.32) Head circumference <35 1.00 1.00 1.00 1.00 >35 1.76 (1.62-1.92) 1.42 (1.29-1.57) 1.95 (1.65-2.31) 1.52 (1.23-1.88) Birth weight <4000 g 1.00 1.00 1.00 1.00 >4000 g 2.16 (1.96-2.39) 1.69 (1.52-1.88) 2.54 (2.22-2.91) 1.86 (1.57-2.21) Length of second stage <90 min 1.00 1.00 1.00 1.00 >90 min 1.36 (1.25-1.47) 1.15 (1.05-1.26) 2.44 (1.98-3.01) 1.93 (1.53-2.45) Length of gestation (weeks) <40 1.00 1.00 1.00 1.00 >40 1.50 (1.38-1.64) 1.20 (1.09-1.31) 1.64 (1.42-1.87) 1.25 (1.04-1.49) Epidural analgesia No 1.00 1.00 1.00 1.00 Yes 1.02 (0.94-1.10) 0.82 (0.74-0.90) 1.28 (1.11-1.47) 1.01 (0.88-1.24) Non-occiput anterior presentation No 1.00 1.00 1.00 1.00 Yes 1.48 (1.20-1.82) 1.30 (1.01-1.68) 1.92 (1.47-2.51) 1.51 (1.10-2.05) Oxytocin augmentation No 1.00 1.00 1.00 1.00 Yes 1.31 (1.21-1.42) 1.21 (1.09-1.35) 1.33 (1.15-1.53) 0.93 (0.78-1.12) *Adjusted for all other variables in table + time in years + in-hospital delivery unit nadjusted and adjusted risk ratios and 95 % CI for OASIS among nulliparous and parous management techniques [40] and such information was not systematically documented. Furthermore, we were unable to analyse the midwives’ experience and training, which could impact on outcome. Strengths and limitations However, all midwives at the hospital delivery units have received similar mid- wifery training even though the attitudes towards en- couraging women to deliver in alternate positions might differ [8, 42]. from prospectively recorded information in antenatal and obstetrical records. We were able to control for a number of possible confounders, including cluster vari- ation between hospitals. The restriction of analyses to women with no episiotomy is a strength as it increases clarity although it limits generalizability. Lack of infor- mation on fundal pressure [26] and perineal protection is a limitation. Midwives may use various perineal Page 8 of 9 Page 8 of 9 Elvander et al. BMC Pregnancy and Childbirth (2015) 15:252 This is an observational study, and the results cannot be causally interpreted. However, the findings are bio- logically plausible and have partially been supported in other studies [4, 8, 43]. 10. Pollack J, Nordenstam J, Brismar S, Lopez A, Altman D, Zetterstrom J. Anal incontinence after vaginal delivery: a five-year prospective cohort study. Obstet Gynecol. 2004;104(6):1397–402. 11. Fenner DE, Genberg B, Brahma P, Marek L, DeLancey JO. Fecal and urinary incontinence after vaginal delivery with anal sphincter disruption in an obstetrics unit in the United States. Am J Obstet Gynecol. 2003;189(6):1543–9. discussion 9–50. References 1. Michel SC, Rake A, Treiber K, Seifert B, Chaoui R, Huch R, et al. MR obstetric pelvimetry: effect of birthing position on pelvic bony dimensions. AJR Am J Roentgenol. 2002;179(4):1063–7. 1. Michel SC, Rake A, Treiber K, Seifert B, Chaoui R, Huch R, et al. MR obstetric pelvimetry: effect of birthing position on pelvic bony dimensions. AJR Am J Roentgenol. 2002;179(4):1063–7. 1. 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Risk factors for anal sphincter tear during vaginal delivery. Obstet Gynecol. 2007;109(1):29–34. 7. Sandin-Bojo AK, Kvist LJ. Care in labor: a Swedish survey using the Bologna Score. Birth. 2008;35(4):321–8. 31. Wheeler 2nd TL, Richter HE. Delivery method, anal sphincter tears and fecal incontinence: new information on a persistent problem. Curr Opin Obstet Gynecol. 2007;19(5):474–9. 8. Author details 1 22. Baghestan E, Irgens LM, Bordahl PE, Rasmussen S. Trends in risk factors for obstetric anal sphincter injuries in Norway. Obstet Gynecol. 2010;116(1):25–33. 1Department of Medicine, Division of Clinical Epidemiology, T2, Karolinska University Hospital, 171 76 Stockholm, Sweden. 2The Department of Health Sciences, Faculty of Medicine, Lund University, Box 117, 221 00 Lund, Sweden. 23. Laine K, Gissler M, Pirhonen J. Changing incidence of anal sphincter tears in four Nordic countries through the last decades. Eur J Obstet Gynecol Reprod Biol. 2009;146(1):71–5. Received: 1 June 2015 Accepted: 5 October 2015 Received: 1 June 2015 Accepted: 5 October 2015 24. Zetterstrom J, Lopez A, Anzen B, Norman M, Holmstrom B, Mellgren A. Anal sphincter tears at vaginal delivery: risk factors and clinical outcome of primary repair. Obstet Gynecol. 1999;94(1):21–8. 25. Raisanen S, Vehvilainen-Julkunen K, Cartwright R, Gissler M, Heinonen S. A prior cesarean section and incidence of obstetric anal sphincter injury. Int Urogynecol J. 2013;24(8):1331–9. Competing interest p g The authors declare that they have no competing interests. 17. Ekeus C, Nilsson E, Gottvall K. Increasing incidence of anal sphincter tears among primiparas in Sweden: a population-based register study. Acta Obstet Gynecol Scand. 2008;87(5):564–73. Authors’ contributions CE d l d th i i CE developed the original idea for the study, performed the analyses and wrote a majority of the manuscript. SC, OS, MA and LTL assisted with design and interpretation of study findings and contributed to writing and revising the manuscript. All authors read and approved the final manuscript. 18. Ampt AJ, Ford JB, Roberts CL, Morris JM. Trends in obstetric anal sphincter injuries and associated risk factors for vaginal singleton term births in New South Wales 2001–2009. Aust N Z J Obstet Gynaecol. 2013;53(1):9–16. 19. Laine K, Gissler M, Pirhonen J. Changing incidence of anal sphincter tears in four Nordic countries through the last decades.Eur J Obstet Gynecol Reprod Biol 2009;146(1): 71-75. Conclusion 12. Andrews V, Thakar R, Sultan AH, Jones PW. Evaluation of postpartum perineal pain and dyspareunia—a prospective study. Eur J Obstet Gynecol Reprod Biol. 2008;137(2):152–6. Compared with sitting position, lateral position has a slightly protective effect in nulliparous whilst an in- creased risk is noted among women in the lithotomy position, irrespective of parity. Births in the lithotomy position were accompanied by other risk factors for OASIS which partly explained the elevated risk. Squatting and birth seat position involved an increase in risk among parous women. 13. Macarthur AJ, Macarthur C. Incidence, severity, and determinants of perineal pain after vaginal delivery: a prospective cohort study. Am J Obstet Gynecol. 2004;191(4):1199–204. 14. Handa VL, Zyczynski HM, Burgio KL, Fitzgerald MP, Borello-France D, Janz NK, et al. The impact of fecal and urinary incontinence on quality of life 6 months after childbirth. Am J Obstet Gynecol. 2007;197(6):636. e1-6. 14. Handa VL, Zyczynski HM, Burgio KL, Fitzgerald MP, Borello-France D, Janz NK, et al. The impact of fecal and urinary incontinence on quality of life 6 months after childbirth. Am J Obstet Gynecol. 2007;197(6):636. e1-6. y 15. Bharucha AE, Zinsmeister AR, Locke GR, Seide BM, McKeon K, Schleck CD, et al. Prevalence and burden of fecal incontinence: a population-based study in women. Gastroenterology. 2005;129(1):42–9. 15. Bharucha AE, Zinsmeister AR, Locke GR, Seide BM, McKeon K, Schleck CD, et al. Prevalence and burden of fecal incontinence: a population-based study in women. Gastroenterology. 2005;129(1):42–9. 16. Evers EC, Blomquist JL, McDermott KC, Handa VL. Obstetrical anal sphincter laceration and anal incontinence 5–10 years after childbirth. Am J Obstet Gynecol. 2012;207(5):425. e1-6. Acknowledgements This study was supported by grants from the Swedish Research Council (2013–2429, OS and 2008–5857, SC) by grants provided by the Stockholm County Council (ALF projects 20130156, OS and 20140105, SC) and by the Swedish National Doctoral School in Health Care Sciences (CE). 20. Ismail SI, Puyk B. The rise of obstetric anal sphincter injuries (OASIS): 11-year trend analysis using Patient Episode Database for Wales (PEDW) data. J Obstet Gynaecol. 2014;34(6):495–8. 21. Baghurst PA. The case for retaining severe perineal tears as an indicator of the quality of obstetric care. Aust N Z J Obstet Gynaecol. 2013;53(1):3–8. Elvander et al. BMC Pregnancy and Childbirth (2015) 15:252 Elvander et al. BMC Pregnancy and Childbirth (2015) 15:252 Elvander et al. BMC Pregnancy and Childbirth (2015) 15:252 randomized controlled trial. Int Urogynecol J Pelvic Floor Dysfunct. 2007;18(2):141–6. randomized controlled trial. Int Urogynecol J Pelvic Floor Dysfunct. 2007;18(2):141–6. 34. Jander C, Lyrenas S. Third and fourth degree perineal tears. Predictor factors in a referral hospital. Acta Obstet Gynecol Scand. 2001;80(3):229–34. 35. Albers LL, Anderson D, Cragin L, Daniels SM, Hunter C, Sedler KD, et al. Factors related to perineal trauma in childbirth. J Nurse Midwifery. 1996;41(4):269–76. 36. Soong B, Barnes M. Maternal position at midwife-attended birth and perineal trauma: Is there an association? Birth-Iss Perinat C. 2005;32(3):164–9. 36. Soong B, Barnes M. Maternal position at midwife-attended birth and perineal trauma: Is there an association? Birth-Iss Perinat C. 2005;32(3):164–9. 37. Laine K, Skjeldestad FE, Sandvik L, Staff AC. Incidence of obstetric anal sphincter injuries after training to protect the perineum: cohort study. BMJ open. 2012;2(5). 38. Richter HE, Brumfield CG, Cliver SP, Burgio KL, Neely CL, Varner RE. Risk factors associated with anal sphincter tear: a comparison of primiparous patients, vaginal births after cesarean deliveries, and patients with previous vaginal delivery. Am J Obstet Gynecol. 2002;187(5):1194–8. 39. Swedish National Board of Health and Welfare. Statistical database of pregnancies, births and newborns. http://www.socialstyrelsen.se/statistik/ statistikdatabas/graviditeter-forlossningarochnyfodda. Accessed 20 May 2015. 40. Aasheim V, Nilsen ABV, Lukasse M, Reinar LM. Perineal techniques during the second stage of labour for reducing perineal trauma. Cochrane Database Syst Rev. 2011;12:CD006672. 41. Raisanen SH, Vehvilainen-Julkunen K, Gissler M, Heinonen S. Lateral episiotomy protects primiparous but not multiparous women from obstetric anal sphincter rupture. Acta Obstet Gynecol Scand. 2009;88(12):1365–72. 42. Kemp E, Kingswood CJ, Kibuka M, Thornton JG. Position in the second stage of labour for women with epidural anaesthesia. Cochrane Database Syst Rev. 2013;1:CD008070. 43. Thies-Lagergren L, Kvist LJ, Christensson K, Hildingsson I. No reduction in instrumental vaginal births and no increased risk for adverse perineal outcome in nulliparous women giving birth on a birth seat: results of a Swedish randomized controlled trial. BMC Pregnancy Childbirth. 2011;11:22. References Gupta JK, Hofmeyr GJ, Shehmar M. Position in the second stage of labour for women without epidural anaesthesia. Cochrane Database Syst Rev. 2012;5:CD002006. 32. Signorello LB, Harlow BL, Chekos AK, Repke JT. Midline episiotomy and anal incontinence: retrospective cohort study. BMJ. 2000;320:86–90. 9. Borello-France D, Burgio KL, Richter HE, Zyczynski H, Fitzgerald MP, Whitehead W, et al. Fecal and urinary incontinence in primiparous women. Obstet Gynecol. 2006;108(4):863–72. 33. Altman D, Ragnar I, Ekstrom A, Tyden T, Olsson SE. Anal sphincter lacerations and upright delivery postures—a risk analysis from a Page 9 of 9 Page 9 of 9 Elvander et al. BMC Pregnancy and Childbirth (2015) 15:252 Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit
https://openalex.org/W2084953613	https://kops.uni-konstanz.de/bitstream/123456789/32531/3/Bossmann_0-313994.pdf	English	null	The Association between Short Periods of Everyday Life Activities and Affective States: A Replication Study Using Ambulatory Assessment	Frontiers in psychology	2,013	cc-by	6,705	INTRODUCTION due to various types of PA (Folkins, 1976; LaFontaine et al., 1992; Fox, 1999; Bartholomew et al., 2005; Blake, 2009). Anxious and sedentary people seem to proﬁt most from PA (Folkins, 1976; LaFontaine et al., 1992). Non-depressive persons often exhibit no or minimal effects due to high baseline values of positive affect (LaFontaine et al., 1992). Other studies support the assumption that PA has a more general protective effect on psychological health (Thayer, 1987; Otto and Stemmann, 1991; Gauvin and Spence, 1996; Ekkekakis et al., 2000). The association between physical activity (PA) and affective states has been of interest for several decades (Arent et al., 2000; Netz et al., 2005). In the ﬁeld of exercise and psychological adapta- tions, it is widely acknowledged that structured and organized sports activities have positive effects on subjective well-being (LaFontaine et al., 1992; Reed and Ones, 2006). A meta-analysis carried out by Reed and Ones (2006) veriﬁes this positive effect on various mood parameters (affective states) for low to mod- erate intensity forms of PA. The meta-analysis found that the effects lasted for at least 30 min and inﬂuenced both valence (pos- itive/negative) and energetic arousal (positive activated affect). Greater enhancements were documented when the baseline mood level was lower. In addition, current research suggests that even short inter- ruptions of sedentary behavior might positively inﬂuence health and affective states (Hamilton et al., 2007; Biddle et al., 2010). For example,Thayer(1987)demonstratedthat10 minof briskwalking raised energy levels and reduced tension in adults. The immedi- ate changes lasted for 2 h. Furthermore, Ekkekakis et al. (2000) found that short walking bouts had a positive effect on affective states. This positive effect on affective states has been shown in sev- eral age and patient groups. For example, some studies, focusing especially on young adults (20–30 years old), showed that acute exercise resulted in improved positive affect and less negative affect (Gauvin et al., 1996; Hausenblas et al., 2008; LePage and Crowther, 2010; Kanning et al., 2012). In addition, numerous studies with older and depressive participants have demonstrated the immediate short-term improvements in depressive symptoms and the lasting improvements in affective states that can occur Consequently,it seems that everyday life activities such as walk- ing can be used to improve people’s subjective well-being and psychological health. ORIGINAL RESEARCH ARTICLE published: 15 April 2013 doi: 10.3389/fpsyg.2013.00102 Erschienen in: Frontiers in Psychology ; 4 (2013). - 102 http://dx.doi.org/10.3389/fpsyg.2013.00102 ORIGIN Erschienen in: Frontiers in Psychology ; 4 (2013). - 102 http://dx.doi.org/10.3389/fpsyg.2013.00102 ORIGINAL RESEARCH ARTICLE The association between short periods of everyday life activities and affective states: a replication study using ambulatory assessment Correspondence: Correspondence: Thomas Bossmann, Department of Sport and Exercise Science, University of Stuttgart, Allmandring 28, 70569 Stuttgart, Germany. e-mail: tbossmann@gmx.de Correspondence: Thomas Bossmann, Department of Sport and Exercise Science, University of Stuttgart, Allmandring 28, 70569 Stuttgart, Germany. e-mail: tbossmann@gmx.de Keywords: ambulatory assessment, affective states, mood regulation, physical activity, well-being Thomas Bossmann1, Martina Kanning 1, Susanne Koudela-Hamila2, Stefan Hey 2 and Ulrich Ebner-Priemer 2,3 Thomas Bossmann1, Martina Kanning 1, Susanne Koudela-Hamila2, Stefan Hey 2 and Ulrich Ebner-Priemer 2,3 1 Department of Sport and Exercise Science, University of Stuttgart, Stuttgart, Germany 2 Department of Sport and Sport Science and House of Competence, Karlsruhe Institute of Technology, Karlsruhe, Germany 3 Department of Psychosomatic Medicine and Psychotherapy, Central Institute of Mental Health, University of Heidelberg, Mannheim, Germany 1 Department of Sport and Exercise Science, University of Stuttgart, Stuttgart, Germany 2 Department of Sport and Sport Science and House of Competence, Karlsruhe Institute of Technology, Karlsruhe, Germany 3 Department of Psychosomatic Medicine and Psychotherapy, Central Institute of Mental Health, University of Heidelberg, Ma Regularly conducted exercise programs effectively inﬂuence affective states. Studies sug- gest that this is also true for short bouts of physical activity (PA) of 10 min or less. Accordingly, everyday life activities of short duration might be used to regulate affective states. However, this association has rarely been studied in reference to unstructured activities in ongoing real-life situations. The current study examined the inﬂuence of var- ious everyday life activities on three dimensions of mood (valence, calmness, energetic arousal) in a predominantly inactive sample. Ambulatory Assessment (AA) was used to investigate the association between actual PA and affective states during the course of 1 day. Seventy-seven students ages 19–30 participated in the study. PA was assessed with accelerometers, and affective state assessments were conducted hourly using an e-diary with a six-item mood scale that was specially designed for AA. Multilevel analyses indicated that the mood dimensions energetic arousal (p = 0.001) and valence (p = 0.005) were positively inﬂuenced by the intensity of the activity carried out in the 10-min prior to the assessment. As their activity increased, the participants’ positive feelings and ener- getic arousal increased. However, the students’ calmness was not affected by their activity levels.The ﬁndings highlight the importance of integrating short activity intervals of 10 min or less into everyday life routines to improve affective states. Reviewed by: Reviewed by: Claudia Voelcker-Rehage, Jacobs University Bremen, Germany Ernst-Joachim Hossner, University of Bern, Switzerland Edited by: y Ernst-Joachim Hossner, University of Bern, Switzerland Reviewed by: Claudia Voelcker-Rehage, Jacobs University Bremen, Germany Ernst-Joachim Hossner, University of Bern, Switzerland Correspondence: Thomas Bossmann, Department of Sport and Exercise Science, University of Stuttgart, Allmandring 28, 70569 Stuttgart, Germany. e-mail: tbossmann@gmx.de Ernst-Joachim Hossner, University of Bern, Switzerland Keywords: ambulatory assessment, affective states, mood regulation, physical activity, well-being Edited by: Ernst-Joachim Hossner, University of Bern, Switzerland Reviewed by: Claudia Voelcker-Rehage, Jacobs University Bremen, Germany Ernst-Joachim Hossner, University of Bern, Switzerland *Correspondence: Thomas Bossmann, Department of Sport and Exercise Science, University of Stuttgart, Allmandring 28, 70569 Stuttgart, Germany. e-mail: tbossmann@gmx.de INTRODUCTION Unfortunately, deﬁnitions of mood, affect, and emotions are quite diverse, and a serious discussion of the differences between these theoretical constructs lies beyond the scope of this paper (for a deeper discussion, see Frijda, 1994; Cabanac, 2002; Scherer, 2009). In the current paper we assume that affective states are volatile and dynamic phenomena (Kuppens et al., 2010). As intro- duced by several authors (Matthews et al., 1990; Steyer et al., 1997; Schimmack and Grob, 2000), we will base our research on a three-dimensional scale model including valence (unwell vs. well), calmness (relaxed vs. tense), and energetic arousal (tired vs. awake). Dunton et al. (2011) assessed PA and mood in 121 chil- dren using e-diaries and activity monitors but did not report on the relationship between the two variables, as they were interested in context-speciﬁc (physical location, social context) interactions. In addition, several studies have assessed the rela- tionship between psychological variables and PA using AA in patients with pediatric affective disorders (Axelson et al., 2003), borderline personality disorder (Ebner-Priemer et al., 2008), and breast cancer (Grossman et al., 2008), as well as those who have just had joint replacement surgery (Powell et al., 2009). Innovative methods are needed to document continuous indi- vidual changes in affective states. Unlike research in traditional laboratory settings, such methods assess affective states and PA in real-life situations. Ambulatory assessment (AA) allows the repeatedassessmentof individualchangesinPAlevelsandaffective states over time in everyday life (Bussmann and Ebner-Priemer, 2011; Trull and Ebner-Priemer, 2013). The natural setting and non-invasive procedure provides a less biased assessment of the participants’ affective states. AA has often been called the gold standard in everyday life because it allows (a) the repeated real- time assessment of subjective experiences over long periods of time without retrospective distortions, (b) the real-life assessment of daily activities, and (c) the objective assessment of behav- ior using accelerometers (Bussmann et al., 2009; Bussmann and Ebner-Priemer, 2011; Trull and Ebner-Priemer, 2013). Accord- ing to the Society for Ambulatory Assessment (SAA), “Ambu- latory assessment comprises the use of ﬁeld methods to assess the ongoing behavior, physiology, experience, and environmen- tal aspects of humans or non-human primates in naturalistic or unconstrained settings. INTRODUCTION The likelihood that such forms of PA will be continuously integrated into the everyday life routines of individu- als seems higher than the likelihood that individuals will routinely participate in structured, organized physical activities that require www.frontiersin.org April 2013 \| Volume 4 \| Article 102 \| 1 Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-0-313994 April 2013 \| Volume 4 \| Article 102 \| 1 www.frontiersin.org Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-0-313994 Activities and affect: a replication Bossmann et al. more time and effort (Ekkekakis et al., 2000; Schwerdtfeger et al., 2008). more time and effort (Ekkekakis et al., 2000; Schwerdtfeger et al., 2008). more time and effort (Ekkekakis et al., 2000; Schwerdtfeger et al., 2008). studies have been conducted with healthy subjects. Schwerdtfeger et al. (2008) recorded the PA of 124 individuals using accelerom- eters and assessed mood hourly via handheld computers. They conﬁrmed that there is a signiﬁcant positive association between PA andenergetic arousaland positiveactivated affect. Signiﬁcantly, PA is typically a global score that includes intensity and duration of PA. Accordingly, higher intensity and/or duration accompany higher energetic arousal and more positive affect in Schwerdt- feger et al.’s (2008) study. Kanning et al. (2012) investigated the interaction between affective changes, PA, and autonomous reg- ulation in 44 university students using 24-h accelerometry and electronic diaries. Like Schwerdtfeger et al. (2008), the authors reported a positive association between PA and energetic arousal. However, contrary to Schwerdtfeger et al. (2008), they found no signiﬁcant effect on valence and a signiﬁcant negative effect on calmness. Only few studies have focused on everyday life activities and their effects on affective states (Axelson et al., 2003; Motl et al., 2004; Grossman et al., 2008; Schwerdtfeger et al., 2008; Ebner-Priemer et al., 2008; Powell et al., 2009; Kanning and Schlicht, 2010; Dunton et al., 2011; Kanning et al., 2012). A majority of studies on PA and mood have been conducted in controlled settings such as sports classes or training sessions (for a discussion of this aspect of the research, see Schwerdt- feger et al., 2008; Kanning et al., 2013). In addition, many of these studies have used retrospective questionnaires to assess affective states. Retrospective reports have been criticized as biased due to systematic recall errors (Trull and Ebner-Priemer, 2013). 1www.ambulatory-assessment.org INTRODUCTION Ambulatory assessment designates an eco- logically relevant assessment perspective that aims at understanding biopsychosocial processes as they naturally unfold in time and in context.”1 As demonstrated in the above discussion, studies that assess the relationship between everyday life PA and affect on the highest methodological level are rare, and their ﬁndings are contradic- tory. Therefore, the current study aims to clarify the associa- tion between PA in everyday life situations and affective states in the course of 1 day. Unlike previous researchers, we exam- ined a sample of students during their exam period; thus, these students are relatively young, ﬁt, and healthy but were inactive at the time of the study. AAs were conducted using electronic diaries and accelerometers to generate objective activity data and avoid retrospective distortions of the affect assessment. Based on recent ﬁndings, we hypothesized that the more active peo- ple are within the 10-min prior to the affect assessment, the better they feel (valence), the more activated and energetic they feel (energetic arousal), and the less calm they feel (calmness). As Gauvin and Rejeski (2000) indicated that changes in affective states at least partly follow diurnal patterns, we controlled for time of day. MATERIALS AND METHODS PARTICIPANTS The current sample originates from a larger study on workload and stress in students. The original study comprised 149 par- ticipants, but PA was assessed in a subsample of 77 students only. We had missing data in 15 subjects, resulting in a ﬁnal data set with 62 subjects. The participants were between 19 and 30 years old (M = 21.4; SD = 1.8); 53 participants were male, and 9 participants were female. The participants’body mass index var- ied between 18 and 29 (M = 22.1; SD = 2.0). The participants There are still few studies that use AA to investigate the asso- ciation between unstructured activities and affective states in real time, in real life, and repeatedly over a period of time (see Kanning et al., 2013 for an overview). To our knowledge, only three such April 2013 \| Volume 4 \| Article 102 \| 2 Frontiers in Psychology \| Movement Science and Sport Psychology Activities and affect: a replication Bossmann et al. (MDMQ; Steyer et al., 1997), which has been explicitly developed and evaluated for use in AA (Wilhelm and Schoebi, 2007). The scale contains six items that measure the basic affective states of valence (unwell vs. well), calmness (relaxed vs. tense), and energetic arousal (tired vs. awake) using two bipolar items for each sub- scale. Homogeneity was assessed at the between-person level and the within-person level. The level-speciﬁc reliability coefﬁcient for the between-person level was 0.92 for valence and 0.90 for energetic arousal and calmness. The reliability coefﬁcient for the within- personlevelwas0.70forvalence andcalmness and0.77forenergetic arousal, resulting in satisfactory internal consistency (see Wilhelm and Schoebi, 2007 for the procedure used). The subjects indicated the extent to which they were experiencing speciﬁc affective states on a six-point scale. Answers were provided by moving a slider from the left (“0,” i.e., “discontent”) to the right (“5,” i.e., “con- tent”) end of the bipolar scale. The scores for each subscale were obtained by summing the item scores, in which generated a range from“0”(low value) to“10”(high value). The electronic diary was provided on HTC Touch 2 smartphones that were programmed using the experience sampling software MyExperience movisens Edition version 594 (movisens GmbH, Karlsruhe, Germany). For data management on the smartphones, the MyExperience IDE program version 1.3.594 (movisens GmbH, Karlsruhe, Germany) was used. were recruited via a lecture on time management. ANALYSES Movement and activity patterns were recorded using an accelerometer (Move I)2. Move I consists of a triaxial acceleration sensor with a range of ±8 g, a resolution of 12 bit and a sampling frequency of 32 Hz. Each participant wore an accelerometer on a belt around his or her chest. The recorded raw data were saved in Unisens format and transferred via a USB 2.0 interface to a com- puter for further analysis. The acceleration sensor can assess both movement and posture (static acceleration due to gravity). The AC (dynamic) and the DC (static) parts of the acceleration were separated by subtracting the mean value of the signal every 4 s. The AC parts of the acceleration were then used to estimate the vector magnitude of the acceleration, and the mean value of every 1 min interval was calculated. PA was quantiﬁed using the unit“milli-g.” To relate PA to the e-diary data, we computed 10-min segments of PA for the 10-min before each e-diary entry. We used 10-min segments to maintain comparability across our studies (Kanning et al., 2012; Ebner-Priemer et al., 2013). The original decision to use a 10-min segment (more details in Ebner-Priemer et al., 2013) was based on the ﬁndings of Schwerdtfeger et al. (2008), which showed promising relationships for two time frames (5, 15 min), and was based on the information that PA for a minimum of 10 min yields health beneﬁts (Haskell et al., 2007). The AA approach produced repeated measurements of PA and affective states (level-1) that were nested within persons (level- 2). To assure chronological comparability, the accelerometers and smartphones were synchronized. We conducted separate multi- level analyses for each affect subscale using the statistical program HLM 6.0 (Raudenbush et al., 2004). We used restricted maximum likelihood estimations for the multilevel analyses. The α-level of the tests was set to p < 0.05. First, we estimated the intra-class coefﬁcient of the three sub- scales with unconditional models where “y” is not modeled as a function of another variable at level-1 or level-2. Second, we consecutively entered the predictor variables PA and time-square into each model (see Eq. 1). We analyzed the linear effect but did not ﬁnd any differences between these results and the ones that we obtained when we controlled for time-square only. Thus, we deleted the linear effect. MATERIALS AND METHODS PARTICIPANTS Accordingly, the sample was entirely composed of students from the Karl- sruhe Institute of Technology (KIT, Germany). The study was performed during the exam period at the end of the semester. We assumed that during this period, students spend nearly all of every day at their writing desks preparing for their upcom- ing exams. Therefore, we expected them to show low PA but felt that this would not indicate that the group is generally inactive. All participants provided informed consent prior to the assess- ment. The subjects received no monetary compensation for their participation. ANALYSES Third, we analyzed whether affective state and the last 10 min of PA before each e-diary entry (level-1) sig- niﬁcantly varied as a function of three level-2 predictors, gender (sex), the average level of the affective state (mean_affst), and the average level of PA (mean_PA). These three level-2 predictors were inserted into each model. Thus,for each subscale,we examined the relationship between the level-1 intercept and the level-2 predic- tors (see Eq. 2) as well as the cross-level interaction between the level-1 slopes and level-2 predictors (see Eqs 3 and 4). AMBULATORY ASSESSMENT PROCEDURE The study was conducted over the course of 1 week. PA was mea- sured continuously for 24 h using an accelerometer. Electronic diary items were completed via smartphones. The students acti- vated their electronic diaries after waking up, and the measure- ments were repeated each full hour thereafter. An acoustic signal prompted the participants to complete the electronic diary entries. If a participant could not use the smartphone at a particular assessment point, no later entries were assigned. Before the study began, all of the participants were fully informed about the content of the study and the handling of the smartphones. After they had received all of the neces- sary equipment, the participants were able to pursue their daily activities. We controlled for time of day. First, we centered the time vari- ables. Then, we added the variables “time” and “time-square” into the analyses to control for the linear and squared effects of time. 2www.movisens.com DISCUSSION The 77 participants provided 807 data points, yielding an aver- age of 10.5 e-diary entries per subject. The overall average level of PA across the 10-min periods prior to the measurements of the affective states was 62.0 mg/min (SD = 64.9) with a range from 12.8 to 765.2 mg/min. For the sake of comparison, note that jogging episodes equal approximately 1000 mg/min, walk- ing episodes approximately 350 mg/min, and pure sitting episodes approximately 10 mg/min. The average levels of valence, ener- getic arousal, and calmness were 8.2, 7.0, and 8.5, respectively, representing medium to high affective states. The results for the intra-classcoefﬁcientwereρI = 0.50forvalence,ρI = 0.40forener- getic arousal, and ρI = 0.46 for calmness. This indicates that 50, 60, and 54%, respectively, of the affect subscales’ variance was caused by intra-individual variation. The distribution of PA, the time-square variable, and the three affect subscales allowed for multilevel analyses. To investigate the association between PA and affect in everyday life and to clarify the contradictory results of previous studies (Schwerdtfeger et al., 2008 vs. Kanning et al., 2012), we used state-of-the-art methodology: namely, AA with real-time, real- life,repeated,objective assessment. In general,the current ﬁndings support the hypothesis that there is a positive association between PA and affective states. We documented the signiﬁcant inﬂuence of PA on energetic arousal. This result conﬁrms previously published ﬁndings that have been obtained using AA (Schwerdtfeger et al., 2008; Kanning et al., 2012). In addition, the data revealed a signiﬁcant association between PA and positive affect (valence). This ﬁnding is consistent with the ﬁndings of Schwerdtfeger et al. (2008) but contrary to the ﬁndings of Kanning et al. (2012). Unlike the ﬁndings of Kanning et al. (2012), the current data analysis did not reveal a negative association between activity and calmness. The random error terms of the PA slopes were not signiﬁcant in the models. They were ﬁxed because the random and ﬁxed vari- ability of the slopes could not be separated reliably. Nevertheless, the slopes of the last 10 min of PA before each diary entry varied between the persons but did not vary randomly as a function of the level-2 predictors gender and the average levels of the affective state and PA. The ﬁxed and random effects on each affective state are shown in Table 1. WITHIN-PERSON EFFECTS Valence was signiﬁcantly predicted by the last 10 min of PA before each diary entry (p = 0.005) but not by time-square (p = 0.419). The more our subjects were physically active during the preceding 10 min, the better and more satisﬁed they felt. The standardized effects were 0.07 for PA. Thus, if PA increased by 1 SD, the valence increased by 0.07 SD. (4) Level-1 represents the participants’ responses (subscript i) on one of the basic affect subscales (Yti) in any given diary entry (sub- scriptt).Yti is deﬁnedas theaverage interceptof thecorresponding affect subscale across all participants (β0i) and the two level-1 pre- dictors PA (β1i PAti) and time-square (β2i time-square). These predictors are group mean-centered, with the group referring to a person (level-2). The intercepts and slopes are conceived as vary- ing randomly. The random effect of the level-1 model is given by rti. It is assumed to be normally distributed with a mean of “0” and a variance of σ2. Level-2 expresses between-subject effects. It includes the ﬁxed effects, γ, as the average intercepts and slopes across all persons,the predictors gender, mean_affst and mean_PA, and the random effects µ0i, µ1i, µ2i, and µ3i. The random effects are assumed to be multivariate and normally distributed, both with expected values of “0.” Energetic arousal was positively affected by the last 10 min of PA before each diary entry (p = 0.001) and negatively affected by time-square (p < 0.001). As PA during the preceding 10 min increased, the students’ sense that they were awake and energized increased. The analyses also revealed that the later it was during the day, the more tired and weak the participants felt. According to the standardized effects, when the PA or time-square ﬁgure increased by 1 SD, energetic arousal increased by 0.1 SD, and decreased by 0.3 SD, respectively. Calmness was not signiﬁcantly predicted by the 10-min of PA preceding each diary entry (p = 0.079; SD = 0.04) but was pre- dicted by time-square (p = 0.018; SD = 0.65). The later it was in the day, the more relaxed and calm the participants felt. AFFECTIVE STATES To assess momentary affective states, we used a short scale with six items based on the Multidimensional Mood Questionnaire Level-1: Yti = β0i + β1i (PA)ti + β2i time - square ti + rti (1) Level-2: b0i = γ00 + γ01 (sex) + γ02 (mean_affst) + γ03 (mean_PA) + µ0i (2) (1) (2) April 2013 \| Volume 4 \| Article 102 \| 3 www.frontiersin.org Activities and affect: a replication Bossmann et al. Level-2: b1i = γ10 + γ11 (sex) + γ12 (mean_affst) + γ13 (mean_PA) + µ1i (3) Level-2: b2i = γ20 + γ21 (sex) + γ22 (mean_affst) + γ23 (mean_PA) + µ2i (4) Level-2: b1i = γ10 + γ11 (sex) + γ12 (mean_affst) + γ13 (mean_PA) + µ1i (3) Level-2: b2i = γ20 + γ21 (sex) + γ22 (mean_affst) + γ23 (mean_PA) + µ2i (4) BETWEEN-PERSON EFFECTS To clarify the magnitude of the effect, we standardized the effects of PA and time on the three mood subscales (see Eq. 5). The standard deviation was taken from the mean of the sample of the last 10 min of PA before each diary entry and from the sample mean for the affective states. We used the level-2 predictors to investigate gender differences. In addition,we controlled for the within-person effect of PA on affec- tive states in determining the mean level of the selected affective state and the mean level of PA. As shown in Table 1, neither the average level of the selected affective states nor the mean level of PA were signiﬁcantly related to the within-person effect of PA. We found signiﬁcant gender differences (p = 0.047) for the subscale valence. Women felt signiﬁcantly better and more satisﬁed than men after they increased their activity levels. Standardized effect β1i ∗SD activity or time - square / SD correspondent affective state (5) Standardized effect β1i ∗SD activity or time - square / SD correspondent affective state (5) Standardized effect β1i ∗SD activity or time - square / (5) DISCUSSION To explain the ﬁndings regarding the three dimensions of affect, we speculate that physiological adaptations due to short bursts of increased PA, may cause the participants to perceive themselves as having enhanced energy levels. In some studies, increased energy levels were associated with a sense of reduced calmness; how- ever, this relationship might not necessarily hold. We speculate Frontiers in Psychology \| Movement Science and Sport Psychology April 2013 \| Volume 4 \| Article 102 \| 4 \| 4 Activities and affect: a replication Bossmann et al. Table 1 \| Fixed and random effects and variance components for valence (model 1), energetic arousal (model 2), and calmness (model 3) on actual physical activity (aPA). Outcome Fixed Random Predictor Coefﬁcient SE t-Value (df ) p-Value Variance estimate SD X (df ) p-Value 95-Predictive intervala Slope > 0b MODEL 1:VALENCE Intercept 8.199 0.123 66.41 (58) <0.001 0.65 0.81 194.98 (57) <0.001 Sex −0.297 0.350 −0.85 (58) 0.399 VA-mean 1.094 0.076 14.42 (58) <0.001 aPA_mean 0.006 0.009 0.70 (58) 0.484 aPA 0.003 0.001 2.83 (798) 0.005 −0.002; 0.007 90% Sex −0.007 0.003 −1.93 (798) 0.047 V_mean 0.001 0.001 1.07 (798) 0.286 aPA-mean 0.001 0.001 0.05 (798) 0.964 Time −0.002 0.002 −0.81 (61) 0.419 0.001 0.012 103.18 (60) 0.001 −0.026; 0.022 43% MODEL 2: ENERGETIC AROUSAL Intercept 7.061 0.142 49.71 (58) <0.001 0.875 0.935 196.38 (57) <0.001 Sex −0.253 0.406 −0.62 (58) 0.536 EA-mean 1.045 0.087 12.04 (58) <0.001 aPA_mean 0.012 0.010 1.14 (58) 0.261 aPA 0.005 0.001 3.49 (798) <0.001 0.002; 0.007 99% Sex −0.005 0.004 −1.21 (798) 0.229 EA_mean 0.001 0.001 0.12 (798) 0.905 aPA-mean 0.001 0.001 0.18 (798) 0.858 Time −0.024 0.003 −7.15 (61) <0.001 0.001 0.018 127.75 (60) <0.001 −0.059; 0.011 99% MODEL 3: CALMNESS Intercept 8.457 0.121 69.86 (58) <0.001 0.647 0.805 214.16 (57) <0.001 Sex 0.243 0.354 0.69 (58) 0.495 C-mean 0.886 0.076 11.88 (58) <0.001 aPA_mean −0.012 0.008 −1.31 (58) 0.197 aPA 0.002 0.001 1.76 (798) 0.079 −0.0011; 0.0043 87% Sex −0.002 0.003 −0.47 (798) 0.641 C_mean −0.001 0.001 −0.66 (798) 0.512 aPA-mean −0.001 0.001 −1.03 (798) 0.303 Time 0.007 0.003 2.45 (61) 0.018 0.001 0.012 142.51 (60) <0.001 −0.0255; 0.0399 67% aBased on the assumption of normally distributed regression coefﬁcients, the 95% predictive interval indicates the range of values between which 95% of the regression coefﬁcients are estimated to lie (Hox, 2010). The intervals were calculated based on a model without level-2 predictors. REFERENCES B., Morrison, D., and Ciccolo, J. T. (2005). Effects of acute exercise on mood and well-being in patients with major depressive dis- order. Med. Sci. Sports Exerc. 37, 2032–2037. Biddle, S., Cavill, N., Ekelund, U., Gorely,T.,Grifﬁths,M.D.,Jago,R.,et al. (2010). Sedentary Behaviour and Obesity: Review of the Current Scien- tiﬁc Evidence. London: Department of Health/Department for Children, Schools and Families. Kanning, M., and Schlicht, W. (2010). Be active and become happy: an ecological momentary assess- ment of physical activity and mood. J. Sport Exerc. Psychol. 32, 253–261. Ebner-Priemer, U. W., Koudela, S., Mutz, G., and Kanning, M. (2013). Interactive multimodal ambulatory monitoring to investigate the asso- ciation between physical activity and mood. Front. Psychol. 3:596. doi:10.3389/fpsyg.2012.00596 Hamilton, M. T., Hamilton, D. G., and Zderic, T. W. (2007). Role of low energy expenditure and sitting in obesity, metabolic syn- drome, type 2 diabetes, and car- diovascular disease. Diabetes 56, 2655–2667. Kuppens, P., Oravecz, Z., and Tuer- linckx, F. (2010). Feelings change: accounting for individual differ- ences in the temporal dynamics of affect. J. Pers. Soc. Psychol. 99, 1042–1060. Blake, H. (2009). How effective are physical activity interven- tions for alleviating depressive symptoms in older people? A systematic review. Clin. Rehabil. 23, 873–887. Ekkekakis, P., Hall, E. E., VanLanduyt, L. M., and Petruzzello, S. J. (2000). Walking in (affective) circles: can short walks enhance affect? J. Behav. Med. 23, 245–275. Haskell, W. L., Lee, I.-M., Pate, R. R., Powell, K. E., Blair, S. N., Franklin,B. A.,et al. (2007). Physical activity and public health: updated recommen- dation for adults from the American College of Sports Medicine and the American Heart Association. Circu- lation 116, 1081–1093. LaFontaine, T. P., DiLorenzo, T. M., Frensch, P. A., Stucky-Ropp, R. C., Bargman, E. P., and McDonald, D. G. (1992). Aero- bic exercise and mood. A brief review, 1985-1990. Sports Med. 13, 160–170. Folkins, C. H. (1976). Effects of physical training on mood. J. Clin. Psychol. 32, 385–388. Bussmann, J. B. J., and Ebner-Priemer, U. W. (2011). “Ambulatory activ- ity monitoring: assessing activ- ity, posture, and motion patterns in daily life,” in Handbook of Research Methods for Studying Daily Life, eds M. Mehl and T. Con- nor (New York: Guilford Press), 235–250. Fox,K. R. (1999). The inﬂuence of phys- ical activity on mental well-being. Public Health Nutr. 2, 411–418. Hausenblas, H. A., Gauvin, L., Symons Downs, D., and Duley, A. R. (2008). REFERENCES posture, and speciﬁc motion pat- terns in daily life. Eur. Psychol. 14, 142–152. Hox, J. (2010). Multilevel Analysis – Techniques and Applications. New York: Routledge. posture, and speciﬁc motion pat- terns in daily life. Eur. Psychol. 14, 142–152. physical activity to explaining diur- nal variations in feeling states in active, middle-aged women. Health Psychol. 19, 365–375. Arent, S. M., Landers, D. M., and Etnier, J. L. (2000). The effects of exercise in mood in older adults: a meta ana- lytic review. J. Aging Phys. Act. 8, 407–430. Cabanac, M. (2002). What is emotion? Behav. Processes 60, 69–83. Kanning, M., Ebner-Priemer, U., and Brand, R. (2012). Autonomous regulation mode moderates the effect of actual physical activity on affective states: an ambu- lant assessment approach to the role of self-determination. J. Sport Exerc. Psychol. 34, 260–269. Gauvin, L., Rejeski, W. J., and Nor- ris, J. L. (1996). A naturalistic study of the impact of acute phys- ical activity on feeling states and affect in women. Health Psychol. 15, 391–397. Dunton, G. F., Liao,Y., Intille, S., Wolch, J., and Pentz, M. A. (2011). Physi- cal and social contextual inﬂuences on children’s leisure-time physical activity: an ecological momentary assessment study. J. Phys. Act. Health 8, 103–108. Axelson, D. A., Bertocci, M. A., Lewin, D. S., Trubnick, L. S., Birmaher, B., Williamson,D. E.,et al. (2003). Mea- suring mood and complex behav- ior in natural environments: use of ecological momentary assessment in pediatric affective disorders. J. Child. Adolesc. Psychopharmacol. 13, 253–266. Axelson, D. A., Bertocci, M. A., Lewin, D. S., Trubnick, L. S., Birmaher, B., Gauvin, L., and Spence, J. C. (1996). Physical activity and psychological well-being: knowledge base, current issues, and caveats. Nutr. Rev. 54, S53–S65. Kanning M. K., Ebner-Priemer, U. W., and Schlicht, W. M. (2013). How to investigate within-subject associations between physical activity and momentary affective states in everyday life: a position statement based on a literature overview. Front. Psychol. 4:187. doi:10.3389/fpsyg.2013.00187 Ebner-Priemer, U. W., Kuo, J., Schlotz, W., Kleindienst, N., Rosenthal, Z., Detterer, L., et al. (2008). Dis- tress and affective dysregulation in patients with borderline per- sonality disorder: a psychophys- iological ambulatory monitoring study. J. Nerv. Ment. Dis. 96, 314–320. Grossman, P., Deuring, G., Garland, S. N.,Campbell,T.S.,andCarlson,L.E. (2008). Patterns of objective physical functioning and perception of mood and fatigue in posttreatment breast cancer patients and healthy controls: an ambulatory psychophysiological investigation. Psychosom. Med. 70, 819–828. Bartholomew, J. physical activity to explaining diur- nal variations in feeling states in active, middle-aged women. Health Psychol. 19, 365–375. DISCUSSION bBased on the assumption of normally distributed regression coefﬁcients, this value indicates true percentage of regression coefﬁcients that are positive (Hox, 2010). The percentages were calculated based on a model without level-2 predictors. Table 1 \| Fixed and random effects and variance components for valence (model 1), energetic arousal (model 2), and calmness (model 3) on actual physical activity (aPA). that individual activity thresholds (for duration and/or intensity) must be exceeded for positive affect to develop. The dimension valence, however, might be positively affected by even short bouts of PA. to positively change a person’s affective state. Our use of a sam- ple that was rather inactive at the time of the study (students during their exam period) further supports this point. However, as our participants were not chronically inactive, but rather cur- rently inactive (because of their exams), generalization of our ﬁndings is limited regarding chronically inactive participants. We might speculate that our only currently inactive sample does proﬁt more from small activity episodes, because of learned connec- tions between PA and affect, compared to chronically inactive samples. This study communicates an important health message, espe- cially for people who sit at work for more than 8 h per day and/or lead an inactive life. The results suggest that short and low-intensity PA, which may be conducted as brief breaks from sedentary time even in such working environments,has the poten- tial to enhance people’s energy levels and make them feel better instantly. Accordingly, jogging intervals of half an hour are not necessary to induce such changes. It might even be speculated that everyday life activities such as stair climbing, taking a short walk around the block or doing jumping jacks may be sufﬁcient Previous ﬁndings indicated that the effects of PA were stronger when initial mood levels were low (Folkins,1976; LaFontaine et al., 1992). However, the current study did not conﬁrm the existence of these ceiling effects. The strength of the association between April 2013 \| Volume 4 \| Article 102 \| 5 www.frontiersin.org www.frontiersin.org Activities and affect: a replication Bossmann et al. PA and valence/energetic arousal was not affected by the students’ average mood level. Therefore,even people who are already experi- encing positive feelings could proﬁt from carrying out short bursts of low-intensity activity. DISCUSSION This ﬁnding is consistent with those of studies that have supported the assumption of a more general pro- tective effect of PA on psychological health (Thayer,1987;Otto and Stemmann,1991;GauvinandSpence,1996;Ekkekakisetal.,2000). 2013). In interactive AA, e-dairy entries are triggered not by time but by the variables of interest, such as activity. Second, only university students were questioned, leaving out less edu- cated or older people with a lower socio-economic status. Third, the assessment period covered only 1 day. Fourth, we primar- ily focused on the association between PA and affective states. Future studies should further analyze the association between PA and the degree of a subsequent change in affective states over a longer period of time. This search for an effective “intensity threshold” could aid in the development of more precise rec- ommendations regarding activities that could interrupt sedentary behavior. Several limitations of the present study must be taken into account. First, the measurement time points were ﬁxed. This can be problematic, if the levels of activity prior to the assess- ments show too little variation, decreasing the power of the assessment approach. However, despite this disadvantage, we showed signiﬁcant associations between activity and two of the three affective dimensions. To overcome the limited variation in the PA conducted directly before the e-diary assessments, interactive AA has been recommended (Ebner-Priemer et al., The key message of the current study is that repeated short periods of everyday life activities have the potential to positively inﬂuence affective states and subjective well-being and, in the long term, support a healthy lifestyle. REFERENCES Effects of abstinence from habitual involvement in regular exercise on feeling states: an eco- logical momentary assessment study. Br. J. Health Psychol. 13, 237–255. Frijda, N. H. (1994). “Emotions are functional, most of the time,” in The Nature of Emotion: Fundamen- tal Questions, eds P. Ekman and R. J. Davidson (New York: Oxford University Press), 127–130. LePage, M. L., and Crowther, J. H. (2010). The effects of exercise on body satisfaction and affect. Body Image 7, 124–130. Bussmann, J. B. J., Ebner-Priemer, U. W., and Fahrenberg, J. (2009). Ambulatory behavior monitoring: progress in measurement of activity, Matthews, G., Jones, D. M., and Cham- berlain, A. G. (1990). Reﬁning the measurement of mood: the UWIST Gauvin,L.,and Rejeski,W. (2000). Con- tributions of acute bouts of vigorous April 2013 \| Volume 4 \| Article 102 \| 6 Frontiers in Psychology \| Movement Science and Sport Psychology Activities and affect: a replication Bossmann et al. mood adjective checklist. Br. J. Psy- chol. 81, 17–42. could be construed as a potential con- ﬂict of interest. could be construed as a potential con- ﬂict of interest. could be construed as a potential con- ﬂict of interest. Monitoring in der Gesundheitspsy- chologie. Z. Gesundheitspsychol. 16, 2–11. mood adjective checklist. Br. J. Psy- chol. 81, 17–42. Raudenbush, S. W., Bryk, A., and Cong- don, R. (2004). HLM6: SSI Scientiﬁc Software International. Available at: http://www.ssicentral.com Motl, R. W., Birnbaum, A. S., Kubik, M. Y., and Dishman, R. K. (2004). Naturallyoccurringchangesinphys- ical activity are inversely related to depressive symptoms during early adolescence. Psychosom. Med. 66, 336–342. Received: 02 October 2012; accepted: 12 February 2013; published online: 15 April 2013. Steyer, R., Schwenkmezger, P., Notz, P., and Eid, M. (1997). Der Mehrdimensionale Beﬁndlichekeits- fragebogen (MDBF). Göttingen: Hogrefe. p February 2013; published online: 15 April 2013. Reed, J., and Ones, D. S. (2006). The effect of acute aerobic exer- cise on positive activated affect: a meta-analysis. Psychol. Sport. Exerc. 477–514. Citation: Bossmann T, Kanning M, Koudela-Hamila S, Hey S and Ebner- Priemer U (2013) The association between short periods of everyday life activities and affective states: a replication study using ambulatory assessment. Front. Psychol. 4:102. doi:10.3389/fpsyg.2013.00102 Thayer, R. E. (1987). Energy, tiredness, and tension effects of a sugar snack versus moderate exercise. J. Pers. Soc. Psychol. 52, 119–125. Netz, Y., Wu, M. J., Becker, B. J., and Tenenbaum, G. (2005). REFERENCES Physi- cal activity and psychological well- being in advanced age: a meta- analysis of intervention studies. Psy- chol. Aging 20, 272–284. Scherer, K. R. (2009). Emotions are emergent processes: they require a dynamic computa- tional architecture. Philos. Trans. R Soc. Lond B Biol. Sci. 364, 3459–3474. Trull, T. J., and Ebner-Priemer, U. W. (2013). Ambulatory assessment. Annu. Rev. Clin. Psychol. 9, 4.1–4.27. This article was submitted to Frontiers in Movement Science and Sport Psychology, This article was submitted to Frontiers in Movement Science and Sport Psychology, a specialty of Frontiers in Psychology. Schimmack, U., and Grob, A. (2000). Dimensional models of core affect: a quantitative comparison by means of structural equa- tion modeling. Eur. J. Pers. 14, 325–345. Otto, J., and Stemmann, O. (1991). Changes in mood caused by staged, moderate physical activ- ity. Z. Exp. Angew. Psychol. 38, 264–278. Wilhelm, P., and Schoebi, D. (2007). Assessing mood in daily life. Struc- tural validity, sensitivity to change, and reliability of a short-scale to measure three basic dimensions of mood. Eur. J. Psychol. Assess. 23, 258–267. Copyright © 2013 Bossmann, Kan- ning, Koudela-Hamila, Hey and Ebner- Priemer. This is an open-access arti- cle distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are cred- ited and subject to any copyright notices concerning any third-party graphics etc. Copyright © 2013 Bossmann, Kan- ning, Koudela-Hamila, Hey and Ebner- Priemer. This is an open-access arti- cle distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are cred- ited and subject to any copyright notices concerning any third-party graphics etc. Powell, R., Allan, J. L., Johnston, D. W., Gao, C., Johnston, M., and Ken- nardy, J. (2009). Activity and affect: repeated within-participant assess- ment in people after joint replace- ment surgery. Rehabil. Psychol. 54, 83–90. Schwerdtfeger, A., Eberhardt, R., and Chmitorz, A. (2008). Gibt es einen Zusammenhang zwischen Bewe- gungsaktivität und psychischem Beﬁnden im Alltag? April 2013 \| Volume 4 \| Article 102 \| 7 REFERENCES Eine Metho- denillustration zum ambulanten Conﬂict of Interest Statement: The authors declare that the research was conducted in the absence of any com- mercial or ﬁnancial relationships that Conﬂict of Interest Statement: The authors declare that the research was conducted in the absence of any com- mercial or ﬁnancial relationships that April 2013 \| Volume 4 \| Article 102 \| 7 www.frontiersin.org
https://openalex.org/W2164291896	https://qmro.qmul.ac.uk/xmlui/bitstream/123456789/12640/1/Screen%20Functionally%20distinct%20tendon%20fascicles%202014%20Published.pdf	English	null	Functionally distinct tendon fascicles exhibit different creep and stress relaxation behaviour	Proceedings of the Institution of Mechanical Engineers. Part H, Journal of engineering in medicine	2,013	cc-by	7,874	Original Article Proc IMechE Part H: J Engineering in Medicine 2014, Vol 228(1) 49–59 IMechE 2013 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0954411913509977 pih.sagepub.com Keywords Keywords Tendon, mechanics, fatigue, human semitendinosus, bovine Keywords Tendon, mechanics, fatigue, human semitendinosus, bovine Date received: 18 April 2013; accepted: 2 October 2013 testing routes are of significant importance for under- standing overuse damage in tendons. 1Institute of Bioengineering, School of Engineering and Materials Science, Queen Mary, University of London, London, UK 2School of Biological Sciences, University of East Anglia, Norwich, UK Abstract Most overuse tendinopathies are thought to be associated with repeated microstrain below the failure threshold, analogous to the fatigue failure that affects materials placed under repetitive loading. Investigating the progression of fatigue damage within tendons is therefore of critical importance. There are obvious challenges associated with the sourcing of human tendon samples for in vitro analysis so animal models are regularly adopted. However, data indi- cates that fatigue life varies significantly between tendons of different species and with different stresses in life. Positional tendons such as rat tail tendon or the bovine digital extensor are commonly applied in in vitro studies of tendon overuse, but there is no evidence to suggest their behaviour is indicative of the types of human tendon par- ticularly prone to overuse injuries. In this study, the fatigue response of the largely positional digital extensor and the more energy storing deep digital flexor tendon of the bovine hoof were compared to the semitendinosus tendon of the human hamstring. Fascicles from each tendon type were subjected to either stress or strain controlled fatigue loading (cyclic creep or cyclic stress relaxation respectively). Gross fascicle mechanics were monitored after cyclic stress relaxation and the mean number of cycles to failure investigated with creep loading. Bovine extensor fascicles demonstrated the poorest fatigue response, while the energy storing human semitendinosus was the most fatigue resistant. Despite the superior fatigue response of the energy storing tendons, confocal imaging suggested a similar degree of damage in all three tendon types; it appears the more energy storing tendons are better able to withstand damage without detriment to mechanics. Functionally distinct tendon fascicles exhibit different creep and stress relaxation behaviour Jennifer H Shepherd1, Kirsten Legerlotz2, Taylan Demirci1, Christian Klemt1, Graham P Riley2 and Hazel RC Screen1 1Institute of Bioengineering, School of Engineering and Materials Science, Queen Mary, University of London, London, UK 2School of Biological Sciences, University of East Anglia, Norwich, UK Corresponding author: Jennifer H Shepherd, Institute of Bioengineering, School of Engineering and Materials Science, Queen Mary, University of London, Mile End Road, London E1 4NS, UK. Email: jennyh.shepherd@gmail.com Corresponding author: Jennifer H Shepherd, Institute of Bioengineering, School of Engineering and Materials Science, Queen Mary, University of London, Mile End Road, London E1 4NS, UK. Email: jennyh.shepherd@gmail.com Introduction Many painful tendon lesions are thought to be the result of a gradual accumulation of micro-injuries.1 Tendinopathy is thus often referred to as an overuse pathology and can, in some ways, be considered analo- gous to the fatigue failure of common engineering materials during cyclic loading.2 This is of course an oversimplification, as tendon is not simply a fibre- reinforced composite, but a living tissue maintained by its resident cell population, the tenocytes. Damage to tendon probably occurs every day as a result of a vari- ety of normal activities, but this damage only accumu- lates when equilibrium cannot be maintained between the rate of damage and the cellular driven rate of repair.3 The progression of this damage can be challen- ging to monitor in situ, and as such, in vitro fatigue Tendon mechanics are defined by the hierarchically organised collagen within the tissue, providing a uniaxi- ally strong material, in which failure of one or a few collagen components within the hierarchy does not Corresponding author: Jennifer H Shepherd, Institute of Bioengineering, School of Engineering and Materials Science, Queen Mary, University of London, Mile End Road, London E1 4NS, UK. Email: jennyh.shepherd@gmail.com Corresponding author: The ability of the fascicles to act as apparently independent struc- tural units, and slide against each other, enables them to transmit tension despite the changing angles of a joint and allows tendons to change shape as their muscle contracts.5 However, sliding is not just limited to between collagen units at the fascicle level; fibre straining and inter-fibre matrix shearing within the fascicle are suggested to be critical for tendon func- tion.6 While fascicle mechanics cannot necessarily be considered to be representative of tendon behaviour as a whole, it can add much to the understanding of tendon mechanics and the role of the hierarchical structure. While all tendons play a positional role, positioning joints to facilitate movement, there are also those which must operate as energy stores to allow for more efficient locomotion.7 Energy-storing tendons experience high strains and consequently high stresses as they stretch during loading, ready to recoil and return energy to the system. These tendons tend to operate with low safety factors and thus have a much higher propensity for injury.8 Prime examples of these energy-storing tendons are the human Achilles or equine superficial digital flexor tendon (SDFT), but evidence would sug- gest that the tendons of the musculotendon hamstring system also exhibit energy-storing capability9,10 with a stretch-shortening cycle observed.11,12 Certainly, there is a high propensity for hamstring injury and in a study that considered 170 consecutive cases of ham- string strain in athletes, 21 exhibited proximal tendon injury, 4 of these being distal tendon avulsions and 3 of which were of the semitendinosus.13 While not on the same scale as the equine SDFT, the peak strain of the semitendinosus musculotendon unit has been observed to be 8.73% and this musculotendon unit also observed the greatest lengthening velocity of three units of the hamstring.11 While positional ten- dons such as rat tail tendon or the bovine digital extensor are commonly applied in in vitro studies of tendon overuse, there is no evidence to suggest that their behaviour is indicative of the types of human tendon particularly prone to overuse injuries. It is likely that more energy-storing tendons from other animal models such as the equine SDFT or bovine digital flexor may prove more comparable. Corresponding author: Proc IMechE Part H: J Engineering in Medicine 228(1) 50 stress relaxation describes a test at constant displace- ment where the reduction in associated load is mea- sured. However, the terms more accurately describe the materials response to the test, that is, a lengthening with constant load in the case of creep or a reduction in stress with constant displacement in the case of stress relaxation. As these responses also occur during cyclic loading,15,16 there has become some precedent to describe cyclic tests under either load or displacement control as cyclic creep or stress relaxation tests, respec- tively,16,17 and it is these terms that will be used here. With different boundary conditions, the two testing routes would be expected to elicit a different response from the loaded tendon, but they are often used inter- changeably with very little literature comparing the two testing techniques directly. Stress relaxation tests of tendon fascicles are typically more straightforward to carry out, but as creep tests may be more physiologi- cally relevant, they cannot simply be neglected. It has been suggested in ligament (where the function is to connect bone to bone) that the in-life loading is a sim- ple creep situation, with the same loads repeatedly applied by the joint. However, tendons transfer the muscle force to the bone in order to facilitate locomo- tion, and as a result, load conditions may be more complex.18 The time-dependent properties of tendon will undoubtedly affect its ability to convert muscle contraction into skeletal movement, and as such, both creep and stress relaxation behaviour must be well defined in order to accurately predict the behaviour of tendon.19 significantly reduce the strength of the whole tendon.4 The fascicular level is particularly critical for the mechanics of tendon and is at a scale that can be mechanically tested with relative ease. Corresponding author: Comparative study on the fatigue behaviour of con- trasting tendon types is limited;20,21 yet, the behaviour of other mammalian tendon of a variety of types is fre- quently used to understand the progression of fatigue damage in human tendon. It is critical to have an understanding of how fatigue behaviour may differ in functionally distinct tendons and also to establish whether we are indeed increasing our understanding of human tendon behaviour by investigating other tendon types whether in vivo or in vitro. While it might be expected that the structure and mechanics of different tendons differ so as to mirror the quantitative func- tional variations, the evidence in the literature does not entirely support this. When Pollock and Shadwick22 measured Young’s modulus (E) and percentage hyster- esis loss (H) for mature tendons from 18 species of mammal, individual tendons were found to differ sig- nificantly, but there was no significant relationship with the type of tendon or mass of the animal. As sum- marised in a review article by Ker et al.,20 numerous studies have also shown a lack of correlation between the ultimate tensile strength (UTS) of tendons and the stresses seen in vivo. High stress-in-life tendons such as the Achilles tendon actually tend to exhibit lower fail- ure strengths than more positional tendons (a compro- mise to allow higher elasticity),20,23–26 meaning a lower safety margin and perhaps the higher propensity for In vivo loading conditions of tendon are complex, and thus, careful consideration of the nature of the fati- gue loading is required, to provide the greatest equiva- lence to the clinical situation. Fatigue testing can be carried out either in the form of repeated loading or in the form of repeated deformation, that is, under load or displacement control.14 In the nomenclature of static testing, a creep test is one where a constant load is applied and the increase in extension monitored, while 51 Shepherd et al. damage. However, when fatigue properties are consid- ered, high stress-in-life tendons show significantly greater lifetimes under cyclic loading when all tendons are fatigued to the same stress.20 tendons in the hoof so these were pooled.28 Fascicle diameters were measured continuously along a 10 mm length in the centre of each fascicle using a laser micro- meter (LSM-501; Mitutoyo, Kawasaki, Japan; resolu- tion of 0.5 mm). Corresponding author: The minimum value of approximately 25 measurements over this 10 mm length was used to calculate the cross-sectional area assuming a circular cross section. Fascicles with a diameter between 0.18 and 0.4 mm were considered for mechanical characterisation. This article investigates the fatigue characteristics of the fascicles from three tendons for the first time: the extensor and deep flexor digital tendons of the bovine hoof and the human semitendinosus tendon. The bovine digital tendons are readily available and provide a straightforward source of tendon material for in vitro investigation. Both the deep digital flexor tendon and human semitendinosus provide some energy-storing capability while the bovine digital extensor tendon plays a purely positional role. Considering fascicles from all three tendons allows the comparison of not only energy-storing and positional tendons from a sin- gle species, but also the comparison of energy-storing tendons across species, comparing the bovine deep digi- tal flexor tendon with the clinically relevant human semitendinosus. It is hypothesised that species variation may be less significant than functional requirement in determining fascicle fatigue behaviour, and that posi- tional tendons such as the bovine digital extensor will not provide an ideal model for investigating energy- storing and injury-prone tendons such as the hamstring semitendinosus or Achilles. Finally, the complex load- ing environment of tendon means that value can be gained by comparing the response to cyclic creep and cyclic stress relaxation in an in vitro study. The micro- structural processes that take place during the two test- ing routes are likely be fundamentally different, and as such, a difference in the rate of damage progression is expected between the two fatigue conditions. Stress relaxation and creep tests Each fascicle was secured in an individual custom-made stainless steel loading chamber,29 with a grip-to-grip distance of 10 mm. The grip system ensured slippage of the fascicles did not occur during testing.29 Each cham- ber was filled with DMEM, to maintain full hydration during testing, and the chambers were secured within a BOSE loading frame (BOSE Corporation, Eden Prairie, MN, USA) housed in an incubator to maintain samples in 5% CO2 at 37 C. Legerlotz et al.29 have previously established that cyclic stress relaxation to 60% of the failure strain resulted in a significant decrease in fascicle mechanical properties in just 15 min. This value was subsequently adopted for testing in the current study, allowing a rapid analysis of damage accumulation across the range of tendon types; 60% strain to failure corresponded to 14% applied strain in all cases. During stress relaxation tests, cyclic loading at a frequency of 1 Hz with a sine wave form was carried out under displacement control, to a fixed peak displacement of 1.4 mm (initial gauge length of 10 mm). Cyclic loading was carried out for 5 min (300 cycles), 15 min (900 cycles) or 30 min (1800 cycles) and the reduction in load over time was moni- tored. Mean stress relaxation (defined as percentage of stress at cycle 1) was determined for the three time points, after which samples were subjected to confocal imaging or quasi-static tensile testing to determine the effects of fatigue loading. Tendon preparation The feet of young healthy bovines (male steers between 18 and 36 months of age), with no observed tendon injury, were sourced from a local abattoir. The ten- sional regions of the medial, lateral and common digi- tal extensor tendons (CDETs) as well as the deep digital flexor tendon were removed within 24 h of slaughter. Ethics permission for the collection of waste human semitendinosus tissue from patients undergoing patellar stabilisation surgery was obtained from Essex 2 Research Ethics Committee, reference 09/H0302/3. All samples were frozen at 220 C until required. Quasi-static tensile testing (Bionix100; MTS, Cirencester, UK; 50 N Load Cell) was carried out using the same DMEM filled stainless steel loading cham- bers. Testing was carried out at an extension rate of 1 mm/s to failure and stress–strain curves derived using the initial measured cross-sectional area. At least 10 fascicles from multiple tendons were tested at each analysis point: 0, 300, 900 and 1800 cycles of stress relaxation. Upon defrosting, sample hydration was maintained with Dulbecco’s modified Eagle’s medium (DMEM) while collagen fascicles of length greater than 20 mm were carefully dissected. A variation in the mechanical properties of fascicles from different regions of the human patellar tendon has previously been observed,27 and as a result, fascicles were only taken from the cen- tral regions of the bovine tendons. Previous studies have shown no difference in the mechanical properties of fascicles from any of the three bovine extensor For creep tests, cyclic loading was again carried out at a frequency of 1 Hz (same sine wave form), but under load control to a constant load of 60% of the UTS. Based on previous quasi-static testing of fascicles from these three tendon types, an average UTS value of 48 MPa was adopted for the bovine extensor and flexor samples and 60.22 MPa for human semitendinosus samples, equating to an applied stress of 28.8 MPa in the case of the bovine fascicles and 36.1 MPa for the Proc IMechE Part H: J Engineering in Medicine 228(1) 52 human tendon. Displacement was monitored through the duration of the test. At least 12 fascicles from four tendons were cyclically tested to failure for each of the three different tendon types. Additional tests were ter- minated after 300, 900 and 1200 cycles and samples were stored for confocal imaging. Figure 1. Confocal imaging At each level of fatigue, fascicles were stained with acri- dine orange and imaged to investigate damage accumu- lation. Acridine orange is a nucleic acid selective fluorescent cationic dye used extensively for a variety of applications including the tracking of tenocyte nuclei during straining experiments under confocal micro- scopy.6,30 For nuclear staining, concentrations of the order of 5 mM are typically used; however, at higher concentrations, the staining becomes non-specific and will stain the collagen and matrix constituents. A 5 mM acridine orange (Invitrogen, Eugene, OR, USA) solu- tion in DMEM was used to stain the fascicles for 40 min, followed by repeated phosphate-buffered saline (PBS) washing to ensure removal of excess stain. Imaging was carried out using a PerkinElmer Ultraview Spinning Disc Confocal system with a Nikon Eclipse TE300 Microscope and 4* and 20* objective apertures. Lower magnification images were used in order to con- struct a composite image of the entire fascicle length, while the higher magnification images enabled qualita- tive investigation of damage accumulation within the tendon matrix. Tendon preparation Typical stress relaxation curves from (a) human, (b) bovine flexor and (c) bovine extensor tendon fascicles. Statistical analysis Statistical significance in percentage stress relaxation as well as UTS values from quasi-static tests after cyclic loading was determined with a single-factor analysis of variance (ANOVA) followed by Tukey’s post hoc test (IBM SPSS software). For investigation of the number of cycles to failure during creep testing, a Kruskal–Wallis test followed by a Mann–Whitney test was used. For all statistical tests, significance was established at p4 0.05. Figure 1. Typical stress relaxation curves from (a) human, (b) bovine flexor and (c) bovine extensor tendon fascicles. only apparent at 1800 cycles and at no time point did a statistically significant variation exist between the human semitendinosus and bovine flexor tendon. Despite considerable stress relaxation after 300 cycles of loading, all samples including the bovine extensor tendon fascicles retained their tensile strength during quasi-static testing at this first time point (Figure 2). However, by 900 cycles of stress relaxation, both bovine tendon types showed a statistically signifi- cant reduction in UTS, and by 1800 cycles, the decrease in UTS was also evident in human semitendinosus fas- cicles. Interestingly, although UTS tapered off most rapidly in the bovine extensor, it actually exhibited the highest mean UTS in control samples pre-fatigue test- ing. If UTS reductions with stress relaxation are expressed as a percentage of the starting value, the dra- matic drop in strength in the bovine extensor becomes evident (66% at 1800 cycles) compared to the human semitendinosus (32% decrease). Stress relaxation Typical stress relaxation curves from each tendon type are shown in Figure 1 with mean data in Table 1. The data indicate markedly greater stress relaxation for the extensor tendon fascicles compared with the other two tendon types. After just 300 cycles, the sample stress had decreased by 48% in extensor fascicles compared to less than 30% in the human semitendinosus. The human semitendinosus and bovine extensor created the two extreme responses, with the energy-storing human semi- tendinosus exhibiting significantly more fatigue resis- tance throughout. However, a significant difference between the response of the two bovine tendons was 53 Shepherd et al. Table 1. Mean stress relaxation data 6 standard deviation (expressed as a percentage of the stress applied in the first cycle) for human semitendinosus, bovine flexor and bovine extensor tendon after 300, 900 and 1800 cycles to a displacement at 60% UTS. human semitendinosus, bovine flexor and bovine extensor tendon after 300, 900 and 1800 cycles to a displacement at 60% UTS. Tendon 0 cycles 300 cycles 900 cycles 1800 cycles Human Semitendinosus 100% 70.89611.82% 64.89612.56% 61.50613.95% Bovine Flexor Tendon 100% 66.26611.09% 57.99615.87% 54.34617.95% Bovine Extensor Tendon 100% 52.12614.91% 42.57616.16% 36.06617.20% Statistical significance (p40.05) was observed between human semitendinosus and bovine extensor samples at all time points and between the bovine flexor and bovine extensor after 1800 cycles; difference between the human semitendinosus and bovine flexor remained insignificant at all time points. Statistical significance (p40.05) was observed between human semitendinosus and bovine extensor samples at all time points and between the bovine flexor and bovine extensor after 1800 cycles; difference between the human semitendinosus and bovine flexor remained insignificant at all time points. Figure 2. Mean failure stress for quasi-static tests to failure, carried out on samples after 300, 900 or 1800 cycles of stress relaxation. ‘’ denotes significant variation at p40.05. The confocal images of Figure 3 show fascicles from the three tendon types before loading. The low magnifi- cation composite images allow imaging of the entire fascicle length. Extensor fascicles could be extracted from the bovine tendon with ease and showed a gener- ally consistent cross section along their length and less evidence of damage than the other two tendon types (Figure 3(c)). Stress relaxation Bovine flexor fascicles appeared to show a much higher degree of interfascicular binding and were dissected with considerably more difficulty than the extensor fascicles (Figure 3(b)), while a degree of surgically induced damage appeared to be present in the semitendinosus fascicles (Figure 3(a)). At a higher magnification, all non-loaded fascicles generally exhib- ited a dense, well-aligned collagen fibrillar arrange- ment, although both human and flexor tendon fascicles exhibited some areas with widening of the inter-fibre space (Figure 3(d) and (e)). Figure 2. Mean failure stress for quasi-static tests to failure, carried out on samples after 300, 900 or 1800 cycles of stress relaxation. ‘’ denotes significant variation at p40 05 ‘’ denotes significant variation at p40.05. ‘’ denotes significant variation at p40.05. Figure 3. Confocal images of fascicles of (a and d) human, (b and e) bovine flexor and (c and f) bovine extensor tendons. All samples were stained with 5 mM acridine orange solution. Figure 3. Confocal images of fascicles of (a and d) human, (b and e) bovine flexor and (c and f) bovine extensor tendons. All amples were stained with 5 mM acridine orange solution. Figure 3. Confocal images of fascicles of (a and d) human, (b and e) bovine flexor and (c and f) bovine extensor tendons. All samples were stained with 5 mM acridine orange solution. Figure 3. Confocal images of fascicles of (a and d) human, (b and e) bovine flexor and (c and f) bovine extensor tendons. All samples were stained with 5 mM acridine orange solution. Proc IMechE Part H: J Engineering in Medicine 228(1) 54 Figure 4. Collagen organisation in tendon fascicles with increasing number of cycles of stress relaxation. Figure 4. Collagen organisation in tendon fascicles with increasing number of cycles of stress relaxation. presented in Figure 6. Large variations in the cycles to failure were evident for all three tendon types (Figures 5 and 6), but this was particularly true for the human semitendinosus where cycles to failure varied from 340 to in excess of 80,000. As a result of the large variances, significant difference in the cycles to failure existed only between the human and bovine extensor tendon sam- ples. Creep curves for the three tendon types showed a similar form, although initial displacement and the length of stage II (secondary) creep varied within and between tendon types. Stress relaxation No significant correlation was observed between initial displacement and number of cycles to failure for any of the tendon types. For bovine samples, the gradient of the secondary creep stage was relatively consistent across all samples; however, in human samples, an elevated gradient was observed in those samples failing at lower numbers of cycles. Although all fascicle types retained their tensile strength after 300 cycles of stress relaxation, some degree of disorganisation within the collagen matrix structure was observed at this time point particularly with the bovine samples (Figure 4). After 900 cycles, significant kinking of the collagen fibrils was observed in all fascicle types, alongside some breakdown of collagen fibril align- ment. While this correlated with the reduction in UTS in bovine samples, no significant decrease in the UTS of human tissue was evident at this time point. By 1800 cycles, the fascicles exhibit a more fibrous structure and spacing between fibres became evident; the clear evidence of matrix breakdown in all samples supports the signifi- cant loss in mechanical properties observed (Figure 4). Creep testing Typical creep curves for the three tendon types are shown in Figure 5, with mean cycles to failure While little accumulated damage was observed after 300 cycles in any of the three tendon types, very 55 Shepherd et al. Figure 5. Typical creep curves for (a) human semitendinosus, (b) bovine flexor and (c) bovine extensor tendon. significant damage was observed in all tendons after 900 and 1200 cycles (Figure 7). Fibril kinking and loss of alignment were observed in all instances along with fibril breakages and widening of the inter-fibre space particularly after 1200 cycles. This damage generally appeared more significant than observed in stress- relaxed samples. Figure 5. Typical creep curves for (a) human semitendinosus, (b) bovine flexor and (c) bovine extensor tendon. Figure 6. Box and whisker plot summarising number of cycles to failure during creep testing for the 3 tendon types. The horizontal line within the box indicates the median, boundaries of the box indicate the 25th (Q1) and 75th (Q3) percentile, the upper whisker indicates the highest data value within the limit Q3+1.5(Q3-Q1) and the lower whisker the lowest data value within Q1-1.5(Q3-Q1). Discussion This is the first study to carry out a full comparison of fascicle level fatigue behaviour across both functionally distinct tendons and species, correlating mechanical changes with structural ones. It additionally provides the first data comparing stress relaxation and creep response in these different tissue types. The bovine extensor tendon fascicles considered in the current study exhibited significantly less fatigue resistance under both cyclic stress relaxation and cyclic creep conditions than either of the other tendon types, but particularly when compared to the human semiten- dinosus. The bovine flexor tendon appeared to exhibit an intermediary response in both instances, although no significant variation was observed between semiten- dinosus and bovine flexor in terms of either stress relaxation or number of cycles to failure during creep. y g p Both the semitendinosus and bovine digital flexor tendons will have a degree of energy-storing capability, helping return energy to the muscle-tendon unit for more efficient movement. The extensor tendon on the other hand acts only to extend the distal limb prior to limb placement. A clear difference in the mechanics of digital flexor and extensor tendons has previously been observed in the horse,8,31 but this is in an animal where strains of 16% have been observed in the superior digi- tal flexor tendon during galloping compared to a maxi- mum peak strain of just 2.5% in the extensor tendon.32 The variation in strains (and associated loads) between flexor and extensor tendons is likely to be less signifi- cant in the cow where sustained high-speed movement is less common, and a less significant adaptation could perhaps be expected in the two tendon types. This sup- ports the absence of significant variation in cycles to failure and stress relaxation at all but the last time point between bovine flexor and extensor fascicles. Much like the equine flexor, the human semitendinosus is expected to be well adapted to cope with the rela- tively high strains and loads it experiences, hence the superior fatigue characteristics. Both the semitendinosus and bovine digital flexor tendons will have a degree of energy-storing capability, helping return energy to the muscle-tendon unit for more efficient movement. The extensor tendon on the other hand acts only to extend the distal limb prior to limb placement. Discussion A clear difference in the mechanics of digital flexor and extensor tendons has previously been observed in the horse,8,31 but this is in an animal where strains of 16% have been observed in the superior digi- tal flexor tendon during galloping compared to a maxi- mum peak strain of just 2.5% in the extensor tendon.32 Figure 5. Typical creep curves for (a) human semitendinosus, (b) bovine flexor and (c) bovine extensor tendon. Figure 6. Box and whisker plot summarising number of cycles to failure during creep testing for the 3 tendon types. The horizontal line within the box indicates the median, boundaries of the box indicate the 25th (Q1) and 75th (Q3) percentile, the upper whisker indicates the highest data value within the limit Q3+1.5(Q3-Q1) and the lower whisker the lowest data value within Q1-1.5(Q3-Q1). Figure 6. Box and whisker plot summarising number of cycles to failure during creep testing for the 3 tendon types. The horizontal line within the box indicates the median, boundaries of the box indicate the 25th (Q1) and 75th (Q3) percentile, the upper whisker indicates the highest data value within the limit Q3+1.5(Q3-Q1) and the lower whisker the lowest data value within Q1-1.5(Q3-Q1). An area of concern in the consideration of fascicle mechanics is the effect of fascicle dissection and the influence of any resultant damage on mechanical char- acteristics. The fascicles of the bovine digital extensor tendon are visible to the naked eye and run in a straight line along the length of the tendon, making for straight- forward dissection.16 Flexor tendon fascicles, on the other hand, are much less visible, are not always straight along the length of the tendon and also appear Figure 6. Box and whisker plot summarising number of cycles to failure during creep testing for the 3 tendon types. The horizontal line within the box indicates the median, boundaries of the box indicate the 25th (Q1) and 75th (Q3) percentile, the upper whisker indicates the highest data value within the limit Q3+1.5(Q3-Q1) and the lower whisker the lowest data value within Q1-1.5(Q3-Q1). Proc IMechE Part H: J Engineering in Medicine 228(1) 56 Figure 7. Collagen organisation in tendon fascicles with increasing number of cycles of creep loading. Figure 7. Collagen organisation in tendon fascicles with increasing number of cycles of creep loading. to exhibit much more interfascicular binding. Discussion These factors can result in the generation of quite significant dissection damage of flexor tendon fascicles and may explain the wider spread of cycles to failure observed with flexor fascicles compared to extensor. It appears that while the flexor tendon exhibits fatigue behaviour most representative of the human semitendinosus, it is also far more likely to suffer from significant dissection damage. Having said this, the range of cycles to failure was even more significant for the human semitendino- sus fascicles, which did not appear to exhibit the same difficulty in dissection. However, these samples may be influenced by the necessary surgical removal. for creep testing. However, when quasi-static testing was carried out on freshly dissected fascicles for the current study, UTS values for both the bovine extensor and human semitendinosus were found to be in excess of 100 MPa and the bovine flexor greater than 75 MPa. Actual UTS values for the three tendon types before loading are included in Table 2, which also includes the percentage of this UTS used during creep testing. These data highlight the potential variation in both quasi-static and fatigue properties that can be induced through sample preparation and may explain the high sample variability. The elevated quasi-static tensile strength of the bovine extensor is at first glance surprising given the low in-life loading of this tendon type. While this may to a degree be the result of the absence of dissection damage in these fascicles, previous studies have also found a lack of correlation between UTS and in-life As a starting point for this study and based upon previous quasi-static testing of fascicles, an average UTS value of 48 MPa was assumed for the bovine ten- don fascicles and 60.22 MPa for the fascicles from the human semitendinosus, with 60% of these values used 57 Shepherd et al. Table 2. Mean UTS Values 6 standard deviation, determined during the course of this work for tendon fascicles without fatigue loading and actual percentage of the UTS applied during creep testing. Table 2. Mean UTS Values 6 standard deviation, determined during the course of this work for tendon fascicles without fatigue loading and actual percentage of the UTS applied during creep testing. Tendon type UTS (MPa) Percentage UTS applied during creep Human Semitendinosus 101.27626.29 35.6% Bovine Flexor 78.78622.24 36.6% Bovine Extensor 107.77655.05 26.7% UTS: ultimate tensile strength. Standard deviations are included in parenthesis. Discussion These stages provide not only a very good descrip- tion of the progression of damage observed here in Figures 4 and 7 for creep but also stress relaxation load- ing. The initial stages of kinked fibres and widening of the inter-fibre space suggest the inter-fibre space forms the weakest link of the tendon fascicle matrix and fati- gue damage initiates here. Indeed, after 300 cycles of stress relaxation, the only damage was generally in the form of kinked fibres, and quasi-static testing would suggest this did not affect the tensile characteristics of the fascicles from any of the three tendon types. Only with high levels of fatigue was evidence of breakdown of the collagen composite structure observed. In the human tendon fascicles, it was not until 1800 cycles of stress relaxation, with quite significant matrix damage, that a statistically significant reduction in UTS was observed. Despite the more prolonged maintenance of mechanical strength in the energy-storing tendons, the nature and rate of matrix breakdown with cyclic defor- mation did not differ significantly between the three tendon types. Table 3. Progression of fatigue damage during controlled in vivo loading: the effect on collagen fibril architecture. Level of fatigue Collagen fibril architecture No fatigue Aligned collagen fibrils Low-level fatigue Kinked fibre deformations Moderate fatigue Kinked fibre deformation with widening of the inter-fibre space High-level fatigue Severe matrix disruption, fibre thinning, angulations and fibre discontinuities Source: Data collated from Neviaser et al.36 Table 3. Progression of fatigue damage during controlled in vivo loading: the effect on collagen fibril architecture. Source: Data collated from Neviaser et al.36 loading.20,26,33 In the equine model, at both the whole tendon and fascicle level, a significantly lower UTS has been observed in the SDFT than the CDET.26 It is sug- gested that in the energy-storing tendon, some compro- mise in UTS is observed in order to allow for the reduced modulus and higher extensibility necessary for energy storage and transfer.26 It should be noted that despite human and bovine flexor tendon fascicles showing a greater degree of ini- tial damage, they still showed superior fatigue charac- teristics; differences may well be more significant in the absence of damage. Furthermore, the use of a lower proportion of the UTS in the case of the bovine exten- sor tendon only highlights its reduced fatigue resistance compared to other two tendon types. References 1. Wang JHC. Mechanobiology of tendon. J Biomech 2006; 39: 1563–1582. 2. Riley G. The pathogenesis of tendinopathy. A molecular perspective. Rheumatology 2004; 43: 131–142. 3. Archambault J. Tendon micromechanics and research methods in tendinopathy. J Musculoskelet Neuronal Interact 2003; 3: 326–328. 4. Ottani V, Raspanti M and Ruggeri A. Collagen structure and functional implications. Micron 2001; 32: 251–260. 5. Benjamin M, Kaiser E and Milz S. Structure-function relationships in tendons: a review. J Anat 2008; 212: 211–228. 6. Screen HRC, Lee DA, Bader DL, et al. An investigation into the effects of the hierarchical structure of tendon fas- cicles on micromechanical properties. Proc IMechE, Part H: J Engineering in Medicine 2004; 218: 109–119. This study provides quantitative data concerning stress relaxation and creep response and a qualitative assessment of the accumulation of damage at the larger hierarchical levels of the tendon structure (primarily at the fibre level). It is deformation and structural changes at this level that are perhaps the most significant for the cell environment. The kinking of the collagen fibres and widening of the inter-fibre space in the earlier stages of fatigue are likely to elicit a response from the resident cells, which may well take an active role in controlling further tendon behaviour and may trigger reactive tendinopathy in vivo.45 An understanding of the inter- actions between collagen fibres, cells and the inter- fibre matrix and how these are influenced by fatigue damage is therefore of critical importance. In an eva- luation of human asymptomatic tendons, it has been found that cell change is always observed alongside matrix change. In addition, the matrix change was found to be largely in the inter-fibre space and only in more extreme damage was a change in the collagen structure observed.46 Fatigue damage will have a sig- nificant effect on the cell environment and biological activity, and fatigue testing of viable samples is thus a critical next step. 7. Thorpe CT, Klemt C, Riley GP, et al. Helical sub- structures in energy-storing tendons provide a possible mechanism for efficient energy storage and return. Acta Biomater 2013; 9: 7948–7956. 8. Batson EL, Paramour RJ, Smith TJ, et al. Are the mate- rial properties and matrix composition of equine flexor and extensor tendons determined by their functions? Equine Vet J 2003; 35: 314–318. 9. Hamner SR, Seth A and Delp SL. Muscle contributions to propulsion and support during running. The authors declare that there is no conflict of interest. The authors declare that there is no conflict of interest. The authors declare that there is no conflict of interest. Funding This study was supported by the Wellcome Trust, Grant No. WT087112. This study was supported by the Wellcome Trust, Grant No. WT087112. Conclusion The bovine extensor tendon, with its positional role, demonstrated a poorer fatigue response than the more highly loaded bovine flexor or the human semitendino- sus tendons, supporting previous data, suggesting that fatigue resistance correlates with in-life loading. Despite the superior fatigue response of the flexor and semiten- dinosus tendon fascicles, a very similar degree of matrix breakdown was observed in the confocal images of all three tendon types. Indeed, some damage to the matrix structure could be observed, without detriment to the quasi-static properties of the fascicle. The assessment of fatigue behaviour in the current study highlighted the care needed in dissection. However, even with appropriate dissection technique, fatigue characteristics should not be assumed to be defined at the fascicular level. In a study of the equine SDFT and CDET that considered both the fascicular and whole tendon level, significantly greater extensions were observed in whole flexor tendons than their con- stitute fascicles, behaviour facilitated by greater capac- ity for sliding at the interfascicular interface.26 The same degree of sliding was not observed in the extensor fascicles. In several studies comparing the behaviour of whole tendon and fascicles, higher failure stress and modulus have been observed in the whole tendon com- pared to the fascicle,26,42 contrary to standard compo- site models that would predict a lower stiffness for the bulk material. While interfascicular behaviour may in part explain differences between whole tendon and fas- cicle behaviour, fascicle isolation is also likely to have an effect. There are, however, distinct advantages to fascicle testing: besides providing an understanding of mechanics at levels below the whole tendon, it also allows for far more straightforward imaging, not only of matrix damage as demonstrated here but also the effect of fatigue on cellular morphology and processes. Fascicular testing also allows for multiple testing condi- tions to be considered with fascicles from the same ani- mal (intra-animal comparison) and has thus been used in a number of studies of mechanobiology and load- induced matrix metabolism.29,43,44 Discussion It has been shown previously that when loading is carried out at levels proportional to in-life loading, then widely varying ten- don types generally exhibit a similar number of cycles to failure.34 The human semitendinosus is likely to see a stress higher than that utilised here and the bovine extensor considerably less; hence, balancing these for in-life load would be expected to result in much more consistent cycles to failure. Although the appearance of the fatigue damage was similar for the two testing techniques, creep tested samples demonstrated more significant damage at a lower number of cycles. This was observed across the three tendon types. It has been speculated that creep is fundamentally more non-linear than relaxa- tion, and that the microstructural processes taking place during the two testing routes could be funda- mentally different.37 During creep, fibre bundles are recruited and stretched out progressively. Clearly, this progressive recruitment would not be expected to occur under stress relaxation, where extension is not increased during the course of the test, perhaps explaining the more significant, widespread matrix damage observed in creep. Nevertheless, with both testing conditions, damage appeared to be initiated in the non-collagenous matrix. The confocal images, although undoubtedly qualita- tive, provide a good insight into the damage accumula- tion that occurred during both creep and stress relaxation tests. In previous studies, the accumulation of matrix damage has been imaged using a second har- monic image technique35,36 and images obtained in this manner are not dissimilar to those obtained here. A useful description of the accumulation of damage in response to creep loading during a controlled in vivo study was included in the article of Neviaser et al.36 and is summarised in Table 3. The matrix changes appear broadly consistent with the fairly limited data from tendinopathic biopsies.38–41 Tendinopathic and ruptured tendons have been observed to exhibit fibres with increased spacing and waviness, as well as disrupted crimp continuity.14 Not only are the microstructural changes in tissue biopsies generally consistent with those in fatigue mod- els, but matrix changes have also been observed to pre- cede cellular, molecular and mechanical changes.14 The matrix changes appear broadly consistent with the fairly limited data from tendinopathic biopsies.38–41 Proc IMechE Part H: J Engineering in Medicine 228(1) 58 Kongsgaard et al.41 observed that while fibril morphol- ogy was abnormal in tendinopathic biopsy specimens, in vivo tendon mechanical properties did not appear altered. References J Biomech 2010; 43: 2709–2716. 10. Clanton TO and Coupe KJ. Hamstring strains in athletes: diagnosis and treatment. J Am Acad Orthop Surg 1998; 6: 237–248. 11. Schache AG, Dorn TW, Blanch PD, et al. Mechanics of the human hamstring muscles during sprinting. Med Sci Sports Exerc 2012; 44: 647–658. 12. Witvrouw E, Mahieu N, Roosen P, et al. The role of stretching in tendon injuries. Br J Sports Med 2007; 41: 224–226. 13. Koulouris G and Connell D. Evaluation of the hamstring muscle complex following acute injury. Skeletal Radiol 2003; 32: 582–589. 59 Shepherd et al. 14. Shepherd JH and Screen HRC. Fatigue loading of ten- don. Int J Exp Pathol 2013; 94: 260–270. 31. Shadwick RE. Elastic energy storage in tendons: mechan- ical differences related to function and age. J App Physiol 1990; 68: 1033–1040. 15. Ker RF. The implications of the adaptable fatigue qual- ity of tendons for their construction, repair and function. Comp Biochem Physiol A Mol Integr Physiol 2002; 133: 987–1000. 32. Thorpe CT, Clegg PD and Birch HL. A review of tendon injury: why is the equine superficial digital flexor tendon most at risk? Equine Vet J 2010; 42: 174–180. 33. Birch HL. Tendon matrix composition and turnover in relation to functional requirements. Int J Exp Pathol 2007; 88: 241–248. 16. Legerlotz K, Riley GP and Screen HRC. GAG depletion increases the stress-relaxation response of tendon fasci- cles, but does not influence recovery. Acta Biomater 2013; 9: 6860–6866. 34. Ker RF. Mechanics of tendon, from an engineering per- spective. Int J Fatigue 2007; 29: 1001–1009. 17. Thornton GM, Shrive NG and Frank CB. Ligament creep recruits fibres at low stresses and can lead to modulus-reducing fibre damage at higher creep stresses: a study in rabbit medial collateral ligament model. J Orthop Res 2002; 20: 967–974. 35. Andarawis-Puri N, Sereysky JB, Sun HB, et al. Molecu- lar response of the patellar tendon to fatigue loading explained in the context of the initial induced damage and number of fatigue loading cycles. J Orthop Res 2012; 30: 1327–1334. 18. Screen HRC. Investigating load relaxation mechanics in tendon. J Mech Behav Biomed Mater 2008; 1: 51–58. 36. Neviaser A, Andarawis-Puri N and Flatow E. Basic mechanisms of tendon fatigue damage. J Shoulder Elb Surg 2012; 21: 158–163. 19. Duenwald S, Vanderby R and Lakes R. Viscoelastic relaxation and recovery of tendon. Ann Biomed Eng 2009; 37: 1131–1140. 37. References Thornton GM, Oliynk A, Frank CB, et al. Ligament creep cannot be predicted from stress relaxation at low stress: a biomechanical study of the rabbit medial collat- eral ligament. J Orthop Res 1997; 15: 652–656. 20. Ker RF, Wang XT and Pike AVL. Fatigue quality of mammalian tendons. J Exp Biol 2000; 203: 1317–1327. 21. Pike AV, Ker RF and Alexander RM. The development of fatigue quality in high- and low-stressed tendons of sheep (Ovis aries). J Exp Biol 2000; 203: 2187–2193. 38. Jo´ zsa L and Kannus P. Histopathological findings in spontaneous tendon ruptures. Scand J Med Sci Sports 1997; 7: 113–118. 22. Pollock CM and Shadwick RE. Relationship between body mass and biomechanical properties of limb tendons in adult mammals. Am J Physiol 1994; 266: R1016–R1021. 39. Movin T, Gad A, Reinholt FP, et al. Tendon pathology in long-standing achillodynia: biopsy findings in 40 patients. Acta Orthop Scand 1997; 68: 170–175. 23. Butler DL, Grood ES, Noyes FR, et al. Effects of struc- ture and strain measurement technique on the material properties of young human tendons and fascia. J Biomech 1984; 17: 579–596. 40. Tallon C, Maffulli N and Ewen SW. Ruptured Achilles tendons are significantly more degenerated than ten- dinopathic tendons. Med Sci Sports Exerc 2001; 33: 1983–1990. 24. Benedict JV, Walker LB and Harris EH. Stress-strain characteristics and tensile strength of unembalmed human tendon. J Biomech 1968; 1: 53–63. 41. Kongsgaard M, Qvortrup K, Larsen J, et al. Fibril mor- phology and tendon mechanical properties in patellar ten- dinopathy: effects of heavy slow resistance training. Am J Sports Med 2010; 38: 749–756. 25. Yamada H. Strength of biological materials (ed FG Evans). Lippincott, USA: The Williams & Wilkins Com- pany, 1970. 42. Yamamoto E, Hayashi K and Yamamoto N. Mechanical properties of collagen fascicles from the rabbit patellar tendon. J Biomech Eng 1999; 121: 124–131. 26. Thorpe CT, Udeze CP, Birch HL, et al. Specialization of tendon mechanical properties results from interfascicular differences. J R Soc Interface 2012; 9: 3108–3117. 43. Screen HRC, Shelton JC, Bader DL, et al. Cyclic tensile strain upregulates collagen synthesis in isolated tendon fascicles. Biochem Biophys Res Commun 2005; 336: 424–429. 27. Haraldsson BT, Aagaard P, Krogsgaard M, et al. 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https://openalex.org/W3028008942	https://www.hal.inserm.fr/inserm-02913696/file/s12885-020-06940-z.pdf	English	null	Quantitative hormone receptor (HR) expression and gene expression analysis in HR+ inflammatory breast cancer (IBC) vs non-IBC	BMC cancer	2,020	cc-by	7,133	To cite this version: Toshiaki Iwase, Kenichi Harano, Hiroko Masuda, Kumiko Kida, Kenneth R Hess, et al.. Quanti- tative hormone receptor (HR) expression and gene expression analysis in HR+ inflammatory breast cancer (IBC) vs non-IBC. BMC Cancer, 2020, 20 (1), pp.430. 10.1186/s12885-020-06940-z. inserm- 02913696 HAL Id: inserm-02913696 https://inserm.hal.science/inserm-02913696v1 Submitted on 10 Aug 2020 Quantitative hormone receptor (HR) expression and gene expression analysis in HR+ inflammatory breast cancer (IBC) vs non-IBC Toshiaki Iwase, Kenichi Harano, Hiroko Masuda, Kumiko Kida, Kenneth R Hess, Ying Wang, Luc Dirix, Steven J van Laere, Anthony Lucci, Savitri Krishnamurthy, et al. To cite this version: Toshiaki Iwase, Kenichi Harano, Hiroko Masuda, Kumiko Kida, Kenneth R Hess, et al.. Quanti- tative hormone receptor (HR) expression and gene expression analysis in HR+ inflammatory breast cancer (IBC) vs non-IBC. BMC Cancer, 2020, 20 (1), pp.430. 10.1186/s12885-020-06940-z. inserm- 02913696 Toshiaki Iwase, Kenichi Harano, Hiroko Masuda, Kumiko Kida, Kenneth R Hess, Ying Wang, Luc Dirix, Steven J van Laere, Anthony Lucci, Savitri Krishnamurthy, et al. Quantitative hormone receptor (HR) expression and gene expression analysis in HR+ inflammatory breast cancer (IBC) vs non-IBC Toshiaki Iwase1,2, Kenichi Harano1,2, Hiroko Masuda1,2, Kumiko Kida1,2, Kenneth R. Hess3, Ying Wang4, Luc Dirix5, Steven J. Van Laere5, Anthony Lucci1,6, Savitri Krishnamurthy1,7, Wendy A. Woodward1,8, Rachel M. Layman1,9, François Bertucci10 and Naoto T. Ueno1,2* HAL Id: inserm-02913696 https://inserm.hal.science/inserm-02913696v1 Submitted on 10 Aug 2020 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Iwase et al. BMC Cancer (2020) 20:430 https://doi.org/10.1186/s12885-020-06940-z Open Access Abstract Background: The purpose of this study was to determine the prognostic role of hormone receptor (HR) on inflammatory breast cancer (IBC) to elucidate its aggressive biological behavior. Methods: We evaluated the expression of estrogen receptor (ER) and progesterone receptor (PR) by immunohistochemical staining and determined the predictive and prognostic role of HR expression on 189 patients with HR+/HER2– IBC and 677 patients with HR+/HER2– stage III non-IBC. Furthermore, we performed gene expression (GE) analyses on 137 patients with HR+/HER2– IBC and 252 patients with HR+/HER2– non-IBC to detect genes that are specifically overexpressed in IBC. Results: The expression of ER% was significantly associated with longer distant disease-free survival and overall survival. However, there was no significant relationship between ER% and neoadjuvant chemotherapy outcome. In the GE study, 84 genes were identified as significantly distinguishing HR+ IBC from non-IBC. Among the top 15 canonical pathways expressed in IBC, the ERK/MAPK, PDGF, insulin receptor, and IL-7 signaling pathways were associated with the ER signaling pathway. Upregulation of the MYC gene was observed in three of these four pathways. Furthermore, HR+/HER2– IBC had significantly higher MYC amplification, and the genetic alteration was associated with poor survival outcome. Conclusions: Higher ER expression was significantly associated with improved survival in both HR+/HER2– IBC and HR+/HER2– stage III non-IBC patients. HR+/HER2– IBC had several activated pathways with MYC upregulation, and the genetic alteration was associated with poor survival outcome. The results indicate that MYC may be a key gene for understanding the biology of HR+/HER2– IBC. Keywords: Inflammatory breast neoplasms, Estrogen receptors, Immunohistochemistry, Gene ex * Correspondence: nueno@mdanderson.org 1 2Section of Translational Breast Cancer Research, Department of Breast Medical Oncology, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030, USA F ll li f h i f i i il bl h d f h i l 1515 Holcombe Blvd, Houston, TX 77030, USA Full list of author information is available at the end of the article Background cancer electronic medical record management system at The University of Texas MD Anderson Cancer Center between January 1, 1989, and April 30, 2015 (n = 1731). A multidisciplinary team, consisting of a medical oncologist, surgical oncologist, radiologist, and nurse, determined the clinical diagnosis of IBC according to the IBC-specific clinical manifestation. This clinical manifestation includes history of rapid onset of breast erythema, edema and/or peau d’orange, and/or warm breast, with or without an underlying palpable mass. A history of flattening, crusting, or retraction of the nipple were also considered. We excluded cases with inflammatory skin change secondary to non-IBC. Inflammatory breast cancer (IBC) is a rare type of breast malignancy characterized by diffuse erythema and edema called peau d’orange without palpable mass. The incidence is approximately 2.0 to 2.5% in a U.S. national survey [1]. This phenotype is also known to have a very aggressive tumor behavior, with a 2.9- to 4.2-year median survival period, which is a significantly poorer survival period than that in locally advanced non-IBC [1, 2]. Estrogen receptor (ER) and progesterone receptor (PR) expression by immunohistochemical (IHC) analysis is commonly used as a predictive marker for endocrine treat- ment as well as a prognostic indicator in non-IBC [3, 4]. Commonly, ER and PR expression by IHC analysis is posi- tively associated with response to endocrine treatment and with better prognosis in early-stage ER-positive (ER+) non- IBC [3, 5]. However, the role of these hormone receptors (HRs) in IBC has been inconsistent, according to a retro- spective analysis based on a large data registry [6, 7]. For patient selection, we first excluded patients who did not have adequate pathological information with which to determine the percentage expression of ER (ER%) and the percentage expression of PR (PR%) (n = 452). Next, we excluded patients who had undergone neoadjuvant endo- crine therapy (n = 59), no definitive surgery (n = 43), or insufficient pathological data for pathological complete response (pCR) (n = 25) or survival (n = 11). We also excluded patients with T stage 0–2 (n = 275) because we did not consider these stages to be locally advanced. Finally, we obtained 866 eligible patients, including 189 with IBC and 677 with case-matched stage III non-IBC (Supplementary Fig. 1). GE evaluation and pathway analysis for GE study We examined GE differences between patients with HR+/HER2– IBC and HR+/HER2– non-IBC by feature- by-feature linear mixture models and then fitting a beta- uniform mixture model to control for multiple testing [11, 12]. The number of significant genes was counted Patient selection Our study population consisted of two groups: (1) the IHC study group, which consisted of 866 patients (189 IBC and 677 non-IBC) and (2) the GE study group, which included 389 patients (137 IBC and 252 non-IBC). Background We previously investigated the prognostic value of HRs in patients with IBC who underwent neoadjuvant chemotherapy (NAC) and found that HR positivity had no prognostic value for survival after NAC among HR- positive (HR+)/human epidermal growth factor receptor 2–positive (HER2+), HR+/HER2-negative (HER2−), and HR-negative (HR−)/HER2+ subtypes [8]. This result was in contrast to those of previous studies showing that the ER+/HER2−subtype demonstrated significantly worse survival outcome compared with ER+/HER2+ or ER- negative (ER−)/HER2+ subtypes [7], or that ER positivity had a significant association with better survival outcome in patients with IBC, regardless of the type of treatment [6]. Although these inconsistencies may be explained by the nature of retrospective analysis, more detailed analysis is needed to understand the mechanism responsible for the differences between HR+ IBC and non-IBC. To eluci- date this mechanism, we applied a two-step approach—an IHC analysis and gene expression (GE) analysis focused on the estrogen signaling pathway in IBC. Pathological evaluation for IHC study We obtained the continuous value of percentage HR expression both in ER and PR. We defined ER as posi- tive if ER expression by IHC was 1% or more. HER2 positivity was determined according to the ASCO/CAP guidelines at the time of pathological evaluation. We defined pCR as no invasive components in residual tumor in the primary site or axillary lymph nodes in the surgical specimen [10]. Our main hypothesis was that HR expression has a prognostic role in HR+/HER2– IBC and that HR+/ HER2– IBC has specific GE in the ER signaling pathway that characterizes aggressive biological behavior. © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Iwase et al. BMC Cancer (2020) 20:430 Page 2 of 9 Page 2 of 9 GE study h For the GE study group, we used mRNA expression data from the World IBC Consortium gene database [9]. The World IBC Consortium is a multicenter collaborative project that explores the biology of IBC based on gene expression by applying whole-transcriptome Affymetrix DNA microarrays. This data set includes the compre- hensive gene set used in our study of 137 IBC patients and 252 non-IBC patients. Patient characteristics Patients with IBC demonstrated significantly higher nuclear grades (P < 0.001) and more frequent ductal histology than did those in the non-IBC group (P = 0.003). In contrast, positivity for lymphatic and vascular invasion was not sig- nificantly different between the non-IBC and the IBC groups. Significantly more patients in the non-IBC group received adjuvant endocrine therapy than did patients in the IBC group (P = 0.007, Table 1). There were no signifi- cant differences regarding to the radiation therapy between two groups. The Mann-Whitney U test showed that the IBC group had significantly lower ER% and PR% compared with the non-IBC group (median ER%: 85% for IBC vs. 90% for non-IBC, P = 0.012; median PR%: 30% for IBC vs. 50% for non-IBC, P = 0.034) (Supplementary Fig. 2). Statistical analysis We compared the clinicopathological characteristics be- tween patients with HR+/HER2– IBC and corresponding non-IBC with use of a chi-square test for categorical data and Student t test for interval-scaled data. We also used a logistic regression model to determine the associ- ation between ER%, PR%, and pCR. IHC study For the IHC study group, we retrospectively reviewed clinical and pathological information from the breast Iwase et al. BMC Cancer (2020) 20:430 Iwase et al. BMC Cancer (2020) 20:430 Page 3 of 9 was approved by the Institutional Review Board at MD Anderson Cancer Center (PA17–0491). was approved by the Institutional Review Board at MD Anderson Cancer Center (PA17–0491). for false discovery rates at 1%. We used the Affymetrix U133 annotation package hgu133a.db (Affymetrix, Santa Clara, CA, USA) to export gene symbols for 22,283 probes. We determined upregulation and downregulation by me- dian value of gene expression. After identifying significantly upregulated/downregulated genes in IBC, we looked for enriched canonical pathways that included these genes by using ingenuity pathway analysis (QIAGEN, Germantown, MD, USA). Next, we investigated the relationship between these canonical pathways and the ER signaling pathway. Survival analysis and setting the cutoff points y We performed a survival analysis with two outcomes for the IHC study (distant disease-free survival [DDFS] and overall survival [OS]) and three outcomes for the GE study (recurrence-free survival [RFS], DDFS, and OS). We defined RFS as the time from the date of definitive surgery to the date of locoregional recurrence or distant metastasis, DDFS as the time from the date of definitive surgery to the date of distant metastasis, and OS as the time from the date of definitive surgery to the date of death due to any causes or the date of last follow-up. Survival rates were calculated by using the Kaplan-Meier method, and curves were compared with the log-rank test. In the Cox proportional hazard model, we adjusted for age, menopausal status, histology, cN stage, cT stage, lymphatic invasion, vascular invasion, grade, and mastec- tomy status. We calculated the hazard ratio for HR ex- pression as 50% increase, which can be thought of as comparing outcomes in two patients, one with ER/PR level X and another with ER/PR level X + 50%. We applied recursive partitioning analysis (RPA) to deter- mine the optimal cutoff points for ER% and PR% that maximized the difference in DDFS. RPA created a re- gression tree that was divided by certain cutoff points that maximized the difference in outcome and then determined the optimal cutoff points [13]. Treatment response, survival analysis, and HR expression Of 677 study patients with non-IBC, 33 (5%) achieved pCR after NAC; of 189 patients with IBC, 13 (7%) achieved pCR. Our logistic regression model showed that the ER% and PR% were not significantly associated with pCR in either non-IBC or IBC (data not shown). The median follow-up for non-IBC and IBC was 4.0 and 3.8 years, respectively. During follow-up, 90 IBC patients (48%) and 226 non-IBC patients (33%) had distant recur- rences; also during this period, 80 IBC patients (42%) and 186 non-IBC patients (27%) died. In the multivariate analysis, expression of ER% was significantly associated with longer DDFS as well as OS for IBC (P = 0.0068 for DDFS and P < 0.001 for OS). However, the effect of the PR% was marginal or non-significant, respectively, for DDFS and OS (P = 0.049 for DDFS and P = 0.14 for OS) (Fig. 1). A similar association between ER expression and survival outcome was observed in non-IBC. ER% and PR% cutoff points RPA showed that the optimal cutoff points for DDFS in ER% and PR% in IBC were 91.5 and 9%, respectively (Fig. 2b). The same cutoff points also distinguished OS for IBC (Fig. 2d). In non-IBC, the survival curves for the group with ER% ≥91.5% and PR% ≥9%, and for the group with ER% < 91.5% and PR% ≥9%, were overlapped for DDFS and OS (Fig. 2a and c). In addition, we performed an external validation ana- lysis by using an external cohort from the Institut Paoli- Calmettes (Marseille, France). The cohort included 57 patients with HR+/HER2– IBC and 78 patients with stage III HR+/HER2– non-IBC who underwent NAC between February 1, 1993, and February 28, 2015. All statistical analyses were performed two-sided, and P < 0.05 was defined as statistically significant. This study We attempted an external validation of the newly established cutoff with use of the external cohort from Institut Paoli-Calmettes. The median follow-up periods for IBC and non-IBC patients were 7.0 and 9.0 years, re- spectively. During follow-up, 28 IBC patients (49%) and 23 non-IBC patients (30%) had distant recurrences; 20 Page 4 of 9 Iwase et al. BMC Cancer (2020) 20:430 Table 1 Patient characteristics Characteristic non-IBC No. IBC No. ER% and PR% cutoff points (n = 677) % (n = 189) % P Age, median (range), y 50 (22–83) 51 (23–75) 0.56 BMI, mean ± SD, m2 29.3 ± 6.8 31.7 ± 7.5 0.93 Menopausal status 0.002 Pre 352 (52) 75 (40) Post 304 (45) 108 (57) Unknown 21 (3) 6 (3) cT stage < 0.001 3 395 (58) 0 (0) 4 282 (42) 189 (100) Unknown 0 (0) 0 (0) cN stage 0.046 0 57 (8) 29 (15) 1 415 (61) 109 (58) 2 69 (10) 18 (10) 3 135 (20) 33 (17) Unknown 1 (1) 0 (0) Histology 0.003 Ductal 507 (75) 160 (85) Lobular 103 (15) 12 (6) Mixed 46 (7) 9 (5) Other 21 (3) 8 (4) Histological grade < 0.001 1 46 (7) 4 (2) 2 313 (46) 67 (35) 3 288 (43) 107 (57) Unknown 30 (4) 11 (6) Lymphatic invasion 0.277 Positive 389 (57) 116 (61) Negative 271 (40) 67 (36) Unknown 17 (3) 6 (3) Vascular invasion 0.512 Positive 400 (59) 106 (56) Negative 260 (38) 77 (41) Unknown 17 (3) 6 (3) NAC regimen 0.308 A 74 (11) 27 (14) T 28 (4) 5 (2) A + T 572 (84) 156 (83) Other 3 (1) 1 (1) Adjuvant chemotherapy 0.001 Yes 120 (13) 54 (28) No 557 (87) 135 (72) Page 5 of 9 Iwase et al. BMC Cancer (2020) 20:430 Table 1 Patient characteristics (Continued) Characteristic non-IBC No. IBC No. (n = 677) % (n = 189) % P Adjuvant endocrine therapy 0.007 Yes 590 (87) 150 (79) No 87 (13) 39 (21) Neoadjuvant radiation therapy 0.056 Yes 6 (1) 5 (3) No 671 (99) 184 (97) Adjuvant radiation therapy 0.100 Yes 612 (90) 163 (86) No 65 (10) 26 (14) Abbreviations; IBC inflammatory breast cancer, BMI body mass index, SD standard deviation, NAC neoadjuvant chemotherapy, A anthracycline, T taxane Fig. 1 Effects of change in ER% and PR% on survival outcomes by multivariate analysis. a Comparison of hazard ratio for distant disease-f according to the change in ER and PR expression. b Comparison of hazard ratio for overall survival according to the change in ER and PR Fig. 1 Effects of change in ER% and PR% on survival outcomes by multivariate analysis. a Comparison of hazard ratio for distant disease-free survival according to the change in ER and PR expression. Gene expression analysis p y Although the validation study on the outside cohort could not determine the universality of newly detected cutoff points, the IHC study suggested that ER% was as- sociated with significantly better survival outcome in ER+/HER2– IBC than in corresponding non-IBC. This result indicated the difficulty in establishing universal cutoff points for HR+ IBC and the need to deeply inves- tigate the role of the ER signaling pathway at the gene level. To this end, we further compared GE between HR+/HER2– IBC patients and the corresponding non- IBC patients to detect the specific genetic alteration in the ER signaling pathway. ER% and PR% cutoff points b Comparison of hazard ratio for overall survival according to the change in ER and PR expression Fig. 1 Effects of change in ER% and PR% on survival outcomes by multivariate analysis. a Comparison of hazard ratio for distant disease-free survival according to the change in ER and PR expression. b Comparison of hazard ratio for overall survival according to the change in ER and PR expression Iwase et al. BMC Cancer (2020) 20:430 Page 6 of 9 Iwase et al. BMC Cancer Fig. 2 Survival outcomes according to newly defined cutoff points for ER and PR expression in ER+/HER2– IBC and corresponding non-IBC. a Distant disease-free survival by ER and PR levels for non-IBC. b Distant disease-free survival by ER and PR levels for IBC c Overall survival by ER and PR levels for non-IBC. d Overall survival by ER and PR levels for IBC IBC patients (35%) and 9 non-IBC patients (12%) died. DDFS and OS rates were lower in IBC than in non- IBC patients. Although a similar pattern of survival curves was observed in DDFS and OS for non-IBC group, the survival analysis with optimal grouping for ER% and PR% identified in the training set showed no significant differences in prognosis in the IBC group (Supplementary Fig. 3a-d). IBC patients (35%) and 9 non-IBC patients (12%) died. DDFS and OS rates were lower in IBC than in non- IBC patients. Although a similar pattern of survival curves was observed in DDFS and OS for non-IBC group, the survival analysis with optimal grouping for ER% and PR% identified in the training set showed no significant differences in prognosis in the IBC group (Supplementary Fig. 3a-d). Discussion To the best of our knowledge, the present study was the first to find that the positivity level of ER expression had a significant prognostic role, even in HR+/HER2– IBC. Furthermore, the GE exploratory analyses indicated that MYC was the key gene in understanding the biological behavior of HR+/HER2– IBC. p Generally, MYC expression was associated with cell cycle activity with increased cyclin B1 and Ki-67 expres- sion [17] and can be a predictive marker for endocrine therapy resistance [20]. Indeed, we observed MYC up- regulation in the ERK/MAPK and PDGF pathways, which have a significant role in endocrine therapy resist- ance [21, 22]. The activation of ERK/mitogen-activated protein kinase induces tamoxifen resistance by altering the level of estrogen-related receptor γ (ERRγ), which is an orphan member of the nuclear receptor superfamily. Furthermore, ERRγ-driven transcriptional activity is im- paired by the mutation of ERK target sites, leading to the tamoxifen resistance [21]. For the PDGF pathway, a clinical study of 45 breast cancer patients treated with an aromatase inhibitor showed that the protein expres- sion of PDGF receptor α and β in tumor was signifi- cantly increased at the point of relapse and the higher expression was correlated with shorter time to treatment failure [22]. Although the detailed mechanism for endo- crine therapy resistance by MYC for HR+/HER2– IBC needs to be further investigated, the results in the present study suggest that MYC possibly contributed to poor prognosis due to either intrinsic characteristics or endocrine treatment resistance. In contrast to the predictive value, we identified the prognostic role of ER in HR+/HER2– IBC. Basically, the HR-positive breast cancer population shows a low per- centage of pCR because of tumor dormancy, and as tumor stage becomes more advanced, pCR can be more difficult to obtain [7, 8]. Indeed, the present study showed that only 13 IBC patients (7%) and 33 non-IBC patients (5%) experienced pCR, which was very small compared with the population of patients with early breast cancer. Notably, patients with HR+/HER2– IBC had a wider range of heterogeneity in survival outcome according to ER expression level, and those with high ER expression had a better prognosis, which was similar to that of non-IBC patients. The results indicated that ER expression level also had an important prognostic role even in patients with HR+/HER2– IBC. Survival analysis based on MYC expression difference in diagnostic criteria for IBC could affect the outcome. Further investigation is needed to establish the globally applicable cutoff point. y p The Wilcoxon test showed no significant differences in MYC expression levels between patients with HR+/ HER2– IBC and those with non-IBC. In IBC patients, a Cox proportional hazard model indicated significant as- sociations between MYC level and RFS (hazard ratio, 1.93; 95% confidence interval, 1.09 to 3.43, P = 0.003) and between MYC level and DDFS (hazard ratio, 2.00; 95% confidence interval, 1.10 to 3.64, P = 0.028), but not between MYC level and OS (hazard ratio, 1.45; 95% con- fidence interval, 0.65 to 3.24, P = 0.38) in HR+/HER2– IBC (Supplementary Table 4). g y pp p In the GE analysis, MYC was found to be upregulated in 3 of 4 pathways overlapping the ER pathway, and the gene had a significant impact on survival outcome in IBC. MYC is a regulator gene coding for transcriptional factors involved in cell cycle and cell growth. Generally, MYC amplification was observed in more aggressive subtypes such as HER2+ and triple-negative types [14], as well as in advanced clinical status [15], leading to poor survival outcome [16, 17]. For IBC, MYC has been investigated mainly in the triple-negative type [18, 19]; however, the present study found that MYC was also up- regulated in HR+/HER2– IBC, leading to a significant association with poor survival outcome. Pathway analysis of significant genes associated with IBC status The distribution of patient characteristics was not signifi- cantly different between the IBC and non-IBC groups (Supplementary Table 1). We identified 97 probe sets that significantly distinguished IBC from non-IBC at a false discovery rate of 1%. Among the 97 probe sets, 13 did not have a gene symbol in the Affymetrix annotation package, and 84 genes remained (Supplementary Table 2). g pp y After the 84 genes associated with IBC were investigated with use of ingenuity pathway analysis, the top 15 canon- ical pathways in which these genes were included were revealed (Supplementary Fig. 4). However, the number of genes included in each pathway was relatively small (1 to 3). Among the top 15 pathways, the extracellular signal- regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) signaling pathway, platelet-derived growth factor (PDGF) pathway, insulin receptor signaling pathway, and interleukin-7 (IL-7) signaling pathway overlapped with the ER signaling pathway. Among the upregulated/downregu- lated genes in those four pathways, MYC was the most frequently observed upregulated gene in three of the path- ways (Supplementary Table 3). Iwase et al. BMC Cancer (2020) 20:430 Page 7 of 9 Page 7 of 9 Acknowledgements We thank Tamara K. Locke for editing the manuscript, Modest G. Patangan Jr. and Limin Hsu for data collection, and Huiming Sun and Jie S Willey for helping with preparing the protocol. Discussion The present study also detected the optimal cutoff points for survival in HR+/HER2– IBC at 91.5% for ER and 9% for PR. Furthermore, these cutoff points were IBC-specific since they could not be applied to corre- sponding non-IBC. Unfortunately, however, the external validation study failed to show the universality of the newly detected cutoff points on prognosis. In fact, the distribution of HR expression was significantly different between MD Anderson’s cohort and the validation co- hort, showing 72.2 and 80.4% in mean ER and 40.4 and 53.8% in PR for MD Anderson’s cohort and the valid- ation cohort, respectively. Accordingly, OS was generally better in the validation cohort than in the MD Anderson cohort (data not shown). The difference in survival was probably due to the fact that most of the patients with IBC at MD Anderson were referred from community clinics and this data set included more complexed or advanced cases with comorbidities. In addition, the Notably, MYC upregulation contributed to survival outcome only in RFS and DDFS but not in OS for HR+/ HER2– IBC. Previous studies had suggested that IBC has a unique metastatic process characterized by higher lymphatic invasion, tumor embolization, activated in- flammatory pathways, and increased growth factors [23]. The MYC gene codes transcription factors and regulates every stage of the metastasis process, including cell pro- liferation, angiogenesis, and epithelial-to-mesenchymal transition [24]. However, it is unclear whether MYC has any specific effect on the metastatic process, especially for IBC. We reported that metastasis for IBC was associ- ated with a risk allele at 8q24 where MYC located [25]. Moreover, we determined that the MYC activation in Page 8 of 9 Page 8 of 9 Iwase et al. BMC Cancer (2020) 20:430 Iwase et al. BMC Cancer (2020) 20:430 Page 8 of 9 IBC was caused by the dysfunctional antagonization of MYC by the activation of SMAD3, which was located downstream of the TGF-beta signaling pathway [26]. Since MYC can be activated by upstream signaling path- ways and codes many transcriptional factors, more com- prehensive gene analysis will be needed to elucidate how MYC affects the metastasis process in HR+/HER2– IBC. growth factor; RFS: Recurrence-free survival; OS: Overall survival; PR: Progesterone receptor; RPA: Recursive partitioning analysis growth factor; RFS: Recurrence-free survival; OS: Overall survival; PR: Progesterone receptor; RPA: Recursive partitioning analysis Author details 1 l h 1Morgan Welch Inflammatory Breast Cancer Research Program and Clinic, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030, USA. 2Section of Translational Breast Cancer Research, Department of Breast Medical Oncology, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030, USA. 3Department of Biostatistics, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030, USA. 4Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030, USA. 5Department of Oncology, University of Antwerp, Prinsstraat 13, 2000 Antwerpen, Belgium. 6Department of Breast Surgical Oncology, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030, USA. 7Department of Anatomical Pathology, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030, USA. 8Department of Radiation Oncology, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030, USA. 9Department of Breast Medical Oncology, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030, USA. 10Laboratory of Predictive Oncology, Centre de Recherche en Cancérologie de Marseille (CRCM), Inserm, U1068, CNRS UMR7258, Institut Paoli-Calmettes, Aix-Marseille Université, F-13009 Marseille, France. Received: 6 February 2020 Accepted: 7 May 2020 Received: 6 February 2020 Accepted: 7 May 2020 Availability of data and materials All analyzed data are included in this published article and its supplementary file. The original data are available upon reasonable request to the corresponding author. Competing interests The authors declare no competing interests. Authors’ contributions The chief limitation of the present study is that we excluded a certain number of patients during the selec- tion process because they did not have a detailed patho- logical report; most of these patients had been evaluated outside of MD Anderson. Although we cannot estimate the result of excluding these patients, it is possible that HR distribution and the cutoff point may have been dif- ferent if all cases had been included in the analysis. Moreover, the antibody used for IHC and the definition of HER2 positivity was not consistent over the study period, which possibly affected the overall results. Concept and design: TI, KH, HM, NTU. Data acquisition: TI, KH, HM, LD, SJVL, FB. Data analysis and interpretation: TI, KH, HM, KRH, YW. Manuscript writing: TI. Critical review of the manuscript: TI, KH, MK, KK, KRH, YW, LD, WAW, RML, SJVL, AL, SK, FB, NTU. All authors read and approved the final manuscript. Funding The gene analysis part was funded by the Morgan Welch Inflammatory Breast Cancer Research Program, the State of Texas Rare and Aggressive Breast Cancer Research Program Grant (1R01CA205043-01A1) (NTU), and MD Anderson’s Cancer Center Support Grant (P30CA016672). Consent for publication Not applicable. Consent for publication Not applicable. Ethics approval and consent to participate The present study was the first to find that higher ER expression level was significantly associated with better survival, even in patients with HR+/HER2– IBC. Gene analysis showed that IBC had several activated pathways with MYC upregulation compared with corresponding non-IBC. The results indicated that MYC may be a key gene for understanding the biological behavior of HR+/ HER2– IBC. This study was approved by the Institutional Review Board at MD Anderson Cancer Center (PA17–0491). Informed consent was waived for this retrospective analysis. 1. Hance KW, Anderson WF, Devesa SS, Young HA, Levine PH. Trends in inflammatory breast carcinoma incidence and survival: the surveillance, epidemiology, and end results program at the National Cancer Institute. J Natl Cancer Inst. 2005;97(13):966–75. https://doi.org/10.1093/jnci/dji172. 2. Gonzalez-Angulo AM, Hennessy BT, Broglio K, Meric-Bernstam F, Cristofanilli M, Giordano SH, Buchholz TA, Sahin A, Singletary SE, Buzdar AU, Hortobagyi GN. Trends for inflammatory breast cancer: is survival improving? Supplementary information f Supplementary information accompanies this paper at https://doi.org/10. 1186/s12885-020-06940-z. Supplementary information accompanies this paper at https://doi.org/10. 1186/s12885-020-06940-z. Additional file 1: Figure S1. CONSORT diagram. Figure S2. Scatter plot of ER% and PR% in IBC and non-IBC. Figure S3. Result of external validation for newly defined cutoff points by the cohort from Institut Paoli-Calmettes. Figure S4. Top canonical pathways including upregu- lated/downregulated genes associated with IBC by ingenuity pathway analysis. After analyzing the 84 genes associated with IBC with use of in- genuity pathway analysis, the top 15 canonical pathways, which included those genes, were revealed. The z-score determines whether an upstream transcription regulator has significantly more “activated” predictions than “inhibited” predictions (z > 0) or vice versa (z < 0). The ratio in the figure means the percentage of included genes in each pathway. All P values were unadjusted. Additional file 2: Table S1. Patient background in gene expression analysis. Table S2. Probes and symbols for gene expression analysis. Table S3. Upregulated and downregulated genes in four pathways Additional file 2: Table S1. Patient background in gene expression analysis. Table S2. Probes and symbols for gene expression analysis. Table S3. Upregulated and downregulated genes in four pathways overlapping the estrogen receptor signaling pathway. Table S4. The result of the Cox proportional hazard model based on MYC expression in ER+/HER2– IBC. Hazard ratio was calculated according to the continuous value of MYC expression. Additional file 2: Table S1. Patient background in gene expression analysis. Table S2. Probes and symbols for gene expression analysis. Table S3. Upregulated and downregulated genes in four pathways overlapping the estrogen receptor signaling pathway. Table S4. The result of the Cox proportional hazard model based on MYC expression in ER+/HER2– IBC. Hazard ratio was calculated according to the continuous value of MYC expression. 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https://openalex.org/W2021541942	https://izajolp.springeropen.com/track/pdf/10.1186/s40173-015-0040-y	English	null	Did the Intergenerational Solidarity Pact increase the employment rate of older workers in Belgium? A macro-econometric evaluation	IZA journal of labor policy	2,015	cc-by	14,023	Correspondence: bruno.vanderlinden@uclouvain.be 1IRES - Université catholique de Louvain, Louvain-la-Neuve, Belgium 2Fonds de la Recherche Scientifique-FNRS, Brussels, Belgium Full list of author information is available at the end of the article © 2015 Dejemeppe et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Did the Intergenerational Solidarity Pact increase the employment rate of older workers in Belgium? A macro-econometric evaluation Muriel Dejemeppe1, Catherine Smith1 and Bruno Van der Linden1,2,3 Correspondence: bruno.vanderlinden@uclouvain.be 1IRES - Université catholique de Louvain, Louvain-la-Neuve, Belgium 2Fonds de la Recherche Scientifique-FNRS, Brussels, Belgium Full list of author information is available at the end of the article Abstract In December 2005, the Belgian government adopted the law on the Intergenerational Solidarity Pact (ISP) aimed at increasing the employment rate of older workers. The main policies of the ISP consist of a pension bonus, reductions in employers’ social security contributions and measures discouraging early retirement while encouraging working time reductions at the end of the career. We aim at evaluating the overall effectiveness of the ISP in raising the employment rate of older workers. To that purpose, we compare the actual evolution of the employment rate after the implementation of the policies to its predicted (counterfactual) evolution based on the estimation of a macro-econometric model in a period prior to the ISP. The results suggest a slight positive impact of the ISP on the employment rate of older workers but to the detriment of the younger workers. However, there is a lack of statistical power to draw firm conclusions on the overall effect of the ISP. JEL classification: J21, J26, H53, E32 JEL classification: J21, J26, H53, E32 Keywords: Aging; Evaluation of labor market policies; Macro-econometrics Dejemeppe et al. IZA Journal of Labor Policy (2015) 4:17 DOI 10.1186/s40173-015-0040-y Dejemeppe et al. IZA Journal of Labor Policy (2015) 4:17 DOI 10.1186/s40173-015-0040-y Open Access 1 Introduction The challenge brought by an aging population on the welfare system of many countries in the world is a well-known fact. In this respect, Belgium is particularly exposed because of policies introduced between the 70’s and the 90’s fostering early exit from the labor market. According to OECD statistics for 2005, in Belgium, the employment rate of men and women aged 55 to 59 years old was, respectively, 55.4 % and 31.8 %, while they were 67.2 % and 48.6 %, on average, in the EU15. In 2012, the situation has improved with an employment rate of 63.9 % and 48.4 % for elderly men and women in Belgium compared to 73.2 % and 59.4 %, on average, in the EU15 countries. Under the impulse of the Lisbon strategy, Belgian policy-makers began to acknowledge the issue of population aging and adopted a package of active aging policies on December 2005, called the law on the Intergenerational Solidarity Pact (Pacte de Solidarité entre les Générations), despite the strong opposition of labor unions. The objective of the Intergen- erational Solidarity Pact (ISP) was to increase the employment rate of older workers. The main policies consist in a reduction in employers’ social security contributions, several reforms intending to discourage access to early retirement, an easier access to working pe et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 Dejemeppe et al. IZA Journal of Labor Policy (2015) 4:17 Page 2 of 23 time reduction with (partial) wage compensation, and a pension bonus. In 2008, the reduction in employers’ social security contributions represented, on average, 0.7 % of total labor cost among workers aged 50 years old and over. The pension bonus amounted to 2 euros per day of full-time work while being at least 62 years old and up to the legal retirement age of 65 years old.1 The aim of our analysis is to evaluate the overall effect of the ISP on the official target of the program, namely the (headcount) employment rate of older workers by gender: did the ISP have any major impact on their employment rate compared to a non-ISP counterfactual? We also aim at evaluating whether the employment rate of a younger age group has been indirectly affected by the ISP through negative or positive spillover effects. The evaluation period ends in 2008q2 since, as of this moment, the effects of the ISP policies cannot be disentangled from the policies taken to counteract the effect of the financial and economic crisis. This paper relies on a macro-econometric evaluation to assess the impact of the ISP policies taken as a whole. Although crucial for policy-making, an assessment of the macro-economic impact of the ISP reform has never been rigorously conducted for Belgium, that is, in searching for a causal link between the ISP and the increasing employ- ment rate among older people from 2007 onwards. How can then citizens be ensured that public funds are spent wisely? Furthermore, most of the evaluation literature on labor market policies takes a micro-econometric approach, focusing on the evaluation of a spe- cific program on its direct (and, at times, indirect) beneficiaries. The drawback of relying only on this approach is that it fails to capture possible general equilibrium impacts. And these impacts are likely to be further amplified if the program is part of a larger set of reforms, as is the case with the ISP. Our evaluation method consists in comparing the actual quarterly evolution of the age and gender-specific employment rates over the period 2007 to mid-2008 to a counterfac- tual evolution. The latter should measure what would have happened in the absence of the ISP. More recently, Dmitrijeva and Hazans (2007), Fahr and Sunde (2009), Hertweck and Sigrist (2013) and Klinger and Rothe (2012) evaluate the macro effect of the Hartz I/II and III reforms in Germany on the efficiency of the match- ing process between job seekers and vacancies. To that purpose, they estimate a matching function on their entire period of observation and make the matching efficiency depen- dent on a constant term, a business cycle indicator (the IFO business expectations index), month fixed effects and a (treatment) dummy equal to 1 after the implementation of the Harz reforms. This treatment dummy is not made time dependent, thus allowing only for a single structural change of the outcome variable. Given the usual lags associated with the implementation of policies and the response of the various parties involved, labor market reforms are expected to have an impact only after some time has passed. This justifies time-varying treatment effects as we do in the present paper. Besides, our eval- uation approach, which consists in comparing actual outcomes to predicted ones, is less parametric and more flexible than estimating treatment effects within a regression model. Another method that was commonly used in the 90’s to evaluate macro impacts of labor market policies uses variations in program scale (spending or number of participants) over time and/or across regional units or countries (Calmfors and Skedinger 1995; de Koning 1993; Hujer et al. 2002; Jackman et al. 1990; Layard et al. 1991; Lehmann 1993; OECD 2011). The fundamental issue of this evaluation approach is to ensure that these variations are exogenous and, if they are not, to rely on credible instruments. For the evaluation of the ISP, this type of method cannot be envisaged in any case because of the high number of policies introduced by the ISP and data limitation. To the best of our knowledge, for Belgium, there is only one paper, Bassilière et al. (2007), that investigates the macro impact of one policy of the ISP, which is the reduc- tion in employers’ social security contributions (henceforth, “RESSC”) for older workers. Bassilière et al. (2007) use simulations of a large annual macro-econometric model with and without the reduction and then compare the two scenarios. They find a positive impact on the employment of the older workers but a negative impact on the employ- ment of younger workers with low wages. It is constructed by first estimating a macro-econometric model explaining the quar- terly age and gender-specific employment rates over the period 1997 (first year where the data are available) to 2007. Next, this estimated model is used to forecast the employment rate from 2007 onwards. A positive difference between the actual value of the employ- ment rate and the predicted, counterfactual value is then interpreted as a positive impact of the ISP. A widely-used major determinant of the employment rate is the current and past lev- els of real GDP. For the forecasts, we assume that the observed level of GDP is the same whether the ISP is in place or not. As this assumption might be questioned, so we check the robustness of our results by considering instead the GDP of the neighboring countries of Belgium. Some other determinants of headcount employment are found in the macro- econometric literature, namely the cost of labor, working hours and labor productivity (Bodart et al. 2009; Mourre 2006). As the forecast has to generate a convincing counter- factual, we cannot consider the cost of labor nor working hours of the older workers in the model specification simply because the ISP is affecting these variables to an extent that can hardly be predicted without a structural model of wages and hours worked. Build- ing such a model is clearly beyond the scope of this paper. Next, determinants of the employment rate other than the ISP could in principle have changed during that period. However, we have found no important shock other than the ISP that could have had a Dejemeppe et al. IZA Journal of Labor Policy (2015) 4:17 Page 3 of 23 major influence on the employment rate of the older workers at that time. Our empirical analysis cannot reject the hypothesis that the ISP had no effect on the employment rate of the salaried workers aged 50 or more and occupied in the private sector. A similar evaluation method was used by Kopits (1978) to assess the employment effect of a temporary wage subsidy in France (incentive bonus for job creation) for the period 1959–1975. The author estimates an employment equation by industry for the period before the subsidy was made available. Then, employment is predicted over two quar- ters and the difference between the actual values and the forecasts are used to evaluate the impact of the wage subsidy. 2.1 Institutional background around the reform of the ISP Belgium is a federal state that has decentralized some policies to regional authorities (Brussels, Flanders and Wallonia). The social security and pension systems are organized at the federal level. The payment of unemployment benefits (UB) and the issuing of sanc- tions in the case of non-compliance with the rules are federal competencies organized by the National Office for Employment (ONEM). The regions, in turn, are responsible for the Public Employment Services, which are in charge of counseling, job search assistance, intermediation services and training of unemployed and employed workers. The policies of the ISP were taken at the federal level. Belgium has a pay-as-you-go public pension system. All employees and self-employed people pay compulsory contributions to the federal state funds for old age and invalidity pensions. The legal retirement age is 65 years old for men and was brought from 60 years old in 1996 to 65 years old in 2009 for women. In this setting, no minimum years of paid contributions is imposed. The basic state pension depends on the occupational status, the gross salary, the number of years worked and the family composition. Anticipated retirement can be claimed up to 5 years before the normal retirement age with at least 35 years of paid contributions (including periods on unemployment or dis- ability benefits). However, four other schemes allow an early withdrawal from the labor market in Belgium. Those mechanisms are described as they were before the introduction of the ISP in December 2005. First, various schemes allowing older workers to withdraw earlier from the labor market coexist. The most important one, the collectively agreed (or “conventional”) early-retirement regime, entitles old workers who loose their jobs to UB with an addi- tional allowance paid by the previous employer. The requirement is a minimum age of 58 with at least 25 years of career. People that benefit from this early retirement scheme are exempted from job search, and pension coverage is continued during that period. The employer has to replace the early retired for a period of at least three years, although exemptions to this obligation are awarded in 57 % of the cases (OECD 2003, p. 75). However, several exceptions to the age requirement exist. For instance, the age threshold can be set to 56 for night work and between 50 and 55 for firms in finan- cial difficulties or in restructuring. 2 Institutional setting, the ISP law and its expected effects This section is divided in two parts. The first one provides a brief description of the orga- nization of the social security and pension systems in Belgium. The second one is devoted to the description of the main policies implemented through the ISP and to the discussion of their expected effects. It goes without saying that these results strongly depend on the underlying assumptions of the simulated model. Turning to micro-econometric analyses, Albanese and Cockx (2015) evaluate RESSC introduced in 2002 and targeted at workers older than 58. They find positive but non–significant effects on their employment rate. This effect is however more important and significant for work- ers employed in sectors with a high incidence of early retirement. Lefevre and Orsini (2011) and López-Novella (2012) conclude that the pension bonus had, if any, a very limited effect on the employment rate of male old workers. This paper proceeds by explaining, in section 2, the institutional setting, the main fea- tures of the ISP and its expected effects on the employment rate of older workers. Section Dejemeppe et al. IZA Journal of Labor Policy (2015) 4:17 Page 4 of 23 Page 4 of 23 3 describes the data. In section 4, we explain the method used to evaluate the overall impact of the ISP on the age and gender-specific employment rates in Belgium. In section 5, the results are presented and interpreted. The final section concludes. 2.1 Institutional background around the reform of the ISP Specific eligibility conditions can also be defined at the sectoral level by collective agreements. There is also an “unofficial early retire- ment scheme.” As in the collectively agreed pre-retirement regime, the employer who lays off a worker under this scheme pays a supplement to UB. This scheme is how- ever more flexible for the employer since there are no specific requirements to fulfill apart from the fact that the worker is at least 45 years old. The conditions of the Dejemeppe et al. IZA Journal of Labor Policy (2015) 4:17 Page 5 of 23 early retirement (such as the minimal notice period, the amount and duration of the lump-side payment) are defined by a private agreement between the employer and the worker. The second scheme is known as the regime of old aged unemployed exempted from job search (chômeur âgé non demandeur d′emploi). Older unemployed workers can be exempted from searching for a job and being available on the labor market. To benefit from these advantages, the unemployed must be either aged between 50 and 57 years old, unemployed for more than one year and have reported a very long career (at least 38 years), or aged at least 58 years old. These workers continue to be eligible for UB and receive in addition a benefit supplement. The third scheme is the full-time time-credit (crédit temps à temps plein). People of all ages can interrupt their career for a period of one year if they have worked for the same employer for at least 12 months out of the 15 last months. The period of full-time time- credit can be extended up to 5 years by means of collective agreements at the sectoral level. People on full-time time-credit receive a lump-sum allowance from the National Office for Employment to compensate partially the loss of salary. Workers at the end of their career can then use this scheme right before being eligible for other routes toward early withdrawal. It should be mentioned that a specific scheme, the part-time time- credit, allows workers above the age of 50 and with 5 years full-time within the same firm, to reduce working time either by 20 % or by 50 % until the age of statutory retirement. The resulting loss of salary is partially offset by a lump-sum (generous) allowance from the National Office for Employment. 2.1 Institutional background around the reform of the ISP Disability benefits constitute the last scheme leading to early withdrawal. It requires that the workers are not able to work. 2.2 The Intergenerational Solidarity Pact Belgium has not waited for the introduction of the Intergenerational Solidarity Pact (ISP) to try to expand the employment rate of the older workers.2 However, the ISP marks a turning point through the number of policies taken on various aspects of older workers’ employability. This sub-section presents the main new policies included in the ISP and the major changes brought to existing policies. Only policies introduced over our evaluation period (2007 to mid-2008) are considered. It begins by describing the policies and then looks at the magnitude of the policies in terms of number of participants and expenditure. The age of eligibility varies across policies. Most of time they target people aged 50 years old and over, and sometimes people aged 45 and over. This sub-section also discussed likely effects of those policies. 2.2.1 Description The four main policies consist in a pension bonus, RESSC, more restrictive access to the early retirement scheme and less restrictive access to part-time time-credit. The pension bonus was introduced in January 2007, so about one year after the adoption of the ISP law. All workers and self-employed aged at least 62 years old or with at least 44 years of paid contributions received a bonus for pensions starting from January 2007 and before 2013. The bonus corresponded to 2 euros for each day worked in a full time position, limited to 624 euros for each full year of work. Dejemeppe et al. IZA Journal of Labor Policy (2015) 4:17 Page 6 of 23 In April 2007, within the framework of the ISP, the government introduced a specific RESSC for workers aged between 50 and 56 years old earning 12,000 euros (gross) or less per quarter. The quarterly reduction amounts to 50 euros per quarter for a worker aged 50 years old and increased by 50 euros every year until the age 56. The reduction is permanent and concerns both the new hires and the incumbent workers. For workers aged at least 58 (respectively, 57), employers’ social security contributions were already reduced by 400 euros per quarter since April 2002 (respectively, January 2004). With the ISP, the government reinforced the RESSC for workers aged 57 with a further reduction of 50 euros per year worked from 57 years old onwards. The maximum quarterly RESSC is reached at the age of 65 years and amounts to 800 euros. RESSC targeted at workers aged 50 or more represented 0.6 % of the total wage cost of these workers in 2007 and 0.7 % in 2008. Since the ISP, the government has also tried to discourage access to early retirement in three ways. First, at the beginning of 2008, the age requirement of the collectively agreed pre-retirement regime increased from 58 to 60, and the required years of career increased from 25 to 30 (26) for men (women). The age threshold remained at 58 for par- ticularly long careers or in the case of heavy work, providing the worker has at least 35 years of career. Second, the government decided to make the unofficial early retirement scheme less attractive. Since April 2006, the allowance paid to the unemployed worker by the previous employer has been made subject to social security contributions. 2.2.1 Description Thirdly, since January 2007, the employer’s allowance under both the official and unofficial pre- retirement regime has to be paid to the old worker even if she finds a job in another firm.3 In case of refusal of the former employer, financial penalties are applied. At the same time, the government made the possibility of gradually reducing working time more appealing for the workers aged 50 or more. Since June 2007, the eligibility condition for the part-time time-credit changed to 3, instead of 5, years of seniority with the current employer. On top of these four main policies, early-retired workers laid-off by restructuring firms have to remain available for work since April 2006. As of January 2008, the ISP constrained an increasing number of early-retired workers to stay available for the labor market until the age of 58. However, on average in 2008, only 1 % of the early-retirees were available for work. Some policies of the ISP were also introduced to promote the adaptation of the workplace to older workers, but they were not widely used. 2.2.2 Participants and expenditure This subsection describes and comments on the data gathered on the number of participants and the program expenditure for a number of policies targeted at older workers in Belgium during the period 2002–2008. Although the ISP law was adopted in December 2005, the graphs are for the period 2002–2008 to be able to see the changes the ISP brought to already existing policies. Figure 1 shows the evolution of real expenses and participants in the collectively agreed early retirement scheme. Both curves are on the rise during the post-2005 period. Since there is no obligation for the worker to report the supplement to the UB under the unofficial early retirement scheme, no figure on the use of this scheme is available. As the ISP reforms affect more substantially this unofficial scheme than the collectively agreed one, the post-2005 evolution on Fig. 1 could capture a substitution between the two schemes. Dejemeppe et al. IZA Journal of Labor Policy (2015) 4:17 Page 7 of 23 Fig. 1 The collectively agreed full-time early retirement scheme. Full-time participants and public expenses, in million constant euros, base year 2010 (Source: ONEM) Fig. 1 The collectively agreed full-time early retirement scheme. Full-time participants and public expenses, in million constant euros, base year 2010 (Source: ONEM) Fig. 1 The collectively agreed full-time early retirement scheme. Full-time participants and public expenses, in million constant euros, base year 2010 (Source: ONEM) Fig. 1 The collectively agreed full-time early retirement scheme. Full-time participants and public expenses, in million constant euros, base year 2010 (Source: ONEM) Figure 2 shows the annual number of participants aged 50 or more by measure. It reveals an increase in the number of participants benefiting from RESSC between 2003 and 2004. Most of this rise is however spurious since there are no data on the number of bene- ficiaries of RESSC for the period 2002–2003.4 The strongest increase in the number of participants is registered between 2006 and 2007. It is mainly due to the extension of the RESSC to people aged 50 or more within the framework of the ISP. In the year following Fig. 2 Recipients by policy from 2002 to 2008a (Sources: ONEM, ONSS, Banque Nationale de Belgique, Office National des Pensions, SPF Emploi, Travail et Concertation sociale). aThe recipients are either measured by their number or by the number of payments (which does not always correspond to the number of recipients). 2.2.2 Participants and expenditure 3 Expenditure by policy from 2002 to 2008, in constant euros, base year 2010 (Sources: ONEM, ONSS, Banque Nationale de Belgique, Office National des Pensions, SPF Emploi, Travail et Concertation sociale). aThis category contains two policies: a temporary hiring subsidy (“Plan Activa”) received by the employer who hires a long-term unemployed person aged at least 45 years old and an in-work benefit (Complément reprise du travail) paid when some unemployed aged 50 years old and over finds a job Fig. 3 Expenditure by policy from 2002 to 2008, in constant euros, base year 2010 (Sources: ONEM, ONSS, Banque Nationale de Belgique, Office National des Pensions, SPF Emploi, Travail et Concertation sociale). aThis category contains two policies: a temporary hiring subsidy (“Plan Activa”) received by the employer who hires a long-term unemployed person aged at least 45 years old and an in-work benefit (Complément reprise du travail) paid when some unemployed aged 50 years old and over finds a job Fig. 3 Expenditure by policy from 2002 to 2008, in constant euros, base year 2010 (Sources: ONEM, ONSS, Banque Nationale de Belgique, Office National des Pensions, SPF Emploi, Travail et Concertation sociale). aThis category contains two policies: a temporary hiring subsidy (“Plan Activa”) received by the employer who hires a long-term unemployed person aged at least 45 years old and an in-work benefit (Complément reprise du travail) paid when some unemployed aged 50 years old and over finds a job the adoption of the ISP law, the policies with the largest number of participants are the RESSC for workers aged 50–56 (198,326 recipients in 2007) and the part-time time-credit (59,741 mean payments per months in 2007). Figure 3 gives the yearly public expenditure in constant euros (base year 2010) for the main policies targeted at older workers in Belgium during the period 2002–2008. The real expenditure went from 42 million euros in 2002 to 634 million in 2008. There is a rise in the expenditure between 2003 and 2004, which is spurious for the same reason as for the participants (cf. above). A sharp increase appears between 2006 and 2007 with the RESSC for workers aged 50–56 decided within the framework of the ISP. In 2007, following the implementation of the ISP, a fundamental change was seen in the public investment for older workers, especially through the RESSC for workers aged 50–56. 2.2.2 Participants and expenditure There is indeed a sharp rise in the number of participants and an increase in the spending at that time. 2.2.2 Participants and expenditure For policies that deliver a permanent monthly or quarterly financial benefit, data are annualized by using the monthly or quarterly mean in order to avoid double counting. bThis category contains two policies: a temporary hiring subsidy (“Plan Activa”) received by the employer who hires a long-term unemployed aged at least 45 years old and an in-work benefit (Complément reprise du travail) paid when some unemployed aged 50 years old and over find a job Fig. 2 Recipients by policy from 2002 to 2008a (Sources: ONEM, ONSS, Banque Nationale de Belgique, Office National des Pensions, SPF Emploi, Travail et Concertation sociale). aThe recipients are either measured by their number or by the number of payments (which does not always correspond to the number of recipients). For policies that deliver a permanent monthly or quarterly financial benefit, data are annualized by using the monthly or quarterly mean in order to avoid double counting. bThis category contains two policies: a temporary hiring subsidy (“Plan Activa”) received by the employer who hires a long-term unemployed aged at least 45 years old and an in-work benefit (Complément reprise du travail) paid when some unemployed aged 50 years old and over find a job Dejemeppe et al. IZA Journal of Labor Policy (2015) 4:17 Page 8 of 23 Fig. 3 Expenditure by policy from 2002 to 2008, in constant euros, base year 2010 (Sources: ONEM, ONSS, Banque Nationale de Belgique, Office National des Pensions, SPF Emploi, Travail et Concertation sociale). aThis category contains two policies: a temporary hiring subsidy (“Plan Activa”) received by the employer who hires a long-term unemployed person aged at least 45 years old and an in-work benefit (Complément reprise du travail) paid when some unemployed aged 50 years old and over finds a job Fig. 3 Expenditure by policy from 2002 to 2008, in constant euros, base year 2010 (Sources: ONEM, ONSS, Banque Nationale de Belgique, Office National des Pensions, SPF Emploi, Travail et Concertation sociale). aThis category contains two policies: a temporary hiring subsidy (“Plan Activa”) received by the employer who hires a long-term unemployed person aged at least 45 years old and an in-work benefit (Complément reprise du travail) paid when some unemployed aged 50 years old and over finds a job Fig. 2.2.3 Expected effects What are the mechanisms through which the policies of the ISP should have an impact on the employment rate of older workers? We look at potential effects in the short run. The adoption of the reform was for long uncertain because of a strong opposition of labor unions. So, economic agents could not easily anticipate the measures actually decided. The first policy consists in a pension bonus received by people continuing to work after the age of 62. This provides an incentive to stay active before that age. From 62 onwards, the expected positive effect is to increase the opportunity cost of early retirement and induce people to work longer (Maes 2008). However, there is also an income effect work- ing in the other direction: the bonus increases the expected income from early retirement and may push some people to retire earlier (Lefevre and Orsini 2011; Maes 2008). At the micro-level, both the difference-in-differences method used by López-Novella (2012) and Dejemeppe et al. IZA Journal of Labor Policy (2015) 4:17 Page 9 of 23 the structural modeling approach of Lefevre and Orsini (2011) conclude that the net effect of the pension bonus is very small. The second policy involves RESSC targeted to older workers. Belgium is a unionized economy with an estimated union coverage in the private sector of 96 % (Source: ICTWSS data base). Under wage bargaining, linear tax cuts and RESSC have a favorable impact on the employment rate if the flow value in unemployment (unemployment benefits plus the value of time) is not indexed on the bargained wage (Pissarides 1998). A lump-sum RESSC increases the total surplus (or value) of a job. Under Nash bargaining, the reduction is shared between workers and firms, and the part accruing to the employers induces a rise in labor demand. In the Belgian context, in particular for older workers, such an effect may be less obvious, and here are some reasons why. Over the period 1998–2006 in Belgium, Cataldi et al. (2012) and Vandenberghe et al. (2013) provide evidence that a rise in the share of occupied workers aged at least 50 reduces the log of firm’s average value added per worker more than the log of its average labor cost per worker. So, the productivity-labor cost gap is significantly and negatively affected by the share of old workers in the firm. 2.2.3 Expected effects Working hours are indeed not completely flexible and people cannot always reduce their working time as they would like (Gustman and Steinmeier 1983; Kahn and Lang 1995; Stewart and Swaffield 1997; Euwals 2001; Bryan Dejemeppe et al. IZA Journal of Labor Policy (2015) 4:17 Page 10 of 23 2007; Gielen 2009). In particular, Decicca (2007) conclude for the US that the “amount by which hours being constrained is estimated to raise retirement probabilities is nearly as large as the effect of being in relatively poor health” (p. 251). A reduction of working time when getting older could also have a beneficial effect on health and hence could prolong the attachment to the labor market. However, if participation to this scheme is prepar- ing a complete withdrawal from the labor market (as suggested by (Looy et al. 2014), for a Belgian region), this effect could be short-lived. On the demand side, the pol- icy could have positive (improved employee satisfaction) as well as negative (signal of a reduced availability to work, organizational difficulties) effects on employment (Devisscher and Sanders 2007). Induced effects on the labor supply of other age groups cannot be excluded. For instance, in countries like Belgium, Spiess and Schneider (2003) find an association between care-giving and reductions in work hours among women aged 45–59. Care-giving by grandparents could have an induced effect of the labor sup- ply of their children (on this possibility see also Gielen 2009). All in all, the international evidence about the causal impact of subsidized part-time schemes on the employment rate of old workers remains mixed, with negative effects in Austria (Graf et al. 2011) and Portugal (Machado and Portela 2012) but positive ones in Germany (Huber et al. 2013). At last, the ISP as a whole could have triggered a change in the perception of older workers on the labor market, which could potentially have a positive impact on both the demand and supply sides of the labor market. However, in view of the foregoing, the effect of the ISP on the employment rate of older workers are expected to be positive but small. 2.2.3 Expected effects Unless an agreement can be found through early retirement schemes, employers are reluctant to fire these workers because of the threat of a labor dispute with the (relatively powerful) Belgian unions.5 In this context, the introduction of RESSC lowers the productivity-labor cost gap. Whether the limited reduction introduced by the ISP does more than compensate this gap is an empir- ical question. If it does, this reduction provides an incentive to keep old workers longer in the firm (potentially inducing a substitution effect detrimental to the employment level of younger workers). Thirdly, we consider the potential impact of the package of reforms affecting early retirement, namely making early retirement more costly to employers (in particular in the unofficial scheme), increasing the earliest age at which early retirement can begin, and raising the minimal required career length. Since, as argued above, employers would like to reduce their old workforce but can hardly do so without the early retirement schemes, the above-mentioned combination of changes could give employers an incentive to keep part of this workforce longer. However, these changes can be seen as a piecemeal reform since the ISP did not modify simultaneously the multiple pathways to retirement. Nei- ther disability insurance (DI) nor the anticipated retirement were reformed. Jousten et al. (2013) provides descriptive evidence that the share of women aged 55–64 on DI increased after 2007. For both genders, the take-up rate of anticipated retirement increased as well. In sum, this package of reforms affecting early retirement can contribute to an improvement in the employment rate and/or it induces substitutions among the differ- ent pathways to retirement. To illustrate these spillovers effects, consider a reform in Austria that increased the age at which anticipated retirement can be claimed. Staubli and Zweimüller (2013) show that this reform had a positive but moderate causal effect on the employment rate, a substantial positive one in the unemployment rate and a small positive effect on the percentage of people on disability benefits. Regarding the fourth policy, allowing a reduction of working time at the end of the career and compensating (partly) the worker for the wage loss are believed to increase the employment rate of older people. 3 Data and sample The outcome variable of our evaluation analysis is a national indicator of the quarterly salaried employment rate by gender and age group in the private sector. This indicator is built using administrative data on national salaried employment and on the population by gender and age group. The quarterly number of private sector salaried employees by gender and age group is obtained from the National Social Security Office (ONSS) and is available since the first quarter of 1997 (1997q1). The analysis is restricted to the private sector for two reasons. First, the private employment is more reactive to fluctuations in the economic activity than the public employment. Second, the private sector, as opposed to the public sector, is targeted by all policies of the ISP. The salaried private employment is available in fourteen age groups.6 The data are adjusted for seasonality7 and corrected for a change in the classification that took place in 2003q1.8 The quarterly employment rate is obtained by dividing the quarterly salaried private employment of a particular gender and age group by the population of that same gender and age group of the corresponding year.9 The target of the ISP is the employment rate of older workers defined, in this paper, as people aged between 50 and 59 years old. Employed people aged 60 years old and over are not taken into account because the main hypothesis of the methodology used in this paper, responsiveness of the employment rate to fluctuation in economic activity, does not hold for them. People aged 45 to 49 years old are also left apart because they are not concerned by all policies of the ISP. A younger age group, formed by people aged 35–44 years old, is taken into account to investigate possible substitution or complemen- tarity effects of the ISP. The private salaried employment rate by gender for the age group Dejemeppe et al. IZA Journal of Labor Policy (2015) 4:17 Page 11 of 23 A B Fig. 4 The quarterly salaried private employment rate of workers aged 50–59 years old (in %), 1997–2008 (Sources: ONSS and SPF Economie). Panel A: men. Panel B: women A B Fig. 4 The quarterly salaried private employment rate of workers aged 50–59 years old (in %), 1997–2008 (Sources: ONSS and SPF Economie). Panel A: men. Panel B: women 50–59 years old is depicted in Fig. 4. 3 Data and sample A slightly stronger rise in the employment rate of older workers appears in the years following the ISP for men. For older women, the overall evolution of the employment rate is dominated by an upward trend. In this paper, the employment rate is explained by an indicator of the economic activity. The obvious candidate is the gross domestic product (GDP) measured by the seasonally adjusted Belgian quarterly real GDP from the National Bank of Belgium. As already mentioned in the introduction, the sample is restricted to the period span- ning from 1997q1 to 2008q2. The analysis stops at the beginning of the financial and economic crisis of 2008 because anti-crisis employment policies were taken by the government at that time. Thus after 2008q2, the effects of the policies of the ISP cannot be disentangled from those taken to counteract the effect of the crisis. Figure 5 shows the evolution of Belgian GDP for the period 1996q1-2008q2. Belgium underwent two Fig. 5 Seasonally adjusted Belgian quarterly real GDP in chained euros, reference year 2009 (million euros), 1996–2008 (Source: BNB) Fig. 5 Seasonally adjusted Belgian quarterly real GDP in chained euros, reference year 2009 (million eur 1996–2008 (Source: BNB) Fig. 5 Seasonally adjusted Belgian quarterly real GDP in chained euros, reference year 2009 (million eu 996–2008 (Source: BNB) Dejemeppe et al. IZA Journal of Labor Policy (2015) 4:17 Page 12 of 23 economic slowdowns, the first at the end of 2001 and the second at the end of 2002. Then, since 2003, the Belgian GDP grew steadily. 4.1 The evaluation strategy in a nutshell The primary purpose of the paper is to measure the overall effectiveness of the ISP in raising the employment rate of workers aged 50–59 years old. To that purpose, we need to prove that without the ISP, i.e., in the counterfactual situation, the employment rate of these workers would have been smaller. In the present context, neither a control exper- iment nor a quasi-experimental design are available because the reforms apply to all workers aged 50 years or more. In the absence of control groups against which the impact of the ISP could be measured, we rely on an alternative strategy to recover the counterfac- tual scenario. Our method consists in comparing the actual evolution of the (gender and age specific) employment rate from the second quarter of 2007 to mid-2008 to a counter- factual evolution. The latter is the predicted evolution of the employment rate based on a macro-econometric model estimated over the period going from the first quarter of 1997 to the first quarter of 2007. The model explains quarterly variations in the employment rate by current and past levels of the real Belgian GDP, taking into account past dynamics of the dependent variable itself. As the forecast has to generate a convincing counter- factual, we cannot consider the cost of labor nor working hours of the older workers in the specification of the model simply because the ISP is affecting these variables (see the discussion of this point in the introduction). The estimation period ends in 2007q1 from which the evaluation period takes over. As illustrated by Figs. 2 and 3, the strongest increase in the number of participants and expenditure is registered between 2006 and 2007, under the impulse of the RESSC for workers aged 50–56 years in April 2007. As a robustness check, we make the evaluation period start earlier in 2006q2 which corresponds to the introduction of the first policy of the ISP. 4 The evaluation method This section is divided in two parts. The first describes the evaluation method, which is based on a counterfactual analysis. The second presents the macro-economeric regression model and discusses the potential simultaneity issue related to the model specification. 4.2 The macro-econometric model The model selection is automated using Autometrics (‘Automatic general-to-specific selection’, see Doornik 2009) on a gen- eral unrestricted model with four lags in the first difference of the employment rate and GDP growth rate: ERg,a,t = α0 + α1t + 4 i=1 φiERg,a,t−i + 4 i=0 γilog[GDP]t−i + ϵt (1) (1) In equation 1, empg,a,t stands for the first difference in the employment rate (ER) where the subscripts g corresponds to gender, a to age and t to time. log[GDP]t is the growth rate of real GDP at time t. For women aged 50–59 and 35–44 years old, a linear trend is added to the regression as the first difference of the employment rate has a trend. We expect the coefficients of the growth rate of real GDP in equation 1 to be positive if the employment rate is to increase when GDP grows. Given that we allow the employment rate at time t to depend on the GDP growth rate at time t, it raises a potential simultaneity issue: GDP determines the employment rate, but the employment rate might also influence GDP. And this reverse causality may be induced by the ISP itself if it is effective in raising the employment rate of older workers. If there is no autocorrelation in the error terms of the models, a simultaneity bias may arise only when the best specification includes the contemporaneous value of GDP growth rate and not when it includes only lagged values. Even when the relationship is contempora- neous, the fact of working by age group and gender weakens the potential causality from the employment rate to GDP. This is particularly true for the main group of interest, the workers aged 50–59, as they constitute only 16 % of total employment in Belgium (figures for 2007q1). As outlined in the next section, the contemporaneous value of GDP only appears in the regression models estimated for men. For those regressions, two robustness checks are implemented. The first one simply consists in removing the contemporaneous value of GDP. The second robustness analysis uses the mean real GDP of five neighboring countries (France, Luxembourg, Germany, the Netherlands and the U.K.) instead of the GDP of Belgium. As Belgium is a small open economy, the GDP of neighboring countries should have a great influence on the Belgian economic activity, and therefore on the employment rate. 4.2 The macro-econometric model As it is well known, most macro-economic variables are non-stationary series. According to the Augmented Dickey-Fuller (ADF) unit root tests, both the employment rate and GDP are integrated of order one in our data.10 Regressions based on variables integrated of order one are spurious unless they involve cointegrated variables. Could it be the case that there exists a long term equilibrium rela- tionship between the employment rate and GDP? Due to the relative short time span of our data, a long term relationship between GDP and the employment rate in Belgium, if it exists, may be hard to identify. Being in the bivariate (two variables) case, the Engle and Granger (1987) test for cointegration is first used. Then as the Engle and Granger (1987) test has some important defects,11 the robustness of the results is checked using the Johansen (1988) cointegration test. The Engle and Granger (1987) and the Johansen Dejemeppe et al. IZA Journal of Labor Policy (2015) 4:17 Page 13 of 23 (1988) tests differ concerning men aged 35–44 years old. The Engle and Granger (1987) test finds cointegration while the Johansen (1988) test does not. For all other age groups and gender, no cointegration is found between the employment rate and GDP. On the basis of those results, we make the assumption that there is no cointegration between the employment rate and GDP.12 These results confirm those of Bodart et al. (2009) who esti- mate an aggregate employment equation for Belgium for the period 1980–2002. They do not find a cointegration relationship between employment, GDP and the labor cost. As no cointegration relationship was found between the employment rate and GDP, the model is estimated in first difference. The analysis is based on an autoregressive dis- tributed lag model with the first difference of the employment rate as dependent variable and the growth rate of GDP as the explanatory variable. We allow past levels of GDP to influence current variations in the employment rate since the latter variable does not adjust immediately to changes in the economic activity. aDurbin’s alternative test for serial correlation without strictly exogenous regressors. bWhite test for heteroscedasticity. significant at 1 % level, 5 % level, 10 % level. The model selection is automated using Autometrics 4.2 The macro-econometric model However, the real GDP of neighboring countries should not be affected (at least directly) by the gender and age specific employment rate in Belgium. We estimate by OLS the model of equation 1 on the period 1997q1 to 2007q1 by age- group and gender. Based on the estimated parameters, dynamic forecasts are generated Dejemeppe et al. IZA Journal of Labor Policy (2015) 4:17 Page 14 of 23 over the period 2007q2 to 2008q2 (5 values are forecasted by age-group and gender). The model specifies the employment rate in first difference while the forecasted values of the employment rate should be in level. To retrieve these values, we rely on the identity equation defining the first difference of the employment rate.13 The forecasts are based on the estimated parameters but also on the lagged values of the employment rate and on the current and past values of GDP. For the lagged values of the employment rate, the actual values are used until 2007q1, after which the predicted values of the employment rate are exploited. For GDP, actual values are always used. The evaluation strategy then consists in comparing, from 2007q2 onwards, the actual employment rate to the counterfactual (predicted) one. The difference between the actual and predicted employment rate represents the part of the employment rate that cannot be explained by its past dynamics and GDP. It is our estimator of the overall impact of the ISP. A 95 % confidence interval around the predicted values is used to assess the significance of the estimated impact. If the actual employment rate is above (below) the upper (lower) bound of the confidence interval, there is a positive (negative) effect of the ISP on the employment rate. The evaluation strategy then consists in comparing, from 2007q2 onwards, the actual employment rate to the counterfactual (predicted) one. The difference between the actual and predicted employment rate represents the part of the employment rate that cannot be explained by its past dynamics and GDP. It is our estimator of the overall impact of the ISP. y g g significant at 1 % level, 5 % level, 10 % level. The model selection is automated using Autometrics 5 Results Table 1 shows the estimated parameters of the macro-econometric model specified in equation 1, by age-group (50–59 years old and 35–44 years old) and gender. The coeffi- cients of the growth rate of GDP are significant at the 1 % or 5 % level for each gender and age group. As expected, an increase in (current or past) GDP has a positive impact on the employment rate. It is worth noting that the estimated effects of GDP are larger for men than for women, irrespective of the age group, and decrease with age. The results of three hypothesis tests (absence of autocorrelation, normality and homoscedasticity of the error terms) are reported at the bottom of Table 1. The null homoscedasticity of the error terms) are reported at the bottom of Table 1. The null Table 1 The quarterly variation of the salaried private employment rate by age group and gender - OLS estimates Dep. var. Employment rate 50–59 years old 35–44 years old Men Women Men Women Employment ratet−1 0.533 0.418* (0.103) (0.117) log[GDP]t 0.087* 0.127* (0.027) (0.036) log[GDP]t−3 0.115* 0.048 0.153* 0.127* (0.028) (0.021) (0.039) (0.041) Constant -0.071* 0.066 -0.054 0.134 (0.026) (0.029) (0.036) (0.056) Trend 0.006* 0.003* (0.001) (0.002) Nbr obs. 39 40 39 40 R2 0.697 0.574 0.633 0.228 Adj. R2 0.671 0.551 0.601 0.187 Serial correlation (AR 1-3)a F(3,32) = 1.112 F(3,34) = 0.559 F(3,32) = 0.998 F(3,34) = 1.566 Normality test Chi2(2) = 1.3 Chi2(2) = 0.725 Chi2(2) = 1.286 Chi2(2) = 0.468 Heteroscedasticity testb F(6,32) = 0.686 F(4,35) = 0.849 F(6,32) = 0.284 F(4,35) = 2.155 aDurbin’s alternative test for serial correlation without strictly exogenous regressors bWhite test for heteroscedasticity Table 1 The quarterly variation of the salaried private employment rate by age group and gender - OLS estimates Dejemeppe et al. IZA Journal of Labor Policy (2015) 4:17 Page 15 of 23 hypothesis of no serial correlation, normality and homoscedasticity cannot be rejected at conventional significance levels. Since there is no evidence of autocorrelation of the error terms, simultaneity is likely to be an issue only if the best specification includes the contemporaneous value of GDP growth rate (i.e., for men). This section proceeds by presenting the estimated effect of the ISP on the quarterly age and gender-specific employment rates. We then investigate the sensitivity of the results to alternative specifications and indicators of the economic activity. 5.1 The effect of the ISP The estimated effect of the ISP on the employment rate can be depicted graphically. In Fig. 6, the solid line corresponds to the observed employment rate. The dashed line corre- sponds to its fitted values up to 2007q1 and to its predicted values from 2007q2 onwards. The vertical bars represent the 95 % confidence interval around the forecasts. For workers aged between 50 and 59 years old, both the observed and predicted values of the employment rate have been increasing since 2007q2 for both genders. So, even in the absence of the ISP, the employment rate of older workers would have increased as the result of the good economic conditions at that time. Still, Fig. 6 indicates that this rise would have been lower in the absence of the ISP, the actual level of the employment rate being above the predicted one for both genders, although less obviously for women. From this figure, we can therefore confirm a positive impact of the ISP on the employment rate of older workers, which seems to have risen steadily since its implementation. Given that the confidence interval of the forecasts includes the observed value of the employment rate for both genders, we can however not conclude that there is a statistically significant impact at a level of 5 %. Fig. 6 The quarterly salaried private employment rate (in %), 1997q2-2008q2, — observed series, - - - fitted values and forecasts from 2007q2, ⊢⊣95 % CI of the forecasts Fig. 6 The quarterly salaried private employment rate (in %), 1997q2-2008q2, — observed series, - - - fitted values and forecasts from 2007q2, ⊢⊣95 % CI of the forecasts Fig. 6 The quarterly salaried private employment rate (in %), 1997q2-2008q2, — observed series, - - - fitted values and forecasts from 2007q2, ⊢⊣95 % CI of the forecasts Page 16 of 23 Dejemeppe et al. 5.1 The effect of the ISP IZA Journal of Labor Policy (2015) 4:17 Table 2 The impact of the ISP on the quarterly salaried private employment (rate) of workers aged 50–59 in 2008q2, by gender Observed Predicted Impact in % value value (s.e.) pointsa (s.e.) Employment rate Men aged 50–59 36.23 % 35.71 % (0.31) 0.52 (0.31) 95 % CI [35.07 ; 36.34] 90 % CI [35.18 ; 36.24] Women aged 50–59 24.20 % 23.94 % (0.18) 0.26 (0.18) 95 % CI [23.58 ; 24.30] 90 % CI [23.64 ; 24.24] Employment Men aged 50–59 258,178 254,439 3,739 Women aged 50–59 172,366 170,553 1,813 aThe impact is measured as the difference between the observed and the predicted value Table 2 The impact of the ISP on the quarterly salaried private employment (rate) of workers aged 50–59 in 2008q2, by gender e 2 The impact of the ISP on the quarterly salaried private employment (rate) of workers aged 9 in 2008q2 by gender In Table 2, we report the estimated impact of the ISP in 2008q2 (i.e., the estimated dif- ference between the observed and predicted value of the employment rate at that date) together with its standard deviation. The impact, although positive, is not significant at conventional levels. In 2008q2, the observed employment rate of men aged 50–59 is 36.23 %, while the predicted one is 35.71 %. The gap, which is our measure of impact, is only 0.52 of a percentage point, which represents only 1.46 % of the predicted value. This corresponds to approximately 3,700 additional jobs due to the ISP. For women aged 50– 59, the observed and predicted employment rates are, respectively, 24.20 % and 23.94 %. So the impact is smaller, 0.26 of a percentage point, which corresponds to 1.09 % of the predicted employment rate for older women and an additional job creation of about 1,800 units. There is an overlap in the 95 % confidence intervals of the estimated effect for men and women. So even if the effect is larger for men than for women, we can not reject that both effects are the same, and equal to zero. For younger men aged 35–44 (see panel (c) in Fig. 6, as well as Table 3), both the observed and predicted values of the employment rate have also been increasing since 2007q2. 5.1 The effect of the ISP However, since the actual level of the employment rate is below the predicted one, the employment rate of men aged 35–44 would have been higher in the absence of the ISP. This result therefore points to a negative impact of the ISP on the younger age group, Table 3 The impact of the ISP on the quarterly salaried private employment (rate) of workers aged 35–44 in 2008q2, by gender Observed Predicted Impact in % value value (s.e.) pointsa (s.e.) Employment rate Men aged 35–44 54.04 % 54.67 % (0.36) -0.63 (0.36) 95 % CI [53.93 ; 55.41] 90 % CI [54.06 ; 55.28] Women aged 35–44 43.81 % 43.91 % (0.34) -0.1(0.34) 95 % CI [43.21 ; 44.61] 90 % CI [43.32 ; 44.49] Employment Men aged 35–44 428,735 433,711 -4,976 Women aged 35–44 338,504 339,263 -759 aThe impact is measured as the difference between the observed and the predicted value Table 3 The impact of the ISP on the quarterly salaried private employment (rate) of workers aged 35–44 in 2008q2, by gender Dejemeppe et al. IZA Journal of Labor Policy (2015) 4:17 Page 17 of 23 suggesting a potential substitution effect. Based on Table 3, this negative effect is how- ever not statistically significant at even 10 %. For men aged 35–44 years old, the observed value of the employment rate in 2008q2 is 54.04 %, while the predicted, counterfactual value is 54.67 %. The gap, which is our measure of impact, is -0.63 of a percentage point, which amounts to about 4,976 jobs lost. For younger women aged 35–44 (see panel (d) in Fig. 6, as well as Table 3), there is nearly no difference between the observed values and the forecasts from 2007q2 onwards. The estimated impact is still negative, -0.10 (759 jobs lost), but not at all significant. In sum, the point estimates suggest that there could be a small positive impact of the ISP on the employment rate of older workers. Consistent with this finding, the results suggest a small negative impact for younger workers, which could be interpreted as a substitution effect. However, since the standard deviation of the estimated effects is quite large in relation to their magnitude, we cannot confirm to a significant impact of the ISP. As explained in sub-section 2.2.3, we expect a positive but small overall impact of the ISP on the employment rate of older workers. 5.1 The effect of the ISP In that context, the probability of detect- ing a statistically significant impact depends crucially on the standard deviations of the forecasts. These in turn depend on the goodness of fit of the macro-econometric model (R2) and the standard errors of the estimated parameters reported in Table 1. For identi- fication purposes, we had to rely on a relatively simple model of the employment rate as reflected in the relatively low R2, especially for women. This is therefore the price we pay for trying to identify a causal effect of the ISP. Although nothing can be done about it, the small size of our sample also induces less precision in the estimated parameters and so widens the confidence intervals around the forecasts. aDurbin’s alternative test for serial correlation without strictly exogenous regressors. bWhite test for heteroscedasticity. y *significant at 1 % level, 5 % level, 10 % level. The model selection is automated using Autometrics 5.2 Robustness checks As a first robustness check, we make the evaluation period start one year earlier, in 2006q2, which corresponds to the introduction of the first policy of the ISP. The results of this robustness analysis are depicted in Fig. 7. For workers aged 50–59, a positive impact of the ISP is still visible in the graphs, more markedly for men. For workers aged 35–44, the conclusion of a negative impact of the ISP is maintained, although this is only visible for men. For both age groups, the divergence between the predicted and observed values of the employment rate appears earlier, at the beginning of 2006 for the older age group, between the second and third quarter of 2006 for the youngest one. These results suggest that the ISP could have had an impact from the beginning of its implementation. Still, the estimated effects are not statistically significant at conventional levels. The second robustness check investigates whether the results are sensitive to a potential simultaneity bias. Such a bias arises only when the contemporaneous GDP growth rate appears in the regressors. This is the case for men of both age groups. Two sensitivity analyses are therefore made for this specific gender. The first one consists in removing the contemporaneous value of GDP of the regression model and the second takes the mean real GDP of France, Luxembourg, Germany, the Netherlands and the U.K. as a regressor instead of Belgian GDP. Table 4 gives the OLS estimates of the two sensitivity analyses. The null hypothesis of no serial correlation, normality and homoscedasticity cannot be rejected at conventional significance levels for both analysis and age groups. The estimated parameters of the model without the contemporaneous value of GDP growth rate are very close to those Dejemeppe et al. IZA Journal of Labor Policy (2015) 4:17 Page 18 of 23 Fig. 7 The quarterly salaried private employment rate (in %), 1997q2-2008q2, — observed series, - - - fitted values and forecasts from 2006q2, ⊢⊣95 % CI of the forecasts Fig. 7 The quarterly salaried private employment rate (in %), 1997q2-2008q2, — observed series, - - - fitted values and forecasts from 2006q2, ⊢⊣95 % CI of the forecasts Fig. 5.2 Robustness checks 7 The quarterly salaried private employment rate (in %), 1997q2-2008q2, — observed series, - - - fitted values and forecasts from 2006q2, ⊢⊣95 % CI of the forecasts Table 4 The quarterly variation of the male salaried private employment rate by age group, with alternative specifications of GDP - OLS estimates Table 4 The quarterly variation of the male salaried private employment rate by age group, with alternative specifications of GDP - OLS estimates Dep. var. Employment rate Men 50–59 years old 35–44 years old Employment ratet−1 0.553 0.461* 0.387* 0.360 (0.116) (0.128) (0.134) ( 0.154) log[GDP]t−3 0.102* 0.142* (0.031) (0.045) log[GDP neighbors]t 0.031 0.053 (0.033) (0.046) log[GDP neighbors]t−3 0.132* 0.125 (0.032) (0.052) Constant -0.015 -0.064* 0.03 -0.010 (0.022) (0.032) (0.03) (0.047) Nbr obs. 39 39 39 39 R2 0.605 0.657 0.502 0.464 Adj. R2 0.583 0.627 0.474 0.418 Serial correlation (AR 1-3)a F(3,33) = 0.317 F(3,32) = 0.468 F(3,33) = 0.115 F(3,32) = 0.148 Normality test Chi2(2) = 0.376 Chi2(2) = 2.867 Chi2(2) = 0.516 Chi2(2) = 8.094 Heteroscedasticity testb F(4,34) = 0.125 F(6,32) = 0.816 F(4,34) = 1.159 F(6,32) = 0.448 aDurbin’s alternative test for serial correlation without strictly exogenous regressors bWhite test for heteroscedasticity Dejemeppe et al. IZA Journal of Labor Policy (2015) 4:17 Page 19 of 23 Fig. 8 The quarterly salaried private employment rate (in %), 1997q2-2008q2, — observed series, - - - fitted values and forecasts from 2007q2 based on lagged values of GDP only, ⊢⊣95 % CI of the forecasts Fig. 8 The quarterly salaried private employment rate (in %), 1997q2-2008q2, — observed series, - - - fitted values and forecasts from 2007q2 based on lagged values of GDP only, ⊢⊣95 % CI of the forecasts including it. Using the GDP of neighboring countries, only the third lag is statistically significant for both age groups, and the point estimates are again not too far from those estimated in the baseline model. However, the R2 decreases for both age groups as compared to the baseline model of Table 1, above all for men aged 35–44 years old. The estimated impact of the ISP under these two alternative specifications are given in Figs. 8 and 9. As the R2 are smaller, the confidence intervals are wider compared to the baseline analysis, but the results are maintained. 5.2 Robustness checks The point estimates indicate a pos- sible small positive impact of the ISP for men aged 50–59 and a small negative impact for younger men. Though, the results are not statistically significant at even 10 %. Table 5 reports the estimated impacts of the ISP in 2008q2 for men aged 50–59 under both specifications. Their 95 % confidence interval overlaps the ones of the baseline estimated impacts. 6 Conclusion The main identification hypothesis of our evaluation method is that, in the absence of the ISP, changes in the employment rate would have been closely linked to the GDP growth rate. As the predicted values have to generate a convincing counterfactual, we can- not consider the cost of labor nor working hours of the older workers in the model specifi- cation simply because the ISP is affecting these variables mainly through two policies, i.e., the reduction in employers’ social security contributions and the working time reduction with (partial) wage compensation. The size of this influence can hardly be predicted with- out a structural model of wages and hours worked. Developing such a model is beyond the scope of this paper. Besides, we did not find any major shock other than the ISP that could have had a substantial influence on the employment rate of the older workers at that time. Now, if the ISP itself is effective in raising the employment rate of older workers, this can induce reverse causality between the employment rate and the GDP growth rate. This is the reason why we use the mean real GDP of five neighboring countries (France, Luxembourg, Germany, the Netherlands and the U.K.) instead of the GDP of Belgium in a sensitivity analysis. The results show that if there is a positive impact of the ISP, it is too small to be statis- tically significant. For men aged 50–59, the impact is only 0.52 (s.e. 0.31) of a percentage point, rising the employment rate from 35.7 % to 36.2 %, while for women aged 50–59, the impact is 0.26 (s.e. 0.18) of a percentage point, increasing the employment rate from 23.9 % to 24.2 %. Moreover, the results suggest a small negative effect on the employ- ment rate of younger workers. For men aged 35–44 years old, the impact is -0.63 (s.e. 0.36) of a percentage point, decreasing the employment rate from 54.7 % to 54.0 %. For younger women, the impact is -0.10 (s.e. 0.34) percentage point. Even if the effects are larger in absolute value for men than for women, we cannot reject that both effects are the same and not significantly different from zero. In sum, the results suggest a slight pos- itive impact of the ISP on the employment rate of older workers at the expense of younger workers. 6 Conclusion The primary aim of this paper is to evaluate the overall impact of the active aging policies introduced by the Intergenerational Solidarity Pact (ISP) in December 2005 on the employment rate of workers aged 50–59 in Belgium. The possible substitution or complementarity effects of the ISP on the employment rate of a younger age group (those aged 35–44) are also investigated. The impacts of the reforms introduced by the ISP are measured over the period 2007q2-2008q2 and assessed through a counterfactual analysis. Fig. 9 The quarterly salaried private employment rate (in %), 1997q2-2008q2, — observed series, - - - fitted values and forecasts from 2007q2 based on GDP of neighbors, ⊢⊣95 % CI of the forecasts Fig. 9 The quarterly salaried private employment rate (in %), 1997q2-2008q2, — observed series, - - - fitted values and forecasts from 2007q2 based on GDP of neighbors, ⊢⊣95 % CI of the forecasts Dejemeppe et al. IZA Journal of Labor Policy (2015) 4:17 Page 20 of 23 Table 5 The impact of the ISP on the quarterly salaried private employment (rate) of male workers aged 50–59 in 2008q2 with alternative specifications of GDP Observed Predicted Impact in % value value (s.e.) pointsa Regressions without the contemporaneous value of GDP Employment rate 36.23 % 35.80 % 0.43 95 % CI [35.06 ; 36.53] 90 % CI [35.18 ; 36.41] Employment 258,178 255,087 3,091 Regressions using GDP of neighbors Employment rate 36.23 % 35.84 % 0.39 95 % CI [35.19 ; 36.49] 90 % CI [ 35.30 ; 36.38] Employment 258,178 255,379 2,799 aThe impact is measured by the difference between the observed and the predicted value Table 5 The impact of the ISP on the quarterly salaried private employment (rate) of male workers aged 50–59 in 2008q2 with alternative specifications of GDP Table 5 The impact of the ISP on the quarterly salaried private employment (rate) of male workers aged 50–59 in 2008q2 with alternative specifications of GDP The observed values of the employment rate are compared to their predicted (counter- factual) values based on a macro-econometric model estimated over a period of time preceding the implementation of the ISP. The motivation of this paper is the scarcity of empirical research about the overall impact of labor market reforms, especially in Belgium, despite their importance for policy making. Endnotes 1 For the period from January 2003 to December 2005, the legal retirement age was 63 for women. It reached 64 in January 2006 and 65 in January 2009. 2Some policies have already been taken before: the above-mentioned increase in the legal retirement age of women; the introduction of RESSC at the age of 58 (respectively, 57) in 2002 (respectively, 2004); the part-time time-credit for people aged at least 50 introduced in 2002; an in-work benefit (Complément reprise du travail) paid when an unemployed aged 50 years old and over finds a job after a sufficiently long career (introduced in 2002 as well); beyond a given unemployment duration, a temporary hiring subsidy for people aged at least 50 (the Plan Avantage à l’embauche) since 1995. The latter policy has been replaced in 2002 by a similar scheme for those at least 45 years old (the Plan Activa). 3The allowance is then not subject to social security contributions as it is the case when the allowance is paid to an unemployed worker. 4A presumably minor part of this increase is driven by the extension of the RESSC to workers aged 57 or more in 2004 (instead of 58 before). 4A presumably minor part of this increase is driven by the extension of the RESSC to workers aged 57 or more in 2004 (instead of 58 before). 5A reputation effect could also play a role. When wages increase with seniority, the deferred payment acts as an incentive device provided that the firm is not publicly known to renege on the promise of higher future wages. Finally, firing an old worker (instead of a younger one) could in some cases lead to an appeal against this decision on the basis of a discriminatory argument. 5A reputation effect could also play a role. When wages increase with seniority, the deferred payment acts as an incentive device provided that the firm is not publicly known to renege on the promise of higher future wages. Finally, firing an old worker (instead of a younger one) could in some cases lead to an appeal against this decision on the basis of a discriminatory argument. 6<18; 18-19; 20-21; 22-24; 25-29; 30-34; 35-39; 40-44; 45-49; 50-54; 55-59; 60-64; ≥65; missing. 6<18; 18-19; 20-21; 22-24; 25-29; 30-34; 35-39; 40-44; 45-49; 50-54; 55-59; 60-64; ≥65; missing. 6 Conclusion However, there seems to be a lack of statistical power to draw firm conclusions Dejemeppe et al. IZA Journal of Labor Policy (2015) 4:17 Page 21 of 23 on the overall effect of the ISP. A low sample size is certainly part of the explanation, as well as a relatively low goodness of fit of the macro-econometric model. For identification purposes, we had indeed to rely on a somewhat simple model of the employment rate. This is the price we pay for trying to identify a causal effect of the ISP. 10The results of ADF tests are available from the authors upon request Endnotes 7For seasonal adjustment, we use the software developed by Eurostat, Demetra+, with the specification X12ARIMA RSA5C. 8During the first semester of 2003, the composition of employment measured by the ONSS changed due to harmonization between the different Belgian social security bodies. The dataset used in this paper is concerned with the introduction of two new categories of workers: disabled workers with more than 12 months of incapacity and people working in childcare. As a consequence, a level shift shows up in the employment data in 2003q1. To prevent those changes from having an impact on this analysis, the data are corrected before the statistical analysis. Because data on disabled workers and people working in childcare are not available by gender and age group for the period 1997–2002, the adjustment is made by replacing the permanent blip in the first difference of the employment by a linear interpolation and then rebuilding the level of employment before 2003 using this new first difference for 2003q1. This approach follows the method proposed by Nielsen (2004) for additive outliers. 9The data on the population by gender and age group are annual and measured as the resident population on the 1st of January of each year (National Register of Population, SPF Economie). The population is available in 12 age groups (15-19; 20-24; 25-29; 30-34; 35-39; 40-44; 45-49; 50-54; 55-59; 60-64; ≥65). As the employment data in the numerator of the employment rate are quarterly data, the annual population data are transformed in quarterly data. The data of January for a given year are assigned to the fourth quarter of the previous year. 10The results of ADF tests are available from the authors upon request. 11 10The results of ADF tests are available from the authors upon request. 11 11The Engle-Granger methodology is limited to a single-equation test, which is problematic as the equilibrium relationship could be expressed in two ways (the employment rate as a function of GDP or GDP as a function of the employment rate). Dejemeppe et al. IZA Journal of Labor Policy (2015) 4:17 Page 22 of 23 Two Engle-Granger tests could therefore be performed (one using the employment rate as a function of GDP and the other GDP as a function of the employment rate). Endnotes As the sample size grows infinitely large, the two tests become equivalent, but in a small sample, the results might differ, which is a very undesirable feature (Enders 1995). Moreover, the Engle-Granger test relies on a two-step estimator. Hence, errors introduced in the first step are carried into the second one (Enders 1995). p ( ) 12The results of the Engle and Granger (1987) and the Johansen (1988) tests are available from the authors upon request. 13ERg,a,t = ERg,a,t −ERg,a, 13ERg,a,t = ERg,a,t −ERg,a,t−1 Competing interests h l f b p g The IZA Journal of Labor Policy is committed to the IZA Guiding Principles of Research Integrity. The authors declare that they observed these principles. Acknowledgements g We are grateful to the referees, the editor, Sophie Béreau, Vincent Bodart, Bart Cockx, Arnaud Dufays and William Parienté for their very useful comments. We thank in particular Fatemeh Shadman. We also thank participants to various seminars in Louvain-la-Neuve and to the Belgian Day of labor Economists in Leuven (December 2012) for their comments and suggestions. The usual caveat applies. We finally thank the Belgian Federal Administration Science Policy for its financial support on the project “Age, women and employment: An evaluation” within the program Society and Future (CONTRACT NR TA/00/044). Responsible editor: Juan F Jimeno Authors’ information C h S h b Catherine Smith contributed to this paper when she was affiliated to IRES, Université Catholique de Louvain. Author details 1 1IRES - Université catholique de Louvain, Louvain-la-Neuve, Belgium. 2Fonds de la Recherche Scientifique-FNRS, Brussels, Belgium. 3IZA. Received: 12 June 2014 Accepted: 2 June 2015 Received: 12 June 2014 Accepted: 2 June 2015 References Albanese A, Cockx B (2015) Permanent wage cost subsidies for older workers: An effective tool for increasing working time and postponing early retirement? 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https://openalex.org/W3097464594	https://www.e3s-conferences.org/10.1051/e3sconf/202019502027/pdf	English	null	Modelling free gas overpressure in peat layers	E3S web of conferences	2,020	cc-by	5,087	1 Introduction Worth mentioning, Vonk [3] observed water spouting out from probing holes after pulling out the penetrometer during a series of CPT tests at the Bleiswijk polder near Rotterdam. Standpipes installed in the holes through the basal peat layer showed a water level raising 2.5 m above the ground surface, which was ascribed to the presence of a cluster of biogenic gas bubbles accumulated in the peat layer over years. Assessment of transport and flood defence infrastructure founded on peat is challenging for geotechnical engineers due to the presence of abundant organic fraction made of partially decomposed roots, stems and fibres in the peats matrix. In the recent years, concerns arose from the decomposition of the organic matter which tends to saturate the pore fluid with gas species as CH4, H2S and CO2. When the concentration of these gases exceeds the equilibrium solubility, dissolved gases can exsolve upon anthropogenic actions as excavation and dewatering, and environmental loads as temperature rise and atmospheric pressure change as schematised in Fig. 1. Gas bubbles trapped in the basal peat were observed by Den Haan and Kruse [1] in the Wilnis dyke by means of CPT camera sounding and, more recently, by Zwanenburg [2], with gas bubbles venting upon dewatering during the field trial test at Uitdam on the Lake Markermeer, north of Amsterdam. Cyclic changes in pore water pressure in peat bogs have been correlated to the natural oscillations of the atmospheric pressure around its average value, 0.1013 MPa ([4-8]). Of particular interest, systematic higher pore pressure in closed piezometers than in open vented standpipes was measured at decreasing atmospheric pressure, which was attributed to the exsolution and expansion of dissolved and free gas in the peat matrix ([5]). Fig. 2 reports the difference in pore pressure measured by non-vented pressure transducers and open piezometers compared to the atmospheric pressure oscillation at three different depths in a fen peatland in the southern Québec in Canada. The data in Fig. 2 show the typical mirror effect in the oscillation of the atmospheric pressure and the variation of the excess pore pressure. The presence of gas bubbles trapped in peat layers is confirmed by pore pressure measurements in buried closed piezometers. Fig. 1. Schematic representation of gas exsolution occurring in the foundation peat layer and interaction with external loads. Modelling free gas overpressure in peat layers Stefano Muraro1,, and Cristina Jommi1,2 Stefano Muraro , and Cristina Jommi 1Delft University of Technology, Stevinweg 1 / PO-box 5048, 2628 CN, Delft, the Netherlands 2Politecnico di Milano, piazza Leonardo da Vinci 32, 20133, Milano, Italy 1Delft University of Technology, Stevinweg 1 / PO-box 5048, 2628 CN, Delft, the Netherlands 2Politecnico di Milano, piazza Leonardo da Vinci 32, 20133, Milano, Italy Abstract. The paper assesses fully coupled hydro-mechanical numerical approaches developed for unsaturated soils to model the effect of free gas overpressure on the response of peat layers. A simple linear model is used for the soil skeleton, however, the global response is non-linear due to changes over time of the compressibility of the solid skeleton over the compressibility of the fluid, and solubility of gas in water. The overpressure generated in foundation peat layers by barometric pressure oscillations is modelled, and the results are compared to literature data. The development of pore overpressure upon unloading is analysed as a function of the soil skeleton compressibility, and the consequences on the average stress acting on the soil skeleton are discussed. 1 Introduction Despite the magnitude of the excess pore pressure being limited to few kPa, its effect on the response of earth embankments on peats should not be disregarded, as shown by Acharya et al. [9] and Jommi et al. [10] in dedicated experimental studies. A fully coupled hydro- mechanical numerical approach is proposed here to quantify the effects of gas on the pore liquid phase response. The sensitivity to gas exsolution of the soil response is evaluated as a function of the ratio between the volumetric stiffness of the pore fluid and the soil skeleton. Fig. 1. Schematic representation of gas exsolution occurring in the foundation peat layer and interaction with external loads. © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). DP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 ses/by/4 0/) https://doi.org/10.1051/e3sconf/202019502027 https://doi.org/10.1051/e3sconf/202019502027 E3S Web of Conferences 195, 02027 (2020) E-UNSAT 2020 Corresponding author: S.Muraro@tudelft.nl SWRC adopted in the numerical analyses. ( ) 1 1− - r S = 1+ λ (1) ( ) 2 1/ rl r r k = S 1- 1-S (2) (1) (2) Fig. 2. Variation of the excess pore pressure at three different depths and deviation of the atmospheric pressure from 101.3 kPa (re-drawn from Kellner et al. [5]). * Corresponding author: S.Muraro@tudelft.nl © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). E3S Web of Conferences 195, 02027 (2020) E-UNSAT 2020 https://doi.org/10.1051/e3sconf/202019502027 ! " ! " ! # " $ % & Fig. 2. Variation of the excess pore pressure at three different depths and deviation of the atmospheric pressure from 101.3 kPa (re-drawn from Kellner et al. [5]). ! " ! " ! # " $ % & Fig. 2. Variation of the excess pore pressure at three different The retention behaviour was characterised in a dedicated experimental activity by Trivellato [12]. The Soil Water Retention Curve (SWRC) and the liquid phase relative permeability, krl, of the peat are displayed in Fig. 4 and Fig. 5. A van Genuchten model ([13]) is adopted to fit the experimental data, with α = 350 kPa and λ = 0.25. Soil Water Retention Curve (SWRC) and the liquid phase relative permeability, krl, of the peat are displayed in Fig. 4 and Fig. 5. A van Genuchten model ([13]) is adopted to fit the experimental data, with α = 350 kPa and λ = 0.25. ( ) 1 1− - r S = 1+ λ (1) ( ) 2 1/ rl r r k = S 1- 1-S (2) Fig. 4. SWRC adopted in the numerical analyses. &'# & &# & &# () $ * + & & & & , - Fig. 5. Liquid phase relative permeability adopted in the numerical analyses. ( ) 1 1− - r S = 1+ λ (1) ( ) 2 1/ rl r r k = S 1- 1-S (2) Fig. 4. 3 Numerical results The contour plots of the gas pressure and the degree of saturation for the liquid phase during the gas injection is reported in Fig. 6. Due to localised, instead of diffused, gas injection, some time is needed for the gas to distribute within the peat layer and for the gas pressure to equalise. At the end of the equalisation time, the gas eventually accumulates under the upper cover layer, as observed in peat layers in the field. The degree of saturation at the end of the equalisation step attains a value of about 0.95. p Fig. 8 reports the dissolved gas fraction defined according to the Henry’s law as g g g w u M = H M (4) (4) where g u is the absolute gas pressure, H is the Henry’s constant of methane equal to 3638 MPa (T=20°C) ([14]), Mg and Mw are the molecular masses of methane 0.016 kg/mol and of water, 0.018 kg/mol, respectively. After equalisation, the atmospheric pressure acting on the ground surface is varied with time as in Fig 2. The numerical results are discussed with reference to the pore liquid pressure, ul, the dissolved gas fraction, χg, and the excess pore liquid pressure, Δul, computed as the difference between the liquid pressure in the presence and in absence of gas in the pore fluid. This simulation strategy allows for a comparison of the numerical results with the field measurements reported by Kellner et al. [5]. All the plots refer to an observation point located in the peat layer, 1 m below the cover (Fig. 3). The comparison between Fig. 7 and Fig. 8 indicates that the occurrence of gas exsolution, promoted by the drop in the liquid pressure, contributes to the total change in pore liquid pressure by increasing the fluid compressibility. Fig. 8 also reveals a dependence of the amount of gas exsolved on the volumetric stiffness ratio, Kw/K, which will be further discussed in the following. The results in Fig. 7 allow estimating the excess pore liquid pressure due to the presence of free gas in the pore fluid upon atmospheric pressure changes. The pore liquid pressure in the peat layer is shown in Fig. 7(b) for the case of Kw/K = 667. The variation of the pore liquid pressure is dampened by the presence of gas, which increases the compressibility of the pore fluid ([10]). As shown in Fig. 2 Finite element model The gas phase relative permeability, krg, is assumed to follow the relationship rg rl k =1-k (3) (3) The retention properties of the upper cover were chosen in order to limit the escape of the gas entrapped in the lower peat layer, as shown in Fig. 4 and Fig. 5. To simulate biogenic gas production, the analyses included the following steps: - initialisation of the geostatic stress conditions; - injection of 4.7 kg of CH4 in the peat layer; - injection of 4.7 kg of CH4 in the peat layer; - equalisation of gas pressure and gas distribution; - variation of the atmospheric pressure at the ground surface (Patm variation in Fig. 7(a)). Two sets of analyses are conducted to mimic the field observations. In the first set, the gas phase is not included, which reproduces the field observations from open vented standpipes. The second series accounts for the presence of entrapped gas in the peat layer to reproduce field measurements with non-vented pore pressure piezometers. Gas was injected through four internal nodes in the lower part of the layer as shown in Fig. 3, allowing for drainage of the necessary amount of water from the top boundary of the model. The variation of the atmospheric pressure replicates the data by Kellner et al. [5] reported in Fig. 2. The properties of methane (density, viscosity, diffusivity and solubility) and those of water were assigned for a reference temperature of 20°C, which was kept constant throughout the analyses. Fig. 6. Contour plots of the absolute gas pressure and the degree of saturation during gas injection and after equalisation. The results in Fig. 7(b) clearly show that the pore liquid pressure remains higher than that of the fully saturated soil upon a decrease in the atmospheric pressure. The opposite is observed when the atmospheric pressure increases. 2 Finite element model Fully coupled two-phase flow with hydro-mechanical coupling was simulated on a 2D plain strain model with CODE_BRIGHT ([11]). For the sake of simplicity, an elastic model is adopted for the soil mechanical behaviour. The assumption does not hinder the validity of the results, as unloading stress paths are of relevance in triggering gas exsolution. The typical soil profile at the Markermeer, consisting of a peat layer underneath a surficial clay cover is considered (Fig. 3). The material properties of relevance for the coupled response are reported in Table 1. Two different values of the water- soil volumetric stiffness are considered (Kw/K). Fig. 4. SWRC adopted in the numerical analyses. Table 1. Material properties of relevance in the analyses. Parameters Symbol Value Ratio between water and soil skeleton stiffness Kw/K 89 - 667 Saturated hydraulic conductivity ksat (isotropic) 7.4e-7 m/s peat 1e-9 m/s clay Fig. 3. Geometry of the problem. &'# & &# & &# () $ * + & & & & , - Fig. 5. Liquid phase relative permeability adopted in the numerical analyses. &'# & &# & &# () $ * + & & & & , - Fig. 5. Liquid phase relative permeability adopted in the numerical analyses. Table 1. Material properties of relevance in the analyses. Parameters Symbol Value Ratio between water and soil skeleton stiffness Kw/K 89 - 667 Saturated hydraulic conductivity ksat (isotropic) 7.4e-7 m/s peat 1e-9 m/s clay Table 1. Material properties of relevance in the analyses. &'# & &# & &# () $ * + & & & & , - Fig. 3. Geometry of the problem. Fig. 5. Liquid phase relative permeability adopted in the numerical analyses. 2 https://doi.org/10.1051/e3sconf/202019502027 E3S Web of Conferences 195, 02027 (2020) E-UNSAT 2020 Fig. 6. Contour plots of the absolute gas pressure and the degree of saturation during gas injection and after equalisation. 3 Numerical results Evolution of the atmospheric pressure imposed to the ground surface (a), and the corresponding pore liquid pressure for a soil layer with and without gas (b). Fig. 9. Excess pore liquid pressure du fluid upon variation of the atmospheri of water-soil volumetric stiffness. 4 Discussion 4.1 Concomitant factors to ga The maximum excess pore liquid Fig. 9 is about 0.8 kPa for a low Kw/K and 0.4 kPa in the case of h of magnitude found in the field by displayed in Fig. 2 for the same oscillation is about 3 kPa. V agreement between numerical observation is achieved. Howev accounted for in the proposed n worth mentioning, which may co observed quantitative differences. Seasonal changes in temperat the major contributing factors pressure often measured during A h l [9] d fi Fig. 9. Excess pore liquid pressure due to free gas in the pore fluid upon variation of the atmospheric for two different values of water-soil volumetric stiffness. Fig. 9. Excess pore liquid pressure due to free gas in the pore fluid upon variation of the atmospheric for two different values of water soil volumetric stiffness ! & & & & & & $ % & Fig. 9. Excess pore liquid pressure due to free gas in the pore fluid upon variation of the atmospheric for two different values of water-soil volumetric stiffness. 3 Numerical results 9, the coupled formulation is capable to reproduce the mirror effect in the oscillation of the pore liquid pressure compared to the atmospheric pressure, as observed in Fig. 2 from field measurements. 3 https://doi.org/10.1051/e3sconf/202019502027 E3S Web of Conferences 195, 02027 (2020) E-UNSAT 2020 ! & & & & & & $ % & Fig. 7. Evolution of the atmospheric pressure imposed to the ground surface (a), and the corresponding pore liquid pressure for a soil layer with and without gas (b). Fig flu of 4 4. Th Fi Kw of dis os ag ob ac wo ob the ! & & & & & & $ % & Fig. 7. Evolution of the atmospheric pressure imposed to the ground surface (a), and the corresponding pore liquid pressure for a soil layer with and without gas (b). Fig. 9. Excess pore liquid pressure due to free gas in the pore fluid upon variation of the atmospheric for two different values of water-soil volumetric stiffness. 4 Discussion 4.1 Concomitant factors to gas overpressure The maximum excess pore liquid pressure displayed in Fig. 9 is about 0.8 kPa for a low relative stiffness ratio Kw/K and 0.4 kPa in the case of higher Kw/K. The order of magnitude found in the field by Kellner et al. [5] and displayed in Fig. 2 for the same atmospheric pressure oscillation is about 3 kPa. Very good qualitative agreement between numerical results and field observation is achieved. However, some aspects not accounted for in the proposed numerical analyses are worth mentioning, which may contribute to explain the observed quantitative differences. Seasonal changes in temperature seem to be one of the major contributing factors to high excess pore pressure often measured during the warmer months. Acharya et al. [9] reported field measurements and laboratory data confirming liquid pressure in excess of the hydrostatic values due to thermal gas exsolution and expansion in peat (i. e. decrease in the gas solubility with increasing temperature). A pore liquid pressure increase of 11 kPa was found resulting from an increase in temperature of 6°C ! & & & & & & $ % & Fig. 7. 4.2 Soil sensitivity to gas exsolution The previous numerical results suggest a dependence of the pore liquid pressure change (Fig. 7) and the dissolved gas fraction (Fig. 8) on the volumetric stiffness ratio Kw/K. The influence of the soil skeleton compressibility on the sensitivity of soils to gas exsolution is further investigated with a single soil element model. To model the mechanical behaviour, the mean soil skeleton stress, ˆp , is chosen as stress variable ([15]) ˆ l r g l p=p-u -(1-S )(u -u ) (5) (5) where p is the mean total stress and ug is the gas pressure. Non-linear isotropic elasticity is considered with a constant slope of the isotropic unloading- reloading line (ISO-URL), κ. Finite compressibility of the fluid and gas exsolution-dissolution and expansion are accounted for in the water mass balance and gas mass balance discretised equations. The retention curve in Fig. 4 is adopted to describe the gas - liquid pressure equilibrium. Methane is considered as gas species and its properties are evaluated at 20°C. Table 2 reports the properties adopted in the analyses. Gas exsolution is triggered by means of isotropic unloading under external undrained conditions. The results are reported in Fig. 10 and Fig. 11 in terms of pore liquid pressure and mean soil skeleton stress respectively. where p is the mean total stress and ug is the gas pressure. Non-linear isotropic elasticity is considered with a constant slope of the isotropic unloading- reloading line (ISO-URL), κ. Finite compressibility of the fluid and gas exsolution-dissolution and expansion are accounted for in the water mass balance and gas mass balance discretised equations. The retention curve in Fig. 4 is adopted to describe the gas - liquid pressure equilibrium. Methane is considered as gas species and its properties are evaluated at 20°C. Table 2 reports the properties adopted in the analyses. Gas exsolution is triggered by means of isotropic unloading under external undrained conditions. The results are reported in Fig. 10 and Fig. 11 in terms of pore liquid pressure and mean soil skeleton stress respectively. Fig. 10. Evolution of the pore liquid pressure during isotropic undrained unloading of gassy soils. Fig. 11. Evolution of the mean soil skeleton stress during isotropic undrained unloading of gassy soils. For an initial part of the unloading path until ul > ul/g, no gas is exsolved and the decrease in the pore liquid pressure equates the reduction in the mean total stress (i.e. 4.1 Concomitant factors to gas overpressure accompanied by gas release, as confirmed by numerous authors ([4-7], [9]). 4.1 Concomitant factors to gas overpressure The maximum excess pore liquid pressure displayed in Fig. 9 is about 0.8 kPa for a low relative stiffness ratio Kw/K and 0.4 kPa in the case of higher Kw/K. The order of magnitude found in the field by Kellner et al. [5] and displayed in Fig. 2 for the same atmospheric pressure oscillation is about 3 kPa. Very good qualitative agreement between numerical results and field observation is achieved. However, some aspects not accounted for in the proposed numerical analyses are worth mentioning, which may contribute to explain the observed quantitative differences. Fig. 7. Evolution of the atmospheric pressure imposed to the ground surface (a), and the corresponding pore liquid pressure for a soil layer with and without gas (b). Seasonal changes in temperature seem to be one of the major contributing factors to high excess pore pressure often measured during the warmer months. Acharya et al. [9] reported field measurements and laboratory data confirming liquid pressure in excess of the hydrostatic values due to thermal gas exsolution and expansion in peat (i. e. decrease in the gas solubility with increasing temperature). A pore liquid pressure increase of 11 kPa was found resulting from an increase in temperature of 6°C. Fi 8 Di l d f ti i th fl id i ti Fig. 8. Dissolved gas fraction in the pore fluid upon variation of the atmospheric pressure. Temperature increase intensifies the biological activity, hence promoting faster peat decomposition. Higher rate of production of CH4, H2S and CO2 may supersaturate the pore fluid, which promotes additional gas exsolution. Eventually, gas expansion and coalescence create local overpressure zones with gas bubbles entrapped in the peat matrix. These zones will experience liquid pressure notably higher than that of the adjacent soil, which results in gas bursting when a pressure threshold across the blocking bubbles is reached ([5]). Evidence of gas bursting is shown in Fig. 2 by a series of sudden drops in the measured pore pressure, which is Fig. 8. Dissolved gas fraction in the pore fluid upon variation of the atmospheric pressure. 4 E3S Web of Conferences 195, 02027 (2020) E-UNSAT 2020 https://doi.org/10.1051/e3sconf/202019502027 Fig. 10. Evolution of the pore liquid pressure during isotropic undrained unloading of gassy soils. Fig. 10. Evolution of the pore liquid pressure during isotropic undrained unloading of gassy soils. accompanied by gas release, as confirmed by numerous authors ([4-7], [9]). 4.2 Soil sensitivity to gas exsolution For soft soils, the stiffness ratio is still in favour of the pore fluid and the total stress 5 https://doi.org/10.1051/e3sconf/202019502027 E3S Web of Conferences 195, 02027 (2020) E-UNSAT 2020 E3S Web of Conferences 195, 02027 (2020) E UNSAT 2020 reduction transferred onto the soil skeleton is limited (Fig. 11). On the contrary, for stiffer soils, a small amount of gas is sufficient to increase significantly the stiffness ratio (Fig. 12), causing a small reduction in the pore liquid pressure (Fig. 10). On the one hand, this limits further gas exsolution, as displayed by the high degree of saturation in Fig. 12. On the other hand, it implies that a significant portion of the total stress reduction is transferred onto the soil skeleton (Fig. 11). As soon as the mean soil skeleton stress reduces, the stiffness of the soil skeleton drops dramatically, and the volumetric stiffness ratio starts decreasing in favour of the fluid phase again. Eventually, the limit condition for which K → 0 is approached, with the stress acting on the soil skeleton tending to zero ˆp → 0. It is worth noting that the stiffer the soil, the lower the amount of gas generated. In the case analysed, the final degree of saturation of the stiffer soil reaches Sr = 0.983, whereas the more compressible soil attains Sr = 0.974. fully saturated soils strongly depends on the ratio of the stiffness of the soil skeleton over that of the pore fluid. fully saturated soils strongly depends on the ratio of the stiffness of the soil skeleton over that of the pore fluid. For the case of stiff soils as sands, a small amount of gas exsolution increases significantly the stiffness ratio leading to a dramatic reduction in the stress acting on the soil skeleton upon undrained unloading. On the contrary, soft soils as peats are capable to exsolve higher amount of gas before experiencing significant loss of stress. The results of this work are of relevance for future assessment of serviceability and stability analyses of earth embankments founded on peats, where a correct estimation of pore pressure is of primary importance due to the low total stresses experienced by peats. For the case of stiff soils as sands, a small amount of gas exsolution increases significantly the stiffness ratio leading to a dramatic reduction in the stress acting on the soil skeleton upon undrained unloading. 4.2 Soil sensitivity to gas exsolution fully saturated response). When the liquid pressure drops below the gas - liquid saturation pressure, gas exsolution and expansion take place and the response starts diverging from that observed in the fully saturated case. At this stage, the decrease in the pore liquid pressure is highly dependent on the relative compressibility of pore fluid - soil skeleton. For stiff soils, the liquid pressure decreases less than for highly compressible soils. As shown in Fig. 10, the pore liquid pressure for κ = 0.012 remains higher than that of the soil having κ = 0.09. This translates into a significant reduction in the mean skeleton stress despite the external undrained conditions. The results displayed in Fig. 11 suggest that stiffer soils may reach the condition ˆp → 0 more likely than softer soils. Fig. 11. Evolution of the mean soil skeleton stress during isotropic undrained unloading of gassy soils. Fig. 12 reports the ratio of the volumetric stiffness of the soil skeleton, K, over that of the pore fluid, Kf, calculated for the mixture of gas and water ([16]): ˆ vp K= (6) ( ) 1 g l f r r r w g u u 1 K =1 S + 1-S +S h K p u p (7) ˆ vp K= (6) (6) Table 2. Material properties and initial variables used in the analyses of gas exsolution. ( ) 1 g l f r r r w g u u 1 K =1 S + 1-S +S h K p u p (7) (7) Parameters Symbol Value Slope of ISO-URL κ 0.09 - 0.045 - 0.012 Initial specific volume v0 3 Bulk modulus of water Kw 2.22 GPa Initial stiffness ratio Kw/K 667 - 333 - 89 Mean total stress p 400 kPa Initial liquid pressure ul0 300 kPa Gas-liquid saturation pressure ul/g 200 kPa where g u is the absolute gas pressure and h is the Henry’s solubility via concentration. Before gas exsolution takes place, Kf = Kw and K/Kf ≅ 0. As the gas starts exsolving, the stiffness of the pore fluid decreases. References 1. E. J. Den Haan, G. A. M. Kruse. Proc. 2nd Int. Workshop on Characterisation and Engineering of Natural Soils, 29, 2101-2133 (Taylor & Francis Group, 2007). . . * & & & & % & % &# % & " / $ & & & & '& '& & & & + 0 &# Fig. 12. Evolution of the volumetric stiffness ratio between the soil skeleton and the pore fluid during isotropic undrained unloading of gassy soils. 2. C. Zwanenburg. Dikes on Peat: analysis of field trials (Internal Report Deltares, 2013). 3. B. F. Vonk. In Advances in understanding and modelling mechanical behaviour of peat, 389-399 (Balkema, Rotterdam, 1994). 4. P. H. Glaser, J. P. Chanton, P. Morin, D. O. Rosenberry, D. I. Siegel, O. Ruud, L. I. Chasar, A. S. Reeve. Global Biogeochem. Cy. 18, 1, 1-15 (2004). 5. E. Kellner, J. M. Waddington, J. S. Price. Water Resour. Res. 41, 8, 1-12 (2005). 6. T. Tokida, T. Miyazaki, M. Mizoguchi. Geophys. Res. Lett. 32, 13 (2005). 7. M. Strack, E. Kellner, J. M. Waddington. Hydrol. Process. 20, 17, 3611-3622 (2006). 8. J. M. Waddington, K. Harrison, E. Kellner, A. J. Baird. Hydrol. Process. 23, 20, 2970-2980 (2009). 9. M. P. Acharya, M. T. Hendry, C. D. Martin. Int. J. Geotech. Eng. 10, 2, 145-154 (2016). Fig. 12. Evolution of the volumetric stiffness ratio between the soil skeleton and the pore fluid during isotropic undrained unloading of gassy soils. 10. C. Jommi, S. Muraro, E. Trivellato, C. Zwanenburg. Géotechnique 69, 9, 753-766 (2019). 11. S. Olivella, A. Gens, J. Carrera, E. E. Alonso. Eng. Computation. 13, 7, 87-112 (1996). 4.2 Soil sensitivity to gas exsolution On the contrary, soft soils as peats are capable to exsolve higher amount of gas before experiencing significant loss of stress. The results of this work are of relevance for future assessment of serviceability and stability analyses of earth embankments founded on peats, where a correct estimation of pore pressure is of primary importance due to the low total stresses experienced by peats. 5 Conclusions The work assesses fully coupled hydro-mechanical numerical approaches developed for unsaturated soils to model the effect of free gas overpressure on the response of peat layers. The presence of gas affects significantly the hydro-mechanical response to external anthropogenic and environmental loads, as several field observations reported. A series of fully coupled two-phase flow with hydro-mechanical coupling numerical analyses were performed accounting for the presence of gas in the soil layer to replicate field observations. The numerical results match qualitatively well the observed field response, confirming that the free gas phase dampens the variation of the pore liquid pressure. However, the magnitude of the pore liquid in excess to the case of 12. E. Trivellato. The effects of partial saturation on the geotechnical properties of peats: an experimental investigation. MSc thesis Politecnico di Milano (2014). 13. M. T. van Genuchten. Soil Sci. Soc. Am. J. 44, 5, 892-898 (1980). 14. R. Sander. Atmospheric Chem. & Phys. 15, 4399- 4981 (2015). 15. C. Jommi. Experimental evidence and theoretical approaches in unsaturated soils, 139-153 (Balkema Rotterdam, 2000). 16. D. G. Fredlund, H. Rahardjo. Soil mechanics for unsaturated soils (John Wiley & Sons, 1993). 6
https://openalex.org/W4234532925	https://www.qeios.com/read/7HMFPY/pdf	English	null	Quasi-Static Compliance	Definitions	2,020	cc-by	67	Qeios · Definition, February 2, 2020 Open Peer Review on Qeios Open Peer Review on Qeios Quasi-Static Compliance National Cancer Institute National Cancer Institute Qeios ID: 7HMFPY · https://doi.org/10.32388/7HMFPY Source National Cancer Institute. Quasi-Static Compliance. NCI Thesaurus. Code C139255. National Cancer Institute. Quasi-Static Compliance. NCI Thesaurus. Code C139255 The static elastic recoil pressure of the lungs at a given lung volume. Qeios ID: 7HMFPY · https://doi.org/10.32388/7HMFPY 1/1
https://openalex.org/W2904716286	https://www.scielo.br/j/sa/a/sqM9fg4kvT3FN8LDtVjmczg/?lang=en&format=pdf	English	null	Automation of lettuce seedlings irrigation with sensors deployed in the substrate or at the atmosphere	Scientia Agrícola	2,019	cc-by	7,819	atmosphere Carlos Chagas Filho, 373 – 21941- 902 – Rio de Janeiro, RJ – Brasil. 3Universidade de São Paulo/ESALQ – Depto. de Genética, Av. Pádua Dias, 11 – 13418-900 – Piracicaba, SP – Brasil. 4Università degli Studi Di Milano – Dipto. di Scienze Agrarie e Ambientali, Via Giovanni Celoria 2 – 20133 – Milano – Italia. 5Universidade Federal Rural do Rio de Janeiro – Depto. de Ciências Fisiológicas. Ambientali, Via Giovanni Celoria 2 – 20133 – Milano – Italia. Corresponding author <anacarolina_mb@yahoo.com.br> Corresponding author <anacarolina_mb@yahoo.com.br> Edited by: Mohammad Valipour Edited by: Mohammad Valipour Received May 14, 2017 Accepted October 23, 2017 Introduction mensions and is suitable for use in the production of seedlings. The production of seedlings is an important step in the vegetable production process (Herrera et al., 2008). During germination and early development, plants are highly sensitive to environmental factors, especially as they are kept in containers with a small amount of soil, which limits the availability of water (and nutri- ents) (Carmona et al., 2012). Moreover, the use of irriga- tion systems is essential to greenhouse production since this is the only way to provide water to the crops. Thus, irrigation management plays a pivotal role, since it is important that water is applied at the proper time and in the appropriate volume, to avoid wasting water and nutrients (Dai et al., 2016). Although there are reports on the use of sensors based on automation of irrigation scheduling (Abdurrah- man et al., 2015; Chaware et al., 2015), to the best of our knowledge, there is no study on the use of these sensors in seedling production, with the exception of our own previous report (Dias et al., 2013). Therefore, this study had as its main aim the evaluation of the effect of water irrigation management offered by this device first, with its sensor installed in the soil (based on the water availability in the substrate) and second, suspended in the atmosphere adjacent to the production trays (based on the variation of water vapor pressure), with differ- ent regulations (tensions), in the production of lettuce seedlings. The performance of the SIC was evaluated by monitoring several plant traits. Due to the increasing scarcity of water and the waste of water practiced by many farmers, there is great concern about the negative impacts of irrigation tech- niques on the productive sector, pointing to the need for a precise balance between agricultural productivity and the preservation of natural resources (Mojid et al., 2012). Given this scenario, studies relating to water eco- nomics are included in most sustainable types of irriga- tion management. Materials and Methods atmosphere Ana Carolina Mendes Bezerra1* , David da Cunha Valença2 , Daniel Fonseca de Carvalho1 , Camila Ferreira de Pinho1 , Fernanda Reinert2 , Daniela Pinto Gomes1 , Fernanda Palmeira Gabetto5 , Ricardo Antunes Azevedo3 , Daniele Masseroni4 , Leonardo Oliveira Medici5 Ana Carolina Mendes Bezerra1* , David da Cunha Valença2 , Daniel Fonseca de Carvalho1 , Camila Ferreira de Pinho1 , Fernanda Reinert2 , Daniela Pinto Gomes1 , Fernanda Palmeira Gabetto5 , Ricardo Antunes Azevedo3 , Daniele Masseroni4 , Leonardo Oliveira Medici5 ABSTRACT: Seedling production is an important step in the lettuce production system. How- ever, there is a tendency to apply excessive irrigation at this stage. The aim of this study was to test the use of the Simplified Irrigation Controller (SIC) under two conditions: first, with the sensor installed in the substrate (measuring the soil water tension) and second, in the atmosphere (“at- mospheric” - responding to vapor demand in the atmosphere) to control the irrigation of lettuce seedlings. The performance of the SIC was evaluated by monitoring plant traits. The lettuce seeds of the Regina cultivar were sown in styrofoam trays with commercial organic substrate. Both experiments were conducted under greenhouse conditions and evaluated under different SIC activation tensions. Shoot fresh weight and dry weight, height, number of leaves, leaf length, leaf area, water use efficiency (WUE) (ratio between the dry mass of plant produced by the vol- ume of water applied), chlorophyll content, stomatal conductance and chlorophyll a fluorescence were assessed. In the experiment with the substrate sensor, the tension of 11.5 kPa caused death in 94 % of the seedlings and was disregarded in the statistical analysis. All biometric and physiological traits evaluated decreased as the SIC tensions increased. Thus, tensions of 4.0 (substrate sensor) and 3.5 kPa (atmospheric sensor) showed greater potential for producing vigorous seedlings, with WUE average values of 1.86 and 1.37 g L–1, respectively. Cultivation of lettuce seedlings proved viable under both conditions when the SIC was used, with the emer- gence of a number of practical advantages of the atmospheric sensor over the substrate one. Keywords: Lactuca sativa L., Simplified Irrigation Controller, water use efficiency 1Universidade Federal Rural do Rio de Janeiro – Depto. de Engenharia, Rod. BR 465, km 7 – 23897-000 – Seropédica, RJ – Brasil. 2Universidade Federal do Rio de Janeiro/Instituto de Biologia – Depto de Botânica, Av. Agricultural Engineering \| Note DOI: http://dx.doi.org/10.1590/1678-992X-2017-0163 Sci. Agric. v.76, n.2, p.179-189, March/April 2019 Irrigation management, treatments and experimental design Irrigation was performed with micro sprinklers installed 0.75 m away from each tray. A 0.5-hp pump picked up water from a 1000-L tank, installed at a height of 1 m. A flow switch installed in the water outlet of the pump ensured its automatic activation at the moment when the SIC’s drive tensions were reached. The experimental design for the substrate sen- sor experiment was block randomization, comprising 6 blocks and four adjustments in the SIC (4.0 kPa, 6.5 kPa, 9.0 kPa and 11.5 kPa), totaling 24 experimental plots (Fig- ure 2A). In the atmospheric sensor experiment with the SIC positioned in the air, a randomized block design was used in a factorial scheme where factor A comprised the four adjustments of the SIC (3.5 kPa, 5.0 kPa, 6.5 kPa and 8.0 kPa) and factor B the two evaluation periods (from 8 July to 8 Aug and 16 Sept to 16 Oct, both in 2016) with four replications, totaling 32 plots (Figure 2B). The greater the vertical distance, the faster the device uptakes water, leading to a faster switch off. Thus, a distance of 30 cm was set and used for the test because it was the shortest practical vertical distance for installing the SIC for the experiments. Conse- quently, in this case the device deactivates in a short- er period of time than at other settings. Furthermore, the vertical distance of 30 cm was the distance used for testing in the previous study by Dias et al. (2013). Throughout this test a shorter response time for the equipment was reached, compared to that previously found by Dias et al. (2013), dropping from 27 to 5 s (Dias et al., 2013). The evaluation periods of the year were chosen because of the interest in evaluating the device with the sensor positioned in the air in different climatic condi- tions throughout the year. Figure 1 – SIC installed with the sensor positioned in the substrate (A) and SIC installed with the sensor positioned in the atmosphere adjacent to the trays (B). The SIC is regulated by the difference between the height of the pressure switch and the candle. The verti- cal distance is proportional to the water tension (Medici et al., 2010; Gomes et al., 2017). For example, 30 cm of vertical distance corresponds to 3.0 kPa; 50 cm to 5.0 kPa; and so on. Site description Two experiments were carried out under green- house conditions in Seropédica, RJ, Brazil (22°48’00’’ S; 43°41’00’’ W; 33.0 m). In the first experiment the SIC sensor was installed in the substrate (Figure 1A) while in the second, the equipment was evaluated with the ceramic sensor positioned above the substrate in the at- mosphere adjacent to the production trays (Figure 1B). From the moment the irrigation is triggered, water enters the sensor so as to bring the negative pressure value into equilibrium with the atmospheric pressure value. Furthermore, the water entering in the sensor also increases the pressure in the pressure switch so that it will turn off the irrigation system. The lettuce seedlings (Lactuca sativa cv. Regina) were sown in styrofoam trays (200 cells) with organic commercial substrate. The dimensions of each tray were: 67.4 cm long, 34.3 cm wide and 5.4 cm high. As previously stated, SIC uses a domestic filter candle as a sensor (Batista et al., 2013; Gomes et al., 2017; Medici et al., 2010); however, in this study the sensors were made with the original candle adapted to smaller dimensions, for the purpose of being used in seedling production systems. The dimensions of the sub- strate sensors were 3 × 1 × 1 cm and conical in shape for better insertion in the tray’s cells, whilst the atmo- sphere sensors had dimensions of 4 × 1 × 1 cm and a parabolic shape. The adaptation consisted of cutting and sanding the original candle. Once the new sensor was ready and the SIC mounted, a test to measure the time it takes for the device to turn off was carried out by simu- lating a 30 cm difference between the pressure switch and the sensor and subsequently verifying how long it takes to react to a possible irrigation. Materials and Methods The use of the SIC ensures greater practicality and economy in irrigation. It is produced with a domestic filter candle (sensor), which is connected to a washing machine pressure switch through a flexible tube. With the tube filled with water, the pressure switch receives water column pressure while keeping the electric cur- rent interrupted. When the sensor humidity is reduced, negative pressure is generated on the filter candle, acting as a signal; this pressure is then transmitted to the pres- The Simplified Irrigation Controller (SIC) (Medi- ci et al., 2010) is presented as an alternative in irriga- tion management, since the version used in the field (Batista et al., 2013; Gomes et al., 2017) can be adapt- ed from the construction of sensors with reduced di- Sci. Agric. v.76, n.2, p.179-189, March/April 2019 Lettuce seeding cultivation Bezerra et al. sure switch through the flexible tube, as per the Pascal Principle, causing the irrigation water supply to be elec- trically engaged. Sci. Agric. v.76, n.2, p.179-189, March/April 2019 Irrigation management, treatments and experimental design Aiming to facilitate the use of the sensor in the irrigation of seedlings the present study tested the use of the SIC by positioning the sensor in the atmosphere. Among the advantages of this positioning is the fact that, in the case of seedling irrigation, due to the re- duced dimensions of the tray cells for the use of the de- veloped equipment, it is necessary to make adaptations and because of the small size of the production cell there may not be adequate contact between the sub- strate and the ceramic, which is essential for correct measuring of the water tension. Another advantage is the greater mobility of the production trays, which would not be possible in the case where the sensor is located in the substrate. Figure 1 – SIC installed with the sensor positioned in the substrate (A) and SIC installed with the sensor positioned in the atmosphere adjacent to the trays (B). 180 Sci. Agric. v.76, n.2, p.179-189, March/April 2019 Lettuce seeding cultivation Bezerra et al. Figure 2 – Sketch of the experimental area. Figure 2 – Sketch of the experimental area. The parcels of the first block received four SICs, each one related to a treatment. In the other plots the amount and time of irrigation were the same for each treatment which were triggered whenever irrigation was applied in the plots of the first block. Since each tray was irrigated by its own sprinkler, there was no over- lap necessary for the application of water to be uniform throughout the tray. Thus, to warrant uniform applica- tion of water in the seedlings, only the 16 central cells were considered as useful plots. Seed germination occurred within the first five days after sowing (DAS). During the first eight DAS, manual and automatic irrigations were similar in all plots for the purpose of ensuring uniform germination. For automatic irrigation, in this period, all SICs were adjusted to 3.0 kPa. Greenhouse climate data were re- corded by a USB temperature and humidity data logger (Impac, São Paulo, Brazil). In the second period of the experiment with the atmospheric sensor, it was necessary to install shading screens (50 %) due to conditions of high temperature and light, which represented a limitation in seed germi- nation and seedling survival. Irrigation management, treatments and experimental design To guarantee uniformity in the application of wa- ter among the trays of the same treatment, a preliminary test on the sowing was carried out. The test consisted of positioning four Falcon tube caps in a quadruple for- mat over the edges of the working area, which contained the 16 production cells monitored in all trays of all the treatments. Next, the irrigation system was activated for 5 min and the volume of water collected in each tray was measured using a graduated cylinder. The volumes were compared with each other. The maximum varia- tion of 10 % between trays under the same treatment was stipulated in order to try to obtain the smallest pos- sible variation. Evaluated traits In both experiments biometric traits of the seed- lings and the amount of water used in their cultivation were evaluated. Therefore, shoot fresh and dry weight (SFW and SDW), height (H) and number of leaves (NL) were analyzed at 28 DAS. With the data of SDW and estimated water volume applied, water use efficiency (WUE) was calculated as a ratio of these two parameters. Additional traits were evaluated for each experi- ment. In the substrate sensor experiment the following traits were also evaluated: leaf length, leaf area and chlo- rophyll content (Chl), whereas in the atmospheric sen- sor experiments stomatal conductance (gs) and analysis of the chlorophyll a fluorescence transient parameters were also evaluated. Next, by using the same collectors that had been used in the previous test, the application uniformity test was carried out based on the test described by Chris- tiansen (1942), aiming to guarantee 90 % of uniformity in the trays. Estimated volume, germination and climate Leaf length and leaf area were evaluated at 15 DAS. This evaluation was carried out on two seedlings per tray. Each experimental plot received two Falcon 50 mL conical tubes. The tubes were placed randomly under two cells in the tray: one empty cell, to quantify the volume of water applied per cell, and another filled with substrate and seedling, in order to quantify drainage. This allowed for estimating the drainage and volume ap- plied daily. Chlorophyll content (Chl) was monitored at 28 DAS. A Chlorophyll content meter, ChlorofiLOG (FALK- ER, Porto Alegre, Rio Grande do Sul, Brazil), was used for the Chl reading and the readings were taken on the expanded leaves. 181 Sci. Agric. v.76, n.2, p.179-189, March/April 2019 Lettuce seeding cultivation Bezerra et al. Stomatal conductance (gs) was measured at 25 DAS using a SC-1 Leaf Porometer (Decagon Devices, Washing- ton, USA). Measurements were taken around 12h00. Statistical analysis The results were submitted to analysis of variance at p ≤ 0.05. In the presence of significant interaction, a sliced ANOVA was carried out for each factor. Scott-Knott’s test was used to determine significance at p ≤ 0.05. The data were analyzed with the aid of the Sisvar statistical program (Federal University of Lavras, Lavras, Brazil). Biometric traits Tests at the lowest tensions tested, 4.0 (substrate sensor) and 3.5 kPa (atmospheric sensor), showed great- er potential for seedling production. SFW, SDW, H and NL presented reductions as the tested tension increased with the substrate sensor (Table 1). Due to the elevat- ed death rate caused by the 11.5 kPa tension with the substrate sensor, the results of this treatment were not considered for statistical analysis. In the experiment with the atmospheric sensor the second period tested produced more vigorous seedlings than the first one, exhibiting higher SFW, SDW, H and NL. On the other hand, greater differentiation between the traits consid- ered according to the increase in tension evaluated in the second period was observed (Table 2). Irrigation management The different uses of SIC (substrate and atmo- sphere) provided different frequency and irrigation re- gimes. In the experiment with the substrate sensor the 4.0 kPa (Figure 4A) tension provided irrigations almost daily. For the 6.5 kPa tension (Figure 4B), irrigation events occurred on average every two days, while at the 9.0 kPa tension (Figure 4C) an average of three days were spent without irrigation, and at the 11.5 kPa ten- sion (Figure 4D) there was a period of five days without irrigation. This fact resulted in only five episodes of ir- rigation by SIC for this last treatment throughout the ex- perimental period. As for the atmospheric sensor experi- ment, the irrigations occurred almost daily at all tension levels and periods tested (Figure 5A, B, C, D, E, F, G and H) and the major difference observed was related to the volumes of water applied. The final higher volume of water applied to the lower tensions treatments may be the result of there being more than one irrigation event the same day. Fluorescence parameters were evaluated at 28 DAS. The measurements of chlorophyll fluorescence were taken using a portable Plant Efficiency Analyzer, the Handy PEA (Hansatech, Norfolk, UK). The measure- ments were taken in the middle third of fully expanded young leaves in the morning with dark adaptation. Fluo- rescence emission was induced in a 4 mm diameter of leaf area by exposing the sample to a pulse of saturating light at an intensity of 3.000 μmol m–2 s–1. From the fluo- rescence intensities, the parameters were calculated by the JIP Test, according to the methodology proposed by Strasser and Strasser (1995). Results We used two modes of detecting water demand by lettuce plants – a substrate sensor and an atmospheric sensor. Both systems proved to be efficient in the irriga- tion of lettuce seedlings, especially for the lowest ten- sions tested. Each seedling production consumed an average of 38.3 (substrate sensor) and 29.4 mL (atmo- spheric sensor). In the first experimental period with the atmospheric sensor no differences were observed in the biometric traits at all tensions tested. Approximately 0% (4.0 kPa), 8% (6.5 kPa), 24% (9.0 kPa) and 94% (11.5 kPa) of the seedlings died be- fore the end of the experiment with the substrate sen- sor (Figure 3), whereas, all seedlings survived under the atmospheric sensor treatments. WUE WUE also showed a reduction as the tested tension increased, except for the first period of the ght; shoot dry weight; height; number of leaves; leaf length; leaf area; water use efficiency and chlorophyll b content of at different tension adjustments of the SIC during the experiment with the sensor positioned in the substrate. ble 1 – Shoot fresh weight; shoot dry weight; height; number of leaves; leaf length; leaf area; water use efficiency and c Lactuca sativa seedlings at different tension adjustments of the SIC during the experiment with the sensor positioned in Table 1 – Shoot fresh weight; shoot dry weight; height; number of leaves; leaf length; leaf area; water use efficiency and chlorophyll b content of Lactuca sativa seedlings at different tension adjustments of the SIC during the experiment with the sensor positioned in the substrate. Traits Adjustment Tension of SIC (Simplified Irrigation Controller) 4.0 6.5 9.0 11.5 -------------------------------------------------------------------- kPa -------------------------------------------------------------------- kPa* Shoot Fresh Weight (g per plant) 0.53 ± 0.044 a 0.13 ± 0.050 b 0.06 ± 0.027 c / Shoot Dry Weight (g per plant) 0.07 ± 0.005 a 0.02 ± 0.008 b 0.01 ± 0.005 c / Height (cm) 5.01 ± 0.280 a 2.77 ± 0.618 b 1.87 ± 0.649 c / Number of leaves (units) 7.47 ± 0.536 a 5.19 ± 0.935 b 3.44 ± 1.087 c / Leaf length (cm) 16.08 ± 3.758 a 10.89 ± 2.276 b 8.31 ± 1.334 c / Leaf area (cm2) 47 ± 17 a 17 ± 5 b 9 ± 3 c / Water Use Efficiency (g L–1) 1.86 ± 0.457 a 0.73 ± 0.226 b 0.45 ± 0.091 c / Chlorophyll b content (FCI - Falker chlorophyll index)** 1.98 ± 0.102 a 3.35 ± 0.968 b 2.58 ± 0.852 a / Volume of water to produce a seedling (L) 0.04 ± 0.006 0.03 ± 0.004 0.03 ± 0.004 / Small caption letters at the rows represent significant differences using Tukey’s test at p < 0.05. 11.5 kPa tension caused death in 94 % of the seedlings and was disregarded in the statistical analysis; no difference was observed for Chlorophyll a (data not shown). Small caption letters at the rows represent significant differences using Tukey’s test at p < 0.05. WUE 11.5 kPa tension caused death in 94 % of the seedlings and was disregarded in the statistical analysis; **no difference was observed for Chlorophyll a (data not shown). 182 Sci. Agric. v.76, n.2, p.179-189, March/April 2019 Bezerra et al. Lettuce seeding cultivation Figure 3 – Development of lettuce seedlings in trays of the first block (SIC block) at 11, 20 and 28 days after sowing (DAS) with the substrate sensor. Variation of shoot fresh weight values of 17.3 %. Figure 3 – Development of lettuce seedlings in trays of the first block (SIC block) at 11, 20 and 28 days after sowing (DAS) with the substrate Figure 3 – Development of lettuce seedlings in trays of the first block (SIC block) at 11, 20 and 28 days after sowing (DAS) with the substrate sensor. Variation of shoot fresh weight values of 17.3 %. atmospheric sensor (Tables 1 and 2). Tensions of 4.0 (substrate sensor) and 3.5 kPa (atmospheric sensor) presented WUE with an average value of 1.86 g L–1 and 1.37 g L–1 (second experimental period), respec- tively. Atmospheric sensor experiment – The gs measured at 25 DAS decreased as a function of the regulated ten- sion for the SIC in the second period. However, the gs values measured in the first period were very close for seedlings at 3.5, 5.0 and 6.5 kPa tensions (Table 2). i No significance was observed in the isolated effect or interaction of the treatments tested in the parameters analyzed by the JIP test (data not show). Other evaluated characteristics Substrate sensor experiment – Leaf length and leaf area data exhibited the same behavior as the previous variables, presenting increased reductions when sub- jected to tensions greater than 4.0 kPa (Table 1). Chlo- rophyll content, measured 28 DAS (Table 1), climbed higher as the tension increased. Discussion Irrigation management and death rate Irrigation management and death rate In both experiments, SIC activation was less fre- quent at higher tensions. It could be assigned to a certain 183 Sci. Agric. v.76, n.2, p.179-189, March/April 2019 Lettuce seeding cultivation Bezerra et al. Table 2 – Shoot fresh weight; shoot dry weight; height; number of leaves; water use efficiency and stomatal conductance of Lactuca sativa seedlings during the experiment with the atmospheric sensor at different tension adjustments and two experimental periods. Table 2 – Shoot fresh weight; shoot dry weight; height; number of leaves; water use efficiency and stomatal conductance of Lactuca sativa seedlings during the experiment with the atmospheric sensor at different tension adjustments and two experimental periods. Table 2 – Shoot fresh weight; shoot dry weight; height; number of leaves; water use efficiency and stomatal conductance of Lactuca sativa seedlings during the experiment with the atmospheric sensor at different tension adjustments and two experimental periods. Irrigation management and death rate Traits Adjustment Tension of SIC (Simplified Irrigation Controller) 3.5 5.0 6.5 8.0 ------------------------------------------------------------------------------------------ kPa ------------------------------------------------------------------------------------------ Periods 1 Shoot Fresh Weight (g per plant) 0.21 ± 0.065 B 0.20 ± 0.064 0.20 ± 0.042 0.19 ± 0.063 2 0.38 ± 0.047 aA 0.24 ± 0.028 b 0.23 ± 0.014 b 0.14 ± 0.021 c 1 Shoot Dry Weight (g per plant) 0.02 ± 0.006 B 0.02 ± 0.005 B 0.02 ± 0.003 0.01 ± 0.004 2 0.04 ± 0.004 aA 0.03 ± 0.002 bA 0.02 ± 0.001 c 0.01 ± 0.006 c 1 Height (cm) 4.06 ± 0.342 3.70 ± 0.533 B 3.69 ± 0.348 4.24 ± 0.445 A 2 4.49 ± 0.318 a 3.96 ± 0.223 aB 3.80 ± 0.235 b 3.39 ± 0.248 bB 1 Number of leaves (units) 5.25 ± 0.614 B 5.43 ± 0.745 5.78 ± 1.026 5.59 ± 0.666 A 2 6.45 ± 0.592 aA 5.60 ± 0.693 b 5.33 ± 0.249 b 4.51 ± 0.382 cB 1 Water Use Efficiency (g L–1) 0.77 ± 0.177 B 0.75 ± 0.129 B 0.74 ± 0.062 0.71 ± 0.170 2 1.37 ± 0.236 aA 1.12 ± 0.083 bA 0.90 ± 0.070 c 0.88 ± 0.270 c 1 Stomatal conductance (mmol m–2 s–1) 261.5 ± 20 aB 228.6 ± 26 aB 221.1 ± 42 a 120.2 ± 32 b 2 444.5 ± 42 aA 296.3 ± 47 bA 178.6 ± 42 c 109.9 ± 40 d 1 Volume of water to produce a seedling (L) 0.025 0.027 0.027 0.014 2 0.029 0.027 0.027 0.011 Small caption letters at the rows represent significant differences using Tukey’s test at p < 0.05 and upper case letters at the columns represent significant differences using the F-test at p < 0.05. Small caption letters at the rows represent significant differences using Tukey’s test at p < 0.05 and upper case letters at the columns represent significant differences using the F-test at p < 0.05. Figure 4 – Volumes of water applied during the experiment with the sensor positioned in substrate in the following tensions: 4.0 kPa (A); 6.5 kPa (B); 9.0 kPa (C) and 11.5 kPa (D). Each bar represents the sum of the volume of water applied in the trays of the respective treatments daily, 16 plants per tray and 6 repetitions (blocks). Irrigation management and death rate Figure 4 – Volumes of water applied during the experiment with the sensor positioned in substrate in the following tensions: 4.0 kPa (A); 6.5 kPa (B); 9.0 kPa (C) and 11.5 kPa (D). Each bar represents the sum of the volume of water applied in the trays of the respective treatments daily, 16 plants per tray and 6 repetitions (blocks). lic conductivity. The rapid decrease in conductivity oc- curs because the larger pores are emptied first. resistance of the ceramic material especially at higher tensions, because the water will find greater resistance to flow between the smaller pores, whereas at lower tensions this would not necessarily occur since due to the greater initial amount of available water, the water would flow through the larger pores facilitating hydrau- This process is similar to the loss of water by the soil itself, with concepts similar to saturated flow, when the bigger pores are filled with water and the hydraulic flow occurs more easily due to the action of the gravita- Sci. Agric. v.76, n.2, p.179-189, March/April 2019 184 Lettuce seeding cultivation Bezerra et al. re 5 – Volumes of water applied during the experiment with the atmospheric sensor at different tensions and two experimental periods. First riod: 3.5 kPa (A) 5.0 kPa (B) 6.5 kPa (C) 8.0 kPa (D). Second period: 3.5 kPa (E) 5.0 kPa (F) 6.5 kPa (G) 8.0 kPa (H). Each bar represents the m of the volume of water applied in the trays of the respective treatments daily, 16 plants per tray and 4 repetitions (blocks). Figure 5 – Volumes of water applied during the experiment with the atmospheric sensor at different tensions and two experimental periods. First period: 3.5 kPa (A) 5.0 kPa (B) 6.5 kPa (C) 8.0 kPa (D). Second period: 3.5 kPa (E) 5.0 kPa (F) 6.5 kPa (G) 8.0 kPa (H). Each bar represents the sum of the volume of water applied in the trays of the respective treatments daily, 16 plants per tray and 4 repetitions (blocks). tional potential, and unsaturated flow, as drainage pro- ceeds the smaller pores empty out and the conductivity decreases (Beven and Germann, 1982; Nielsen et al., 1986). flow will be subjected to different resistances from the substrate, added to the fact that at higher tensions the porous nature of the sensor material also leads to greater resistance, as discussed above. Sci. Agric. v.76, n.2, p.179-189, March/April 2019 Biometric traits Lettuce seedlings presented superior performance at the 4.0 kPa with the substrate sensor tension in the traits of SFW, SDW, H and NF, when comparing data from all experiments. However, there was a marked re- duction for all biometric traits as the water tension in the substrate sensor increased, which was not observed with the atmospheric sensor. Thus, the use of tensions higher than 4.0 kPa with a substrate sensor would not be ideal, as it can impair the growth and survival of seed- lings. Figure 6 – Mean values of temperature and relative humidity: experiment with the substrate sensor (A); experiment with the atmospheric sensor in the first (B) and second (C) experimental periods. In view of the specificity of the vegetable seed- ling production system, with small volume production cells and water retention capacity, the use of an atmo- spheric sensor can act by binding the irrigation system and maintaining seedlings with high water content in the substrate. of the experiment. In fact, the irrigation regime with the highest tension tested in this study (11.5 kPa) seriously impaired the development of lettuce seedlings. Further- more, the seedlings of the cells with the sensor (trays of block I) exhibited greater vigor in relation to the others and this difference increased as the tension escalated. This evidence could be explained by the fact that the diaphragm of the pressure switch stores water and re- leases it to the cell where the sensor is installed as the tension is raised throughout its operation. This would act like a buffering of the tension in the sensor cell and prevent these seedlings from suffering the effects of the water restriction that is observed in the other cells. SFW, SDW, H and NL showed better performance for the 4.0 kPa (substrate sensor) and 3.5 kPa tensions (atmospheric sensor) in relation to the other tested ten- sion levels. These results were relatively higher or very close to those reported by Leal et al. (2011) and lower than in other studies (Castoldi et al., 2014; Gonçalves et al., 2014). Such larger production in other studies can be related to the use of trays with larger cells. Usually the use of trays with fewer cells is associated with larg- er seedlings being obtained as reported by Costa et al. (2012), Leal et al. (2011) and Oliveira et al. Irrigation management and death rate Differential climatic con- ditions between experiments may also be responsible for the differences observed (Figure 6A, B and C). When comparing atmospheric with substrate sensors, a lower frequency of water application and re- duction in the seedlings growth were observed as the tension increased when the substrate sensor was used. When the sensor is installed in the substrate the loss of water from the SIC is slower compared to the sensor placed in the atmosphere due to the fact that the water The experiment with the substrate sensor tensions greater than 4.0 kPa also interfered negatively in seed- ling growth (Table 1). About 0 % (4.0 kPa), 8 % (6.5 kPa), 24 % (9.0 kPa) and 94 % (11.5 kPa) of the seedlings did not complete the 28-day period and died before the end 185 Sci. Agric. v.76, n.2, p.179-189, March/April 2019 Lettuce seeding cultivation Bezerra et al. Figure 6 – Mean values of temperature and relative humidity: experiment with the substrate sensor (A); experiment with the atmospheric sensor in the first (B) and second (C) experimental periods. this environment, and would communicate the reduc- tion in internal pressure to the pressure switch, which would act by activating the irrigation. However, since the trend of water movement is always towards the most negative potential, which ends up being the potential of the atmosphere, it would be possible for the equipment to remain connected continuously. Yet, this fact was not observed in practice and still no drainage was observed during the whole experimental period, demonstrating that the system was functioning adequately. Such a fact can also be attributed to the storage of water in the pressure switch that generates tension buffering. Thus, when receiving the water, the pressure switch holds it inside for a certain period, until the ten- sion regulated by the SIC is reached, when it is greater than the tension in question. Sci. Agric. v.76, n.2, p.179-189, March/April 2019 Atmospheric sensor experiment WUE was also reduced at tensions greater than 4.0 kPa (substrate sensor) and 3.5 kPa (atmospheric sen- sor), presenting values of 1.86 g L–1 and 1.37 g L–1, re- spectively. Volumes of 38.3 (substrate sensor) and 29.4 mL (atmospheric sensor) were required for the produc- tion of a seedling. The value is close to those found by Santos et al. (2015) for the cultivation of adult lettuce plants in pots. Stomatal conductance (gs) – Stomatal conductance (gs) exhibited reduction in the second experimental pe- riod, declining as irrigation levels decreased (Table 2). This fact can be seen in both time periods. However, in the first experimental period with the atmospheric sen- sor it is notable that although there was a substantial decrease in gs among the 3.5 kPa, 5.0 kPa and 6.5 kPa treatments, it was significant only when compared to the 8.0 kPa tension. In the first experimental period, WUE was around 0.75 g L−1 for all the tensions tested. This was expected since there was no great variation in the growth rate of the seedlings nor the quantity of water supplied to the seedlings for the traits evaluated. Since the plants re- ceived daily amounts of water, the milder climate with less intensity of incident solar radiation allowed the plants to have similar growth conditions even if some of them received less water. i The higher water availability provided greater sto- matal opening, offering better conditions for physiologi- cal processes. This result is also reflected in the results observed in the analyzed biometric traits, showing the negative influence of the stomatal closure due to lower water availability in the growth of the seedlings. The results are also in agreement with several reports in the literature (Anjum et al., 2011; Chartzoulakis et al., 2002; Kato and Okami, 2011; Kim et al., 2004). Few studies have assessed the efficient applica- tion of water in the production of seedlings of any species, as presented by Scalon et al. (2011) who eval- uated the physiological WUE in mutambo (Guazuma ulmifolia Lam.) seedlings. Scalon et al. (2011) calcu- lated WUE based on the ratio between the photosyn- thetic rate and the transpiration rate, but in this study the calculation was made on the dry mass of plant produced by the volume of water applied, which rep- resents WUE in agronomic terms. For this reason, any comparison would be inappropriate. Atmospheric sensor experiment No literature was found to allow comparison of WUE values in lettuce seedlings. Analysis of the chlorophyll a fluorescence transient Analysis of the chlorophyll a fluorescence transient – The 3.5 kPa treatment which provides greater volume of water was adopted as control in both experimental times. No significance was observed for the isolated ef- fect or for interaction for the treatments tested in the pa- rameters analyzed through the JIP test. Thus, the results do not suggest any damage to the photosynthetic appa- ratus, although photosynthetic performance appears to improve as the irrigation volumes tested increased (data not shown). Bezerra et al. Similarly, the results indicate that high tempera- ture and radiation effects can be minimized when a shade screen is used during the periods where these fac- tors tend to be harmful to seedling development. Screen shading protection produced more vigorous seedlings when production should be reduced. Although no com- parison was made between shade and non-shade pro- duction at the same time of production, the combined effect of the use of shading screen and the choice of climate-adapted cultivar may have been responsible for the better result observed in this period when the atmo- spheric sensor was in use. Chlorophyll content – Chlorophyll b content increased at higher tensions. The higher tensions led to the pro- duction of smaller seedlings and consequently smaller leaf area (Table 1). The magnitude of the leaf area is directly related to the arrangement of the chlorophyll pigment in the leaves, interfering in the concentration of these molecules when considering a particular reading area. The increase in chlorophyll concentration through water stress was also observed for Plantago ovata Forsk by Rahimi et al. (2013) and for Salix spp. by Weih et al. (2011). No difference was observed for chlorophyll a (data not show). Chlorophyll content – Chlorophyll b content increased at higher tensions. The higher tensions led to the pro- duction of smaller seedlings and consequently smaller leaf area (Table 1). The magnitude of the leaf area is directly related to the arrangement of the chlorophyll pigment in the leaves, interfering in the concentration of these molecules when considering a particular reading area. The increase in chlorophyll concentration through water stress was also observed for Plantago ovata Forsk by Rahimi et al. (2013) and for Salix spp. by Weih et al. (2011). No difference was observed for chlorophyll a (data not show). Biometric traits (2012), where the largest seedlings were obtained in cells of higher vol- ume. Additionally, differences can be attributed to dif- ferent climatic conditions or to varietal differences in morphological characteristics among cultivars. This fact is important for the other applications of the SIC, since it emphasizes that the cells of the first block in which the sensors are installed will have a vol- ume of stored water that will not be easily lost to the at- mosphere, while in the other cells this same volume will be available only in the substrate. Due to these facts, even though the SIC is regulated for a certain tension, the other plants will actually be receiving a higher ten- sion, which can lead to different WUE values (Gomes et al., 2017). It is worth noting the need to install a 50 % shading screen in the second period analyzed with the atmospher- ic sensor, due to the conditions of higher temperature and radiation. With the use of shading screens, it is possible to observe productivity increases in regions where tem- perature and light conditions tend to be limiting. These responses, if the reduction in radiation is not excessive, may possibly enhance crop productivity and its quality (Ahemd, 2016; Gent, 2007; Sinclair et al., 1992). ) With the sensor positioned in the atmosphere adja- cent to the production trays, the sensor would lose mois- ture as a function of the vapor pressure deficit (VPD) in 186 Sci. Agric. v.76, n.2, p.179-189, March/April 2019 Lettuce seeding cultivation Bezerra et al. Substrate sensor experiment The substrate sensor measured the water tension in the root system, which is the well-established tradi- tion. However, the use of an atmospheric sensor can act by turning on the irrigation system and maintaining seedlings with higher water content in the substrate, which would be more highly recommended due to its more regular operation, which applies lower volumes of water at a time and activates more times over the production cycle. The practical use of the system with the atmospheric sensor is also a positive point in its favor. Leaf length and leaf area – Leaf length and leaf area data also showed a decrease when subjected to tensions greater than 4.0 kPa (Table 1). The value of leaf area found for the 4.0 kPa (47 cm2) tension is close to the highest values found by Trani et al. (2004) for the same development time of the seedlings. These same authors reported a reduction in leaf area values with the use of trays with more cells (288 cells) compared to trays with fewer cells (200 cells). 187 Sci. Agric. v.76, n.2, p.179-189, March/April 2019 Lettuce seeding cultivation Bezerra et al. The tensions of 4.0 (substrate) and 3.5 (atmo- sphere) kPa showed higher potential for the production of more vigorous seedlings. 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(Bulletin, 670). The authors thank the Programa de Pós-Grad- uação em Engenharia Agrícola e Ambiental at Uni- versidade Federal Rural do Rio de Janeiro (UFRRJ) for their support of this research. The Governmental agencies Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) for research funding and Con- selho Nacional de Desenvolvimento Científico e Tec- nológico (CNPq) for the research fellowships (D.F.C., L.O.M. and R.A.A.). Dai, J.; Cui, Y.; Cai, X.; Brown, L.C.; Shang, Y. 2016. Influence of water management on the water cycle in a small watershed irrigation system based on a distributed hydrologic model. Agricultural Water Management 174: 52-60. Dias, G.C.O.; Medici, L.O.; Vasconcellos, M.A.S.; Carvalho, D.F.; Pimentel, C. 2013. Papaya seedlings growth using a low-cost automatic watering controller. Revista Brasileira de Fruticultura 35: 40-46. Authors’ Contributions Gent, M.P.N. 2007. Effect of degree and duration of shade on quality of greenhouse tomato. HortScience 42: 51. Conceptualization: Bezerra, A.C.M., Valença, D.C., Medici, L.O. Data acquisition: Bezerra, A.C.M., Valença, D.C., Gabetto, F.P. Data Analysis: Bezerra, A.C.M., Valença, D.C., Gomes, D.P. Design of meth- odology: Medici, L.O. Writing and Editing: Bezerra, A.C.M., Valença, D.C., Carvalho, D.F.C., Pinho, C.F., Reinert, F., Gomes, D.P., Azevedo, R.A., Masseroni, D., Medici, L.O. Conceptualization: Bezerra, A.C.M., Valença, D.C., Medici, L.O. Data acquisition: Bezerra, A.C.M., Valença, D.C., Gabetto, F.P. Data Analysis: Bezerra, A.C.M., Valença, D.C., Gomes, D.P. Design of meth- odology: Medici, L.O. Writing and Editing: Bezerra, A.C.M., Valença, D.C., Carvalho, D.F.C., Pinho, C.F., Reinert, F., Gomes, D.P., Azevedo, R.A., Masseroni, D., Medici, L.O. Gomes, D.P.; Carvalho, D.F.; Pinto, M.F.; Valença, D.C.; Medici, L.O. 2017. Growth and production of tomato fertilized with ash and castor cake and under varying water depths, cultivated in organic potponics. 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https://openalex.org/W2135063439	https://europepmc.org/articles/pmc1249580?pdf=render	English	null	Relationship between visual field loss and contrast threshold elevation in glaucoma	BMC ophthalmology	2,005	cc-by	9,542	BioMed Central BioMed Central Op Research article Relationship between visual field loss and contrast threshold elevation in glaucoma CM Tochel1, JS Morton1, JL Jay2 and JD Morrison1 Open Access Address: 1Institute of Biomedical & Life Sciences, West Medical Building, University of Glasgow, Glasgow, Scotland, UK and 2Tennent Institute of Ophthalmology, Gartnavel General Hospital, 1053 Great Western, Road, Glasgow, Scotland, UK ddress: 1Institute of Biomedical & Life Sciences, West Medical Building, University of Glasgow, Glasgow, Scotland, U phthalmology, Gartnavel General Hospital, 1053 Great Western, Road, Glasgow, Scotland, UK Email: CM Tochel - jdm1u@udcf.gla.ac.uk; JS Morton - jdm1u@udcf.gla.ac.uk; JL Jay - jdm1u@udcf.gla.ac.uk; JD Morrison - jdm1u@udcf.gla.ac.uk Email: CM Tochel - jdm1u@udcf.gla.ac.uk; JS Morton - jdm1u@udcf.gla.ac.uk; JL Jay - jdm1u@udcf.gla.ac.uk; JD Morrison* - jdm1u@udcf.gla.ac.uk * Corresponding author Received: 09 May 2005 Accepted: 13 September 2005 Received: 09 May 2005 Accepted: 13 September 2005 Received: 09 May 2005 Accepted: 13 September 2005 Published: 13 September 2005 BMC Ophthalmology 2005, 5:22 doi:10.1186/1471-2415-5-22 p BMC Ophthalmology 2005, 5:22 doi:10.1186/1471-2415-5-22 This article is available from: http://www.biomedcentral.com/1471-2415/5/22 © 2005 Tochel et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Methods Patients The experiments were undertaken with the informed con- sent of the participants and with the approval of the Ethics Committee of the North Glasgow University Hospitals NHS Trust and were performed in conformity with the Declaration of Helsinki. All subjects and patients under- went the Snellen test to determine their best visual acuity with refraction if necessary. A total of 26 patients ages 37 to 83 yr (mean 69 ± 10 S.D. yr) were recruited from the Tennent Institute of Ophthal- mology, Gartnavel General Hospital. They were diag- nosed according to their intra-ocular pressure (IOP) determined by applanation tonometry, the appearance of the optic disc on ophthalmoscopic examination and the extent of visual field loss determined with the Friedmann Visual Field Analyzer Mark II (Clement Clarke Interna- tional Ltd., London). Twenty one patients were diagnosed as having glaucoma (Table 1): the most common condi- tion was primary open angle glaucoma (POAG) in which IOP exceeded 22 mmHg on at least 2 occasions before treatment, there was cupping of the optic disc and visual field defects characteristic of glaucoma were present. Nor- mal pressure glaucoma (NPG) was diagnosed according to the same criteria as POAG except that IOP was less than 22 mmHg. In addition, one patient had each of primary chronic angle closure glaucoma, post-traumatic glaucoma and sarcoid-induced glaucoma. Within the glaucoma group, since 6 patients had a fellow eye which showed no visual field defects or definite pathological cupping of the optic disc and had an IOP of less than 22 mmHg, these eyes were designated normal, though were not used as controls. The 5 ocular hypertensives (OHT) were defined on the basis of an IOP which exceeded 21 mmHg but with no visual field loss and were subdivided into those with and without pathological cupping of the optic disc (Table 1). All patients were selected on the basis of having stable visual fields through being superimposable in terms of their position, extent and depth, tested usually on a six monthly basis over the 2 years preceding the study. Five patients who were subsequently retested over intervals of 1 day to 6 months showed reproducible visual fields. We have therefore set out to answer the question whether regions of glaucomatous visual field loss are associated with elevations of contrast threshold which was employed rather than its reciprocal, the contrast sensitivity. Background conventional manner. A preliminary report of our results has been made previously [23]. g The visual field loss caused by glaucoma has been divided into 5 stages ranging from relative defects in sensitivity to almost complete visual loss [1]. The pattern of loss which alerts awareness of the disease, however, is often an arcu- ate scotoma occurring with approximately equal fre- quency in the superior or inferior hemifield within 20 degrees of the fovea and with or without a nasal step. These losses are generally detected in the clinical environ- ment by visual field tests involving presentation of spots of light within a hemi-spherical dome at various locations within the central visual field. However, by the time visual field defects are detected, considerable loss of retinal gan- glion cells may already have occurred, as exemplified by the case of a glaucoma suspect who, despite an absence of local field defects prior to death, showed ganglion cell losses post-mortem of 63% and 44% in left and right reti- nae, respectively [2]. Accordingly, there has been an impe- tus to develop alternative tests to visual field testing capable of detecting glaucoma at earlier stages. One such test is the measurement of contrast sensitivity in response to sinusoidal grating patterns generated by a variety of methods-oscilloscope, TV or computer monitor, printed paper, laser interferometry, or Snellen letters. The most usual protocol has involved measurements of contrast sensitivity in response to foveal viewing of the display [3- 14], though more involved studies have additionally determined contrast sensitivities at peripheral locations in the visual field [15-22]. The consensus has been, with one exception [13], that contrast sensitivity in response to low spatial frequencies is impaired in glaucoma, leading to strong advocacy of its usefulness in glaucoma screening. While most studies reported the results of visual field tests, correlations between impaired contrast sensitivity and the location of visual field loss were generally not undertaken. In the one case in which this was done on a limited scale [17], contrast sensitivity deficits were described as occurring in both the visual hemifield show- ing no visual loss and the hemifield which did show vis- ual loss. Abstract Background: There is a considerable body of literature which indicates that contrast thresholds for the detection of sinusoidal grating patterns are abnormally high in glaucoma, though just how these elevations are related to the location of visual field loss remains unknown. Our aim, therefore, has been to determine the relationship between contrast threshold elevation and visual field loss in corresponding regions of the peripheral visual field in glaucoma patients. Methods: Contrast thresholds were measured in arcuate regions of the superior, inferior, nasal and temporal visual field in response to laser interference fringes presented in the Maxwellian view. The display consisted of vertical green stationary laser interference fringes of spatial frequency 1.0 c deg-1 which appeared in a rotatable viewing area in the form of a truncated quadrant extending from 10 to 20° from fixation which was marked with a central fixation light. Results were obtained from 36 normal control subjects in order to provide a normal reference for 21 glaucoma patients and 5 OHT (ocular hypertensive) patients for whom full clinical data, including Friedmann visual fields, had been obtained. Results: Abnormally high contrast thresholds were identified in 20 out of 21 glaucoma patients and in 2 out of 5 OHT patients when compared with the 95% upper prediction limit for normal values from one eye of the 36 normal age-matched control subjects. Additionally, inter-ocular differences in contrast threshold were also abnormally high in 18 out of 20 glaucoma patients who had vision in both eyes compared with the 95% upper prediction limit. Correspondence between abnormally high contrast thresholds and visual field loss in the truncated quadrants was significant in 5 patients, borderline in 4 patients and absent in 9 patients. Conclusion: While the glaucoma patients tested in our study invariably had abnormally high contrast thresholds in one or more of the truncated quadrants in at least one eye, reasonable correspondence with the location of the visual field loss only occurred in half the patients studied. Hence, while contrast threshold elevations are indicative of glaucomatous damage to vision, they are providing a different assessment of visual function from conventional visual field tests. Page 1 of 12 (page number not for citation purposes) BMC Ophthalmology 2005, 5:22 http://www.biomedcentral.com/1471-2415/5/22 Page 2 of 12 (page number not for citation purposes) Methods Patients For this purpose, we have designed a streamlined method of measuring contrast thresholds at different peripheral loca- tions in the visual field based on the advantages offered by viewing laser interference fringes in the Maxwellian view. This provides a large visual field which can be viewed without the need for refraction of the viewer and has a sense of proximity, rather than the unavoidable remote- ness of an externally generated grating display. Our results were then compared with visual field data obtained in the As controls, we examined 36 non-glaucoma subjects ages 48–78 yr (mean 62 ± 8 S.D. yr.) who were recruited from within the University and from personal acquaintances. They were deemed to be normal on the basis of a reported absence of visual problems, a Snellen acuity of 6/6 or bet- ter in each eye and a report of normal vision from a recent visit to their optometrist. While 28 subjects had 2 normal eyes, 8 subjects had one normal eye and a non-glaucoma- Page 2 of 12 (page number not for citation purposes) Page 2 of 12 (page number not for citation purposes) http://www.biomedcentral.com/1471-2415/5/22 BMC Ophthalmology 2005, 5:22 Table 1: Clinical data for glaucoma and OHT patients Pat no. Methods Patients Age (yr) LE Condition LE VA LE VF loss LE CTmax RE Condition RE VA RE VF loss RE CTmax I-O diff R2 1 72 POAG 6/5 74% 0.051 POAG 6/4 7% 0.023 0.025 79%* 2 73 POAG 6/6 60% 0.098 POAG 6/7.5 51% 0.168 0.045 0%° 3 67 POAG 6/6 4% 0.099 NPG 6/9 53% 0.107 0.004 48%+ 4 76 POAG 6/6 7% 0.075 POAG 6/7.5 44% 0.078 0.001 1%° 5 71 POAG 6/6 36% 0.142 POAG 6/6 30% 0.067 0.045 0%° 6 66 POAG 6/6 17% 0.075 POAG 6/12 7% 0.091 0.014 -25%° 7 77 POAG 6/24 55% 0.055 POAG 6/6 67% 0.044 0.012 14%° 8 68 POAG 6/6 23% 0.069 normal 6/5 4% 0.049 0.019 55%* 9 75 POAG 6/9 44% 0.102 normal 6/5 1% 0.041 0.034 85%* 10 72 normal 6/6 2% 0.023 POAG 6/9 42% 0.031 0.006 47%+ 11 37 POAG 6/18 14% 0.089 blind nil 100% n/s na na 12 83 POAG pxf 6/9 1% 0.066 POAG pxf 6/9 51% n/s na na 13 83 POAG pxf 6/18 69% 0.058 POAG pxf 6/12 46% 0.074 0.025 -24%° 14 74 POAG pxf 6/5 26% 0.067 OHT 6/6 3% 0.056 0.008 9%° 15 73 NPG 6/5 17% 0.055 NPG 6/5 47% 0.033 0.014 0%° 16 76 NPG 6/9 18% 0.150 NPG 6/12 47% 0.060 0.021 46%+ 17 73 NPG 6/9 53% 0.048 normal 6/9 4% 0.027 0.010 49%+ 18 54 RD 6/12 2% 0.046 NPG 6/5 41% 0.083 0.023 85%* 19 61 PCACG 6/9 11% 0.078 ACG 6/9 3% 0.072 0.012 na 20 79 PTG 6/9 55% 0.147 normal 6/9 6% 0.077 0.055 26%° 21 55 normal 6/6 7% 0.044 SIG 6/6 52% 0.058 0.017 73%* 22 70 OHT 6/5 0%(2%) 0.031 OHT 6/5 0% (6%) 0.018 0.002 na 23 78 OHT 6/5 0% (7%) 0.022 OHT 6/9 3% (8%) 0.033 0.0064 na 24 57 OHT 6/5 6% (13%) 0.054 OHT 6/5 4%(13%) 0.051 0.0006 na 25 62 OHT 6/5 2% (2%) 0.034 OHT pdc 6/12 4% (6%) 0.042 0.010 na 26 72 A OHT pdc 6/60 8% (29%) 0.155 OHT pdc 6/12 8% (35%) 0.059 0.068 na Pat no. Methods Patients Patient number, LE left eye, RE right eye, A amblyopic, CTmax highest contrast threshold value (to be compared against the upper prediction limit of 0.045), I-O diff inter-ocular difference (to be compared against the upper prediction limit of 0.006), NPG normal pressure glaucoma, OHT ocular hypertensive, na not applicable, n/s not seen, PCACG primary chronic angle closure glaucoma, pdc pathological cupping of optic disc, POAG primary open angle glaucoma, PTG post-traumatic glaucoma, pxf pseudoexfoliation, RD retinal detachment, SIG sarcoid-induced glaucoma, VA visual acuity, VF visual field. Negative R2 values denote inverse relationship. p < 0.05, +0.05 <p < 0.10, °p > 0.10. Some eyes denoted normal show visual field loss due to the blind spot while some glaucomatous eyes showing minimal visual loss had additional loss either more peripheral or more central to the truncated quadrant. OHT patients also show visual field loss score in parenthesis at 12 months follow-up examinations. tous problem in the fellow eye, which is dealt with sepa- rately. Of the 36 subjects, 19 were emmetropic while the remainder had refractive errors ranging from +1.50 DS to -7.00 DS with astigmatism of up to +5.00 DC and all were presbyopic to varying extents. Twenty three subjects ages 55–78 yr, including all subjects above 69 yr, were also examined with the Central 24-2 threshold test with the Humphrey II Visual Field Analyzer model 750 (Hum- phrey Instruments, San Leandro, CA, USA) to which we had ready access at times convenient to our subjects. Methods Patients Patient number, LE left eye, RE right eye, A amblyopic, CTmax highest contrast threshold value (to be compared against the upper prediction limit of 0.045), I-O diff inter-ocular difference (to be compared against the upper prediction limit of 0.006), NPG normal pressure glaucoma, OHT ocular hypertensive, na not applicable, n/s not seen, PCACG primary chronic angle closure glaucoma, pdc pathological cupping of optic disc, POAG primary open angle glaucoma, PTG post-traumatic glaucoma, pxf pseudoexfoliation, RD retinal detachment, SIG sarcoid-induced glaucoma, VA visual acuity, VF visual field. Negative R2 values denote inverse relationship. p < 0.05, +0.05 <p < 0.10, °p > 0.10. Some eyes denoted normal show visual field loss due to the blind spot while some glaucomatous eyes showing minimal visual loss had additional loss either more peripheral or more central to the truncated quadrant. OHT patients also show visual field loss score in parenthesis at 12 months follow-up examinations. Table 1: Clinical data for glaucoma and OHT patients Pat no. Patient number, LE left eye, RE right eye, A amblyopic, CTmax highest contrast threshold value (to be compared against the upper prediction limit of 0.045), I-O diff inter-ocular difference (to be compared against the upper prediction limit of 0.006), NPG normal pressure glaucoma, OHT ocular hypertensive, na not applicable, n/s not seen, PCACG primary chronic angle closure glaucoma, pdc pathological cupping of optic disc, POAG primary open angle glaucoma, PTG post-traumatic glaucoma, pxf pseudoexfoliation, RD retinal detachment, SIG sarcoid-induced glaucoma, VA visual acuity, VF visual field. Negative R2 values denote inverse relationship. *p < 0.05, +0.05 <p < 0.10, °p > 0.10. Some eyes denoted normal show visual field loss due to the blind spot while some glaucomatous eyes showing minimal visual loss had additional loss either more peripheral or more central to the truncated quadrant. OHT patients also show visual field loss score in parenthesis at 12 months follow-up examinations. Pat no. Page 3 of 12 (page number not for citation purposes) Apparatus Contrast thresholds were measured with a modification of a laser interferometer described previously [24,25] and is shown schematically in Figure 1. Monochromatic green light (λ = 543 nm) from a 0.95 mW He-Ne laser (Uni- phase 1652P) was passed through a spatial filter (SF), a - 10 DS lens (L1) to expand the beam and a +10 DS lens (L2) to collimate the beam. The beam was then divided by a 2 inch cube beamsplitter (B1), the 2 beams reflected from λ/20 front silvered mirrors (M1 and M2) and recombined by a second 2 inch cube beamsplitter (B2). The two beams were equated in intensity by the neutral density filter N1 and the polarization shift caused by the Page 3 of 12 (page number not for citation purposes) Page 3 of 12 (page number not for citation purposes) BMC Ophthalmology 2005, 5:22 http://www.biomedcentral.com/1471-2415/5/22 Schematic representation of optical apparatus used to generate vertical sinusoidal laser interference fringes which are observed in the Maxwellian view Figure 1 Schematic representation of optical apparatus used to generate vertical sinusoidal laser interference fringes which are observed in the Maxwellian view. Abbreviations: A aperture, B beam splitter, C interference filter, I iris diaphragm, L lens, M front silvered mirror, N neutral density filter, P polarizer, SF spatial filter. Further explanation is given in Methods. BMC Ophthalmology 2005, 5:22 http://www.biomedcentral.com/1471-2415/5/22 Schematic representation of optical apparatus used to generate vertical sinusoidal laser interference fringes which are observed in the Maxwellian view Figure 1 Schematic representation of optical apparatus used to generate vertical sinusoidal laser interference fringes which are observed in the Maxwellian view. Abbreviations: A aperture, B beam splitter, C interference filter, I iris diaphragm, L lens, M front silvered mirror, N neutral density filter, P polarizer, SF spatial filter. Further explanation is given in Methods. p p pp g g g Schematic representation of optical apparatus used to generate vertical sinusoidal laser interference fringes which are observed in the Maxwellian view. Abbreviations: A aperture, B beam splitter, C interference filter, I iris diaphragm, L lens, M front silvered mirror, N neutral density filter, P polarizer, SF spatial filter. Further explanation is given in Methods. beamsplitters was compensated by the rotatable sheet polarizer (P1, extinction = 10-4). Contrast threshold determinations Prior to the determinations, each subject or patient was given a standard explanation with the aid of diagrams as to the nature of the test, which was then carried out under standardized subdued illumination. The subject or patient viewed the display through the Maxwellian lens without external refraction. With the direction of gaze determined by the central fixation spot, the subject or patient increased the contrast of the grating display in the peripherally-located truncated quadrant until the pattern became just visible and no more. This gives results for contrast thresholds similar to those for the more time con- suming 50% of seeing [27]. The angle of rotation was recorded and readings repeated. First, the subject or patient had practice runs with the truncated quadrant in each of its 4 positions. This was then followed by 6 con- trast threshold determinations for each of the 4 truncated quadrant positions for each eye in turn. The tests includ- ing the preliminary Snellen test and explanation took 20– 40 min to complete. Repeat determinations in 2 normal subjects and one glaucoma patients on different days con- firmed the consistency of the contrast threshold values. The determinations for the glaucoma patients were under- taken prior to the release of the visual field charts for anal- ysis and the time interval between the tests ranged from the same day to 36 weeks (mean 64 ± 55 S.D. days). The truncated quadrant extended from 10° to 20° from the central fixation point. The angular dimensions were calibrated from determination of the location of the nasal and temporal margins of the blind spot from the axis of fixation. These were determined first in angular subtenses for viewing a sheet of graph paper from 25 cm. The linear dimensions of the blind spot from the axis of fixation were then obtained for viewing a needle mounted on a micrometer, coplanar with the iris diaphragm, through the Maxwellian lens. The factor to convert linear dimen- sions into angular dimensions was then used to convert the linear dimension of the Maxwellian display into angu- lar subtenses. The procedure was undertaken in both eyes in 3 subjects with close agreement between the results. By translation of mirror M1 to increase the pathlength of the laser beam reflected from M1, interference fringes were generated at a spatial frequency directly related to the pathlength difference. Apparatus high refractive index lenses mounted in the same housing as the rotatable aperture. The total power of the Max- wellian lens assembly was +42 DS. The subject or patient looked into the Maxwellian lens with the chin supported by a chin rest. The observed visual display consisted of an attenuated green central fixation light of subtense 2° and the truncated quadrant which was rotatable to the 4 cho- sen visual field positions viz. temporal, superior, inferior and nasal, as shown for right eye viewing in Figure 2. also equalized. The final viewing intensity was deter- mined psychophysically to be 3.2 log units above foveal threshold. high refractive index lenses mounted in the same housing as the rotatable aperture. The total power of the Max- wellian lens assembly was +42 DS. The subject or patient looked into the Maxwellian lens with the chin supported by a chin rest. The observed visual display consisted of an attenuated green central fixation light of subtense 2° and the truncated quadrant which was rotatable to the 4 cho- sen visual field positions viz. temporal, superior, inferior and nasal, as shown for right eye viewing in Figure 2. Contrast threshold determinations By adjustment, the spatial fre- quency was set to 1.0 c deg-1 which is readily detected for peripheral viewing at our eccentricities [26]. The contrast of the display was varied by rotation of the polarizer P4, since contrast is proportional to sin2θ where θ is the angle of rotation from the position of zero contrast. The inten- sities of the laser beam and background beam were equated using a UDT S370 Optometer at the position of the eye. Likewise, the intensities of the 4 quadrants were Apparatus The combined beams were passed through a second polarizer (P2) to sharpen the polarization prior to combination at the plate beam- splitter B3 with the background beam which consisted of non-coherent light from a tungsten filament microscope lamp. The latter beam was passed through heat absorbing filters, a green interference filter (C, peak λ = 546 nm), a polarizer (P3) to polarize the light at 90° to the laser beamsplitters was compensated by the rotatable sheet polarizer (P1, extinction = 10-4). The combined beams were passed through a second polarizer (P2) to sharpen the polarization prior to combination at the plate beam- splitter B3 with the background beam which consisted of non-coherent light from a tungsten filament microscope lamp. The latter beam was passed through heat absorbing filters, a green interference filter (C, peak λ = 546 nm), a polarizer (P3) to polarize the light at 90° to the laser beams and a neutral density filter (N2) to equate the intensity to that of the laser beams. The combined laser and background beams were passed through a rotatable polarizer of 2 inch diameter (P4, Coherent-Ealing 22- 9161). The emergent beam passed through an iris dia- phragm (I) which controlled the overall field size, an aperture consisting of a rotatable truncated quadrant (Q) and the Maxwellian lens assembly which consisted of 38 mm diameter +20 DS plano-convex and +32 DS biconvex Page 4 of 12 (page number not for citation purposes) Page 4 of 12 (page number not for citation purposes) BMC Ophthalmology 2005, 5:22 http://www.biomedcentral.com/1471-2415/5/22 Schematic representation of the truncated quadrants in the temporal, superior, inferior and nasal positions as seen by the right eye Figure 2 Schematic representation of the truncated quadrants in the temporal, superior, inferior and nasal positions as seen by the right eye. The central fixation light was of subtense 2 deg and the truncated quadrant extended from 10–20 deg. The sinusoidal interference fringe pattern had a spatial frequency of 1.0 c deg-1. Schematic representation of the truncated quadrants in the temporal, superior, inferior and nasal positions as seen by the right eye Figure 2 Schematic representation of the truncated quadrants in the temporal, superior, inferior and nasal positions as seen by the right eye. The central fixation light was of subtense 2 deg and the truncated quadrant extended from 10–20 deg. The sinusoidal interference fringe pattern had a spatial frequency of 1.0 c deg-1. http://www.biomedcentral.com/1471-2415/5/22 http://www.biomedcentral.com/1471-2415/5/22 lar comparisons were made between the mean values for left and right eyes in the group of 28 subjects with 2 nor- mal eyes. The mean of the modulus of the difference was 0.0029 ± 0.0020 S.D. contrast units, giving a 95% upper prediction limit of 0.0060 contrast units. These upper pre- diction limits were then used to assess the contrast thresh- olds of the glaucoma patients and of the abnormal eyes of the non-glaucoma group. lar comparisons were made between the mean values for left and right eyes in the group of 28 subjects with 2 nor- mal eyes. The mean of the modulus of the difference was 0.0029 ± 0.0020 S.D. contrast units, giving a 95% upper prediction limit of 0.0060 contrast units. These upper pre- diction limits were then used to assess the contrast thresh- olds of the glaucoma patients and of the abnormal eyes of the non-glaucoma group. against which the results for the glaucoma patients were compared. After conversion of the data to cumulative probabilities, the sensitivity (the percentage of correctly classified glaucoma patients) was plotted against 1 – spe- cificity (the percentage of incorrectly classified normal subjects) to obtain the Receiver Operating Characteristic (ROC) curve [29,30]. The area under the ROC curve is a measure of the sensitivity over a range of criterion levels and varies from 0.5 when there is an equal likelihood of glaucoma patients and normal subjects exceeding the specified criterion to 1.0 when there is perfect discrimina- tion of glaucoma patients from normal subjects. The Humphrey Central 24-2 test for the 40 normal eyes in 23 subjects (the remaining 6 eyes had non-glaucomatous abnormalities), missed 5 blindspots, gave mean devia- tions outside normal limits in 8 eyes and gave abnormal or borderline results with the glaucoma hemifield test in 12 eyes. In all cases, the abnormal results were explained by drooping eyelids, obstruction by spectacles or trial frames and subject error, which are common hazards of automated perimetry [32]. Comparisons between the mean contrast threshold for each truncated quadrant against visual field loss were undertaken by linear regression analysis after quantifying the visual field loss from the patient's Friedmann visual field chart [31]. Control group The contrast thresholds for the 64 normal eyes were nor- mally uniformly low for each of the superior, temporal, inferior and nasal truncated quadrants with no significant differences between the mean values of the 4 quadrants (p = 0.81). The mean contrast threshold and the 95% upper prediction limit were then calculated for the 4 truncated quadrants for the normal eyes of the group of 8 subjects together with one eye chosen at random from the remain- ing 28 subjects to give equal numbers of left and right eyes. The contrast threshold values for the group con- formed closely to a normal distribution and had a mean value of 0.027 ± 0.011 S.D. contrast units, giving a 95% upper prediction limit of 0.045 contrast units. Inter-ocu- http://www.biomedcentral.com/1471-2415/5/22 A template containing an aperture corre- sponding to the dimensions of the truncated quadrant was laid over the Friedmann visual field chart and the fol- lowing scores allocated to each point tested according to whether the point was visible through the applied neutral density filter: 3 – densest filter appropriate to age (usually 1.2 log units attenuation), 2 – next densest filter (usually 0.8 log units attenuation), 1 – no filter and 0 – not visible. The summed values were expressed as a percentage of the maximum possible score for that truncated quadrant and the difference from 100% was the visual field loss score. This was repeated for each of the 4 positions shown in Fig- ure 2 and no allowance was made for the presence of the blind spot in the temporal quadrant. From the regression analysis, the value of R2, the coefficient of determination, was obtained. This gives as a percentage the amount of the variation in contrast threshold against visual field loss score which is accounted for by the line of best fit (regres- sion line); so R2 ranges from 0% when the data are ran- domly distributed to 100% when the data points fall exactly on the regression line. The value of R2 is amenable to statistical testing and significance was taken as p < 0.05. Glaucoma patients h fi i The first comparison was to determine the extent to which the contrast threshold for a truncated quadrant exceeded the upper prediction limit of 0.045 contrast units, irre- spective of the location of the quadrant. Abnormally high values occurred in 29 out of 33 glaucomatous eyes (87%) and, on a patient by patient basis, in 20 out of 21 patients (95%). There were no apparent differences among the dif- ferent types of glaucoma (Table 1). Examples of patients with elevated contrast thresholds are shown in Figures 3 &4. Second, a comparison was made of the modulus of the inter-ocular difference of the mean contrast threshold for left and right eyes against the upper prediction limit of 0.0060 contrast units. This was abnormally high in 18 out of 20 glaucoma patients (90%) for whom the comparison was possible, including the patient with bilateral normal contrast thresholds. However, when identification was made on the basis of the combination of either an abnor- mal contrast threshold or abnormal inter-ocular differ- ence, 100% correct identification was achieved. The optimal sensitivity and specificity and the area under the ROC curve are given in Table 2 which also shows data for tests of contrast threshold restricted to the superior and inferior truncated quadrants and to the nasal and tempo- ral truncated quadrants. Both these assessments show reduced sensitivity compared with testing of all 4 trun- cated quadrants. It is thus clear contrast threshold eleva- tions, especially in combination with intra-ocular differences were effective in detecting glaucoma. Data analysis i i l l Statistical analyses were undertaken with the Minitab sta- tistical package [28]. The basic method of analysis involved the calculation of 95% prediction limits for the contrast thresholds and for the inter-ocular differences in contrast threshold between companion normal eyes, Page 5 of 12 (page number not for citation purposes) Page 5 of 12 (page number not for citation purposes) BMC Ophthalmology 2005, 5:22 http://www.biomedcentral.com/1471-2415/5/22 OHT patients h In the 5 OHT patients, contrast thresholds were elevated in 4 out of 10 eyes, corresponding to 2 patients, with no differences between those with and without pathological Page 6 of 12 (page number not for citation purposes) Page 6 of 12 (page number not for citation purposes) http://www.biomedcentral.com/1471-2415/5/22 BMC Ophthalmology 2005, 5:22 Patient 9 (Table 1) A & B Histograms of contrast threshold (mean ± S.E.M.) for superior (s), temporal (t), inferior (i) (n) truncated quadrants in the left and right eyes showing upper 95% prediction limit of 0.045 contrast units as broken tal line Figure 3 Patient 9 (Table 1) A & B Histograms of contrast threshold (mean S.E.M.) for superior (s), temporal (t), inferior (i) and truncated quadrants in the left and right eyes showing upper 95% prediction limit of 0.045 contrast units as broken h line. Contrast thresholds were abnormal in the left eye. C & D Friedmann visual fields showing: unmarked letters-no detection (1.2 logarithmic units attenuation), reduced sensitivity (❍ 0.8 logarithmic units attenuation and ø zero atte and ● zero detection. The main visual field loss was in the inferior hemifield of the left eye. E Contrast threshold aga field loss score taken from Friedmann charts for each truncated quadrant, showing best fitting relationship as broken tionship was significant: R2 = 85%, p = 0.001. OHT patients h Patient 9 (Table 1) A & B Histograms of contrast threshold (mean ± S.E.M.) for superior (s), temporal (t), inferior (i) and nasal (n) truncated quadrants in the left and right eyes showing upper 95% prediction limit of 0.045 contrast units as broken horizon- tal line Figure 3 Patient 9 (Table 1) A & B Histograms of contrast threshold (mean S.E.M.) for superior (s), temporal (t), inferior (i) and nasal (n) truncated quadrants in the left and right eyes showing upper 95% prediction limit of 0.045 contrast units as broken horizontal line. Contrast thresholds were abnormal in the left eye. C & D Friedmann visual fields showing: unmarked letters-normal detection (1.2 logarithmic units attenuation), reduced sensitivity (❍ 0.8 logarithmic units attenuation and ø zero attenuation) and ● zero detection. The main visual field loss was in the inferior hemifield of the left eye. E Contrast threshold against visual field loss score taken from Friedmann charts for each truncated quadrant, showing best fitting relationship as broken line. Rela- tionship was significant: R2 = 85%, p = 0.001. Patient 9 ( (n) truncat tal line Figure 3 Patient 9 (Table 1) A & B Histograms of contrast threshold (mean ± S.E.M.) for superior (s), temporal (t), inferior (i) and nasal (n) truncated quadrants in the left and right eyes showing upper 95% prediction limit of 0.045 contrast units as broken horizon tal line Figure 3 Patient 9 (Table 1) A & B Histograms of contrast threshold (mean S.E.M.) for superior (s), temporal (t), inferior (i) and nasal (n) truncated quadrants in the left and right eyes showing upper 95% prediction limit of 0.045 contrast units as broken horizontal line. Contrast thresholds were abnormal in the left eye. C & D Friedmann visual fields showing: unmarked letters-normal detection (1.2 logarithmic units attenuation), reduced sensitivity (❍ 0.8 logarithmic units attenuation and ø zero attenuation) and ● zero detection. The main visual field loss was in the inferior hemifield of the left eye. E Contrast threshold against visual field loss score taken from Friedmann charts for each truncated quadrant, showing best fitting relationship as broken line. Rela- tionship was significant: R2 = 85%, p = 0.001. Page 7 of 12 (page number not for citation purposes) http://www.biomedcentral.com/1471-2415/5/22 BMC Ophthalmology 2005, 5:22 Page 8 of 12 Patient 6 Figure 4 Patient 6. Same conventions as in Figure 3 but with ❍ as 1.0 logarithmic units attenuation. A & B Contrast thresholds were abnormal in left and right eyes. C & D Visual field loss was diffuse in the left eye and occurred in inferior temporal quadrant of right eye. E Relationship between contrast threshold and visual field loss score was not significant: R2 = 25%, p = 0.12. Discussion Contrast thresholds were readily measured with our Max- wellian view interferometer in normal subjects, even in the presence of refractive errors as large as -7.00 DS and +5.00 DC, which is a positive advantage over externally viewed displays which depend on the accuracy of the refraction and factors like spectacle slippage or obstruc- tion by the edges of frames. In a limited number of cases, contrast thresholds were readily obtained in the presence of minor lens opacities and in amblyopes, though the method is impracticable in cases where fixation cannot be achieved. Generally, the contrast thresholds of normal subjects were uniformly low. While evidence of the blind spot in the form of elevated contrast thresholds in the temporal truncated quadrant occurred infrequently, this was probably the result of stimulation of a large area of retina compared with the well circumscribed extent of the blind spot of which there is a perceptual lack of awareness [33]. The use of relatively large truncated quadrants may suggest intuitively that localisation of visual loss was being sacrificed though there is evidence to suggest that this is not the case. Threshold values between neighbour- ing test points tend to be correlated with the result that the central visual field is organized into discrete clusters or sectors which are related to the projection of retinal gan- glion cell axons [34,35]. Another issue is the variation in the number of cycles between superior/inferior and nasal/ temporal truncated quadrants. We chose the stimulus spa- tial frequency to be 1.0 c deg-1 to ensure high sensitivity in the peripheral retina [26] and a number of cycles which did not limit sensitivity, at least for foveal viewing [36]. It is conceivable that, at a peripheral location, differences in the number of cycles may affect contrast sensitivity though such an effect is likely to be small [37]. Frequency Doubling Technology (FDT) perimetry employs stimulus fields not dissimilar to those employed in the present study though the grating pattern is phase modulated at a Comparison of locations of contrast threshold elevation and visual field loss Our main assessment was to determine whether those parts of the visual field which showed abnormally high contrast thresholds also corresponded to the location of the visual field loss. This was undertaken by regression analysis of contrast threshold against the corresponding visual field loss score for each of the truncated quadrants in both eyes. In order to achieve a reasonable distribution of data points, patients were required to have a range of visual field loss scores in excess of 20% together with an absence of visibility in no more than one of the truncated quadrants. This provided 18 glaucoma patients whose results are shown in Table 1. Contrast threshold and vis- ual field loss were strongly related in 5 patients (R2 = 55– 85%, p < 0.05) as illustrated by Figure 3, showed a sem- blance of s direct relationship in 4 patients (R2 = 46–49%, 0.05 <p < 0.10) and showed an absence of a relationship in 9 patients (R2 = 0–26%, p > 0.1) as illustrated by Figure 4. There was no correlation between the form of the rela- tionship and the type of glaucoma (Table 1). We also repeated the analysis in 8 patients with minimal visual field loss in one eye for which a single mean value was cal- culated (i.e. giving n = 5 for regression analysis). The R2 values were unaffected in the new analysis (p = 0.84, paired t-test) and a significant or borderline relationship was still present in 5 patients. Patient 9 ( (n) truncat tal line Figure 3 Patient 6 Figure 4 Patient 6. Same conventions as in Figure 3 but with ❍ as 1.0 logarithmic units attenuation. A & B Contrast thresholds were abnormal in left and right eyes. C & D Visual field loss was diffuse in the left eye and occurred in inferior temporal quadrant of right eye. E Relationship between contrast threshold and visual field loss score was not significant: R2 = 25%, p = 0.12. Page 8 of 12 (page number not for citation purposes) Page 8 of 12 (page number not for citation purposes) http://www.biomedcentral.com/1471-2415/5/22 BMC Ophthalmology 2005, 5:22 Table 2: Signal detection data Criterion n Sensitivity Specificity Area under ROC CTmax by eye 33 93.9% 91.0% 0.956 CTmax S/I 33 87.8% 91.0% 0.953 CTmax N/T 33 81.8% 91.0% 0.923 CTmax by patient 21 90.4% 95.0% 0.954 I-O diff 20 89.5% 85.7% 0.897 CTmax or I-O diff 20 95.0% 100% 0.993 CTmax highest contrast threshold value, S/I superior and inferior truncated quadrants, N/T nasal and temporal truncated quadrants, I-O diff inter- ocular difference. CTmax highest contrast threshold value, S/I superior and inferior truncated quadrants, N/T nasal and temporal trunc ocular difference. ment, mild cataract and solar damage and for 2 amblyopes were normal. The third amblyope had abnor- mally high contrast thresholds while the subject with macular degeneration was unable to fixate with that eye. cupping of the optic disc. The inter-ocular difference was elevated in 3 out of 5 OHT patients. Follow-up of the OHT patients over the next 12 months showed that those with normal contrast thresholds retained normal visual fields while the 2 patients with elevated thresholds developed marked visual field loss in both eyes (Table 1). Page 9 of 12 (page number not for citation purposes) http://www.biomedcentral.com/1471-2415/5/22 BMC Ophthalmology 2005, 5:22 that it was less disconcerting to elderly patients and is con- sistent with the transferability of visual field data between different machines [45]. While our identification of the misalignment with visual field loss relates to the detection of a stationary grating pattern, allusions have also been made to FDT error scores which occurred in the hemifield opposite to that containing the visual field loss [46,47]. This lack of correspondence between contrast threshold elevation and visual field loss can reasonably be attrib- uted to the different attrition rates for the different modal- ities of visual function in different individuals which is well known in glaucoma [12,38,48-53]. To this, we can further add that different modalities, at least in terms of light detection and contrast detection, are affected differ- ently in different parts of the retina. The sentiment has been expressed that it does not matter whether two tests do not agree so long as the disease is detected during screening [47]. To an extent this is understandable though it would clearly be desirable to have a rational basis for whatever testing regime is adopted. One possible explana- tion is that conventional perimetry detects light detection defects [54] while tests like FDT, SWAP and contrast threshold measurements detect different manifestations of ganglion cell damage [40,41]. Certainly there are sev- eral tests, particularly FDT and SWAP, which could be used profitably alongside visual field testing [48] and, to these, contrast threshold determinations in the peripheral visual field may be added. high temporal frequency to produce an illusory doubling of the spatial frequency [38]. Reduction of the dimension of the square stimulus field from 10° to 4° produced only a modest increase in contrast threshold [39]. These results are thus not inconsistent with the absence of a significant differences between the contrast thresholds for the differ- ent truncated quadrants. high temporal frequency to produce an illusory doubling of the spatial frequency [38]. Reduction of the dimension of the square stimulus field from 10° to 4° produced only a modest increase in contrast threshold [39]. These results are thus not inconsistent with the absence of a significant differences between the contrast thresholds for the differ- ent truncated quadrants. Authors' contributions JLJ carried out the clinical examination of the patients. The scientific experiments were carried out and analyzed by CMT, JSM and JDM who also undertook the construction of the apparatus and drafting of the manu- script which all authors approved. http://www.biomedcentral.com/1471-2415/5/22 In terms of correct identification of our glaucoma patients, contrast thresholds were elevated in one or more truncated quadrants in all but one of our 21 patients while inter-ocular differences were elevated in all but 2 out of 20 patients, which is consistent with previous studies [4,15- 18,21]. These elevations were present irrespective of the type of glaucoma, which is consistent with a common mechanism for ganglion cell loss in chronic glaucoma [40]. In terms of detection of glaucoma, our method has performed soundly with an area under the ROC curve of 0.956 which compares well with other methodologies employed in the early detection of glaucoma viz. short wave length automated perimetry (SWAP), frequency doubling technology, resolution acuity perimetry, detec- tion acuity perimetry and temporal modulation perimetry [41] and considerably better than for contrast sensitivities with central viewing [42]. Of especial interest was the per- formance of our test with the OHT patients. Initially, it seemed that two patients would be incorrectly diagnosed in both eyes which had markedly elevated contrast thresholds; however within the next 12 months they each developed appreciable visual field loss in their eyes (Table 1). While the sample size is small, it indicates a potential predictive function which has also been described for FDT, high-pass resolution perimetry and SWAP [43,44]. A further improvement in the area under the ROC curve to 0.993 occurred when the assessment of contrast thresh- olds was combined with that of the inter-ocular difference in contrast threshold. This is a simple extension of the analysis which uses pre-existing data and its value has been remarked upon previously [38]. Competing interests The author(s) declare that they have no competing interests. Conclusion l h Our results therefore do confirm that contrast thresholds in response to sinusoidal grating patterns presented to peripheral regions of the visual field are invariably abnor- mally elevated in cases of glaucomatous visual field loss. The regions of threshold elevations, however, did not map onto the regions of visual field loss, which indicates con- trast threshold testing cannot be considered as a substitute for visual field tests. It may however provide valuable sup- plementary information which may be obtained simply and rapidly with the type of interferometric apparatus we have described. The central aim of our study, however, was whether the contrast threshold results reflected the amount of visual field loss in the same region of the visual field. While there was a reasonable correspondence in 50% of our 18 glau- coma patients (Figure 3), in the other half of the patients, there was a marked mismatch so that some patients had normal contrast thresholds in quadrants showing high visual field loss while others had elevated contrast thresh- olds in seemingly normal parts of the visual field (Figure 4). We are confident that the results were not caused by abrupt shifts of visual field loss between quadrants since the visual fields were stable over the 2 years preceding our tests and indeed subsequent to our tests. While automated perimetry is widely used to ascertain visual field loss, our preference was firmly for the Friedmann test on the basis Control subjects with abnormal fellow eye There were 8 subjects in the non-glaucoma control group with an abnormality of one eye. In the subject with the ret- inal scar, the contrast thresholds were elevated in the trun- cated quadrant containing the scar as confirmed by the Humphrey Visual Field. On the other hand, the contrast thresholds for the subjects with repaired retinal detach- Page 9 of 12 (page number not for citation purposes) Page 9 of 12 (page number not for citation purposes) http://www.biomedcentral.com/1471-2415/5/22 References 26. Kelly DH: Retinal inhomogeneity. I Spatiotemporal contrast sensitivity. J Opt Soc Am A 1984, 1:107-113. 1. Aulhorn E, Karmeyer H: Frequency distribution in early glauco- matous visual field defects. Doc Ophthalmol Proc Ser 1977, 14:75-83. 27. 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https://openalex.org/W4280539981	https://europepmc.org/articles/pmc9114343?pdf=render	English	null	The impact of age, performance status and comorbidities on nab-paclitaxel plus gemcitabine effectiveness in patients with metastatic pancreatic cancer	Scientific reports	2,022	cc-by	6,128	Martina Catalano1, Giuseppe Aprile2, Raffaele Conca3, Roberto Petrioli4, Monica Ramello5 & Giandomenico Roviello6 Martina Catalano1, Giuseppe Aprile2, Raffaele Conca3, Roberto Petrioli4, Monica Ramello5 & Giandomenico Roviello6 Few studies have evaluated the impact of risk factors such as performance status (PS) and comorbidities on overall survival (OS) in patients with metastatic pancreatic cancer (mPC). We investigated the influence of comorbidity, PS and age on nab-paclitaxel and gemcitabine (NabGem) effectiveness profile in naive patients with mPC. 153 patients with mPC treated with NabGem upfront was divided in three groups (score 0 to 3) based on the absence or the presence of one or more risk factors among: age ≥ 70 years, PS 1 and comorbidities and the clinical outcomes was compared. Fifty- five patients were elderly (≥ 70 years), 80 patients have PS 1, whereas the other have PS 0. Patients with no risk factors (score 0) had an overall survival higher (20 months) than patients with one or two risk factors (score 1–2) (OS 11 months) and with three risk factors (score 3) (OS 8 months) (p < 0.01). The difference in OS was also statistically significant in patients without comorbidities (OS 15 months) compared to those with ≥ 1 comorbidity (OS 10 months) (p < 0.001). NabGem chemotherapy represent an effective treatment in naive patients. Age, PS, and comorbidities were prognostic factors in patients with metastatic pancreatic cancer. Pancreatic cancer (PC) is the fourth leading cause of cancer-related death in Europa and United States1,2. Most patients with PC are diagnosed with advanced stage, and the 5-year survival ranged from 5 to 10%3. With the aging of population, the number of elderly cancer patients has increased worldwide4. Pancreatic cancer often affects elderly patients with an average age at diagnosis of around 72 years5. An ever-increasing part of these elderly patients is affected by comorbidities and impaired organ function that often lead to inappropriately treat- ment in clinical practice based on the perception of reduced life expectancy and the ability to tolerate potential therapies side effects. pf Therefore, sometimes less aggressive treatment options or best supportive care alone is offered to elderly patients due to the potential increase in toxicity. In systematic reviews, the use of chemotherapy has been shown to improve survival in advanced PC6. However, data on the benefits and toxicity of chemotherapy in elderly patients are very limited due to the low enrollment of this population in clinical trials7. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports The impact of age, performance status and comorbidities on nab‑paclitaxel plus gemcitabine effectiveness in patients with metastatic pancreatic cancer Martina Catalano1, Giuseppe Aprile2, Raffaele Conca3, Roberto Petrioli4, Monica Ramello5 & Giandomenico Roviello6 Martina Catalano1, Giuseppe Aprile2, Raffaele Conca3, Roberto Petrioli4, Monica Ramello5 & Giandomenico Roviello6 All patients (N = 153) Subgroup patients Score = 0 (N = 28) Score = 1–2 (N = 98) Score = 3 (N = 27) Age, years Median (range) 67 (50–84) 59.5 (49–69) 66 (50–83) 74 (70–84) ≥ 70 55 (35.9%) 0 28 (28.6%) 27 (100%) ECOG PS 1 80 (51.2%) 0 53 (54.1%) 27 (100%) Sex Male 88 (57.5%) 14 (50%) 57 (58.2%) 17 (63%) N of comorbidities ≥ 1 comorbidities 95 (62.3%) 0 68 (69.4%) 27 (100%) CA 19.9—U/ml Median Range 547 (0.8–700,000) 178 (15.7–13,027) 640 (0.8–700,000) 616 (26–182,922) Number of metastatic sites ≥ 3 61 (39.9%) 10 (35.7%) 30 (30.6%) 8 (29.6%) Previous Surgery Surgery 37 (24.2%) 11 (39.3%) 23 (23.5%) 3 (11.1%) Type of comorbidities’ Cardiovascular 69 (45.1%) – – – Diabetes mellitus 52 (34%) Dyslipidemia 29 (18.9%) Respiratory 13 (8.5%) Genitourinary 15 (9.8%) Table 1. Patient characteristics. Number (N); Eastern Cooperative Oncology Group Performace Status (ECOG PS); carbohydrate antigen 19.9 (CA 19.9). combination regimens seem to be effective and well tolerated in these patients, and new combination regimens such as nab-paclitaxel and gemcitabine (NabGem) are evaluating11. Chronological age alone, although associated with age-related impairment in organ function, does not well reflect the general physical status of patients. To date, is controversial which of the factors such as PS, comor- bidity, age, have the most relevant impact on the treatment results in the elderly7. The interplay between age, comorbidity, and PS in predicting outcome in metastatic cancer is poorly understood. The Charlson Comorbidity Index (CCI) is the most widely used tool to measure comorbidity in patients with a prognostic implication in the adjuvant setting8–11. Therefore, an appropriate patient selection including multiple factors (e.g., age, PS, comor- bidities) and a proper balance of potential treatment benefits and side effects represent a crucial point for manag- ing patients with PC to maximize the therapeutic benefit. The aim of this retrospective study was to clarify the impact of age, performance status and comorbidities on prognosis in patients with advanced pancreatic cancer. Martina Catalano1, Giuseppe Aprile2, Raffaele Conca3, Roberto Petrioli4, Monica Ramello5 & Giandomenico Roviello6 p y p p For more than a decade, gemcitabine has been the standard chemotherapy against advanced pancreatic cancer inasmuch it showed to prolong survival and improved clinical benefit response8. Two studies evaluated gemcitabine-containing regimens in elderly patients with pancreatic cancer, which however, have included only patients with good performance status (PS) and few comorbidities, rarely corresponding to patients in clini- cal practice9,10. Although with discordant results, gemcitabine-based treatment and dose-adapted fluorouracil 1Department of Human Health Sciences, School of Human Health Sciences, University of Florence, Largo Brambilla 3, 50134 Florence, Italy. 2Department of Oncology, San Bortolo General Hospital, AULSS8 Berica, Vicenza, Italy. 3Division of Medical Oncology, Department of Onco‑Hematology, IRCCS-CROB, Referral Cancer Center of Basilicata, via Padre Pio 1, 85028 Rionero, Vulture, PZ, Italy. 4Department of Medicine, Surgery and Neurosciences, Medical Oncology Unit, University of Siena, VialeBracci–Policlinico“LeScotte”, 53100 Siena, Italy. 5Oncology Unit, Department of Medical, Surgical and Health Sciences, University of Trieste, Piazza Ospitale, Trieste, Italy. 6Department of Health Sciences, University of Florence, viale Pieraccini, 6, 50139 Florence, Italy. email: catalanomartina8@gmail.com \| https://doi.org/10.1038/s41598-022-12214-4 Scientific Reports \| (2022) 12:8244 www.nature.com/scientificreports/ Table 1. Patient characteristics. Number (N); Eastern Cooperative Oncology Group Performace Status (ECOG PS); carbohydrate antigen 19.9 (CA 19.9). All patients (N = 153) Subgroup patients Score = 0 (N = 28) Score = 1–2 (N = 98) Score = 3 (N = 27) Age, years Median (range) 67 (50–84) 59.5 (49–69) 66 (50–83) 74 (70–84) ≥ 70 55 (35.9%) 0 28 (28.6%) 27 (100%) ECOG PS 1 80 (51.2%) 0 53 (54.1%) 27 (100%) Sex Male 88 (57.5%) 14 (50%) 57 (58.2%) 17 (63%) N of comorbidities ≥ 1 comorbidities 95 (62.3%) 0 68 (69.4%) 27 (100%) CA 19.9—U/ml Median Range 547 (0.8–700,000) 178 (15.7–13,027) 640 (0.8–700,000) 616 (26–182,922) Number of metastatic sites ≥ 3 61 (39.9%) 10 (35.7%) 30 (30.6%) 8 (29.6%) Previous Surgery Surgery 37 (24.2%) 11 (39.3%) 23 (23.5%) 3 (11.1%) Type of comorbidities’ Cardiovascular 69 (45.1%) – – – Diabetes mellitus 52 (34%) Dyslipidemia 29 (18.9%) Respiratory 13 (8.5%) Genitourinary 15 (9.8%) Table 1. Patient characteristics. Number (N); Eastern Cooperative Oncology Group Performace Status (ECOG PS); carbohydrate antigen 19.9 (CA 19.9). Results P ti t All patients (N = 153) Subgroup patients Score = 0 (N = 28) Score = 1–2 (N = 98) Score = 3 (N = 27) Cycles Median (range) 5 (1–17) 6 (1–17) 5 (1–17) 4 (1–10) Dose reduction 88 (57.5%) 18 (64.3%) 56 (57.1%) 14 (51.8%) Treatment delay 51 (33.5%) 8 (28.6%) 36 (37.1%) 7 (25.9%) Treatment interruption 51 (33.5%) 13 (46.4%) 32 (32.6%) 6 (22.2%) GCF-Prophylaxis 25 (16.4%) 4 (14.8%) 12 (12.4%) 9 (33.3%) Subsequent line of therapy 71 (46.4%) 17 (60.7%) 45 (45.9%) 9 (33.3%) Table 2. Dose reduction, treatment delay, treatment interruption and GCF- prophylaxis according to score population. Number (N); granulocyte-colony stimulating factor (GCF). Table 2. Dose reduction, treatment delay, treatment interruption and GCF- prophylaxis according to score population. Number (N); granulocyte-colony stimulating factor (GCF). Table 3. Best response, PFS and OS according to score population. Number (N); partial response (PR); stable disease (SD); disease control rate (DCR); progression disease (PD); not evaluable (NE); mMedian (median); progression free survival (PFS); overall survival (OS); confidence interval (CI). All patients (N = 153) Subgroup patients Score = 0 (N = 28) Score = 1–2 (N = 98) Score = 3 (N = 27) PR 58 (37.1%) 16 (57.1%) 34 (34.7%) 8 (29.6%) SD 44 (28.8%) 7 (25%) 26 (26.5%) 11 (40.7%) DCR (PR + SD) 102 (66.7%) 23 (82.1%) 60 (61.2%) 19 (70.4%) PD 42 (27.4%) 5 (17.9%) 31 (31.6%) 6 (22.2%) NE 9 (5.9%) 0 7 (7.1%) 2 (7.4%) PFS months (95% ICI) (number of events) 6 (5–6) 132 7 (5–9) 25 6 (5–7) 85 6 (4–7) 22 OS months (95% ICI) (number of events) 11 (10–13) 121 20 (12–22) 18 11 (9–13) 78 8 (6–12) 25 Table 3. Best response, PFS and OS according to score population. Number (N); partial response (PR); stable disease (SD); disease control rate (DCR); progression disease (PD); not evaluable (NE); mMedian (median); progression free survival (PFS); overall survival (OS); confidence interval (CI). Results P ti t Patients’ characteristics. Between January 2015 and December 2018, 153 patients diagnosed with mPC and treated with NabGem have been identified. Baseline characteristics are shown in Table 1.h i The mean age at diagnosis was 67 years with a prevalence of the male gender (57.5%); Eastern Cooperative Oncology Group-Performance Status (ECOG-PS) was 1 in 80 (51.2%) patients and 0 in the remaining. Median baseline carbohydrate antigen (CA) 19.9 was 547 U/ml (range 0.8–700,000 U/ml). The 39.9% of the patients had more than 3 metastatic sites; 37 patients have been previously treated with surgery. Regarding comorbidities, 95 (62.3%) presented ≥ 1 comorbidity, and the most frequent (69, 45.1%) was cardiovascular; diabetes mellitus and dyslipidemia were presented in 52 (34%) and 29 (19.9%) patients, respectively, whereas respiratory and genitourinary comorbidities in the 8.5% and 9.8%, respectively. Population subgroups. Twenty-eight patients had a score = 0, 98 patients had a score = 1–2, and 74 a score = 3 (Table 1). In the group with score = 1–2, 28 (28.6%) patients were over 70 years old, 53 (54.1%) had ECOG-PS 1 and 68 (69.4%) presented ≥ 1 comorbidity. Male gender was represented mainly in patients with score = 3 (63%), whereas ≥ 3 metastatic sites were present in patients with score = 1. 39.3% of patients in the score = 0, 23.5% in the score = 1–2 and 11.1% in the score = 3, had previously received surgical treatment. Chemotherapy regimens. During the study, patients received a median of five cycles (range 1–17) of treatment; with a starting dose of nab-paclitaxel 125 mg/m2 plus gemcitabine 1000 mg/m2. Dose reduction has been necessary in 88 (57.5%) patients, 18 (64.3%) in the score = 0, 56 (57.1%) in the score = 1–2 and 14 (51.8%) in the score = 3, without significant difference between gemcitabine and nab-paclitaxel. Dose delays occurred in https://doi.org/10.1038/s41598-022-12214-4 Scientific Reports \| (2022) 12:8244 \| www.nature.com/scientificreports/ Table 2. Dose reduction, treatment delay, treatment interruption and GCF- prophylaxis according to score population. Number (N); granulocyte-colony stimulating factor (GCF). Results P ti t Over half of the patients with score = 0 received a subsequent line of therapy, while less than half in the other two groups (45.95% in score = 1–2 and 33.3% in score = 3) (Table 2). Efficacy outcomes. The efficacy of chemotherapy was compared between the three groups (Table 3). Dis- ease control rate (DCR) by Response Evaluation Criteria in Solid Tumors (RECIST) was 82.1% in score = 0 group, 61.2% in score = 1–2 group and 70.4% in patients with score = 3. Progression disease (PD) was recorded in 42 (27.4%) patients, with prevalence in score = 1–2 patients (31.6%) vs 22.2% and 17.9% in score = 3 and score = 0, respectively. , p y Progression free survival (PFS) was not significantly affected by age, PS, or comorbidity: 7 months in score = 0 vs 6 months in score = 1–2 (hazard ratio [HR] 1.18, 95% confidence interval [CI] 0.98–1.42) (p = 0.09) and score = 3 (HR 1.32, 95%CI 0.83–2.10) (p = 0.2) groups (Table 3) (Fig. 1).i p g p g Contrariwise, overall survival (OS) was significantly higher in patients with score = 0 (20 months) compared to patients with score = 1–2 (11 months) (HR 1.40, 95%CI 1.17–1.68) (p < 0.001) and patients with score = 3 (8 months) (HR 1.91, 95%CI 1.22–2.99) (p < 0.001) (Table 3) (Fig. 2).fi p g Efficacy outcomes were estimated according to the patients score (0 or ≥ 1) and according to number of comorbidities (0 or ≥ 1). PFS was 7 and 6 months in patients with score = 0 and patients with score ≥ 1, respectively (HR 1.5, p = 0.1). OS was of 20 months in score = 0 group and 10 months in score ≥ 1 patients, with a difference statistically significant (HR 2.12, p < 0.001).hfi y gi ( p ) The difference in OS was also statistically significant in patients without comorbidities compared to patients with > 1 comorbidities (15 months vs 10 months) (HR 1.6, p < 0.001). Whereas PFS was the same between comor- bidities groups (6 months) (HR 1.1, p = 0.5) (Table 1S).fi Finally, a subgroup efficacy analysis according to the type of comorbidities was performed. A statistically significant difference in OS was recorded in patients with cardiovascular comorbidities (10 months) compared with patients without cardiovascular comorbidities (13 months) (HR 1.5, p = 0.02) (Table 2S). Results P ti t All patients (N = 153) Subgroup patients Score = 0 (N = 28) Score = 1–2 (N = 98) Score = 3 (N = 27) PR 58 (37.1%) 16 (57.1%) 34 (34.7%) 8 (29.6%) SD 44 (28.8%) 7 (25%) 26 (26.5%) 11 (40.7%) DCR (PR + SD) 102 (66.7%) 23 (82.1%) 60 (61.2%) 19 (70.4%) PD 42 (27.4%) 5 (17.9%) 31 (31.6%) 6 (22.2%) NE 9 (5.9%) 0 7 (7.1%) 2 (7.4%) PFS months (95% ICI) (number of events) 6 (5–6) 132 7 (5–9) 25 6 (5–7) 85 6 (4–7) 22 OS months (95% ICI) (number of events) 11 (10–13) 121 20 (12–22) 18 11 (9–13) 78 8 (6–12) 25 Table 3. Best response, PFS and OS according to score population. Number (N); partial response (PR); stable disease (SD); disease control rate (DCR); progression disease (PD); not evaluable (NE); mMedian (median); progression free survival (PFS); overall survival (OS); confidence interval (CI). Table 3. Best response, PFS and OS according to score population. Number (N); partial response (PR); stable disease (SD); disease control rate (DCR); progression disease (PD); not evaluable (NE); mMedian (median); progression free survival (PFS); overall survival (OS); confidence interval (CI). Table 3. Best response, PFS and OS according to score population. Number (N); partial response (PR); stable disease (SD); disease control rate (DCR); progression disease (PD); not evaluable (NE); mMedian (median); progression free survival (PFS); overall survival (OS); confidence interval (CI). 51 (33.5%) patients with higher prevalence in the score = 1–2 (37.1%); treatment interruption occurred always in 51 (33.5%) patients with greater occurrence in patients with score = 0 (46.4%). GCF prophylaxis was required in 9 (33.3%) patients with score = 3 and in 4 (14.8%) and 12 (12.4%) in patients with score = 0 and score = 1–2, respectively. Over half of the patients with score = 0 received a subsequent line of therapy, while less than half in the other two groups (45.95% in score = 1–2 and 33.3% in score = 3) (Table 2). 51 (33.5%) patients with higher prevalence in the score = 1–2 (37.1%); treatment interruption occurred always in 51 (33.5%) patients with greater occurrence in patients with score = 0 (46.4%). GCF prophylaxis was required in 9 (33.3%) patients with score = 3 and in 4 (14.8%) and 12 (12.4%) in patients with score = 0 and score = 1–2, respectively. Results P ti t No differences in OS and PFS have been shown for other types of comorbidities. https://doi.org/10.1038/s41598-022-12214-4 Scientific Reports \| (2022) 12:8244 \| www.nature.com/scientificreports/ Figure 1. Progression free survival according to the score. Figure 2. Overall survival according to the score. Figure 1. Progression free survival according to the score. Figure 1. Progression free survival according to the score. Figure 2. Overall survival according to the score. Figure 2. Overall survival according to the score. Scientific Reports \| (2022) 12:8244 \| Discussion Pancreatic cancer is primarily a disease of the elderly patients with a median age at diagnosis of 72 years. Although geriatric age is characterized by organic changes that can interfere with the oncological decision-making pro- cess, the therapeutic choice should be evaluated on the basis of the biological age of each patient defined by the performance status and the comorbidities12. Therefore, in elderly patients, and in the presence of comorbidities, the selection of the patient is fundamental to define the best therapeutic choice.f i In elderly patients with resectable pancreatic cancer, surgical resection seems to be effective and safe13–15, while in patients with advanced or metastatic disease the options are palliative chemotherapy or best supportive care, but few studies discuss the better choice in this population16. Literature-based evidence and guidelines support the use of single-agent therapy as the optimal treatment for elderly patients with advanced PC17. In a prospective observational study by Locher et al. conducted in patients aged 70 years and older a fixed-dose rate gemcitabine treatment was feasible in those with good PS and without major comorbidity9. Similarly, in a retrospective study by Marechal et al. gemcitabine-based regimens were effective and well tolerated in patients aged 70 years and older, although, only patients with good PS have been enrolled10.l However, in the last years other chemotherapy regimens, such as FOLFIRINOX (5-fluorouracil/leucovorin plus irinotecan and oxaliplatin) and NabGem have become part of the advanced/metastatic PC treatment. While the unfavorable risk-to-benefit ratio contraindicates the triplet in elderly patients, NabGem was used in several study with good efficacy and safety results18,19. The NAPOLEON study, examined the efficacy and safety of gem- citabine plus nab-paclitaxel in older patients with mPC, especially those ≥ 75 years old. Patients were divides in two group “older” if ≥ 75 years old and “not older” if < 75 years old. The initial dose and relative dose intensities of NabGem were significantly lower in the older group. There were no significant differences in the adverse event and antitumor response rates between the two groups. Median PFS was 5.5 months and median OS was 12.0 months in the older group, versus 6.0 and 11.1 months in the non-older group, respectively20. www.nature.com/scientificreports/ These data, although very limited, confirmed the necessity of develop new prognostic scales for patient with PC by considering the side effects of chemotherapy.f f For some reasons, including a small sample size and differences in inclusion/exclusion criteria, a direct com- parison between the pivotal trial and our real-world experience, was not fully possible. However, despite the limitations, we were able to confirm that the combination regimen with NabGem has resulted effective in our population compared with the randomised controlled trial sample. Specifically, we observed 11 months median OS and 6 months median PFS with a DCR of 66.7%.iffi Notable, the statistically significance difference in efficacy outcome observed in patients with one or more among: age ≥ 70 years old, PS = 1 and presence of at least comorbidity (score = 1–2 and score = 3) compared to < 70 years old patients with PS = 0 and without comorbidities (score = 0). In fact, OS was significantly higher in patients with score = 0 (20 months) compared to patients with score ≥ 1 (10 months) (p < 0.001). Conversely, no difference was recorded in PFS between two groups: 7 months in patients with score = 0 vs 6 months in patients with score ≥ 1 (p = 0.09). Furthermore, regarding comorbidities, a significant difference in OS was observed between patients with ≥ 1 comorbidities (10 months) and without comorbidities (15 months) (p < 0.001). Patients with cardiovascular comorbidities seem to correlate with worse overall survival (p = 0.02). Thus, assessment of comorbidity is important in treating patients with advanced pancreatic cancer, and stratification by dedicate index should be considered in prospective trials, especially in trials including elderly patients. p p p y g y p Our study has some limitation mainly owing to its retrospective nature and the absence of a standardized comparison score. On the other hand, the large sample of patients with only metastatic disease, represent a strength of the study. Moreover, in the absence of clear established criteria, we have presented a score based on three simple criteria that can be easily and quickly evaluated, which could be used in clinical practice to predict the response to treatment with NabGem. This could be useful to evaluate patients in their complexity and not simply on individual risk factors such as age, to ensure the most effective treatment available. www.nature.com/scientificreports/ of a 28-day cycle) to evaluate efficacy and toxicity. The median OS and PFS were 9.1 months and 4.8 months, respectively. Dose reductions of gemcitabine/nab-paclitaxel were required in 10% and 4% patients, respectively21.fii y g q y Ibusuki et al. examined the efficacy and safety of modified NabGem for 34 older patients (≥ 75 years) with advanced PC22. The median OS and PFS were 15.4 months and 5.9 months, respectively. The best response was partial response (PR) in 29% (10/34), stable disease (SD) in 53% (18/34), and progression disease (PD) in 15% of patients (5/34). Early discontinuation owing to intolerable adverse events occurred in one patient, and there were no chemotherapy-related deaths. The present study demonstrated that modified NabGem showed good efficacy with acceptable toxicity and that initial dose reduction may be a good option for older patients with PC to avoid early discontinuation and to maintain dose intensities23. However, in addition to chronological age, comorbidity is a recurring problem in the treatment of elderly patients. Comorbidities are common in cancer patients and the prevalence increases with age. Charlson Comorbidity Index (CCI), which combines age and comorbidity, is the most used index in longitudinal studies for estimating the relative risk of death from prog- nostic clinical covariates24. Although the significant effect of comorbidity on the overall survival was observed in the cancer subtypes with generally longer expected survival time (such as prostate and breast cancers), no statistically significant correlation was found in the cancers with lower life expectancy (such as pancreatic and lung cancers). Nakai et al. showed in their multivariate analysis, that CCI and PS are prognostic factors for sur- vival in 183 patients with advanced pancreatic cancer treated with gemcitabine-based chemotherapy, contrary to age25. Recently, Bagni et al. showed that CCI and other factors such as diabetes, tobacco smoking, alcohol abuse, and body mass index (BMI) had no significant prognostic effect on overall survival in PC patients that received least one cycle of adjuvant or palliative chemotherapy. They confirmed instead that advanced cancer stage and poor PS were associated with increased mortality in patients with pancreatic adenocarcinoma in accordance with previous studies26,27. www.nature.com/scientificreports/ Indeed, as showed in our analysis also patients with ≥ 70 years and in good condition, often excluded from clinical trials, can receive treatment with NabGem with the same effectiveness as the younger ones. Conclusionh This study showed that the combination of nab-paclitaxel and gemcitabine has a similar efficacy profile for not pretreated patients aged ≥ 70 and < 70 years. Especially elderly patients with reduced performance status (PS = 1) and comorbidities can be treated effectively with NabGem and should not be excluded from appropriate treatment based on perceptions about their life expectancy and comorbidities. However, we observed that age, performance status and comorbidities, mainly cardiovascular comorbidities, were associated with a lower OS in patients with PC compared to patients without risk factors. Therefore, given the increasing proportion of those elderly and/or comorbid patients with PC, further investigations are warranted on different risk factors (e.g., age and comorbidity) in the era of aggressive cancer treatment. Discussion Another study analyzed patients > 65 years of age with advanced PC who received a modified of gemcitabine/nab-paclitaxel in a biweekly regimen (gemcitabine 1000 mg/m2 and nab-paclitaxel 125 mg/m2 every 2 weeks on days 1 and 15 https://doi.org/10.1038/s41598-022-12214-4 Scientific Reports \| (2022) 12:8244 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ excluded28.Patient characteristics, including age at diagnosis, gender, performance status, comorbidities, CA, number of metastases, and previous treatment. excluded28.Patient characteristics, including age at diagnosis, gender, performance status, comorbidities, CA, number of metastases, and previous treatment. Patients were divided into three groups by age, ECOG-PS and comorbidities. Patients presented with 1 or 2 factors between age ≥ 70 years; ECOG-PS 1; presence of at least one comorbidity were assigned a score = 1–2; to patients who had all three risk factors a score = 3, whereas a score = 0 to patients without any risk factors. In our analysis score 0 group used as reference.h y g p This study was approved by the Local Institutional Review Board for clinical experimentation of Tuscany (Italy)—“area vasta centro” section, with the number:14565_oss. Written informed consent was obtained from all patients. Chemotherapy. Nab-paclitaxel 125 mg/m2, followed by gemcitabine 1000 mg/m2 were administered intra- venously on days 1, 8 and 15 every 4 weeks according to the pivotal trial19. Recombinant human GCF factor and erythropoietin were administered as needed. In the event of unacceptable toxicity, doses could be reduced up to two times per therapeutic agent (to 100 or 75 mg/m2 for nab-paclitaxel and to 800 or 600 mg/m2 for gemcit- abine)19. A missing dose within four days of the scheduled administration were considered dose delays. Tumor response. Before the start of treatment, a full medical history, physical examination with assessment of ECOG-PS, complete blood count with differential, full serum chemistry profile, and cardiologic assessment (e.g., electrocardiogram, echocardiogram and cardiologic visit) were performed for each patient. Blood tests were performed before each therapy cycle, while measurement of the CA 19-9 serum level was performed at baseline and every 12 weeks. Tumor response was assessed via computed tomography using RECIST version 1.129. The evaluation was repeated every three months or more frequently in patients with clinically suspected progression. Efficacy has been evaluated as overall survival and progression free survival. OS was defined as a time from the diagnosis of advanced pancreatic cancer to death from any cause or the date of the last follow-up visit. PFS was defined as time from the initial assessment at the cancer centre to the date of the disease progres- sion as reported by the clinician. Disease control rate was defined as the proportion of patients with the best overall response determined as CR, PR or SD30. Statistical analysis. www.nature.com/scientificreports/ OS and PFS were estimated using the Kaplan–Meier method and compared using the log-rank test. Parameters with a statistically significant log-rank test were considered independent variables and included in the multivariate Cox proportional hazard regression linear model to compare HR and 95% CI. All reported p-values are the result of two-sided tests; p-values < 0.05 were supposed to indicate statistical signifi- cance. STATA v.2012 was used for statistical analysis. Prognostic factors included age (< 70 or ≥ 70 years old), PS (0 or ≥ 1) and comorbidities (0 or ≥ 1). Ethical approval. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional (Local Institutional Review Board for clinical experimentation of Tuscany (Italy)—“area vasta centro” section, with the number:14565_oss) and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent. Informed consent was obtained from all individual participants included in the study. nformed consent. Informed consent was obtained from all individual participants included in the study. Data availabilityh y The data used to support the findings of this study are available from the corresponding author upon reque Received: 14 October 2021; Accepted: 27 April 2022 Received: 14 October 2021; Accepted: 27 April 2022 Received: 14 October 2021; Accepted: 27 April 2022 Patients and methods Study design and patients. Study design and patients. Patients diagnosed with metastatic pancreatic cancer in four Italian centers between January 2015 and December 2018 were retrospectively included. All patients were deemed eligible for NabGem therapy according to the following criteria: histologically or cytologically confirmed PC; radiographi- cally confirmed metastatic disease; no previous chemotherapy; ECOG-PS ≤ 1; adequate hemopoietic function (absolute neutrophil count [ANC] ≥ 1.5 × 109/L, hemoglobin ≥ 10 g/dL, and platelet count ≥ 100 × 109/L), liver function (bilirubin level ≤ 1.5 mg/dL, aspartate/alanine aminotransferase concentrations ≤ 2.5 times the upper limit of normal in the absence of liver metastases or less than 5 times in case of liver metastases), and renal function (serum creatinine ≤ 1.5 mg/dL or creatinine clearance ≥ 30 mL/min). Patients with history of signifi- cant cardiac disease (e.g., unstable angina, uncontrolled arrhythmias, or myocardial infarction < 3 months) were https://doi.org/10.1038/s41598-022-12214-4 Scientific Reports \| (2022) 12:8244 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ Gemcitabine plus nab-paclitaxel with initial dose reduction for older patients with advanced pancreatic cancer - PubMed. https:// pubmed.ncbi.nlm.nih.gov/32576518/. p g 3. Ibusuki, M. et al. Gemcitabine plus nab-paclitaxel with initial dose reduction for older patients with advanced pancreatic cancer J. Geriatr. Oncol. 12, 118–121 (2021). 24. Charlson, M. E., Pompei, P., Ales, K. L. & MacKenzie, C. R. A new method of classifying prognostic comorbidity in longitudinal studies: development and validation. J. Chronic Dis. 40, 373–383 (1987). 5. Nakai, Y. et al. Comorbidity, not age, is prognostic in patients with advanced pancreatic cancer receiving gemcitabine-based chemotherapy. Crit. Rev. 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Competing interests h g The authors declare no competing interests. Author contributions G.R. had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: R.G., P.R. Acquisition of data: M.C. Analysis and interpretation of data: R.G., G.A. Drafting of the manuscript: R.G., M.C. Critical revision of the manuscript for important intellectual content: G.A., M.R., R.C. Statistical analysis: R.G. Obtaining funding: None. Administra- tive, technical, or material support: None. Supervision: G.A. Financial disclosures: None. Funding/Support and role of the sponsor: None. References 1. Maisonneuve, P. Epidemiology and burden of pancreatic cancer. Presse Medicale 48, e113–e123 (2019). l l d d l f d p gy p 2. Ferlay, J. et al. Cancer incidence and mortality patterns in Europe: estimates for 40 countries and 25 major cancers in 2018. Eur. J Cancer 103, 356–387 (2018). 3. Pancreatic Cancer Prognosis \| Johns Hopkins Medicine. https://www.hopkinsmedicine.org/health/conditions-and-diseases/pancr eatic-cancer/pancreatic-cancer-prognosis. 3. Pancreatic Cancer Prognosis \| Johns Hopkins Medicine. https://www.hopkinsmedicine.org/health/conditions-and-diseases/pancr eatic-cancer/pancreatic-cancer-prognosis. p p g 4. Sung, H. et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA A Cancer J. Clin. 71, 209–249 (2021). 5. SEER Cancer Statistics Review 1975–2006 - Previous Version - SEER Cancer Statistics. https://seer.cancer.gov/archive/csr/1975_ 2006/. 6. Yip, D., Karapetis, C., Strickland, A., Steer, C. B. & Goldstein, D. Chemotherapy and radiotherapy for inoperable advanced pancre- atic cancer. in Cochrane Database of Systematic Reviews (John Wiley & Sons, Ltd, 2006). https://doi.org/10.1002/14651858.cd002 093.pub2. p 7. Hutchins, L. F., Unger, J. M., Crowley, J. J., Coltman, C. A. & Albain, K. S. Underrepresentation of patients 65 years of age or older in cancer-treatment trials. N. Engl. J. Med. 341, 2061–2067 (1999).ii g ( ) 8. Burris, H. A. et al. Improvements in survival and clinical benefit with gemcitabine as first- line therapy for patients with advanced pancreas cancer: a randomized trial. J. Clin. Oncol. 15, 2403–2413 (1997). p 9. Locher, C. et al. Fixed-dose rate gemcitabine in elderly patients with advanced pancreatic cancer: an observational study. Crit. Rev. Oncol. Hematol. 68, 178–182 (2008).fi 10. Maréchal, R. et al. Tolerance and efficacy of gemcitabine and gemcitabine-based regimens in elderly patients with advanced pan- creatic cancer. Pancreas 36, (2008).f 10. Maréchal, R. et al. Tolerance and efficacy of gemcitabine and gemcitabine-based regimens in elderly patients with advanced pan- creatic cancer. Pancreas 36, (2008).f , ( ) 1. Von Hoff, D. D. et al. Increased survival in pancreatic cancer with nab-paclitaxel plus gemcitabine. N. Engl. J. Med. https://doi.org/ 10.1056/NEJMoa1304369 (2013). https://doi.org/10.1038/s41598-022-12214-4 Scientific Reports \| (2022) 12:8244 \| © The Author(s) 2022 Additional informationh Supplementary Information The online version contains supplementary material available at https://doi.org/ 10.1038/s41598-022-12214-4. Correspondence and requests for materials should be addressed to M.C. Correspondence and requests for materials should be addressed to M.C. Reprints and permissions information is available at www.nature.com/reprints. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2022 https://doi.org/10.1038/s41598-022-12214-4 Scientific Reports \| (2022) 12:8244 \|
https://openalex.org/W2265870732	https://www.nature.com/articles/ncomms8626.pdf	English	null	Correction: Corrigendum: Ventral hippocampal afferents to the nucleus accumbens regulate susceptibility to depression	Nature communications	2,015	cc-by	147	DOI: 10.1038/ncomms8626 NATURE COMMUNICATIONS \| 6:7626 \| DOI: 10.1038/ncomms8626 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. Corrigendum: Ventral hippocampal afferents to the nucleus accumbens regulate susceptibility to depression Rosemary C. Bagot, Eric M. Parise, Catherine J. Pen˜a, Hong-Xing Zhang, Ian Maze, Dipesh Chaudhury, Brianna Persaud, Roger Cachope, Carlos A. Bolan˜os-Guzma´n, Joseph F. Cheer, Karl Deisseroth, Ming-Hu Han & Eric J. Nestler Nature Communications 6:7062 doi: 10.1038/ncomms8062 (2015); Published 8 May 2015; Updated 17 Jun 2015 & 2015 Macmillan Publishers Limited. All rights reserved. Nature Communications 6:7062 doi: 10.1038/ncomms8062 (2015); Published 8 May 2015; Updated 17 Jun 2015 In the original version of this Article, the middle initial of the author Joseph F. Cheer was omitted from the author information. The afﬁliation details for this author were also incorrect. These errors have now been corrected in both the PDF and HTML versions of the article.
https://openalex.org/W2790914538	https://europepmc.org/articles/pmc6816246?pdf=render	English	null	A Novel Substrate Radiotracer for Molecular Imaging of SIRT2 Expression and Activity with Positron Emission Tomography	Molecular imaging and biology	2,018	cc-by	8,935	1Karmanos Cancer Institute, Wayne State University, Detroit, MI, 48202, USA 1Karmanos Cancer Institute, Wayne State University, Detroit, MI, 48202, USA 2PET C t W St t U i it D t it MI 48202 USA Author Manuscript 2PET Center, Wayne State University, Detroit, MI, 48202, USA 3National Yang-Ming University School of Medicine, Taipei, Taiwan Published in final edited form as: Published in final edited form as: Mol Imaging Biol. 2018 August ; 20(4): 594–604. doi:10.1007/s11307-017-1149-8. p creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriatecredit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11307–017-1149–8) contains supplementary material, which is available to authorized users. This article is distributed under the terms of the Creative Commons Attribution 4. 0 International License ( http:// HHS Public Access Author Manuscript A Novel Substrate Radiotracer for Molecular Imaging of SIRT2 Expression and Activity with Positron Emission Tomography Robin E. Bonomi1, Maxwell Laws1, Vadim Popov1, Swatabdi Kamal1, Shreya Potukutchi1, Aleksandr Shavrin1, Xin Lu2, Nashaat Turkman1, Ren-Shyan Liu3, Thomas Mangner2, Juri G. Gelovani1 1Karmanos Cancer Institute, Wayne State University, Detroit, MI, 48202, USA 2PET Center, Wayne State University, Detroit, MI, 48202, USA 3 Introduction Over the past two decades, epigenetic regulation has become a rapidly growing, highly innovative and influential field of biology and medicine. One key epigenetic regulatory mechanism involves acetylation and deacetylation of lysine residues of histone core proteins and other critical proteins involved in regulation of gene expression and cell function. Protein acetylation and deacetylation is mediated by histone acetylase transferases (HATs) and histone deacetylases (HDACs), respectively. There are currently 18 HDAC proteins identified in the human genome, which are subdivided into 4 main classes [1]. Classes I, II (a and b), and IV are Zn2+-dependent enzymes [2] and include HDACs 1–11, while class III is comprised of seven isoforms of NAD+-dependent HDAC enzymes. HDAC class III enzymes are termed the “silent information regulators” or “sirtuins” (SIRTs), originating from the yeast SIR2 homologue, and consist of SIRT1–7 [1]. The SIRT isoforms vary in their cellular localization, with SIRTs 1, 6, and 7 being primarily located in the nucleus, SIRTs 3, 4, and 5 located in mitochondria. SIRT2 is primarily localized in the cytoplasm, but can translocate into the nucleus [3] and deacetylase of cytoplasmic alpha-tubulin and nuclear H4K16 proteins, thereby acting as a mitotic exit and cell cycle regulator [3–8]. Author Manuscript Several studies have revealed important roles of SIRT2 in the pathogenesis of various diseases. SIRT2 can play a neuroprotective role in neuorodegenerative diseases, such as Parkinson’s, Alzheimer’s [9], and Huntington’s [10]. SIRT2 has been identified as having both cancer-promoting functions (i.e., through stabilization of Myc oncoproteins in breast cancer [11, 12]), as well as cancer-suppressing functions (i.e., through tubulin regulation [4]). Therefore, SIRT2 has been identified as a potential drug target for treatment of various neurodegenerative and inflammatory diseases and cancer. However, it is important to assess its activity during pharmacomodulation (i.e., activation or inhibition) due to pleiotropic roles of SIRT2 under normal and pathologic conditions. The development of non-invasive molecular imaging approaches for monitoring SIRT2 expression and activity in vivo using PET with SIRT2-selective substrate radiotracer may aid in the development of therapies targeting SIRT2. Author Manuscript Understanding structure-activity relationships (SAR) of SIRT2 and its endogenous substrates is important for the development of SIRT2-specific substrate-type radiotracers. The best-studied endogenous substrates of SIRT2 are acetyl-α-tubulin and acetyl-H4K16. SIRT2, similar to other SIRT isoforms namely 4, 5, and 6, is able to cleave larger moieties outside of the acetyl. eywords SIRT2; Epigenetic Regulation; F-18; Radiotracer; Positron Emission Tomography; Brain SIRT2; Epigenetic Regulation; F-18; Radiotracer; Positron Emission Tomography; Brain chain of DDAHA. A SIRT2-selective radiotracer may provide information about SIRT2 expression and activity in tumors and normal organs and tissues, which may help to better understand the roles of SIRT2 in different diseases. Author Manuscript Mol Imaging Biol. Author manuscript; available in PMC 2019 October 28. Abstract Purpose: The purpose of this study was to develop a SIRT2-specific substrate-type radiotracer for non-invasive PET imaging of epigenetic regulatory processes mediated by SIRT2 in normal and disease tissues. Procedures: A library of compounds containing tert-butyloxycarbonyl- Procedures: A library of compounds containing tert-butyloxycarbonyl- lysineaminomethylcoumarin backbone was derivatized with fluoroalkyl chains 3–16 carbons in length. SIRT2 most efficiently cleaved the myristoyl, followed by 12-fluorododecanoic and 10fluorodecanoic groups (Kcat/Km 716.5 ± 72.8, 615.4 ± 50.5, 269.5 ± 52.1/s mol, respectively). Radiosynthesis of 12- [18F]fluorododecanoic aminohexanoicanilide (12-[18F]DDAHA) was achieved by nucleophilic radiofluorination of 12-iododecanoic-AHA precursor. Author Manuscript Author M Author Manuscript Results: A significantly higher accumulation of 12-[18F]DDAHA was observed in MCF-7 and MDA-MB-435 cells in vitro as compared to U87, MiaPaCa, and MCF10A, which was consistent with levels of SIRT2 expression. Initial in vivo studies using 12-[18F]DDAHA conducted in a 9L glioma-bearing rats were discouraging, due to rapid defluorination of this radiotracer upon intravenous administration, as evidenced by significant accumulation of F-18 radioactivity in the skull and other bones, which confounded the interpretation of images of radiotracer accumulation within the tumor and other regions of the brain. Conclusions: The next generation of SIRT2-specific radiotracers resistant to systemic defluorination should be developed using alternative sites of radiofluorination on the aliphatic Author Manuscript This article is distributed under the terms of the Creative Commons Attribution 4. 0 International License ( http:// creativecommons org/licenses/by/4 0/) which permits unrestricted use distribution and reproduction in any medium ( p creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriatecredit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Correspondence to: Juri Gelovani; juri.gelovani@wayne.edu. Compliance with Ethical Standards The authors declare that they have no conflict of interest. Page 2 Bonomi et al. chain of DDAHA. A SIRT2-selective radiotracer may provide information about SIRT2 expression and activity in tumors and normal organs and tissues, which may help to better understand the roles of SIRT2 in different diseases. chain of DDAHA. A SIRT2-selective radiotracer may provide information about SIRT2 expression and activity in tumors and normal organs and tissues, which may help to better understand the roles of SIRT2 in different diseases. Keywords eywords SIRT2; Epigenetic Regulation; F-18; Radiotracer; Positron Emission Tomography; Brain Synthesis of ω-Fluorinated Acyl Chains Details of chemical synthesis and analytical procedures can be found in Electronic Supplementary Materials (ESM) online. Bonomi et al. Furthermore, the SAR of these compounds with SIRT2 was investigated using in silico modeling to better understand the observed trends (Fig. S1 Electronic Supplementary Material (ESM)). Based on these studies, the lead compound, the Boc-Lys(12- Author Manuscript Author Manuscript SIRT Enzyme Assay Recombinant SIRT1–7 enzymes and BPS1 reference substrate for SIRT2, an oligopeptide corresponding to 379–382 of p53 (Arg-His-Lys-Lys(Ac)-AMC) [16, 17] were purchased from BPS Bioscience (San Diego, CA) and tested using fluorogenic assay. Further details can be found in ESM online. Author Manuscript Bonomi et al. Bonomi et al. To explore the efficiency with which SIRT2 cleaves larger leaving groups, a focused library of potential substrates was synthesized with tert-butyloxycarbonyl-lysine- Author Manuscript Author Ma Author Manuscript To explore the efficiency with which SIRT2 cleaves larger leaving groups, a focused library of potential substrates was synthesized with tert-butyloxycarbonyl-lysine- aminomethylcoumarin (Boc-Lys-AMC) backbone derivatized on the free amino terminus of lysine with a fluoroalkyl chain varying between 3 and 16 carbons in length. Additionally, the influence of fluorine substitution on the ω-carbon in the alkyl chain on substrate affinity and specificity for SIRT2 was evaluated as well. The library of compounds was studied using a fluorogenic enzyme assay with recombinant SIRT2 to determine trends in catalytic efficiency. Furthermore, the SAR of these compounds with SIRT2 was investigated using in silico modeling to better understand the observed trends (Fig. S1 Electronic Supplementary Material (ESM)). Based on these studies, the lead compound, the Boc-Lys(12- fluorododecanoyl)-AMC, was identified and its analogue with an aminohexanoicanilide (AHA) backbone was synthesized (12-fluorododecanoicAHA, 12-FDDAHA). The AHA backbone has been used previously to generate effective HDAC class IIa-specific radiotracers: [18F]FAHA and [18F]TFAHA [14, 15]. Additionally, the radiolabeled analogue of the lead compound, 12-[18F]DDAHA, was synthesized from 12-iodododecanoic and 12- bromododecanoic precursors. Finally, the uptake of 12-[18F]DDAHA was studied in vitro to determine the magnitude of radiotracer accumulation in cells with high versus low SIRT2 expression. However, initial in vivo studies using 12-[18F]DDAHA conducted in a 9L gliomabearing rats were discouraging, due to rapid defluorination of this radiotracer upon intravenous administration, as evidenced by significant accumulation of F-18 radioactivity in the skull and other bones, which confounded the interpretation of images of radiotracer accumulation within the tumor and other regions of the brain. Due to these observations, full characterization of this radiotracer in vivo (i.e., blocking studies) has not been performed. The next generation of SIRT2-specific radiotracers with reduced systemic defluorination should be developed using alternative sites of radiofluorination on the aliphatic chain of DDAHA (i.e., the 3-position). aminomethylcoumarin (Boc-Lys-AMC) backbone derivatized on the free amino terminus of lysine with a fluoroalkyl chain varying between 3 and 16 carbons in length. Additionally, the influence of fluorine substitution on the ω-carbon in the alkyl chain on substrate affinity and specificity for SIRT2 was evaluated as well. The library of compounds was studied using a fluorogenic enzyme assay with recombinant SIRT2 to determine trends in catalytic efficiency. Introduction In particular, it has been demonstrated that SIRT2 can cleave a myristoyl group from the myristoyl(K19 and K20)TNFα [13]. Mol Imaging Biol. Author manuscript; available in PMC 2019 October 28. Page 3 Mol Imaging Biol. Author manuscript; available in PMC 2019 October 28. Histology and Immunohistochemistry After the completion of in vivo imaging studies, animals were euthanized under anesthesia and the brain was extracted and prepared for immunohistochemical staining to visualize SIRT2 expression. Details can be found in ESM online. Results The library of compounds with terminally fluorinated acyl chains was synthesized through traditional coupling methods of the fluorinated carboxylic acid chain with the lysine amino derivative via in-situ formation of an acetyl chloride in moderate yields of 20–55 %. Synthesis of the fluorinated carboxylic acid chains employed a novel approach: diethylaminosulfur trifluoride was used for non-selective fluorination of the n- hydroxycarboxylic acid forming the acetyl fluoride and fluorination of the terminal carbon. Selective hydrolysis of the acetyl fluoride was achieved using flash chromatography over silica gel to form a mono-ω-fluorinated chain due to the abrupt change in pH. The purity of final compounds in the library was 9 95 %, as determined by 1H-NMR, 13C-NMR, 19F- NMR, and HRMS (Fig. 1a). Author Manuscript Compound 7 was synthesized from Cbz-Lys-OH (Bachem) and Boc-Lys-AMC (Bachem), which was derivatized to Boc-Lys(myr)-AMC. Following myristoylation, the Boc protecting group was removed and the resulting free lysine amino terminus was coupled with the Cbz- Lys-OH group. The four-step synthesis yielded 7 in a yield of 12 % with 9 95 % purity as determined by 1HNMR, 13C-NMR, and HRMS (Fig. 1b). Author Manuscript PET/CT/MR Imaging g g PET imaging with 12-[18F]DDAHA was conducted in a similar manner as previously published [15]. Details can be found in the ESM online. g g PET imaging with 12-[18F]DDAHA was conducted in a similar manner as previously published [15]. Details can be found in the ESM online. Author Manuscript Cell Cultures and In Vitro Radiotracer Uptake Studies Cell Cultures and In Vitro Radiotracer Uptake Studies The U87MG, MCF-7, MDA-MB-435, and MiaPaca cell lines were used to demonstrate differential levels of 12[18F]DDAHA cellular accumulation. Further details can be found in ESM online. Author Manuscript Author Manuscript Mol Imaging Biol. Author manuscript; available in PMC 2019 October 28. Computational Docking Studies Schrodinger Suite (New York, NY) with the Glide [18] docking program was used for simulating ligand-protein interactions in silico. Further details can be found in ESM online. Page 4 Page 4 Bonomi et al. Radiosynthesis 12-Fluorododecanoyl-AHA (12-FDDAHA) and 12-[18F]dodecanoylAHA (12- [18F]DDAHA) were synthesized as non-radiolabeled and radiolabeled analog of 4 (Fig. 1c– e, S1 ESM). The initial radiosynthesis of 12-[18F]DDAHA (11) was performed using a brominated precursor, 12Br-DDAHA (9); however, due to low radiochemical yield, the iodinated precursor, 12I-DDAHA (10), was used for optimization of the radiolabeling procedure and improvement of radiosynthetic yield. The iodinated precursor (10) was synthesized from the brominated precursor in a high yield of 73 % using literature procedure Mol Imaging Biol. Author manuscript; available in PMC 2019 October 28. Bonomi et al. Page 5 [19]. The radiochemical yield achieved with precursor 10 was 5-fold higher than that with precursor 9. The highest radiofluorination yields were achieved using precursor 10 in acetonitrile at 85–90 °C. 12[18F]DDAHA was obtained with purity of > 95 % (Fig. 1d, and S3 ESM) and a decay-corrected radiochemical yield of 12 % (total preparation time 66 min) with specific activity of 259 GBq/mmol. Author Manuscript In Silico Modeling In Silico Modeling In silico modeling studies demonstrated that positioning of compounds 4 and 5 in the SIRT2 active site resulted in a smaller distance between the carbonyl carbon and the nicotinamide- ribose ester linkage (Fig. S4a and S4b ESM). This may contribute to enhanced substrate efficiency of these compounds. Biochemical Characterization Results from in vitro characterization of this library of compounds using fluorogenic assay demonstrated a positive correlation between the substrate efficiency for SIRT2 and the increasing length of the leaving alkyl chain. Compound 5 containing the myristoyl group was the most efficient substrate of SIRT2 with Kcat/Km = 715.6 ± 72.8/s mol followed by compound 4 (fluorododecanoyl) and compound 3 (fluorodecanoyl) with Kcat/Km of 615.4 ± 50.5/s mol and 269.5 ± 52.1/s mol, respectively. In contrast, compound 6 containing the fluoropalmitoyl leaving group exhibited a significant decrease in substrate efficiency for SIRT2 (30.4 ± 7.3/s mol), as compared to compounds containing shorter fluoroalkyl chains. The reference substrate, p53(379–382)K382ac-AMC (BPS1) exhibited Kcat/Km = 275 ± 42/s mol (Fig. 2, Table 1). Author Manuscript To assess the influence of a “cap” modification on SIRT2 selectivity, compounds 4, 5, and 7 were evaluated against a panel of recombinant sirtuins (SIRT1–7). Compound 7 was cleaved efficiently by SIRT2, SIRT3, and, to a lesser extent, by SIRT6. In contrast, 4 and 5 were cleaved efficiently only by SIRT2 (Fig. S2 ESM). To validate the substrate efficiency of 4 with SIRT2, a competitive inhibition assay with AGK2 (SIRT2-specific inhibitor) was performed, which yielded an IC50 of 28.21 μmol of AGK2 (Fig. S2 ESM). Author Manuscript Mol Imaging Biol. Author manuscript; available in PMC 2019 October 28. In Vitro Radiotracer Accumulation Studies MDA-MB-435 and MCF-7 with high levels of SIRT2 expression [20] demonstrated significantly higher levels of 12-[18F]DDAHA accumulation (Fig. 3), as compared to U87MG > MiaPaCa > MCF10A cells, which are known to express lower levels of SIRT2 [20] (very low levels of SIRT2 in U87 reported in Maxwell, et al. 2011 [21]). The extracellular-to-intracellular radioactivity concentration equilibrium was achieved in about 1 min of incubation with 12[18F]DDAHA, as the result of high lipophilicity of this radiotracer cLogP = 5.32 which facilitates passive diffusion of intact (parent) compound across the cell membranes in and out of the cells (data not shown). The accumulation and retention of 12- [18F]DDAHA-derived radioactivity is due to much higher negative polarity of ADPR-O- [18F]dodecanoate, which is the theoretical product of enzymatic reaction of SIRT2 and 12- [18F]DDAHA. Author Manuscript Mol Imaging Biol. Author manuscript; available in PMC 2019 October 28. Page 6 Page 6 Bonomi et al. Immunohistochemical Validation of SIRT2 Expression in 9L Glioma and Normal Brain Structures Author Manuscript Fluorescence microscopy of sections of rat brains revealed heterogeneously distributed patchy appearing circular-shaped regions of SIRT2 expression in 9L gliomas (Fig. 5a, b), as well as in the peripheral infiltrating zones. Immunihistochemical staining for HIF-1a expression in adjacent tissue sections confirmed that the areas of reduced SIRT2 expression in tumors co-localized with areas of increased HIF-1a expression (Fig. 5c, d). In the cerebral cortex, SIRT2 expression was expressed mainly in the neurons, with the highest concentrations in perinuclear regions of pyramidal neurons and lower levels in the axons (Fig. 5e, f). In the hippocampus, the level of SIRT2 expression was highest in CA2 and CA3 neurons and had a similar sub-cellular perinuclear localization pattern, as in cortical neurons (Fig. 5g, h). In the cerebellum, SIRT2 was strongly overexpressed in Purkinje cells, with perinuclear localization stronger than in cortical and hippocampal neurons; SIRT2 was also observed in neurons in the molecular layer at somewhat lower levels of expression than in Purkinje cells, but was almost entirely absent in the granular layer neurons (Fig. 5i, j). Author Manuscript Mol Imaging Biol. Author manuscript; available in PMC 2019 October 28. PET/CT/MR Imaging Author Manuscript Dynamic PET/CT/MR imaging of a 9L glioma tumor-bearing rat with [18F]-12-DDFAHA demonstrated increased tracer derived-radioactivity accumulation and retention within the tumor versus normal brain structures between 15 and 20 min post i.v. administration (Fig. 4). However, interpretation of PET/CT/MR images of [18F]-12-DDFAHAderived radioactivity accumulation in 9L gliomas and in the brain cortex was challenging due to high levels of radioactivity accumulation in the adjacent scull bones, which created “spillover” artifacts (Fig. 4b, c). High levels of radioactivity in the bone were caused by rapid defluorination of [18F]-12-DDFAHA after i.v. administration. To reduce the appearance of bone radioactivity- causing artifacts in brain PET images, we used the NIH + white (max) lookup table for color coding the range of radioactivity concentrations in the images (Fig. 4d). Discussion To develop the first-generation substrate-type radiotracer selective to SIRT2, it was necessary to understand the structure-activity relationships of SIRT2 with various natural and synthetic substrates. Previously, it has been reported that in addition to an acetyl group, SIRT2 can also cleave long acyl chains from lysine residues, including a myristoyl moiety [22]. Other HDAC class III enzymes, such as SIRT3 and SIRT6, have also been reported to cleave myristoylated lysines of the H3K9 peptide [23, 24] and K19–20TNF-α [22]. However, there are no data on the effects of fluorine substitution on the ω-carbon in alkyl chains on different lengths on SIRT2 catalytic efficiency. Author Manuscript Our current studies demonstrate that catalytic efficiency of SIRT2 gradually increases with the elongation of fluoroalkyl chain from 3 to 12 carbons. The catalytic efficiency of SIRT2 for fluorododecanoyl chain (Kcat/Km = 615.4 ± 50.5/s mol) is comparable (difference is not statistically significant) with that of the non-fluorinated myristoyl chain (reference compound, BPS1), which is cleaved most efficiently (Kcat/Km = 716.5 ± 72.8/s mol). In contrast, the catalytic efficiency of SIRT2 was significantly lower for the fluoropalmitoyl Mol Imaging Biol. Author manuscript; available in PMC 2019 October 28. Page 7 Bonomi et al. group (Kcat/Km = 60.4 ± 7.3/s mol). These observations agree with previous reports, demonstrating a similar trend in SIRT2 catalytic efficiency for the H3K9 peptide backbone derivatized with hexanoyl, octanoyl, decanoyl, dodecanoyl, and myristoyl groups, but significantly less efficiently the palmitoyl and lipoyl-derivatized H3K9 peptide [23, 24]. Similarly, using TNF-α peptide backbone with varying chain lengths to a fluorophore or quencher moiety the longer chain length (11 carbons) resulted in much improved catalytic efficiency over the shorter (6 or 8 carbons) or very long (13 carbons) chain lengths [25]. However, despite the similarities in trends, the absolute Kcat/Km values obtained in the current study for different alkyl chains with Boc-Lys-AMC backbone are significantly lower than those reported previously using a H3K9 or TNF-α peptide [23–25], which can be explained, at least in part, by the higher binding efficiency of a peptide “cap” group, as compared to Boc-Lys-AMC. Similarly, reports of a “cap” group containing either p53 or TNF-α peptide residues on one side and an AMC group on the other demonstrate reduced Kcat/Km as compared to compounds with a full peptide cap [23, 25, 26]. Also, the observed differences may be due to the presence of electronegative ω-terminal fluorine. Mol Imaging Biol. Author manuscript; available in PMC 2019 October 28. Discussion In the current study, the Kcat/Km values were determined by measuring the rate of the AMC cleavage product formation [26], whereas in some of the previously reported data, Kcat/Km values were determined by measuring the rate of NAD+ consumption, which may also explain the differences in absolute Kcat/Km values between studies [23]. Author Manuscript Auth Author Manuscript To examine the selectivity of compounds for SIRT2 among all SIRT enzymes, three of the most efficient substrates, 4, 5, and 7 were screened against a panel of recombinant SIRT enzymes (SIRT1–7). The aim was to elucidate the differences in SIRT selectivity that may occur as a result of altering the cap (mono- vs di-lysine cap), the leaving group (12- vs 14- carbon chain), and last the terminal fluorine atom (12F-carbon vs 14-carbon). Our results indicate that compound 7, with a di-lysine cap, was cleaved more effectively by SIRT3 and SIRT6 than compound 5, with a mono-lysine cap. Therefore, 5 is more selective to SIRT2 as it shows very little cleavage by other SIRT enzymes. Both 4 (12F-carbon chain) and 5 (14carbon chain) are cleaved effectively by SIRT2 and not by any other SIRT enzymes, indicating that both leaving groups are selective for SIRT2. These findings are consistent with other reports in the literature [22, 24, 27] and demonstrate that leaving group specificity may not entirely define the selectivity of a substrate to a certain SIRT enzyme. Altering the cap group can have a significant impact on the efficiency with which the SIRT enzyme cleaves a substrate. Also, we assessed the effectiveness of the lead compound 4 as a synthetic substrate for SIRT2 using a potent selective SIRT2 inhibitor AGK2 [28]. A competitive inhibition study [28] was performed using increasing concentrations of AGK2 with 4 at its apparent Km concentration (8 μM) for SIRT2. The observed inhibition of SIRT2 by AGK2 at IC50 of 28.21 μM using 4 as substrate was comparable to previously reported IC50 values of 3– 5 μM obtained with myristoylated or acetylated substrates of SIRT2 [29, 30]. As demonstrated by the comparable catalytic efficiencies of 4 and BPS1 (p53 (379–382)K382ac-AMC) this data further confirm that 4 is an efficient substrate of SIRT2. Author Manuscript Based on the results of biochemical and in silico modeling studies (additional discussion can be found in Supplemental Materials), we have chosen the dodecanoyl chain fluorinated on Page 8 Page 8 Bonomi et al. Discussion the ω-position for coupling to an AHA backbone (Fig. S2 ESM) to generate the SIRT2specific 18F-labeled radiotracer termed 12-[18F]DDAHA. We did not study the 14- fluoromyristoyl-Boc-Lys-AMC and 14-[18F]fluoromyristoyl-AHA, because Boc- Lys(12fluorododecanoyl)-AMC exhibits 2/3 efficiency of Boc-Lys(myristoyl)-AMC as selective substrate for SIRT2 and because radiotracers with smaller molecular weight have pharmacokinetic advantages over radiotracers with larger molecular weight. The AHA backbone was chosen to increase the lipophilicity of the radiotracer and permeability across cell membranes, which would not be possible with a larger peptidic backbone. Accordingly, the radiotracer 12-[18F]DDAHA has a cLogP value of 5.32 indicating a strong ability to passively cross the cell membrane in and out of the cell. While the change in “cap” group may alter the selectivity to SIRT2, it has a lesser effect on the catalytic efficiency, as this is determined largely by the leaving group structure rather than the cap group. Nevertheless, the synthesis and evaluation of these compounds are currently underway and will be reported separately. For synthesis of 12-[18F]DDAHA, we adapted methods for radiolabeling of ω-[18F]substituted long-chain fatty acids [19] that are used for PET imaging of cardiac metabolism [31]. Our studies confirmed previous reports that ω-iodinated dodecanoyl precursor provides a 3-fold higher radiosynthetic yield than the ω-brominated precursor [19]. The radiosynthetic yield for 12-[18F]DDAHA could be optimized further by using microwave-assisted heating [19]. Author Manuscript Aut Author Manuscript In vitro radiotracer uptake studies in cell lines with varying degrees of SIRT2 expression provided initial evidence for the efficacy of 12-[18F]DDAHA as a radiotracer for quantification of SIRT2 expression-activity. The level of 12-[18F]DDAHA-derived radioactivity accumulation was significantly increased in MDA-MB-435 melanoma and MCF-7 breast adenocarcinoma and to a much lesser degree, in U87MG glioma, as compared to MiaPaCa pancreatic carcinoma and MCF10A immortalized breast epithelial cells. MCF-7 cells are known to have increased SIRT2 expression [20, 32]. Also, MiaPaCa cells have demonstrated increased levels of SIRT2 expression via Western blot analysis [11]. The non- tumorigenic epithelial cells, MCF10A, exhibit significantly lower levels of SIRT2 expression [20] and served as a lower-expressing control for this study. These results indicate levels of 12-[18F]DDAHA-derived radioactivity accumulation correspond to levels of SIRT2 expression as reported elsewhere for MCF-7, MDA-MB-435, U87MG, MiaPaCa, and MCF10A cell lines. Despite the high lipophilicity of the fluoroalkyl leaving group, the mechanism of substrate cleavage by SIRT enzymes allows for a radioactive leaving group to be entrapped in the cell, at least transiently. Mol Imaging Biol. Author manuscript; available in PMC 2019 October 28. Discussion Specifically, SIRT2 enzymatic cleavage mechanism results in formation of an ester linkage between the alkyl leaving group and O-ADPR (Fig. S1 ESM), resulting in 18F-fluoroalkyl-O-ADPR product which cannot cross a cell membrane because it is quite large and very polar (containing 2 negatively charged phosphate groups) (Fig. S1 ESM). Therefore, SIRT2-mediated cleavage of 18F-fluoroalkyl group from 12[18F]DDAHA with formation of 18F-fluoroalkyl-O-ADPR complex represents the rate-limiting step in the transient cellular entrapment of 12-[18F]DDAHA-derived radioactivity. The eventual efflux of 12-[18F]DDAHA-derived radioactivity from intracerebral 9L glioma also suggests that 18Ffluoroalkyl-O-ADPR is degradable and that 18F-fluoroalkyl moiety eventually diffuses out of the cells. The removal of the acetyl or longer acyl chain moieties from O-ADPR Author Manuscript Bonomi et al. Page 9 Page 9 group has been hypothesized by others to occur by way of ARH3 cleavage or possible methanolysis to form 1′O-methyl′ ADPR [33, 34]. Additionally, a nucleophilic attack from lysine side chains of other proteins in close proximity may also de-acetylate the O-AADPR. Further studies are needed to fully characterize the half-life of O-12-[18F]DD-ADPR and residence time for the 12-[18F]dodecanoyl moiety within the cell. Although this entrapment may be transient, it provides a sufficient retention time within the cells with high SIRT2 expression-activity for PET imaging. Author Manuscript Author Manuscript Furthermore, our initial in vivo PET imaging studies using 12-[18F]DDAHA conducted in a 9L glioma-bearing rats were discouraging, due to rapid systemic defluorination of this radiotracer upon intravenous administration. This was evidenced by significant accumulation of F-18 radioactivity in the skull and other bones, which confounded the interpretation of images of radiotracer accumulation within the tumor and other regions of the brain. Therefore, additional PET/CT MR images in panel Fig. 4d are shown in NIH + white (max) look-up table, and the resulting PET/ MRI fusion images revealed 12-[18F]DDAHA-derived radioactivity accumulation in 9L gliomas and normal brain regions, such as the hippocampus. This proof of concept imaging study demonstrated adequate BBB permeability, as evidenced by the rapid equilibration of 12-[18F]DDAHA radioactivity between blood and normal brain tissue (Fig. 4). Progressive accumulation of 12- [18F]DDAHA-derived radioactivity in normal brain structures with high levels of SIRT2 expression and especially in 9L tumor lesions (Fig. 4) during the first 15-min post intravenous administration supports the proposed mechanism of SIRT2-mediated accumulation and transient retention in tissues. Static images of 12-[18F]DDAHA accumulation/retention (SUV) obtained between 15 and 20 min post i.v. Mol Imaging Biol. Author manuscript; available in PMC 2019 October 28. Bonomi et al. Bonomi et al. In the normal brain structures, the highest level of SIRT2 expression was observed in hippocampal CA2 and CA3 neurons, followed by pyramidal neurons in cerebral cortex, and cerebellar Purkinje cells. The subcellular localization of SIRT2 was predominantly perinuclear with lower levels of diffuse distribution in the rest of the neuronal cytoplasm and axons. These observations of neuronal-specific subcellular localization of SIRT2 in the brain are in agreement with previous reports [21] which demonstrated that SIRT2 expression in the murine brain is higher in hippocampal CA2–3 neurons, cortical neurons, and in cerebellar Purkinje cells and molecular layer neurons, but not in the granular layer neurons or other cells of oligedendroglial lineage. Thus, in situ microscopy results can serve as the basis for interpretation of in vivo PET/CT/MRI images and confirm that the increased levels of radiotracer accumulation in intracerebral 9L gliomas and in certain normal brain structures are mediated by increased SIRT2 expression-activity levels, as compared to other brain structures expressing lower levels of SIRT2. Author Manuscript Autho Author Manuscript Author Manuscript Also, this study provides grounds for development of the next generation of SIRT2-specific radiotracers. Several previous studies have reported defluorination of ω[18F]substituted fatty acids in rodents following intravenous injection [31] but significantly lower levels of defluorination occurred in higher order mammals (i.e. pigs and dogs) and in humans, due to a decrease in level of expression of defluorinating enzymes [31]. An alternative approach to decrease defluorination in a second-generation tracer would be to radiolabel at the ω−3 position of the acyl chain. Additionally, carbon-11 labeling of the dodecanoyl chain could be accomplished similar to 11C-palmitate [37, 38]. Another approach to decrease the magnitude of systemic defluorination could involve pre-treatment with miconazole (CYP450 2E1 inhibitor) which was shown effective for 18F-FCWAY [39], which, however, is not practical for clinical translation because of potential for high patient to patient variability. Further radiotracer development is underway and will be reported elsewhere. Author Manuscript SIRT2 is an emerging target for therapy of various neurologic diseases, including neurodegeneration [10], as well as cardiac [40] and metabolic diseases [40, 41]. Many SIRT2-targeted inhibitors are currently in development and entering the early-stage clinical trials, further driving the need for continuation of this work towards development and clinical translation of second-generation SIRT2-selective radiotracer. Mol Imaging Biol. Author manuscript; available in PMC 2019 October 28. Discussion injection with highest tumor-to-normal brain and tumor-to-muscle SUV ratios may provide more specific images of SIRT2 expression-activity in target tissues, if defluorination would be minimized in the next generation of radiotracers. Because of the observed excessive defluorination of 12-[18F]DDAHA upon intravenous administration, a more complete characterization of this radiotracer in vivo (i.e., blocking studies) has not been performed. Author Manuscript To validate the results of PET/CT/MR imaging with 12[18F]DDAHA and to assess the cell specificity and heterogeneity of SIRT2 expression-activity in 9L gliomas and in different brain structures, we used immunofluorescent microscopy (IFM) in 9L glioma-bearing rat brain tissue sections. Fluorescence microscopy of tissue sections of intracerebral 9L gliomas growing in the rat brains stained with anti-SIRT2 fluorescent antibodies revealed heterogeneously distributed patchy-appearing circular regions of SIRT2 expression in core regions in peripheral infiltrating zones of 9L gliomas. Such pattern of expression suggested that SIRT2 is upregulated in well-perfused regions of 9L gliomas, whereas the expression of SIRT2 in hypoxic-appearing regions is significantly reduced, which was confirmed immunohistochemically by presence of HIF-1α-expression in the same regions in adjacent tissue sections. These results are consistent with previous reports of regulatory loops of HIF-1a-SIRT2, involving HIF-1α-mediated transcriptional repression of SIRT2 expression [35], as well as SIRT2-mediated deacetylation of Lys709, which increases prolyl- hydroxylase binding and ubiquitin-mediated degradation of HIF-1α [36]. Author Manuscript Mol Imaging Biol. 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Conclusions In summary, guided by the results of biochemical and in silico modeling studies of structure- activity relationships between SIRT2 and a peptide-mimetic backbone containing a lysine derivatized with fluoroalkyl chains of different length, we have developed and tested in vitro and in vivo a novel, first-generation, SIRT2-selective substrate-type radiotracer, 12- [18F]DDAHA. Current proof of principal studies, demonstrated suitability for radiofluorinated DDAHA assessment of SIRT2 expression-activity using in vitro radiotracer uptake assays. Molecular imaging with PET using next-generation SIRT2-specific radiofluorinated DDAHA-based radiotracer, which is resistant to defluorination in vivo, should provide valuable information about the location and magnitude of SIRT2 expression and activity in the brain and in tumors. PET imaging may help to determine the mechanistic, Author Manuscript Bonomi et al. 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Page 14 Fig. 1. a The focused library of compounds synthesized with fluroalkyl chain-derivatized lysines. The backbone used tert-butyloxycarbonyl-L-lysine-7-amino-3-methylcoumarin (Boc-Lys- AMC) was coupled to fluoroalkyl chains, varying in length from 3 carbons to 16 carbons using acetyl chloride coupling. Mol Imaging Biol. Author manuscript; available in PMC 2019 October 28. Mol Imaging Biol. Author manuscript; available in PMC 2019 October 28. References b To experimentally determine the effect of the cap on the selectivity of a substrate for SIRT2, we developed a di-lysine substrate, with a more peptidic backbone, Cbz-lys-lys(myr)-AMC. This allows for head-to-head comparison between the Boc-lys(myr)-AMC. c The synthesis of both the cold (non-radiolabeled) analogue (9) and d Bonomi et al. Page 14 Author Manuscript Author Manuscript Author Manuscript Author Manuscript g a The focused library of compounds synthesized with fluroalkyl chain-derivatized lysines. The backbone used tert-butyloxycarbonyl-L-lysine-7-amino-3-methylcoumarin (Boc-Lys- AMC) was coupled to fluoroalkyl chains, varying in length from 3 carbons to 16 carbons using acetyl chloride coupling. b To experimentally determine the effect of the cap on the selectivity of a substrate for SIRT2, we developed a di-lysine substrate, with a more peptidic backbone, Cbz-lys-lys(myr)-AMC. This allows for head-to-head comparison between the Boc-lys(myr)-AMC. c The synthesis of both the cold (non-radiolabeled) analogue (9) and d The backbone used tert-butyloxycarbonyl-L-lysine-7-amino-3-methylcoumarin (Boc-Lys- AMC) was coupled to fluoroalkyl chains, varying in length from 3 carbons to 16 carbons using acetyl chloride coupling. b To experimentally determine the effect of the cap on the selectivity of a substrate for SIRT2, we developed a di-lysine substrate, with a more peptidic backbone, Cbz-lys-lys(myr)-AMC. This allows for head-to-head comparison between the Boc-lys(myr)-AMC. c The synthesis of both the cold (non-radiolabeled) analogue (9) and d Mol Imaging Biol. Author manuscript; available in PMC 2019 October 28. precursors for 12-[18F]DDAHA, 12-BrDDAHA (9), and 12-IDDAHA (10). e The 12- [18F]DDAHA (11) radiotracer was synthesized from the iodinated precursor and the in dry acetonitrile in 85 °C for 15 min. et al. Page 15 Bonomi et al. Page 15 Page 15 precursors for 12-[18F]DDAHA, 12-BrDDAHA (9), and 12-IDDAHA (10). e The 12- [18F]DDAHA (11) radiotracer was synthesized from the iodinated precursor and the in dry acetonitrile in 85 °C for 15 min. precursors for 12-[18F]DDAHA, 12-BrDDAHA (9), and 12-IDDAHA (10). e The 12- [18F]DDAHA (11) radiotracer was synthesized from the iodinated precursor and the in dry acetonitrile in 85 °C for 15 min. Author Manuscript Author Manuscript Author Manuscript Fig. 2. Biochemical assay data from Fluor de Lys assay®. a The catalytic efficiency (Kcat/Km) for SIRT2 against different length fluoroalkyl chain leaving groups to assess the structure activity relationship between these compounds and SIRT2. The error bars represent the standard deviation of N = 6 experiments. These values were taken from 15-min incubation to capture linear phase kinetics. Mol Imaging Biol. Author manuscript; available in PMC 2019 October 28. References b Compound 7 is not selective for SIRT2 whereas compounds 4 and 5 are selective for SIRT2. These results were taken from 40-min incubation of substrate with enzyme. The error bars represent the standard deviations of the means with significance of P < 0.05 denoted by , as determined by two-way ANOVA. c The table details the effect on Km (amount of substrate necessary for half of Vmax), Vmax (the maximal turnover velocity for a given substrate in SIRT2), and Kcat (the catalytic efficiency for a given substrate with SIRT2). This data is determined in linear phase kinetics of SIRT2 with 15-min of incubation; therefore, this data is slightly different than the values in Fig. 2b. Bonomi et al. Page 16 Page 16 Bonomi et al. Bonomi et al. Page 16 Bonomi et al. Author Manuscript Fig. 2. Fig. 2. Biochemical assay data from Fluor de Lys assay®. a The catalytic efficiency (Kcat/Km) for Fig. 2. Biochemical assay data from Fluor de Lys assay®. a The catalytic efficiency (Kcat/Km) for SIRT2 against different length fluoroalkyl chain leaving groups to assess the structure activity relationship between these compounds and SIRT2. The error bars represent the standard deviation of N = 6 experiments. These values were taken from 15-min incubation to capture linear phase kinetics. b Compound 7 is not selective for SIRT2 whereas compounds 4 and 5 are selective for SIRT2. These results were taken from 40-min incubation of substrate with enzyme. The error bars represent the standard deviations of the means with significance of P < 0.05 denoted by , as determined by two-way ANOVA. c The table details the effect on Km (amount of substrate necessary for half of Vmax), Vmax (the maximal turnover velocity for a given substrate in SIRT2), and Kcat (the catalytic efficiency for a given substrate with SIRT2). This data is determined in linear phase kinetics of SIRT2 with 15-min of incubation; therefore, this data is slightly different than the values in Fig. 2b. Author Manuscript Aut g Biochemical assay data from Fluor de Lys assay®. a The catalytic efficiency (Kcat/Km) for SIRT2 against different length fluoroalkyl chain leaving groups to assess the structure activity relationship between these compounds and SIRT2. The error bars represent the standard deviation of N = 6 experiments. These values were taken from 15-min incubation to capture linear phase kinetics. Mol Imaging Biol. Author manuscript; available in PMC 2019 October 28. References b Compound 7 is not selective for SIRT2 whereas compounds 4 and 5 are selective for SIRT2. These results were taken from 40-min incubation of substrate with enzyme. The error bars represent the standard deviations of the means with significance of P < 0.05 denoted by , as determined by two-way ANOVA. c The table details the effect on Km (amount of substrate necessary for half of Vmax), Vmax (the maximal turnover velocity for a given substrate in SIRT2), and Kcat (the catalytic efficiency for a given substrate with SIRT2). This data is determined in linear phase kinetics of SIRT2 with 15-min of incubation; therefore, this data is slightly different than the values in Fig. 2b. Author Manuscript Author Manuscript Author Manuscript Page 17 Bonomi et al. Fig. 3. The radiotracer 12-[18F]DDAHA (11) was used in cellular uptake studies for tumor cell lines. The error bars represent the standard deviation for N = 4 for each cell line. Where * represents P < 0.05 as determined by one-way ANOVA and Tukey’s multiple comparisons test, this value is significantly larger than the other three values. Page Author Manuscript Author Manuscript Fig. 3. The radiotracer 12-[18F]DDAHA (11) was used in cellular uptake studies for tumor cell lines. The error bars represent the standard deviation for N = 4 for each cell line. Where represents P < 0.05 as determined by one-way ANOVA and Tukey’s multiple comparisons test, this value is significantly larger than the other three values. g. 3. The radiotracer 12-[18F]DDAHA (11) was used in cellular uptake studies for tumor cell lines. The error bars represent the standard deviation for N = 4 for each cell line. Where ** represents P < 0.05 as determined by one-way ANOVA and Tukey’s multiple comparisons test, this value is significantly larger than the other three values. Author Manuscript Author Manuscript Bonomi et al. Page 18 Fig. 4. a T2-weighted MR image sliced coronally through the 9L glioma lesion and hippocampus with red arrows indicating areas of tumor lesion. b Dynamic PET images at 15-min post administration of 12-[18F]DDAHA NIH lookup table (LUT). c Dynamic PET images post administration of 12-[18F]DDAHA overlaid onto T2-weighted MR images using the NIH LUT. Mol Imaging Biol. Author manuscript; available in PMC 2019 October 28. References d The same images as in c, but with the NIH + white LUT, to better visualize the internal structures due to the high degree of defluorination occurring, which is seen as F-18 accumulation in the bone causing a “halo” effect around the cortical region due to partial volume effect of bone signal into cortex. e Time activity curves for each region of interest within the brain over 60 min of dynamic PET images displayed as log (SUV) vs time. The error bars represent standard deviation of the voxel values within each ROI. f A visual representation of differential washout between tumor and other regions of the brain at 15- min post i.v. administration of 12-[18F]DDAHA, where error bars represent standard deviations and * or ** represent P < 0.05 as found from one-way ANOVA. Author Manuscript Author Manuscript Author Manuscript Author Manuscript g a T2-weighted MR image sliced coronally through the 9L glioma lesion and hippocampus with red arrows indicating areas of tumor lesion. b Dynamic PET images at 15-min post administration of 12-[18F]DDAHA NIH lookup table (LUT). c Dynamic PET images post administration of 12-[18F]DDAHA overlaid onto T2-weighted MR images using the NIH LUT. d The same images as in c, but with the NIH + white LUT, to better visualize the internal structures due to the high degree of defluorination occurring, which is seen as F-18 accumulation in the bone causing a “halo” effect around the cortical region due to partial volume effect of bone signal into cortex. e Time activity curves for each region of interest within the brain over 60 min of dynamic PET images displayed as log (SUV) vs time. The error bars represent standard deviation of the voxel values within each ROI. f A visual representation of differential washout between tumor and other regions of the brain at 15- min post i.v. administration of 12-[18F]DDAHA, where error bars represent standard deviations and * or ** represent P < 0.05 as found from one-way ANOVA. Author Manuscript Mol Imaging Biol. Author manuscript; available in PMC 2019 October 28. Bonomi et al. Page 19 Fig. 5. Fluorescence and light microscopy images of different regions of immunostained tissu sections of the rat brain with 9L gliomas obtained at × 200 (a, c, e, g, i) and × 400 (b, j). Mol Imaging Biol. Author manuscript; available in PMC 2019 October 28. Mol Imaging Biol. Author manuscript; available in PMC 2019 October 28. high levels of HIF-1α expression (c, d) corresponding to regions of decreased SIRT2 expression in panel. Mol Imaging Biol. Author manuscript; available in PMC 2019 October 28. References Immunofluorescent images of SIRT2 expression in (a, b) 9L glioma, (e, f) contralat brain cortex, (g, h) CA2–3 region of contralateral hippocampus, and (i, j) cerebellum. Immunohistochemically stained adjacent tissue sections of intracerebral 9L gliomas re Author Manuscript Author Manuscript Fig. 5. Fluorescence and light microscopy images of different regions of immunostained tissue sections of the rat brain with 9L gliomas obtained at × 200 (a, c, e, g, i) and × 400 (b, d, f, h, j). Immunofluorescent images of SIRT2 expression in (a, b) 9L glioma, (e, f) contralateral brain cortex, (g, h) CA2–3 region of contralateral hippocampus, and (i, j) cerebellum. Immunohistochemically stained adjacent tissue sections of intracerebral 9L gliomas reveal g Fluorescence and light microscopy images of different regions of immunostained tissue sections of the rat brain with 9L gliomas obtained at × 200 (a, c, e, g, i) and × 400 (b, d, f, h, j). Immunofluorescent images of SIRT2 expression in (a, b) 9L glioma, (e, f) contralateral brain cortex, (g, h) CA2–3 region of contralateral hippocampus, and (i, j) cerebellum. Immunohistochemically stained adjacent tissue sections of intracerebral 9L gliomas reveal Mol Imaging Biol. Author manuscript; available in PMC 2019 October 28. high levels of HIF-1α expression (c, d) corresponding to regions of decreased SIRT2 expression in panel. Page 20 Page 20 Bonomi et al. high levels of HIF-1α expression (c, d) corresponding to regions of decreased SIRT2 expression in panel. Author Manuscript Author Manuscript Author Manuscript Page 21 Bonomi et al. Author Manuscript Kinetic parameters for SIRT2 and various chain length compounds Compound Km (μM) SD Vmax (μM/s) SD Kcat (/sec) SD Kcat/Km (/s mol) SD Boc-Lys(3-F-propanoyl)-AMC (1) 55.53 11.4 0.15 0.016 1.41 × 10−3 5.0 × 10−4 26.3 5.1 Boc-Lys(6-F-hexanoyl)-AMC (2) 44.89 4.56 0.28 0.013 2.58 × 10−3 4.1 × 10−4 57.4 5.2 Boc-Lys(10-F-decanoyl)-AMC (3) 18.03 4.07 0.52 0.041 4.86 × 10−3 1.3 × 10−4 269.5 52 Boc-Lys(12-F-dodecanoyl)-AMC (4) 8.07 0.79 0.54 0.014 4.97 × 10−3 4.2 × 10−4 615.4 50 Boc-Lys(myristoyl)-AMC (5) 2.36 0.22 0.26 0.010 2.38 × 10−3 3.0 × 10−4 715.5 73 Boc-Lys(palmitoyl)-AMC (6) 51.15 6.91 0.33 0.022 3.09 × 10−3 6.8 × 10−4 60.4 7.3 Cbz-Lys-Lys(myr)-AMC (7) 10.36 1.30 0.50 0.022 5.51 × 10−3 6.9 × 10−4 532.3 56 BPS1 31.73 5.57 0.95 0.022 8.78 × 10−3 7.0 × 10−4 275.0 43 M l I i Bi l A h Author Manuscript Author Manuscript

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